Abstract
The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John’s wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR.
SIGNIFICANCE STATEMENT Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.
Introduction
Nuclear receptors (NRs) function as transcriptional regulators and play a central role in maintaining homeostasis in living beings. Upon ligand activation, NRs exert transcriptional activity on their target genes, adapt their expression level, and thus their activity to the present requirements, including the state of health versus disease, exposure to exogenous compounds, or developmental processes (Sladek, 2011; Delfosse et al., 2015). One NR of extraordinary relevance in drug metabolism is the xenosensor pregnane X receptor (PXR; NR1I2), which acts as a transcriptional modulator of a variety of genes involved in drug metabolism and transport (Sladek, 2011). One characteristic of PXR is its species specificity (Jones et al., 2000). In detail, comparing PXR in humans and rodents in terms of target genes shows a broad overlap (Ihunnah et al., 2011); therefore a comparable function in the regulation of drug metabolism is expected in all species. The overlap in target genes is assumed to be explained by the highly conserved DNA binding domain of PXR, where the rodent and human PXR share a 96% sequence homology (Kliewer et al., 2002). Nevertheless, there are species-specific differences in the activating ligands of human and rodent PXR particularly of rat and mouse. Accordingly, it is challenging to translate findings in these preclinical species to the human system. The differences in activating ligands may be due to the low sequence similarity within the ligand-binding domain (LBD) of the protein, where the human LBD shares only 77% or 76% sequence similarity with the mouse or rat LBD, respectively (Ma et al., 2008; AbdulHameed et al., 2016). Known examples of human specific PXR ligands are rifampicin and hyperforin (Tirona et al., 2004).
Hyperforin is found in St. John’s wort (SJW) extracts, which have been successfully used as herbal remedies in the treatment of mild to moderate depression (Friede et al., 2001). However, herbal remedies containing hyperforin bear the potential for drug-herb interactions. Indeed, in humans it is well established that coadministration of SJW with substrates of the cytochrome P450 enzyme CYP3A4 or the efflux transporter MDR1 (ABCB1, P-glycoprotein) can result in severely altered bioavailability of their substrates, as most impressively shown for cyclosporine and digoxin (Johne et al., 1999; Ruschitzka et al., 2000). Despite the in vitro data on the lack of activation of rPXR by hyperforin (Tirona et al., 2004), there are reports showing an influence of SJW on known PXR target genes in rodents. Ho et al. tested the impact of a 15-day SJW treatment (150 or 300 mg/d) on the pharmacokinetics of indinavir, a known CYP3A and MDR1 substrate in rats. They observed a reduction in systemic indinavir exposure (AUC0-inf, indinavir alone vs. indinavir plus SJW 150 mg/kg vs. indinavir plus SJW 300 mg/kg; 5.20 ± 1.09 vs. 0.76 ± 0.50 vs. 1.07 ± 0.26 µgh/ml), which is most likely explained by a change in oral bioavailability as observed in an in situ single-pass intestinal perfusion model. Moreover, the rats treated with 150 mg/kg SJW exhibited an approximately twofold higher CYP3A activity as determined by measuring the N-demethylation of erythromycin in isolated liver microsomes (Ho et al., 2009). An induction of CYP3A-mediated 1-hydroxylation in liver microsomes isolated from rats treated with SJW (1000 mg/kg) was also observed by Qi et al. They tested the time dependency of changes in midazolam pharmacokinetics in rats and reported an approximately 2.6-fold increase in oral clearance of midazolam in rats treated with SJW for 7 days (Qi et al., 2005).
Importantly, SJW formulations differ in hyperforin content (Schäfer et al., 2019). Moreover, clinical data in humans show that SJW-associated interactions are clearly linked to the hyperforin content of the respective formulation (Arold et al., 2005). One low-hyperforin SJW extract approved in various countries for the short-term treatment of depression is Ze117. This formulation with the brand name Rebalance exhibits pharmacological efficacy (Schrader, 2000; Friede et al., 2001) but no relevant impact on the pharmacokinetics of substrates of drug-metabolizing enzymes and P-gp (Bosilkovska et al., 2016; Zahner et al., 2019). Considering the availability of the SJW formulations of differing hyperforin content, we sought to directly compare their in vitro and in vivo effect on the PXR-target gene CYP3A23-3A1 in rats. With this we intended to further contribute to the understanding of the inconsistent findings previously made in vitro and in vivo for rat versus human PXR. To improve readability, CYP3A23-3A1 (NCBI reference sequence NM_013105.2) will be named Cyp3a1 when describing mRNA data and CYP3A1 when reporting on protein expression throughout the manuscript.
Material and Methods
Materials
The low-hyperforin containing Hypericum extract (Rebalance 250, LOT200658 with 500 mg extract) was kindly provided by Zeller AG (Romanshorn, Switzerland). The high-hyperforin containing Hypericum extract (Hyperiplant Rx, LOT1250620, 600 mg, Schwabe Pharma AG, Küssnacht, Switzerland) was commercially obtained. The formulated tablets (of Rebalance and Hyperiplant) were pulverized using the Mixer Mill MM400 (Retsch GmbH, Düsseldorf, Germany). If not otherwise stated, all other chemicals including the pure hyperforin were obtained from Sigma-Aldrich (Buchs, Switzerland).
Molecular Modeling
The rat PXR primary sequence was obtained from the Uniprot server (ID: Q9R1A7) (https://www.uniprot.org/). The sequence was used as input for the SWISS-MODEL protein modeling server to create a rat homology model (https://swissmodel.expasy.org/). A homology model based on the human ortholog template cocrystallized with hyperforin (ID: 1M13) was used for further analyses. The human crystal structure and the rat homology model were superimposed using backbone atoms. The hyperforin molecule from the human PXR binding site along with all resolved water molecules were copied to the rat model. The binding site residues of the rat model were optimized by selecting the best interacting rotamer from the library (H-bonding satisfied, no steric clashes if possible). Finally, both protein-ligand complexes were fully preprocessed using Protein Preparation Wizard in Schrodinger Maestro (Maestro, Schrödinger, LLC, New York, NY, 2020) assuming physiologic pH of 7.4. Next, the interaction patterns in the hyperforin binding sites were compared. Finally, protein-ligand complexes were placed in the cubic periodic boundary system filled with TIP3P water molecules and molecular dynamics (MD) simulations were run using Desmond software [version 2019-1 (Bowers et al., 2006)]. For each species, five independent MD simulations were performed using different random number generator seeds. The OPLS_2005 force field was selected along with the time-step of the RESPA integrator set to 2 fs. After the default equilibration protocol, the production simulations with a duration of 4.8 ns were conducted in an NPT ensemble at 310 K regulated by a Nosé–Hoover thermostat with the atmospheric pressure maintained by a Martyna–Tobias–Klein barostat. The u-series algorithm was selected by default to treat long-range interactions, while bonds to hydrogen atoms were treated with the M-SHAKE algorithm (Shaw et al., 2014). PDB-formatted coordinate files for the human PXR-hyperforin complex (based on PDB entry ID 1M13) and the rat PXR-hyperforin homology model can be found in the Supplemental data 9 and 10, respectively.
Cell Culture
HepG2 (HB-8065; American Tissue Culture Collection ATCC, Manassas, VA, USA; RRID: CVCL_0027) were cultured in Dulbecco’s modified Eagle’s medium (with 4.5g/mL glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate; Sigma-Aldrich) supplemented with 10% fetal calve serum (FCS; BioConcept Ltd, Allschwil, Switzerland) and 1% L-glutamine (200 mM, BioConcept Ltd). H4IIE cells were kindly provided by Prof. Alex Odermatt (Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel). The rat hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium (4.5g/mL glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate; Sigma-Aldrich) supplemented with 10% FCS, 1% minimum Eagle’s medium nonessential amino acids solution (BioConcept Ltd), 1% L-glutamine (200 mM, BioConcept Ltd.), and 1% 1 M HEPES-Buffer (BiConcept Ltd.). HepaRG cells were kindly provided by Dr. Jamal Bouitbir (Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel). These cells were cultured in William’s Medium E (1X, Thermo Fisher Scientific, without L-glutamine), supplemented with 10% FCS, 1% L-glutamine (200 mM, BioConcept Ltd), 5 µg/ml human insulin (Sigma-Aldrich) and 50 µM hydrocortisone hemisuccinate (Sigma-Aldrich), and 1% penicillin/streptomycin (BioConcept Ltd.). HepaRG cells were cultured for 14 days as progenitor cells prior to a 14-day differentiation into biliary cells and hepatocytes, which was induced by addition of 2% DMSO (Guillouzo et al., 2007). All cells were kept at 37°C in a humidified atmosphere supplemented with 5% CO2 and routinely monitored for possible mycoplasma contamination.
Cell-Based Reporter Gene Assay Performed in Human and Rat Liver Cells
In the cell-based reporter gene assays we used the previously reported synthetic reporter gene construct XREM-CYP3A4-PREM-pGL3basic (Meyer zu Schwabedissen et al., 2008), the commercially available pRL-TK (Promega, Dübendorf, Switzerland), and pEF6-based (invitrogen) expression plasmids encoding for human (Ferreira et al., 2019) or rat PXR. The latter was cloned from rat liver cDNA using the primer pair: rPXR-cds-for 5′-CAGTCCAGGACACACAGATGTAAACCTG -’3 and rPXR-cds-rev 5′-CTGCTCCGTGAGATCTCCACTCAG-’3. The resulting 1361bp-amplicon was ligated into pEF6/V5-His TOPO (invitrogen). After amplification of the plasmid in E. coli, the insert was controlled by Sanger Sequencing (Microsynth, Balgach, Switzerland). For the cell-based reporter gene assay, 50,000 HepG2 cells were seeded in 24-well plates. One day after seeding, the cells in one well were transfected with 250 ng of the respective expression plasmid, 250 ng of the reporter gene construct, and 25 ng of the pRL-TK. 2 µl jetPRIME (Chemie Brunschwig, Basel, Switzerland)/1 µg of DNA were used as transfection reagent. The medium was changed 4 hours after transfection. On the next day, the transfected cells were treated with hyperforin (1 µM), pregnenolone 16α-carbonitrile (PCN; 10 µM), rifampicin (10 µM), Rebalance (2.5 mg/ml), or Hyperiplant (4.5 mg/ml). The latter were prepared as a stock solution, where 1% of the respective milled tablets was dissolved in 2 ml DMSO prior to addition to the culture medium (1/100) (Schäfer et al., 2019). Concentrations of the formulations in the cell-based experiments were calculated as previously described (Schäfer et al., 2019) considering the currently clinically used antidepressant treatment regimens. Treatment was performed in the dark to avoid phototoxicity. After 24 hours, cells were lysed and activities of the Firefly and the Renilla luciferase were measured using the Dual-Luciferase Assay System (Promega) and the plate reader Infinite M200 Pro (Tecan, Männedorf, Switzerland) according to the manufacturer’s instructions.
Animal Study
All animal procedures were approved by the cantonal veterinary authority Basel-Stadt, Switzerland (license number 3092). Male Wistar rats obtained from Janvier-Laboratories (Le Genest-Saint-Isle, France) were housed in group cages, at a 12-hour light cycle and 24°C with water and food ad libitum. Experiments were performed following the ARRIVE 2.0 guideline for care and use of laboratory animals (Percie du Sert et al., 2020) and the principles of the CRUS Policy for Animal Research by swissuniversities (2013). Animal group sizes were first estimated using the resource equation approach with a degree of freedom between 10 (minimum) and 20 (maximum) for the error term in an ANOVA (Arifin and Zahiruddin, 2017), which resulted in an expected number of five to seven animals per group (Hyperiplant, Rebalance, or control). Six animals per group were selected, as this group size was expected to allow us to detect differences of around 35% to 55% assuming a standard deviation of 27.5% with the type 1 error (p) at the level of 5% (p = 0.05) with a power of the study at 80%. The treatments were prepared by suspending the respective amount of the SJW powder in the suspension mixture consisting of 0.5% methylcellulose and 0.1% Tween 80 in water. Suspensions were stirred overnight and kept in the dark to avoid degradation of light-sensitive ingredients. Before the start of the experiment, rats aged 7 weeks were randomly assigned to the different treatment groups (Hyperiplant, Rebalance, or control). Blinding was not possible during the in vivo phase of the experiment. Rats received 400 mg/kg body weight of the respective SJW formulation in a volume of 2.8 ml/kg body weight by oral gavage. This dosage was shown to have an antidepressant effect in rats and to induce genes involved in drug metabolism (Rezvani et al., 1999; Shibayama et al., 2004). The suspended formulations or the suspension mixture alone (control) were orally administered with a feeding tube (Instech Laboratories, Leipzig, Germany) on 10 consecutive days between 9 and 10 am. On day 11, the rats were sacrificed by CO2 with subsequent organ harvest. Tissue samples were snap-frozen in liquid nitrogen before storage at −80°C until further use.
Gene Expression Analysis of PXR Target Genes
H4IIE and HepaRG cells were seeded in 6-well plates (Sarstedt, Sevelen, Switzerland) at a density of 300,000 cells per well. H4IIE cells were cultured for 24 hours before treatment was started, while HepaRG cells were differentiated as described previously. For the treatment, hyperforin, PCN, and rifampicin were used at a concentration of 1, 1, and 20 μM, respectively. The SJW formulations Rebalance (2.5 mg/ml or 4.5 mg/ml for H4IIE and 0.25 mg/ml for HepaRG cells) or Hyperiplant (4.5 mg/ml for H4IIE, 0.45 mg/ml for HepaRG cells) were prepared as described previously. Due to the high sensitivity of HepaRG cells, which exhibited a pronounced reduction in viability upon exposure to the formulation suspensions, a 10 times lower concentration was used for the herein reported treatment. H4IIE cells were treated for 48 hours, and HepaRG cells were treated for 72 hours with a change of treatment medium every day. Frozen liver tissue pieces were milled using the Mixer Mill MM400. Extraction of mRNA was performed using TRI Reagent (1 ml/100 mg tissue or 1 ml/well; Sigma-Aldrich) according to the manufacturer’s instructions. Reverse transcription was performed with 1000 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Reinach, Switzerland). The mRNA expression of rCyp3a1 (5′-TCTGTGCAGAAGCATCGAGT-‘3 and 5′-GGCTGTGATCTCCATATCG-‘3), CYP3A4 (5′-ATCATTGCTGTCTCCAACCTTCAC-’3 and 5′-TGCTTCCCGCCTCAGATTTCTC-’3) was measured with the SYBR-Green RT-PCR-Kit (Thermo Fisher Scientific). β-Actin (rat: 5′-GGAGATTACTGCCCTGGCTCCTA-‘3 and 5′-GACTCATCGTACTCCTGCTTGCTG-‘3; human: 5′-CCAACCGCGAGAAGATGA-’3 and 5′- CCAGAGGCGTACAGGGATAG-’3) was used as housekeeping gene. The analysis was performed with the QuantStudio 5 (Applied Biosystems, Thermo Fisher Scientific) using the QuantStudio Design & Analysis Software 1.5.1. Gene expression was evaluated using the 2-ΔΔCT method described by Livak and Schmittgen (Livak and Schmittgen, 2001). The Ct-value of the gene of interest was normalized to the mean of β-actin, and the results are presented as a fold of study mean.
Isolation of Liver Microsomes
Rat liver microsomes of five animals per treatment were isolated as previously described (Hiroi et al., 1998; Haduch et al., 2018). Briefly, 1 g of liver was rinsed with Tris/KCl (20 mM/0.15 M) buffer before homogenization with the Homogenizer Potter S (Sartorius, Göttingen, Germany). The homogenate was centrifuged for 20 minutes at 10,000 × g and 4°C before centrifugation of the supernatant at 100,000 × g and 4°C for 1 hour. The pellet was suspended in Tris/KCl (20 mM/0.15 M) buffer and homogenized using the Polytron PT1200E (Kinematica AG, Malters, Switzerland). After an additional centrifugation at 100,000 × g and 4°C for 1 hour, the pellet was suspended in the Tris/Sucrose buffer (20 mM/0.25 M) using the Polytron PT1200E prior to storage at −80°C until further experiments. Protein content was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and the Microplate Reader Infinite M2000 Pro (Tecan).
Western Blot Analysis
For protein expression analysis, 20 to 40 mg of each liver tissue was suspended in subcellular fractionation buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA) containing the proteinase inhibitors phenylmethylsulfonylfluorid 1 mM, leupeptin 5 μg/ml, and aprotinin 2 μg/ml before homogenization using the Dounce tissue grinder from Wheaton (Millville, NJ, USA). After centrifugation at 720 × g and 4°C for 5 minutes, the supernatant was centrifuged at 10,000 × g and 4°C for 10 minutes. The pellet containing the liver protein lysate was resuspended in lysis buffer (50 mM Tris HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) followed by quantification of protein content as described previously. Prior to SDS 10%-polyacrylamide gel electrophoresis using the Mini-PROTEAN Tetra cell system (Bio-Rad Laboratories AG, Cressier, Switzerland), 10 μg of liver protein lysate supplemented with 4 x Laemmli-solution were incubated at 95°C and 300 rpm for 60 minutes. After the separation the proteins were transferred onto a nitrocellulose membrane using the BlotL1 system (GenScript, Piscataway, NJ, USA). After a 1-hour incubation with 5% nonfat dry milk (Blotto, Carl Roth GmbH, Karlsruhe, Germany) in Tris-buffered saline/Tween 20 (TBS-T; NaCl 140 mM, KCl 2.5 mM, Tris-Ultra 25 mM 0.1% Tween 20) at room temperature, the membranes were exposed to the respective primary antibody solution [antibody diluted in 1% bovine serum albumin (BSA)-TBS-T] overnight at 4°C (anti-CYP3A1, ab1253, 1:10,000; Merck Millipore, Darmstadt, Germany, RRID: AB_90531, or anti-calnexin, ADI-SPA-865, 1:1,000, Enzo Life Sciences, Lausen, Switzerland, RRID: AB_10618434). Thereafter the membranes were washed several times with TBS-T, followed by incubation with the secondary antibody at room temperature for 1 hour (horseradish peroxidase-labeled anti-rabbit, 1:2,000, Cell Signaling Technology, Danvers, MA, USA, RRID: AB_2099233). Immobilization of the secondary antibody was visualized using the ChemiDocTM XRS imaging system equipped with the Image Laboratory software (Version 6.0) and a 1:1-mixture of the Clarity Western ECL Substrate Peroxide solution and the Luminol/enhancer solution (Bio-Rad Laboratories, Cressier, Switzerland). Band intensity was densitometrically analyzed.
Immunohistochemical Staining
Tissue slides were deparaffinized using Ottix plus (DiaPath, Martinengo, Italy) before rehydration in a decreasing ethanol series. Heat-induced epitope retrieval was performed with the citrate buffer (BioSB Inc., Santa Barbara, CA, USA; pH 6.1) in the TintoRetriever pressure cooker. After a 2-hour incubation with Tris-buffered saline 0.025% Triton × 100 (TBS-T) containing 5% donkey serum and 1% BSA, the anti-CYP3A1 antibody (ab1253, Merck Millipore diluted 1:500, in 1% BSA/Tris-buffered saline) was added to the slides and incubated at 4°C overnight in a humidified atmosphere. After several washings in TBS-T, the tissue sections were exposed to 3% H2O2 for 15 minutes before an additional wash and an incubation with the horseradish peroxidase-labeled secondary antibody (A16035, Life Technologies diluted 1:200 in 1% BSA/TBS-T, RRID: AB_2534709) for 2 hours. After washings with TBS-T, the tissue sections were incubated in 50 mM Tris-buffer (pH 7.6) at 37°C before adding the 3,3′-diaminobenzidine -staining solution consisting of 10 mg/ml 3.3-diaminobenzidine in 50 mM Tris-buffer supplemented with 0.02% H2O2. Nuclei were stained with Hemalum solution acid according to Mayer (Carl Roth) before dehydration in an increasing ethanol series. After fixation with Roti-Seal (Carl Roth), the Leica DMi8 Microscope (Carl Zeiss, Microscopy, Munich, Germany) was used for image acquisition. Images were taken by an experimenter who was blinded to group allocation.
Quantification of Hyperforin in Hyperiplant and Rebalance
One hundred mg of the pulverized tablet was suspended in 0.5% methylcellulose and 0.1% Tween 80 in water and stirred overnight. On the next day, suspensions were centrifuged at 13,000 rpm for 5 minutes. Supernatants were filtered through a disposable syringe filter (Micropur PTFE, pore-size: 0.45 µm, Altmann Analytik, Munich, Germany) of which 100 µl were combined with 900 µl DMSO. Ten µl of these solutions were injected for quantification of hyperforin using high-performance liquid chromatography with UV detection. Conditions for high-performance liquid chromatography analysis were the same as previously reported (Schäfer et al., 2019). The sum of hyperforin and adhyperforin was considered when calculating the hyperforin content. For both hyperforin and adhyperforin, a calibration curve was prepared ranging from 1.0 to 0.007 mg/ml. Analysis was performed on an Alliance 2690 chromatographic system coupled to a PDA996 detector (Waters, Milford, MA, USA). The mobile phase consisted of water (A) and acetonitrile (B) both containing 0.1% trifluoroacetic acid. Hyperforin was separated using a 115 Zorbax Eclipse XDB-C8 Narrow-Bore column (2.1 × 150 mm, 3.5 μm; Agilent, Santa Clara, CA, USA) with a gradient of 50% to 100% B in 20 minutes, then 100% B for 15 minutes, followed by 50% B for 10 minutes at a flow rate of 0.4 ml/min Separations were performed at 30°C and detection was at 272 nm. During preparation, the samples were kept in the dark to avoid degradation of light-sensitive ingredients.
In Vitro Assessment of CYP3A Activity in Liver Microsomes
The activity of the CYP enzymes was determined by measuring the rate of 2β- and 6β-hydroxylation of testosterone in rat liver microsomes. For in vitro activity measurement an incubation mixture containing 50 µl of the microsomal suspension (1 mg protein/ml), 30 µl phosphate buffer (50 mM Na2HPO4 adjusted to pH 7.4 with 50 mM KH2PO4), and 900 µl incubation buffer consisting of phosphate buffer supplemented with MgCl2 (3.0 mM), EDTA (1 mM), NADP (1 mM), glucose-6-phosphate (5 mM), and glucose-6-dehydrogenase phosphate (1.7 U/ml) were prepared. The reaction began after adding 20 µL testosterone (100 µM) and placing the samples in a water bath at 37°C. The reaction was stopped after 15 minutes of incubation by adding 200 µl of methanol and transferring the samples on ice. Testosterone and its metabolites 2β- and 6β-hydroxytestosterone were extracted from the incubation mixture to chloroform (6 ml). After evaporation under nitrogen and drying, the residue was dissolved in 100 µl methanol. Ten µl of the resulting solution was loaded onto the C18 SunFire (Waters) column (3.5 µm, 3.0 × 150 mm) with a precolumn (VanGuard from Waters). The mobile phase consisted of methanol-water-acetonitrile (39:60:1) (A) and methanol-water-acetonitrile (80:18:2) (B) with a gradient of 0% to 100% B in 13 minutes and 100% B from 13 to 15 minutes. The gradient started after 2 minutes at a flow rate of 0.3 ml/min. The column temperature was 40°C. Absorbance of testosterone and its metabolites was measured at a wavelength of 254 nm. Retention times for the reaction products were 8.6, 10.4, and 12.2 minutes for 6β-hydroxytestosterone, 2β-hydroxytestosterone, and testosterone, respectively. For calculating the absolute values of metabolites formed, the external standard method was applied, according to Sonderfan et al. (Sonderfan et al., 1987). Briefly, five mixtures containing increasing concentrations of 6β-hydroxytestosterone and 2β-hydroxytestosterone were used (0.01, 0.02, 0.05, 0.1, 0.2 mM). The standard-containing mixtures were treated the same way as the samples; however, they were not incubated at 37°C.
Statistical Analysis
The herein reported data sets were analyzed using Microsoft Excel (Microsoft, Redmond, WA, USA) and the GraphPad Prism software 9.3.1 (GraphPad Software, San Diego, CA, USA). All error bars are represented as a mean ± S.D. Tests for statistical analysis are described in the context of data presentation. A P value below 0.05 was considered statistically significant. We report an exploratory data analysis. Consequently, calculated P values should be interpreted as descriptive.
Results
Hyperforin Exhibits Different Binding Modalities for the Rat or the Human PXR
At first we analyzed in silico the binding of hyperforin to the LBD of human or rat PXR. The structure of the LBD of rat PXR was based on a homology model, and we focused on residues with immediate contacts to the ligand (within 5 Å distance). In accordance with previous findings (Watkins et al., 2003; Ngan et al., 2009), hyperforin finds a well-suited binding pocket within the human PXR. However, in the rat PXR there are differences in amino acid residues, of which some severely decrease the binding affinity of hyperforin to the rat PXR. As shown in Fig. 1A, the rat PXR contains an isoleucine (ILE144) instead of the glutamine (GLN285) in the human PXR (N.B. X-ray structure residue numbering is used for the human structure, but a full sequence numbering is used for the rat homology model). The GLN285 allows a strong and stable binding to the hyperforin by means of an H-bond, which is not present in the rat PXR. Instead, ILE144 in rat PXR forms compensating hydrophobic interactions but also leaves nearby histidine (HIS186) without an interaction partner. Next, the glutamine (GLN266) in the rat PXR—due to its larger size and flexibility—cannot form an analogous stable H-bonding interaction like the histidine HIS407 in the human PXR to one of the carbonyl groups of hyperforin (Fig. 1B). In rat PXR, the H-bond is lost in four of five MD simulations, whereas in human PXR it can be maintained in four of five simulations (see Supplemental Fig. 1, A and B). These simulations confirm the low relative stability of the rat PXR-hyperforin complex and the favorable interaction in the human PXR-hyperforin complex. Differences in case of lipophilic amino acid residues (rMET68 vs. hourLEU209; rILE70 vs. hourVAL211; rLEU102 vs. hourMET243; rVAL105 vs. hourMET246; rLEU182 vs. hourMET323) do not seem to affect the binding mode, as they still preserve the lipophilic character of the hyperforin-receptor interaction. Even if positioned at the outer layer of the binding pocket, the change of the amino acid residue from leucine LEU206 in human PXR to serine SER65 in rat PXR further reduces the binding affinity as a hydrophobic interaction is preferred to a polar one (Fig. 1C). Overall, despite the fact that hyperforin can be sterically accommodated in the binding site of the rat PXR ligand binding domain, the interaction pattern—especially with regard to H-bonding interactions—is much weaker when compared with the human PXR.
Interaction between human (red) and rat (orange) PXR amino acid residues and hyperforin (green). A rat homology model was created with the crystallized human PXR-hyperforin structure (ID: 1M13) using the SWISS-MODEL protein modeling server. Comparison and analysis of rat and human amino acid residues in the PXR binding pocket and their positions were fully preprocessed using Protein Preparation Wizard in Schrodinger Maestro. (A) Human glutamine (GLN) 285 versus rat isoleucine (ILE) 144; (B) human histidine (HIS) 407 versus rat glutamine (GLN) 266; (C) human leucine (LEU) 206 versus rat serine (SER) 65.
Cell-Based Reporter Gene Assays Support That Hyperforin Is Not Involved in Rat PXR-Mediated Transactivation of the CYP3A4-Reporter
Despite the in silico finding suggesting low interaction of hyperforin with the rat PXR, we sought to assess the impact of hyperforin and the two SJW formulations in cell-based reporter gene assays monitoring the activation of the synthetic XREM-CYP3A4-PREM-promoter construct in cells cotransfected with a plasmid encoding for the Renilla luciferase. We determined transactivation of the CYP3A4-reporter (Firefly luciferase) in HepG2 cells transfected with either the human PXR (Fig. 2A) or the rat PXR (Fig. 2B). In cells expressing human PXR, rifampicin, hyperforin, and Hyperiplant led to a 17.2-, 44.5-, and 35.8-fold increase in the normalized luciferase activity, respectively. No activation was observed in Rebalance-treated cells. In HepG2 cells expressing rat PXR, we observed much higher basal activity of the CYP3A4-reporter compared with cells transfected with human PXR. Regardless of this observation, we detected a 4.4-fold, 3.8-fold, and 2.8-fold induction of the reporter activity after treatment with PCN, Hyperiplant, and Rebalance. Exposure to hyperforin did not affect the reporter activity.
Cell-based reporter gene assay testing the transactivation of CYP3A4 in hepatoma cells of human origin. HepG2 cells were transfected with the synthetic reporter construct XREM-CYP3A4-PREM and either the human (A) or the rat (B) PXR. After 24 hours cells were treated with hyperforin (1 μM), Hyperiplant (4.5 mg/ml), Rebalance (2.5 mg/ml), or a prototypical inducer. For human PXR (hPXR) we used rifampicin (10 μM) and for rPXR PCN (10 μM). Luminescence was measured 48 hours after treatment was initiated. Data are reported as the ratio of luminescence of the Firefly luciferase normalized to that of the Renilla luciferase shown as mean ± S.D.. Experiments were performed three times. Each data point represents the mean of three replicates in one independent measurement. * p ≤ 0.05, one-way ANOVA corrected for multiple comparisons (Dunnett’s test).
No Impact of Hyperforin on Endogenous rCyp3a1 Expression in Rat Hepatoma Cells
In a next step we assessed the impact of hyperforin and the SJW formulations on the rCyp3a1 mRNA expression in rat hepatoma cells. Differentiated HepaRG cells served as a model for human hepatocytes. In HepaRG cells we observed a 3.4-fold induction of the CYP3A4 mRNA expression after exposure to hyperforin and a 5.9-fold induction after exposure to Hyperiplant, while no change was observed after a 72-hour exposure to Rebalance (Fig. 3A). In accordance with our findings, in the cell-based reporter gene assay we observed induction of the rCyp3a1 mRNA by about five- and twofold in H4IIE cells treated with Hyperiplant and Rebalance, respectively, while hyperforin did not exert an effect on rCyp3a1 mRNA in these cells (Fig. 3B). Inducibility was controlled with PCN (rCyp3a1 mRNA expression as fold of study mean ± S.D.; 27.7 ± 5.2) and rifampicin (CYP3A4 mRNA expression as fold of study mean ± S.D.; 3.7 ± 0.2) for the rat and the human system, respectively (Fig. 3, C and D). As shown in Supplemental Fig. 2, we also observed differences in the inductive efficacy of Hyperiplant and Rebalance in H4IIE cells after exposure to the same concentration of the formulations. Supplemental Fig. 3, A and B show quantitative expression levels of b-Actin (raw Ct values) measured in H4IIE and HepaRG cells, respectively.
Impact of hyperforin, Hyperiplant, or Rebalance on the CYP3A-transcription in cells of human or rat origin. (A) Differentiated HepaRG cells were treated with hyperforin (1 μM), Hyperiplant (0.45 mg/ml), or Rebalance (0.25 mg/ml) for 72 hours. The amount of CYP3A4 mRNA was measured by quantitative real-time polymerase chain reaction. (B) H4IIE cells were exposed to hyperforin (1 μM), Hyperiplant (4.5 mg/ml), or Rebalance (2.5 mg/ml) for 48 hours prior to mRNA extraction and quantification of rCyp3a1. Rifampicin served as inducibility control for HepaRG cells (C) and PCN for H4IIE cells (D). Data are shown as the mean fold of study mean ± S.D.. Experiments were performed three times. Each data point represents the mean of three replicates in one independent measurement. * p ≤ 0.05, one-way ANOVA corrected for multiple comparisons (Dunnett’s test) (A, B); unpaired t-test (C, D).
Long-Term Oral Treatment of Rats with Hyperiplant or Rebalance Increases CYP3A1 Expression
Observing induction of rCyp3a1 in rat hepatoma cells by both Hyperiplant and Rebalance, we next sought to investigate the influence of the two SJW formulations on the hepatic rCyp3a1 mRNA expression and CYP3A1 abundance in rats. To ensure induction, we treated rats orally on 10 consecutive days with formulations characterized for their hyperforin content (Supplemental Figs. 4 and 5) and probed their livers for rCyp3a1/CYP3A1. Immunohistochemical staining confirmed the presence of CYP3A1 in the livers of the differently treated animals as shown in Fig. 4A, with no obvious change in distribution. Assessing the amount of rCyp3a1, mRNA we observed a 1.8-fold increased transcript level in rats treated with Hyperiplant. However, no change was detected for Rebalance compared with control animals (Fig. 4B). Moreover, no change in mRNA expression was observed for rCyp3a2, which is also considered an ortholog of CYP3A4. Supplemental Fig. 3C shows the quantitative expression levels of b-Actin (raw Ct values) measured in the liver tissue of the treated animals. Finally, we assessed the amount of CYP3A1 by Western blot analysis of rat liver lysates (representative image in Fig. 4C). As shown in Fig. 4D, densitometric analysis revealed that animals treated with either Hyperiplant (mean CYP3A1 protein expression as fold of solvent control; 2.65 ± 0.35; n = 5) or Rebalance (1.83 ± 0.26; n = 5) showed a higher amount of CYP3A1 in liver compared with control rats (1.00 ± 0.21; n = 6 animals). Western blots used for densitometric analysis are summarized in Supplemental Fig. 6.
Hepatic Cyp3a1/CYP3A1 expression in rats treated with Hyperiplant or Rebalance. Rats were orally treated for 10 consecutive days with 400 mg/kg of the respective formulation or the suspension mixture alone as control. (A) Immunohistochemical staining of livers of differently treated animals. (B) The mRNA expression of rCyp3a1 was detected by real-time polymerase chain reaction. Data are reported as fold of solvent control. Indicated is the mean ± S.D. of n = 5 to 6 animals; each value was measured in duplicates. (C) Representative Western blot of 3 randomly selected control-, Hyperiplant-, and Rebalance-treated animals with calnexin used as loading control. (D) Densitometric analysis of CYP3A1 protein expression in rat liver microsomes obtained by Western blot analysis. * p ≤ 0.05, Kruskal–Wallis Test followed by Dunn’s multiple comparisons was used for n = 5 to 6 animals in mRNA expression analysis. Densitometric testing of n = 5 to 6 animals was analyzed with one-way ANOVA followed by Dunnett’s multiple comparisons test; both calnexin lines were taken into consideration for the calculation.
Hyperiplant Increased CYP3A Activity in Microsomes Isolated from Rat Livers
Activity of CYP3A was measured in microsomes isolated from livers of the treated rats by determining the in vitro formation of the testosterone metabolites 6β- or 2β-OH-testosterone. Formation of 6β-OH-testosterone was 547.8 ± 171.3, 852.6 ± 197.7, or 548.4 ± 180.8 nmol mg protein – 1 min −1 in liver microsomes isolated from animals treated for 10 days with solvent control, Hyperiplant, or Rebalance (n = 5 animals; mean ± S.D., Fig. 5A), respectively. A statistically significant increase was observed in in vitro 6β-OH-testosterone formation in liver microsomes isolated from animals treated with Hyperiplant (1.6-fold increase) compared with those isolated from rats treated with Rebalance. Analyzing the data as percentage of solvent control revealed a mean ± S.D. of 155.7 ± 36.1% for Hyperiplant compared with Rebalance (100.1 ± 20.6%) or solvent control (100.0 ± 31.3%). As shown in Fig. 5B, no such effect was observed for the formation of 2β-OH-testosterone. We observed a formation rate of 1911 ± 762.8, 2189 ± 613.2, and 1782 ±775.1 nmol mg protein −1 min −1 2β-OH-testosterone in liver microsomes isolated from animals treated for 10 days with solvent control, Hyperiplant, or Rebalance (each n = 5 animals; mean ± S.D.), respectively. The chromatograms of standard testosterone metabolites and of testosterone metabolites in microsomes of differently treated animals can be found in Supplemental Figs. 7 and 8, respectively.
rCYP3A activity in microsomes isolated from livers of animals after 10 days of treatment with Hyperiplant or Rebalance. CYP activity was determined by measuring the in vitro formation of 6β-OH-testosterone (A) and 2β-OH-testosterone (B) in liver microsomes isolated from rats. Livers were harvested from rats treated on 10 consecutive days with solvent control, Hyperiplant (400 mg/kg), or Rebalance (400 mg/kg). Data are shown as mean ± S.D. of n = 5 animals. Each data point is the mean of three independent measurements. * P ≤ 0.05; one-way ANOVA corrected for multiple testing (Dunnett’s test).
Discussion
In this study we report on the direct comparison of two SJW formulations and their impact on the expression of the metabolizing enzyme CYP3A1 in rats. We compared the formulation Hyperiplant, which contains a high amount of hyperforin (3–6 mg/100 mg dry extract) and Rebalance. Importantly, the latter formulation contains only a low amount of hyperforin (0.2 mg/100 mg dry extract) and has been verified for the lack of hyperforin-mediated interactions in a clinical study in humans (Zahner et al., 2019). Here, Rebalance (aka Ze117) affected activity neither of drug-metabolizing enzymes nor P-glycoprotein as determined using a probe drug cocktail (Zahner et al., 2019). The clinical assumption of Rebalance not affecting the hPXR-mediated transactivation of CYP3A4 is in line with the in vitro findings of our study, where we compared the impact of Hyperiplant and Rebalance on the transactivation of the CYP3A4-promoter in cell-based reporter gene assays (Fig. 2A), or the effect on the CYP3A4 mRNA expression in HepaRG cells (Fig. 3A). However, comparing the two formulations for their impact on rat PXR-mediated transactivation and on the rCyp3a1 mRNA expression in the rat hepatoma H4IIE cells revealed a rather unexpected result. Indeed, we observed for both Rebalance and Hyperiplant increased transactivation of the promoter (Fig. 2B) and enhanced expression of the rPXR target gene (Fig. 3B). However, in line with our in silico findings and previous reports on rPXR and hyperforin (Tirona et al., 2004), there was no effect of this constituent of SJW on the experimental endpoints we measured for rPXR in vitro. Our in silico analysis involving molecular modeling of the LBD confirmed the good embedding of hyperforin in the large and flexible binding pocket of human PXR. In contrast to the rat PXR, there are discrepancies in some amino acid residues within the LBD that lead to a considerable loss in binding affinity. This statement mainly refers to the amino acids GLN285, HIS407, MET243, and LEU206 in the human PXR and ILE144, GLN266, LEU102, and SER65 in the LBD of rat PXR. The amino acids HIS407 and LEU206 in the LBD of human PXR are also of importance for the binding affinity of the cholesterol-lowering drug SR12813. Indeed, Watkins et al. compared the human and mouse ortholog of PXR identifying the amino acids SER208 (P205 in mouse), HIS407 (Q404 in mouse), and ARG410 (GLN407 in mouse) defining the species-different SR12813 binding affinity. Their in silico finding was further validated experimentally showing that humanized mouse PXR gained activation by SR12813, while transaction by PCN was lost (Watkins et al., 2001). Accordingly, slight amino acid variations modify the binding of ligands to PXR (Heuvel, 2020), thereby contributing to the observed species differences in the spectrum of activating ligands.
Nevertheless, there are multiple studies reporting increased function of PXR-target genes in rats treated with SJW (Qi et al., 2005; Ho et al., 2009). For rCyp3a1 mRNA expression, Shibayama et al. specifically report an about 3.5-fold increase in rats treated with 400 mg/kg for 10 consecutive days (Shibayama et al., 2004). In our study we compared two formulations. For a 10-day treatment with 400 mg Hyperiplant per day, we observed increase of not only the mRNA level of rCyp3a1 but also the CYP3A1 protein amount in liver, which translates into higher enzyme activity as measured determining the formation of 6β-hydroxytestosterone in liver microsomes. No impact was observed for the formation of 2β-hydroxycholesterol. Notably, CYPs of the 3A family are highly involved in the 6β-hydroxytestosterone formation while they weakly catalyze 2β-hydroxycholesterol formation (Waxman et al., 1991; Yamazaki and Shimada, 1997). For the treatment with 400 mg/kg Rebalance per day, we observed higher levels of CYP3A1 protein in the livers but no impact on rCyp3a1 mRNA or the enzyme activity. Taken together, our findings on Hyperiplant and Rebalance indicate a mimicking effect on a PXR-regulated drug-metabolizing enzyme, hence comparable to the expected effect of SJW formulations differing in hyperforin content in humans. Nonetheless, compared with a remarkable influence in humans, as for example reported by Markowitz et al. showing a 50% area under the curve decrease and a twofold clearance increase of the CYP3A4 substrate alprazolam after SJW treatment (Markowitz et al., 2003), results of our in vivo study are rather discreet. Furthermore, our in silico and in vitro findings on hyperforin clearly support that this constituent is not driving the observed in vivo changes in rats. However, the in vitro data indicated a difference in efficacy of Hyperiplant and Rebalance in H4IIE cells. The difference in effects may be linked to other constituents of the extracts. Formulated SJW extracts are, according to the pharmacopeia, normalized to the naphthodianthrones calculated as total hypericins. No information is provided for other constituents including flavonol derivatives, biflavones, and proanthocyanidin present in the formulations (Nahrstedt and Butterweck, 1997). However, the manufacturers of Hyperiplant and Rebalance are reporting both the hypericin and the hyperforin content of their product (Hyperiplant, Schwabe Pharma AG; Rebalance, Zeller AG). Despite those constituents, the composition of Rebalance and Hyperiplant may greatly vary even if they are extracts of the same plant material, due to the fact that the two extracts are generated by applying different extraction methods (Nicolussi et al., 2020). Indeed, for Hyperiplant the extractant is methanol, while ethanol is used for SJW extraction in Rebalance. The impact of different extraction methods on the composition of extracts from SJW has nicely been shown by Avato and Guglielmi. They report not only differences in hypericin and hyperforin content but also for other components (Avato and Guglielmi, 2004). Some of these components belong to the class of flavonoids including rutoside or quercetin for which interaction with nuclear receptors is assumed (Avior et al., 2013). In this context it seems noteworthy, that early data from Moore et al., who tested several of these flavonoids for human PXR-mediated transactivation, rather suggested that those molecules are not the driving force of hPXR-mediated interactions (Moore et al., 2000). Nevertheless, for rutoside a twofold increase of the reporter gene was observed in the presence of human PXR (Moore et al., 2000). Furthermore, data from Lau et al. suggest binding at least of quercetin to human PXR and transactivation of CYP3A4 at high concentrations, but the group did not see a similar effect when testing quercetin for the rPXR-mediated transactivation (Lau and Chang, 2015). The impact of quercetin on human PXR is further supported by clinical findings in which a 13-day treatment of 18 healthy male volunteers reduced the systemic exposure of the CYP3A substrate midazolam (Duan et al., 2012).
Even though we are not able to report on the constituent and therefore the mechanism underlying the in vitro activation of rPXR or in vivo induction of rat CYP3A1, we conclude that hyperforin is indeed not the driving constituent for the changes in rCyp3a1/CYP3A1 expression and function in vivo. This differs from the situation in humans, where hyperforin activates PXR and induces PXR target genes (e.g., CYP3A4). This effect depends on the concentration of hyperforin. Consequently, the formulation-specific effect in humans is due to the changes in hyperforin content of the different commercial SJW formulations. Nevertheless, our data support that SJW extracts exert an inducing effect in rat liver but to a lower extent than expected from the findings in humans and most likely by a hyperforin-independent mechanism.
Data Availability
The authors declare that all processed data supporting the findings of this study are available within the paper and its Supplemental Material. Raw data are available on request from the corresponding author.
Authorship Contributions
Participated in research design: Schäfer Meyer zu Schwabedissen.
Conducted experiments: Schäfer, Rysz, Schädeli, Hübscher, Khosravi, Fehr, Seibert, Smieško, Meyer zu Schwabedissen.
Contributed new reagents or analytic tools: Potterat, Smieško, Meyer zu Schwabedissen.
Performed data analysis: Schäfer, Rysz, Schädeli, Hübscher, Khosravi, Fehr, Smieško, Meyer zu Schwabedissen.
Wrote or contributed to the writing of the manuscript: Schäfer, Rysz, Schädeli, Hübscher, Khosravi, Fehr, Potterat, Smieško, Meyer zu Schwabedissen.
Footnotes
- Received May 23, 2023.
- Accepted September 21, 2023.
This work received no external funding.
No author has an actual or perceived conflict of interest with the contents of this article.
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This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- BSA
- bovine serum albumin
- FCS
- fetal calve serum
- LBD
- ligand-binding domain
- MD
- molecular dynamics
- NR
- nuclear receptor
- PCN
- pregnenolone-16α-carbonitrile
- PXR
- pregnane X receptor
- SJW
- St. John’s wort
- TBS-T
- Tris-buffered saline/Tween 20
- Copyright © 2023 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.
