TY - JOUR
AU - Styrishave, Bjarne
AB - Abstract The potential endocrine disrupting effects of the commonly prescribed anti-epileptic drug lamotrigine (LAM) were investigated using the H295R steroidogenic in vitro assay and computational chemistry methods. The H295R cells were exposed to different concentrations of LAM, and a multi-steroid LC-MS/MS method was applied to quantify the amount of secreted steroid hormones. LAM affected several steroid hormones in the steroidogenesis at therapeutic concentrations. All progestagens as well as 11-deoxycorticosterone and corticosterone increased 100–200% with increasing concentrations of LAM suggesting a selective inhibitory effect of LAM on CYP17A1, in particular on the lyase reaction. Recombinant CYP17A1 assay confirmed the competitive inhibition of LAM toward the enzyme with IC50 values of 619 and 764 μM for the lyase and the hydroxylase reaction, respectively. Levels of androstenedione and testosterone decreased at LAM concentrations above the therapeutic concentration range. The ability of LAM to bind to CYP17A1, CYP19A1, and CYP21A2 was investigated using docking and molecular dynamics simulations. This in silico study showed that LAM was able to bind directly to the heme iron in the active site of CYP17A1, but not CYP21A2, thus supporting the results of the in vitro studies. The molecular dynamics simulations also suggested binding of LAM to the heme iron in the CYP19A1 active site. No inhibition of the aromatase enzyme was, however, observed in the H295R assay. This could be due to a sequential effect within the steroidogenesis caused by the inhibition of CYP17A1, which reduced the amounts of androgens available for CYP19A1. Introduction Epilepsy is a neurological disorder affecting about 1% of the population worldwide. The aim of pharmacological treatment with anti-epileptic drugs (AEDs) is to prevent seizures or at least to minimize their frequency. Several new AEDs have been developed and approved during the last 20 years, including lamotrigine (LAM, Figure 1). The new AEDs are characterized by having the same efficacy as the old AEDs, but with fewer adverse effects [1]. The degree of prescribed new AEDs is therefore increasing. The main indication is treatment of epilepsy, but LAM is also approved for treatment of bipolar disorder and as migraine prophylaxis [2]. LAM is recommended as first choice among the AEDs in the treatment of epilepsy because of its efficacy, low risk of adverse effects, and low commercial price. Figure 1. View largeDownload slide Structure of lamotrigine (LAM) including numbering of N-atoms. Figure 1. View largeDownload slide Structure of lamotrigine (LAM) including numbering of N-atoms. LAM functions by blocking the voltage-gated Na+ channels and voltage-gated Ca2+ channels, which stabilizes the neuronal membranes and consequently modulates the presynaptic release of the excitatory amino acids, particularity glutamate [3]. There is no clear-cut relationship between clinical response and serum LAM concentrations, but optimal LAM serum concentrations are around 10–50 μM [4]. Serum concentration >75 μM is not recommended due to the risk of adverse effects. The relationships between endogenous hormones, epilepsy, and AEDs are complex, with several interactions that can affect the endocrine system in both men and women. Patients diagnosed with epilepsy often show hormonal abnormalities, i.e. altered levels of sex steroid hormones (androgens, estrogens, and progestagens), which may lead to different endocrine syndromes and diseases, including reduced fertility in both men and women [5, 6]. Studies have shown that people diagnosed with epilepsy have fewer children compared to the general population and in addition, the prevalence of malformations in children born by epileptic-diagnosed women is higher [7]. These findings can be caused by either the epilepsy itself or by the AEDs, as both can provoke endocrine disruption [5, 8]. Seizures may affect the hypothalamus and disrupt the regulation of steroid hormones. Studies have shown that levels of circulating GnRH, LH, FSH, and levels of sex steroid hormones are altered in patients with epilepsy [8]. Menstrual disorders, polycystic ovary syndrome (PCOS), hypothalamic amenorrhea, premature menopause, and reduced fertility have been described in women with epilepsy, whereas reduced potency and sperm abnormalities are common for men with epilepsy [9]. Both sexes experience reduced sexual desire and responsiveness. In women, endocrine disruptions have been found to be related to lateralized effects of the epilepsy. Left temporal lobe epilepsy is associated with a higher pulse frequency of GnRH secretion, which is associated with higher LH/FSH ratios and higher testosterone (TS) levels. This may result in PCOS, where the high levels of TS and the disturbed LH/FSH ratio lead to high levels of estrogens during the female menstrual cycle, thus infrequent menstrual periods, and the high levels of LH contribute to the formation of cysts in the ovaries [8, 10]. On the other hand, right temporal lobe epilepsy is associated with a reduced GnRH pulse frequency and lower LH and 17β-estradiol (β-E2) levels, which may result in hypothalamic amenorrhea [8]. There seem to be no clear relationship between the different types of epilepsy and reproductive endocrine disorders in men. However, a small study has shown disturbances on the pulsatile secretion of LH in men with temporal epilepsy: delayed LH peaks are observed interracially (between seizures), which alters the male steroid hormone secretion rhythm. Postictally (after a seizure), LH peaks seem to be randomly dispersed, which also disturbs the steroid hormone secretion [8, 11]. The aim of this study was to investigate the potential endocrine disrupting effects of LAM using two in vitro models: the H295R steroidogenesis assay and the recombinant CYP17A1 assay. The former elucidated how LAM affected the overall steroid hormone production and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed quantification of 17 steroid hormones. The recombinant CYP17A1 assay was used to investigate direct effects of LAM on this enzyme. Additionally, to support the results obtained from the in vitro studies and suggest potential mechanisms of action, interactions of LAM with CYP17A1, CYP19A1, and CYP21A2—the three major CYPs involved in the steroidogenesis—were investigated with computational chemistry methods. Materials and methods H295R steroidogenesis assay Chemicals and cells H295R cells were cultured and subcultured as described in the Organisation for Economic Co-operation and Development [12] guideline. Cells were cultured in 75 cm2 flasks in 30 mL Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient mixture (DMEM/F12) media (cell medium) supplemented with 1% ITS-premix and 2,5% Nu-serum at 37°C with 5% CO2 atmosphere. Cell medium was refreshed every 2–3 days and cells were subcultured (splitted) every 5–7 days when confluence levels of 75–95% were reached. Cells were used for experiments from passage 4–12 where levels of all steroids are assumed to be constant [12]. The H295R assay is the most suitable assay for this type of studies since the H295R cells are the only cells capable of de novo synthesis of all steroids in the mammalian steroidogenesis. Furthermore, a standardized OECD guideline is available, making comparisons between laboratories and experiments relatively straightforward. In the present study, all H295R experiments were performed following the descriptions in OECD [12] guideline, with some minor modifications. Reaching a confluency of 75–95%, cells were seeded into 24-well plates at a density of 3 × 105 cells/mL on day 1. After 24 h, cell medium was refreshed and cells were exposed to the test compounds. Each plate included seven concentrations of test compound and a solvent control (SC), all in triplicates. After 48 h of exposure, 950 μL cell medium was carefully transferred from each well to vials. Samples were stored at –20°C if not analyzed immediately after terminating the assay [13]. In total, we conducted three independent experiments with three replicates for each of the seven concentrations in each experiment. Prior to conducting the assay, stock solutions of all test compounds were prepared in DMSO which is the preferred solvent in the H295R assay [12]. LAM was diluted in 100% DMSO to obtain final concentrations in the range 0.3–450 μM with final DMSO concentrations of 0.5% in the wells. Due to precipitation, it was not possible to reach LAM concentrations higher than 450 μM. Quality control and viability assay To verify the performance of the cells, a quality control (QC) plate was included every time the H295R assay was performed. Forskolin (FOR), a known stimulator of β-E2 and TS synthesis, and prochloraz (PRO), a known inhibitor of β-E2 and TS synthesis, were used as positive and negative controls in the assay [12, 14]. A viability assay was conducted as recommended in the OEDC [12] guideline. This was conducted to ensure that potential changes in steroid hormone concentrations were not caused by cytotoxic concentrations of the test compound. The Alamar Blue viability assay [15] was performed immediately after completion of the H295R assay. After removing cell medium for further analysis, 950 μL medium and 50 μL resazurin solution were added to each well. Methanol was added to three blank controls and three SCs to provide a positive control for cytotoxicity. The plate was incubated at 37°C and 5% CO2 for 3 h. Flourescense was monitored at 590 nm emission wavelength and at 560 nm excitation wavelength. The blue rezasurin was reduced to the pink and highly fluorescent resorufin in living cells, meaning that viability could be calculated as the number of living cells present was proportional with the fluorescent of resorufin [15]. Cells with viability lower than 80% compared to the average viability of SCs were not included in the data analysis. We only observed viability below 80% in samples with LAM concentrations higher than 450 μM in which we also observed precipitation. Consequently, these samples were excluded from analysis. Online sample clean-up and LC-MS/MS analysis Steroids were extracted and analyzed using a fully validated method developed by Weisser et al [16] in accordance with the ICH [17] recommendations. This method allows for simultaneous analysis of the 17 major steroids in the steroidogenesis; progesterone (PROG), pregnenolone (PREG), 17α-hydroxyprogesterone (17-OH-PROG), 17α-hydroxypregnenolone (17-OH-PREG), dehydroepiandrosterone (DHEA), androstenedione (AN), androstenediol (ADIOL), TS, dihydrotestosterone (DHT), 11-deoxycorticosterone (11-deoxy-COS), 11-deoxycortisol (11-deoxy-COR), corticosterone (COS), cortisol (COR), aldosterone (ALDO), cortisone (CORNE), estrone (E1), and β-E2. Prior to LC-MS/MS analysis, protein precipitation was performed. Fifty microliters of internal standard (0.1 ng/μl) containing deuterated analogs (d7-AN, d4-E1, d5-β-E2, d9-PROG, d4-PREG, d3-DHT, d3-TS, d6-DHEA, d5–11-deoxy-COR, d8–11-deoxy-COS, d8-COS, d7-ALDO) and 900 μL acetonitrile were added to every vial containing cell samples. Samples were mixed well, left to stand at room temperature for 10 min, and then centrifuged at 10,000 rpm for 10 min. The supernatant was evaporated at 60°C using N2 and hereafter reconstituted in 200 μL 40% MeOH in water (v/v). A binary 1290 Aglient Infinity Series system combined with a binary 1100 Aglient HPLC Series pump was used for online clean up and chromatographic separation of steroids in the samples. The LC set up was composed of a thermostated autosampler set at 8°C (1290 series), a thermostated column oven set at 40°C (1290 series) with two positions, a six port switching valve connected to an enrichment column (XTerra MS C18, 2.1 × 20 mm, 3.5 μm), and an analytical column (Kinetex, 2.6 μm C18, 100A, 75 × 2.1 mm, Phenomenex) with a guard column in front (C18 for 2.1 mm, Phenomenex). Steroids were retained on the enrichment column while salts, proteins, etc. were washed into waste by using an isocratic MeOH: H2O (10:90 v/v) mobile phase, which was allowed to run for 2 min with a flow 1 ml/min. Steroids were then analyzed on the analytical column. Two mobile phases were used: mobile phase A composed of H2O with 0.1% formic acid (v/v) and mobile phase B consisting of 100% MeOH. By changing the elution gradient of mobile phase A and B during the run, it was possible to separate all steroid hormones. The elution gradient was held at 10% B for the first 2 min, 10–30% B from 2.0 to 2.2 min, 30–60% B from 2.2 to 8.0 min, held at 60% B from 8.0 to 10.0 min, 60–99.5% B from 10.0 to 13.5 min and held at 99.5% B from 13.5 to 14.8 min, before re-equilibrating the column. The column oven switching valve was positioned left from 0 to 2 min, right 2 to 13.8 min, and left 15.2 to 16 min. The MS switching valve directs the flow to waste from 0 to 5.5 min and from 12.5 to 16 min. The total run time was 16 min. Mass spectrometry was performed with an AB Sciex 4500 Qtrap mass (Applied Biosystems, Foster City, CA) equipped with an atmospheric pressure chemical ionization Turbo V source. The total run time was 16 min. Collection of data was conducted using Analyst 1.6.2 software package (AB Sciex). All details concerning extraction, clean-up, and analysis of steroids are found in Weisser et al [16]. This includes information on spike-recovery experiments, precission and accuracy, data on calibration curves, and absolute and relative recoveries for individual steroids. Recombinant CYP17A1 assay Recombinant Escherichia coli pJL17/OR coexpressing human CYP17 and rat NADPH-P450-reductase were used in the present study (Hutschenreuter et al [18]). The CYP17 assay was conducted in accordance with Islin et al [19]. Experiments were conducted with the natural CYP17A1 substrates PRO and OH-PRO and with the well-known CYP17A1 inhibitor abiraterone (ABI) as positive control. Furthermore, zero inhibition control samples and absolute inhibition control samples were included in every run to ensure that the enzyme was active. In order to ensure a stable concentration of natural substrate and a maximum velocity of the enzyme (Vmax), an excess of substrate was used in the assay. The amount of PRO was 150 μg/mL, whereas the concentration of OH-PRO was 100 μg/mL. A detailed description of the CYP17A1 assay is found in Islin et al [19] including dose–response curves and IC50 values for ABI. In short, ABI and LAM were diluted in DMSO with final concentrations of 0.16% (v/v) for ABI and 1.25% (v/v) for LAM. Test compounds were then diluted in phosphate buffer (50 mM potassium phosphate, 1 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, pH 7.4). The final concentrations for the test compounds were as follows: ABI: 0, 0.00001, 0.0001, 0.001, 0.010, 0.031 μM; LAM: 0, 1, 10, 31, 100, 450, and 865 μM. Fifty microliters of each test solution in phosphate buffer was transferred to 600 μL tubes. For every test compound and substrate, control samples were prepared with 50 μL phosphate buffer to perform zero inhibition and absolute inhibition controls. All samples were carried out in triplicates. An enzyme-substrate (E/S) solution was prepared in phosphate buffer by the addition of natural CYP17 substrate and CYP17 enzyme in concentrations corresponding to 1.2 μg/mL PRO or 0.8 μg/mL OH-PRO and 40 μg protein/mL enzyme (Islin et al [19]). Fifty microliters of E/S solution was transferred to all test tubes and pre-incubated for 10 min at 37°C. The CYP17 enzyme was activated by the addition of 50 μL 750 μM NADPH in phosphate buffer to each test tube. In control tubes, absolute inhibition of the enzyme was obtained by adding 75 μL of 2 M NaOH immediately after the addition of NADPH. The reaction mixture was incubated for 30 min at 37°C. The reaction was stopped by adding 75 μL of 2 M NaOH to each test tube and then neutralized by adding 75 μL of 2 M HCl. IS mixture (200 μL) containing d9-PRO and d7-AN (0.025 μg/mL) in heptane was transferred to each test tube and liquid–liquid extraction was performed by shaking test tubes for 1/2–1 min on a whirl mixer and centrifuging for 5 min at 9500 G. Heptane phase (100 μL) was transferred to a LC vial with insert. Samples were then evaporated to dryness under a stream of nitrogen at 60°C, re-dissolved in 200 μL 1:9 (v/v) methanol in water, and analyzed using the LC-MS/MS procedure described above. Data processing Peak areas in the chromatograms obtained by LC-MS/MS analysis were manually inspected and corrected if necessary in MultiQuant 3.0 Software. Microsoft Excel was used to process raw data. Ratio between areas of every steroid hormone and area of corresponding deuterated steroid hormone (internal standard) was calculated. Standard curves for each deuterated steroid hormone were made and used to calculate the amount of steroid hormone in each sample. To normalize data, relative steroid hormone concentrations were calculated in proportion to mean of SCs. GraphPad Prism 6 was used for further data treatment. A t-test was used to determine if two data sets were significantly different. Data were presented as a log dose–response relationship, and a nonlinear trend line including 95% confidence interval (presented as dotted lines on each side of the trend line) was plotted. One-way analysis of variance (ANOVA) followed by a two-sided Dunnett multiple comparisons test was conducted to determine if test concentrations were significantly different (P < 0.05) from SCs. Computational chemistry Proteins and ligands preparation Crystal structures of CYP17A1 (PDB ID 3SWZ), CYP19A1 (PDB ID 3S79), and CYP21A2 (PDB ID 4Y8W) were retrieved form the Protein Data Bank [20]. Missing protein residues, hydrogen atoms, and bond orders were added using the Protein Preparation Wizard [21] implemented in Maestro [22]. Water molecules and counter ions present in the original PDB structures were deleted. The formal charge of the heme iron was set to +3 and the network of H-bonds in the protein was optimized at a pH of 7. A restrained minimization of the protein atoms, allowing the heavy atoms to move 0.3 Å, was performed using the OPLS_2005 force field [23, 24]. Protonation states of LAM and co-crystallized ligands were generated at pH 7 ± 2 using Epik [25]. Docking Docking was carried out using the program GOLD Suite version 5.2.2 [26] with the heme-tailored ChemScore scoring function [27]. The protein was kept rigid, while all single bonds of the ligands were treated as rotatable bonds. The radius of the docking was set to 15 Å around the center of mass of the co-crystallized ligand and 50 independent runs were performed. The protocol was validated by comparing the docked poses of the co-crystallized ligands with the conformations originally provided in each crystal structure. Heavy atom RMSD values of 0.82 Å, 0.53 Å, and 0.78 Å were obtained for CYP17A1, CYP19A1, and CYP1A2, respectively [24]. Molecular dynamics simulations Molecular dynamics (MD) simulations were performed in Desmond version 4.0 using the OPLS2005 force field available in Maestro [24]. Complexes of LAM and each CYP were neutralized by adding chlorine anion and solvated in SPC water molecules, using a truncated orthorombic box with a size determined by a 10 Å buffer from the molecule. All simulations were run in the NPT ensemble at 300 K and 1.01325 bar. The systems were equilibrated before simulation by using the default Desmond protocol. Twenty ns trajectories were produced and the most stable 10 ns according to the protein backbone RMSD were analyzed in terms of ligand–protein interactions, ligand-heme distances, and angles. Binding energy calculations on substituted triazine rings. Density functional theory (DFT) method was used to calculate the binding energy of substituted triazine rings to a simplified heme group, modeled as a ferric (FeIII) ion ring with an axial methyl mercaptide group that mimics the Fe-coordinating cysteine residue. Calculations were carried using B3LYP level with unrestricted formalism for open-shell systems in the doublet spin state [28–30]. Geometry optimizations were performed in the gas phase using the 6-31G(d) basis set [31] for all atoms except iron, for which the double-ζ basis set of Schäfer et al. [32] enhanced with a p function was applied. Solvation effect calculations were carried out at the same level of theory with the continuum conductor-like screening model (COSMO, [33]) using an effective dielectric constant of 4. The final binding energy was calculated as the difference of the interaction between the triazine ring alone and in complexes with water and the heme, respectively. Further details are described by Bonomo et al. [23]. The DFT calculations were performed using the software package [34]. Results H295R steroidogenesis assay The cell viability assay was performed for all concentrations of LAM analyzed in the H295R assay and none of the concentrations were cytotoxic. In addition, visual inspections in microscope were performed in all H295R experiments to check conditions of the cells. These inspections also confirmed that no cytotoxicity occurred. Mean basal steroid hormone concentrations of ADIOL and ALDO were not calculated as concentrations were below level of detection (LOD), and it was not possible to identify peaks in the chromatograms either. The changes in relative concentration of steroid hormones as a function of LAM concentrations ranging from 0.3 to 450 μM are presented in Figure 2. Increasing concentrations were observed for all progestagens when compared to SCs, corresponding to an approximately 100% increase for PREG and 17-OH-PREG, and an approximately 200% increase for PROG and 17-OH-PROG. Increases were statistically significant (P < 0.05) at concentrations of LAM ≥30 μM for PREG and PROG when compared to SC, and increases were significant (P < 0.01) at concentrations ≥10 μM and ≥3 μM for 17-OH-PREG and 17-OH-PROG, respectively. A significant increase (P < 0.05) was also seen for 11-deoxy-COS at concentrations ≥3 μM. The increase was approximately 200% when compared to SC. The amount of COS was close to limit of quantification (LOQ), but nevertheless, COS showed a significant increase (P < 0.05) of approximately 100% at 450 μM when compared to SC. The rest of the corticosteroids showed no changes in relative steroid hormone concentrations compared to SC. Figure 2. View largeDownload slide Effects of lamotrigine (LAM) on the H295R steroidogenesis. Relative steroid hormone concentrations when compared to solvent controls (100%) are plotted as a function of increasing LAM concentrations (μM). A trend line including 95% confidence intervals shows steroid hormone levels as a function of LAM concentrations. Significant values are marked with red stars, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). Data from three experiments are presented (n = 3–9). Therapeutic concentration range: 10–50 μM. PREG: Pregnenolone; PROG: Progesterone; 17-OH-PREG: 17-hydroxypregnenolone; 17-OH-PROG: 17-hydroxyprogesterone; DHEA: Dehydroepiandrosterone; AN: Androstendione, ADIOL: Androstenediol; TS: Testosterone; E1: Estrone; 17β-E2: 17β-estradiol; 11-deoxy-COS: 11-deoxycorticosterone; 11-deoxy-COR: 11-deoxycortisol; COS: Corticosterone; COR: Cortisol; ALDO: Aldosterone: CORNE: Cortisone; CYP17: 17α-hydroxylase/17,20-lyase; CYP21: 21α-hydroxylase; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase, CYP19: Aromatase; CYP11β1/2: 11β-hydroxylase; 11β-HSD: 11β-hydroxysteriod dehydrogenase. Figure 2. View largeDownload slide Effects of lamotrigine (LAM) on the H295R steroidogenesis. Relative steroid hormone concentrations when compared to solvent controls (100%) are plotted as a function of increasing LAM concentrations (μM). A trend line including 95% confidence intervals shows steroid hormone levels as a function of LAM concentrations. Significant values are marked with red stars, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). Data from three experiments are presented (n = 3–9). Therapeutic concentration range: 10–50 μM. PREG: Pregnenolone; PROG: Progesterone; 17-OH-PREG: 17-hydroxypregnenolone; 17-OH-PROG: 17-hydroxyprogesterone; DHEA: Dehydroepiandrosterone; AN: Androstendione, ADIOL: Androstenediol; TS: Testosterone; E1: Estrone; 17β-E2: 17β-estradiol; 11-deoxy-COS: 11-deoxycorticosterone; 11-deoxy-COR: 11-deoxycortisol; COS: Corticosterone; COR: Cortisol; ALDO: Aldosterone: CORNE: Cortisone; CYP17: 17α-hydroxylase/17,20-lyase; CYP21: 21α-hydroxylase; 3β-HSD: 3β-hydroxysteroid dehydrogenase; 17β-HSD: 17β-hydroxysteroid dehydrogenase, CYP19: Aromatase; CYP11β1/2: 11β-hydroxylase; 11β-HSD: 11β-hydroxysteriod dehydrogenase. When focusing on the androgens, a LAM concentration at 450 μM resulted in a 50% decrease of the steroid hormones AN and TS. The decrease of AN was significant (P < 0.01) at 450 μM and the decrease in TS was significant (P < 0.05) at concentrations ≥225 μM. No significant effects were observed in DHEA and DHT, and ADIOL was below LOQ. As for the estrogens, no significant effects were seen in either β-E2 or E1. β-E2 showed a decreasing tendency, but as steroid hormone levels were below estimated LOQ and LOD conclusions could not be drawn on the basis of this tendency. Figure 3 shown the ratios between products and substrates for the three major CYP enzymes (CYP17A1, CYP19A1, CYP21A2) in the H295R steroidogenesis. For CYP17A1, both the hydroxylase and the lyase reactions are shown. In this plot, a ratio significantly higher than 1 indicates a stimulatory effect on the steroid production for that particular CYP, whereas a significant decrease indicates an inhibition. The CYP17A1 lyase reaction was the most affected enzymatic reaction with a decreased androgen/OH-progestagen ratio around 0.3, whereas no significant effect was observed on the CYP17A1 hydroxylase. The figure also indicates a relative increase in the activity of the aromatase but this was not significant. Is should be underlined that the absolute amounts of estrogens decreased due to a prior sequential decrease in androgens. Finally, a minor decrease was observed for the CYP21A2 product/substrate ratio. Figure 3. View largeDownload slide Product/substrate ratios for the three main CYP enzymes in the H295R steroidogenesis assay. Increased ratios indicate a stimulatory effect, whereas a decrease in the ratio indicates an inhibition. For CYP17, both the hydroxylase and the lyase reactions are shown. Significant values are marked with colored stars, * (P < 0.05), **** (P < 0.0001). Figure 3. View largeDownload slide Product/substrate ratios for the three main CYP enzymes in the H295R steroidogenesis assay. Increased ratios indicate a stimulatory effect, whereas a decrease in the ratio indicates an inhibition. For CYP17, both the hydroxylase and the lyase reactions are shown. Significant values are marked with colored stars, * (P < 0.05), **** (P < 0.0001). Recombinant CYP17A1 assay The results for the recombinant CYP17A1 assay using LAM as test compound are illustrated in Figure 4. For both hydroxylase and lyase reaction, a concentration-dependent inhibition was observed. The IC50 values were 764 and 619 μM for the hydroxylase and the lyase reaction, respectively, indicating an affinity for the lyase reaction, although the difference in affinity was not significant (P = 0.22). Figure 4. View largeDownload slide Inhibition of LAM in the recombinant CYP17A1 assay using progesterone (top) and 17OH-progesterone (bottom) as substrates, respectively. Figure 4. View largeDownload slide Inhibition of LAM in the recombinant CYP17A1 assay using progesterone (top) and 17OH-progesterone (bottom) as substrates, respectively. Computational chemistry The binding modes of LAM in CYP17A1, CYP19A1, and CYP21A1 were initially determined by docking. In all the three CYP enzymes, the 1,2,4-triazine-3,5-diamine moiety of LAM was pointing toward the heme iron. The nitrogen atoms N1 and N2 have the shortest distance to the CYP17A1 and CYP19A1 cofactors, while N3 is the closest atom to the heme iron in CYP21A2 (Table 1). Table 1. Comparison between the distances from each N-atom of LAM to the heme iron in the three CYP enzymes after docking and MD simulations. CYP17A1 CYP19A1 CYP21A2 docking MDa docking MDa docking MDa N1 2.56 3.0 ± 0.3 2.55 2.40 ± 0.03 5.99 6.0 ± 0.4 N2 2.54 3.1 ± 0.4 2.31 2.77 ± 0.06 4.68 7.1 ± 0.4 N3 4.45 4.1 ± 0.3 4.05 4.00 ± 0.08 3.27 5.5 ± 0.4 N4 4.86 5.1 ± 0.3 4.60 4.73 ± 0.06 5.52 7.4 ± 0.5 N5 6.19 6.8 ± 0.4 6.07 6.27 ± 0.08 7.84 9.5 ± 0.5 CYP17A1 CYP19A1 CYP21A2 docking MDa docking MDa docking MDa N1 2.56 3.0 ± 0.3 2.55 2.40 ± 0.03 5.99 6.0 ± 0.4 N2 2.54 3.1 ± 0.4 2.31 2.77 ± 0.06 4.68 7.1 ± 0.4 N3 4.45 4.1 ± 0.3 4.05 4.00 ± 0.08 3.27 5.5 ± 0.4 N4 4.86 5.1 ± 0.3 4.60 4.73 ± 0.06 5.52 7.4 ± 0.5 N5 6.19 6.8 ± 0.4 6.07 6.27 ± 0.08 7.84 9.5 ± 0.5 All distances are in Ångström. aAverage value ± standard deviation over the most stable10 ns of each trajectory. View Large Table 1. Comparison between the distances from each N-atom of LAM to the heme iron in the three CYP enzymes after docking and MD simulations. CYP17A1 CYP19A1 CYP21A2 docking MDa docking MDa docking MDa N1 2.56 3.0 ± 0.3 2.55 2.40 ± 0.03 5.99 6.0 ± 0.4 N2 2.54 3.1 ± 0.4 2.31 2.77 ± 0.06 4.68 7.1 ± 0.4 N3 4.45 4.1 ± 0.3 4.05 4.00 ± 0.08 3.27 5.5 ± 0.4 N4 4.86 5.1 ± 0.3 4.60 4.73 ± 0.06 5.52 7.4 ± 0.5 N5 6.19 6.8 ± 0.4 6.07 6.27 ± 0.08 7.84 9.5 ± 0.5 CYP17A1 CYP19A1 CYP21A2 docking MDa docking MDa docking MDa N1 2.56 3.0 ± 0.3 2.55 2.40 ± 0.03 5.99 6.0 ± 0.4 N2 2.54 3.1 ± 0.4 2.31 2.77 ± 0.06 4.68 7.1 ± 0.4 N3 4.45 4.1 ± 0.3 4.05 4.00 ± 0.08 3.27 5.5 ± 0.4 N4 4.86 5.1 ± 0.3 4.60 4.73 ± 0.06 5.52 7.4 ± 0.5 N5 6.19 6.8 ± 0.4 6.07 6.27 ± 0.08 7.84 9.5 ± 0.5 All distances are in Ångström. aAverage value ± standard deviation over the most stable10 ns of each trajectory. View Large To assess the stability of the binding modes from docking, MD simulations were carried out with 20 ns of production phase of which only the most stable 10 ns were analyzed. Results are summarized in Table 1 and Figure 4, which shows the orientation of LAM in the last frame of each simulation. The binding modes of LAM in CYP17A1 and CYP19A1 were very stable. N1 and N2 had the same distance to the heme iron in CYP17A1, while N1 moved closer to the cofactor in CYP19A1 during the simulation. The distances allowed these sp2-hybridized nitrogen atoms to coordinate the heme iron, thereby blocking the catalytic activity of the enzyme as already shown by other N sp2-containing heterocyclic compounds ([22, 35]; DeVore et al [36]). During the MD simulation, LAM interacted with these two enzymes mainly with hydrophobic interactions, especially with ALA-113 and ALA-302 in CYP17A1 and VAL-370 and LEU-477 in CYP19A1 (Figure 5A and B, respectively). Additionally, LAM was also hydrogen-bonded to the backbone of VAL-482 in CYP17A1. Figure 5. View largeDownload slide Last frame of the MD simulation of LAM (cyan sticks) in CYP17A1 (A), CYP19A1 (B), and CYP21A2 (C). The heme is colored in green with the central iron ion shown in orange. ASP-288 in CYP21A2 was considered protonated due to hydrogen bonding to the closely located ASP-107 and, accordingly, shown as ASH-288. Figure 5. View largeDownload slide Last frame of the MD simulation of LAM (cyan sticks) in CYP17A1 (A), CYP19A1 (B), and CYP21A2 (C). The heme is colored in green with the central iron ion shown in orange. ASP-288 in CYP21A2 was considered protonated due to hydrogen bonding to the closely located ASP-107 and, accordingly, shown as ASH-288. A general increase in the distance between the 1,2,4-triazine-3,5-diamine moiety and the heme iron was observed in CYP21A2. N3 was still the closest atom to the cofactor, but the Fe-N3 distance increased from 3.3 Å of the docked pose to an average of 5.5 Å during the MD simulation (Table 1). The dichlorophenyl moiety was responsible for hydrophobic and π-π interactions of LAM with LEU-110, ILE-291, and TRP-202 in the CYP21A2 binding pocket (Figure 5C). The amino groups on the 1,2,4-triazine ring accounted for hydrophilic interactions, especially a hydrogen bond with ASP-288 and several water-mediated hydrogen bonds with ASP-107, SER-109, GLY-292, and THR-296. Lamotrigine contains a substituted 1,2,4-triazine ring and in order to determine the ability of this ring system and the effect of the substituents on the binding to the heme group, complexes of a total of 13 1,2,4-triazines, including LAM, were constructed and geometry optimized by DFT calculations with N1, N2, or N4, respectively, coordinating to the Fe atom in the heme group. Whereas any of the three nitrogen atoms of the parent ring system, 1,2,4-triazine, interacts favorably with the heme group, none of the nitrogen atoms in LAM is able to contribute favorably to the binding of this compound (Figure 6A). The effect of the different substituents is illustrated in Figure 6B (see also Supplementary Material, Table S1 for energies for all 13 compounds). A substituent in position 6 of the triazine ring hinders the coordination to the heme group for N1 to coordinate, whereas the effect on N2 and N4 is negligible. An amino substituent in position 5 impairs both N1 and N4 from coordinating to the heme group, probably due to steric effects, whereas it is still possible to obtain a favorable binding by coordination of N2 to the heme group. With substituents in both position 3 (amino group) and 6 (phenyl group), the compound resembles LAM with none of the ring nitrogen atoms accessible for coordination. Thus, the 1,2,4-triazine-5-amine moiety represents an interesting possibility for improving the binding ability of compounds relative to LAM. Figure 6. View largeDownload slide DFT calculated relative binding energies for coordination of 1,2,4-trizaines by the N1, N2, or N4 nitrogen atoms, respectively, to a heme group illustrating (A) the difference between, 1,2,4-triazine, and lamotrigine and (B) the effect of substituents on the 1,2,4-triazine ring system. See Supplementary Material Table S1 for energies and computational details. Figure 6. View largeDownload slide DFT calculated relative binding energies for coordination of 1,2,4-trizaines by the N1, N2, or N4 nitrogen atoms, respectively, to a heme group illustrating (A) the difference between, 1,2,4-triazine, and lamotrigine and (B) the effect of substituents on the 1,2,4-triazine ring system. See Supplementary Material Table S1 for energies and computational details. Discussion When exposed to H295R cells, significant increases were seen for all the progestagens and 11-deoxy-COS, whereas a decrease in AN and TS levels was observed. This indicated an inhibition of CYP17A1 with a preference toward the 17,20-lyase reaction, clearly demonstrated by the relative changes in the product/substrate ratios. The recombinant CYP17A1 assay confirmed the ability of LAM to competitively inhibit this enzyme, and also indicated a minor preference for the lyase reaction in this experimental setup. Since the two in vitro assays showed significant effects of LAM on the steroidogenesis, computational chemistry studies were carried out to elucidate the interactions of this drug with the major CYPs involved in the biosynthesis of hormones, namely CYP17A1, CYP19A1, and CYP21A2. The in silico modeling further supported the in vitro observations, and suggested that the inhibition of CYP17A1 is due to a coordination of the sp2-hybridized N-atom in the 1,2,4-triazine to the heme iron. It is interesting to compare the present results with the study by Sørensen et al. [24], who investigated the in vitro and in silico effects of the proton-pump inhibitor omeprazole on the three major steroidogenic CYP enzymes. This study demonstrated significant inhibition of CYP17A1 and CYP21A2 due to interactions of the sp2-hybridized nitrogen in the pyridine ring, with a DFT-calculated binding energy of approximately –10 kJ/mol. LAM also contains sp2-hybridized nitrogen atoms in a heterocyclic ring system, but our DFT calculations show that LAM is not an optimal heme coordinating compound and that the major part of binding affinity may arise from interactions involving other parts of the molecule. These two studies also show that binding of potent endocrine disruptors is rather complex and not only depends on coordination to the heme group but also depend on other interactions such as the presence of H-donors, H-acceptors, and aromatic rings for π-π stacking. In a recombinant enzyme assay with yeast microsomes containing the human CYP17A1 and 3β-HSD enzymes, Flück et al. [37] investigated the effect of LAM on CYP17A1 and 3β-HSD activity. They found that concentrations of LAM higher than 1000 μM significantly decreased both 17-hydroxylase and 17,20-lyase activities with IC50 values of approximately 3000 μM and 1100 μM, respectively. They concluded that LAM did alter 17-hydroxylase, 17,20-lyase, and 3β-HSD activities, but only at concentrations above the therapeutic range [37]. However, it should be noted that recombinant assays may be significantly less sensitive than that of cell assays (Jacobsen et al [38]). This could be due to competing reactions in the living cells which are obviously not present in the recombinant enzyme assays. Also, Flück et al. [37] analyzed steroid conversion using thin-layer chromatography based on the conversion of 13C-pregnenolone and thus the steroid analysis cannot be regarded as quantitative per se. Furthermore, the study by Flück et al. [37] also indicated an effect of LAM on 3β-HSD, which is not supported by our studies. Nevertheless, Flück et al [37] found the 17,20-lyase reaction to be more sensitive to LAM exposure than the 17-hydroxylase reaction, which was also observed in the present study. Docking and MD simulations also demonstrated the stability of LAM in the CYP19A1-binding cleft and suggested that LAM was able to block the active site of CYP19A1 by coordinating directly to the heme iron atom. These results are in accordance with a study by Jacobsen et al. [39] testing the ability of LAM to inhibit CYP19A1 in a recombinant assay. Significant inhibition of CYP19A1 was already observed at a LAM concentration of 78 μM. However, these observations could not be confirmed in the present H295R assay. We suggest this to be due to the sequential nature of the steroidogenesis. An inhibition of CYP17A1 could mask the potential effects of LAM on CYP19A1 in the H295R assay due to decreasing androgen levels. The MD simulation of LAM docked into CYP21A2 revealed that LAM do not bind directly to the heme iron in CYP21A2 as all Fe–N distances are >5.5 Å. The H295R assay did not show any inhibition of activity of CYP21A2, in agreement with the computational chemistry data. This indicates that other CYP enzymes in the steroidogenesis, in particular CYP17A1, are more likely to be targeted during therapeutic LAM treatment. A clinical study by Svalheim et al. [9] compared levels of steroid hormones in male and female patients treated with LAM with an untreated nonepileptic control group. Results showed significantly lower levels of AN in both male and female patients treated with LAM. Overall levels of TS were not different, but in men treated with LAM, the level of free TS was significantly decreased compared to healthy controls. Level of PROG was measured in female patients, but no differences in levels were observed among the groups. The authors of this clinical study were not able to conclude whether the significant differences of AN levels were caused by LAM or by the epilepsy itself. The differences in AN and TS levels might also be due to confounding factors, e.g. increased physical activity in control males, which have been proven to increase levels of androgens [9]. Another clinical study tested the effect of LAM on serum androgens (TS, AN, DHEA, DHEA sulfate) in males with epilepsy during pubertal maturation. No significant changes in serum androgen levels were found when compared to control males. However, the study group was very small (n = 5), so the authors suggested further studies to confirm their findings [40]. On the basis of the results of the present in vitro study, no clinical effects of LAM on AN and TS levels would be expected as the decreases were only significant at LAM concentrations larger than 450 and 225 μM, respectively. This is well above the therapeutic plasma Cmax levels of 50 μM [3]. The observed decrease in AN and TS concentrations could also very likely be due to secondary sequential effects resulting from CYP17A1 inhibition. Increasing levels of progestagens were, however, seen at LAM concentrations within the therapeutic range. Based on these findings, it is possible that LAM may cause increased levels of PROG in clinical conditions. However, in the single study conducted [8], no clinical change of PROG levels was found, indicating that the female hypothalamic–pituitary–gonadal (HPG) axis was not affected or might be able to compensate for increased PROG levels. The high levels of PROG may initiate a negative feedback to the hypothalamus and pituitary and suppress release of GnRH and FSH/LH. The highly regulated female menstrual cycle could consequently be disturbed. It is also possible that the increased levels of PROG seen in the in vitro assay contribute to an anticonvulsive effect of LAM. In the brain, excess PROG may be converted to the anticonvulsive metabolite allopregnanolone via the enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase [8]. The present in vitro and in silico studies showed no inhibiting effect of LAM on CYP21A2, suggesting that excess levels of progestagens might also be converted to corticosteroids [41]. The clinical study by Svalheim et al. [9] did not examine levels of corticosteroids and it was not possible to find any clinical studies examining levels of corticosteroids in patients treated with LAM. The hypothalamic–pituitary–adrenal (HPA) axis may be able to compensate for changing COR levels. High levels of COR would initiate a negative feedback to inhibit secretion of CRH and ACTH from the hypothalamus and the pituitary glands, thus inhibit synthesis of COR in the adrenal glands. If compensation is not enabled, high levels of the stress hormone could potentially result in a decreased sex drive and a disturbance in the female menstrual cycle [42]. Based on the present study, the effects of LAM on steroidogenesis, in particular CYP17A1, during therapeutic treatment cannot be excluded. LAM may exert steroidogenic effects not only on the progestagens, but also on glucocorticoids and androgens produced for which the progestagens are precursors. This aspect should be investigated further, for example by investigating complete steroids profiles in patients treated with LAM. Using the UK Epilepsy and Pregnancy Register, Morrow et al [43] investigated the risks of major congenital malformation (MCM) of AEDs in pregnancy. Morrow et al [43] reported a significant increase in the MCM rate with increasing dose of LAM. Thus, LAM doses >200 mg increased the MCM rate more than three times compared to LAM doses <100 mg. Investigating the dose-dependent risk of MCM with AEDs using the EURAP Epilapsy and Pregnancy Registry, these observations were confirmed by Tomson et al [44] reporting increased odds ratios for MCM in pregnant women using LAM doses >300 mg. Overall, these studies indicate that LAM may exert endocrine disrupting effects in humans including pregnant women. This aspect could be investigated further by collecting blood samples from epileptic patients and analyze the complete steroidogenic profile in association to dosing regimens. Potential feedback mechanisms on the HPA and HPG axes may also be investigated by analyzing LH, FSH, and ACTH levels in these patients. Conclusions LAM affected the levels of several steroid hormones produced in the H295R cells by inhibiting CYP17A1. All progestagens increased with increasing LAM concentrations, and changes in product/substrate ratios demonstrated specific inhibition of the 17,20-lyase activity. The recombinant CYP17A1 assay confirmed the direct inhibition of this enzyme, with a slight selectivity for the lyase reaction compared to the hydroxylase reaction. CYP21A2 was not inhibited as increasing levels of LAM also increased levels of 11-deoxy-COS and COS. Effects of LAM on CYP19A1 were not observed, potentially due to sequential effects of the CYP17A1 inhibition, leading to decreased amounts of synthesized androgens. Docking and MD simulations of LAM into CYP17A1, CYP19A1, and CYP21A2 suggested that LAM may bind in the active sites of both CYP17A1 and CYP19A1, whereas LAM does not seem to bind to the heme group in CYP21A1. This is in agreement with our in vitro experiments and with the available data on LAM. Since the effects on steroid production in the present study were observed at therapeutic concentrations, further studies should be conducted to clarify any effects that LAM may have on steroid production and feedback mechanisms in patients suffering from epileptic seizures. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Information contains Supplemental Experimental Procedures, one table are available online. Supplemental Table S1. DFT calculated relative binding energies (kJ/mol) for triazines by the N1, N2, or N4 nitrogen atoms, respectively, to a heme group. Notes Edited by Dr. Jodi Flaws, PhD, University of Illinois Footnotes † Grant Support: The Drug Research Academy at the University of Copenhagen is acknowledged for funding CHM. References 1. Neels HM , Sierens AC , Naelaerts K , Scharpe SL , Hatfield GM , Lambert WE . Therapeutic drug monitoring of old and newer anti-epileptic drugs . Clin Chem Lab Med 2004 ; 42 ( 11 ): 1228 – 1255 . Google Scholar CrossRef Search ADS PubMed 2. Tsiropoulos I , Gichangi A , Andersen M , Bjerrum L , Gaist D , Hallas J . Trends in utilization of antiepileptic drugs in Denmark . Acta Neurol Scand 2006 ; 113 ( 6 ): 405 – 411 . Google Scholar CrossRef Search ADS PubMed 3. Johannessen SI , Battino D , Berry DJ , Bialer M , Kramer G , Tomson T , Patsalos PN . Therapeutic drug monitoring of the newer antiepileptic drugs . Ther Drug Monit 2003 ; 25 ( 3 ): 347 – 363 . Google Scholar CrossRef Search ADS PubMed 4. Patsalos PN , Berry DJ , Bourgeois BF , Cloyd JC , Glauser TA , Johannessen SI , Leppik IE , Tomson T , Perucca E . Antiepileptic drugsbest practice guidelines for therapeutic drug monitoring: A position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies . Epilepsia 2008 ; 49 ( 7 ): 1239 – 1276 . Google Scholar CrossRef Search ADS PubMed 5. Isojarvi JI , Tauboll E , Herzog AG . Effect of antiepileptic drugs on reproductive endocrine function in individuals with epilepsy . CNS Drugs 2005 ; 19 ( 3 ): 207 – 223 . Google Scholar CrossRef Search ADS PubMed 6. Pennell PB . Hormonal aspects of epilepsy . Neurol Clin 2009 ; 27 ( 4 ): 941 – 965 . Google Scholar CrossRef Search ADS PubMed 7. Danish Health and Medicines Authority . Valproate ; 2014 http://pro.medicin.dk/. Accessed 20 July 2015 . 8. Taubøll E , Sveberg L , Svalheim S . Interactions between hormones and epilepsy . Seizure 2015 ; 28 : 3 – 11 . Google Scholar CrossRef Search ADS PubMed 9. Svalheim S , Taubøll E , Luef G , Lossius A , Rauchenzauner M , Sandvand F , Bertelsen M , Mørkrid L , Gjerstad L . Differential effects of levetiracetam, carbamazepine, and lamotrigine on reproductive endocrine function in adults . Epilepsy Behav 2009 ; 16 ( 2 ): 281 – 287 . Google Scholar CrossRef Search ADS PubMed 10. Hadley ME , Levine JE . Endocrinology , 6th edn . New Jersey : Pearson Prentice Hall , 2006 . 11. Quigg M , Kiely JM , Johnson ML , Straume M , Bertram EH , Evans WS . Interictal and postictal circadian and ultradian luteinizing hormone secretion in men with temporal lobe epilepsy . Epilepsia 2006 ; 47 ( 9 ): 1452 – 1459 . Google Scholar CrossRef Search ADS PubMed 12. OECD . Test No. 456: H295R Steroidogenesis Assay . Paris : OECD Publishing , 2011 . http://www.oecd.org/env/test-no-456-h295r-steroidogenesis-assay-9789264122642-en.htm . 13. Nielsen FK , Hansen CH , Fey JA , Hansen M , Jacobsen NW , Halling-Sorensen B , Bjorklund E , Styrishave B . H295R cells as a model for steroidogenic disruption: a broader perspective using simultaneous chemical analysis of 7 key steroid hormones . Toxicol in Vitro 2012 ; 26 ( 2 ): 343 – 350 . Google Scholar CrossRef Search ADS PubMed 14. Hecker M , Newsted JL , Murphy MB , Higley EB , Jones PD , Wu R , Giesy JP . Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: hormone production . Toxicol Appl Pharmacol 2006 ; 217 ( 1 ): 114 – 124 . Google Scholar CrossRef Search ADS PubMed 15. O’Brien J , Wilson I , Orton T , Pognan F . Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity . Eur J Biochem 2000 ; 267 ( 17 ): 5421 – 5426 . Google Scholar CrossRef Search ADS PubMed 16. Weisser JJ , Hansen CH , Poulsen R , Larsen LW , Cornett C , Styrishave B . Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays . Anal Bioanal Chem 2016 ; 408 ( 18 ): 4883 – 4895 . Google Scholar CrossRef Search ADS PubMed 17. ICH . International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedure: Text and Methodology Q2(R1) 2005 ; 1 – 17 . 18. Hutschenreuter TU , Ehmer PB , Hartmann RW . Synthesis of hydroxy derivatives of highly potent non-steroidal CYP 17 inhibitors as potential metabolites and evaluation of their activity by a non cellular assay using recombinant human enzyme . J Enzyme Inhibit Med Chem , 2004 ; 19 (1) : 17 – 32 . Google Scholar CrossRef Search ADS 19. Islin J , Munkboel CH , Styrishave B . Steroidogenic disruptive effects of the serotonin-noradrenaline reuptake inhibitors duloxetine, venlafaxine and tramadol in the H295R cell assay and in a recombinant CYP17 assay . Toxicol In Vitro 2018 ; 47 : 63 – 71 . Google Scholar CrossRef Search ADS PubMed 20. Berman HM , Westbrook J , Feng Z , Gilliland G , Bhat TN , Weissig H , Shindyalov IN , Bourne PE . The protein data bank . Nucleic Acids Res 2000 ; 28 ( 1 ): 235 – 242 . Google Scholar CrossRef Search ADS PubMed 21. Schrödinger Suite 2014-2 Protein Preparation Wizard; Epik version 2.8, Schrödinger, LLC, New York, NY, 2014; Impact version 6.3, Schrödinger, LLC, New York, NY, 2014; Prime version 3.6, Schrödinger, LLC, New York, NY , 2014 . 22. Maestro , version 9.8, Schrödinger, LLC, New York, NY , 2014 . 23. Bonomo S , Hansen CH , Petrunak EM , Scott EE , Styrishave B , Jørgensen FS , Olsen L . Promising tools in prostate cancer research: selective non-steroidal cytochrome P450 17A1 inhibitors . Sci Rep 2016 ; 6 ( 1 ): 29468 . Google Scholar CrossRef Search ADS PubMed 24. Sørensen AM , Hansen CH , Bonomo S , Olsen L , Jorgensen FS , Weisser JJ , Kretschmann AC , Styrishave B . Enantioselective endocrine disrupting effects of omeprazole studied in the H295R cell assay and by molecular modeling . Toxicol In Vitro 2016 ; 34 : 71 – 80 . Google Scholar CrossRef Search ADS PubMed 25. Epik , version 2.8, Schrödinger, LLC, New York, NY , 2014 . 26. Jones G , Willett P , Glen RC , Leach AR , Taylor R . Development and validation of a genetic algorithm for flexible docking . J Mol Biol 1997 ; 267 ( 3 ): 727 – 748 . Google Scholar CrossRef Search ADS PubMed 27. Kirton SB , Murray CW , Verdonk ML , Taylor RD . Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins . Proteins 2005 ; 58 ( 4 ): 836 – 844 . Google Scholar CrossRef Search ADS PubMed 28. Becke AD . A new mixing of Hartree–Fock and local density-functional theories . J Chem Phys 1993 ; 98 ( 2 ): 1372 – 1377 . Google Scholar CrossRef Search ADS 29. Becke AD . Density-functional thermochemistry. III. The role of exact exchange . J Chem Phys 1993 ; 98 ( 7 ): 5648 – 5652 . Google Scholar CrossRef Search ADS 30. Lee C , Yang W , Parr RG . Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density . Phys Rev B 1988 ; 37 ( 2 ): 785 – 789 . Google Scholar CrossRef Search ADS 31. Henre J , Radom L , Schleyer von Ragué P , Pople , JA . Ab Initio Molecular Orbital Theory , Wiley : New York 1986 . 32. Schäfer A , Horn H , Ahlrichs R . Fully optimized contracted Gaussian basis sets for atoms Li to Kr . J Chem Phys 1992 ; 97 ( 4 ): 2571 – 2577 . Google Scholar CrossRef Search ADS 33. Klamt A , Schuurmann G . COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient . J Chem Soc Perkin Trans 2 1993 ; 2 ( 5 ): 799 – 805 . Google Scholar CrossRef Search ADS 34. TURBOMOLE , version 6.3 http://www.turbomole.com 2011 . 35. Ortiz de Montellano PR . Cytochrome P450: Structure, Mechanism and Biochemistry , 3rd ed. New York : Kluwer Academic/Plenum Publishers , 2005 . Google Scholar CrossRef Search ADS 36. DeVore NM , Scott EE . Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001 . Nature 2012 ; 482 (7383) : 116 – 119 . Google Scholar CrossRef Search ADS PubMed 37. Flück CE , Yaworsky DC , Miller WL . Effects of anticonvulsants on human p450c17 (17alpha-hydroxylase/17,20 lyase) and 3beta-hydroxysteroid dehydrogenase type 2 . Epilepsia 2005 ; 46 ( 3 ): 444 – 448 . Google Scholar CrossRef Search ADS PubMed 38. Jacobsen NW , Hansen CH , Nellemann C , Styrishave B , Halling-Sorensen B . Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay . Toxicol in Vitro , 2015 ; 29 (7) : 1729 – 1735 . Google Scholar CrossRef Search ADS 39. Jacobsen NW , Halling-Sorensen B , Birkved FK . Inhibition of human aromatase complex (CYP19) by antiepileptic drugs . Toxicol in Vitro 2008 ; 22 ( 1 ): 146 – 153 . Google Scholar CrossRef Search ADS PubMed 40. Mikkonen K , Tapanainen P , Pakarinen AJ , Paivansalo M , Isojarvi JI , Vainionpaa LK . Serum androgen levels and testicular structure during pubertal maturation in male subjects with epilepsy . Epilepsia 2004 ; 45 ( 7 ): 769 – 776 . Google Scholar CrossRef Search ADS PubMed 41. Miller WL , Auchus RJ . The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders . Endocr Rev 2011 ; 32 ( 1 ): 81 – 151 . Google Scholar CrossRef Search ADS PubMed 42. Baynes JW , Dominiczak MH . Medical Biochemistry . 3rd ed . Edinburgh : Mosby Elsevier , 2009 . 43. Morrow J , Russell A , Guthrie E , Parsons L , Robertson I , Waddell R , Irwin B , McGivern RC , Morrison PJ , Craig J . Malformation risks of antiepileptic drugs in pregnancy: a prospective study from the UK Epilepsy and Pregnancy Register . J Neurol Neurosurg Psychiatry 2006 ; 77 ( 2 ): 193 – 198 . Google Scholar CrossRef Search ADS PubMed 44. Tomson T , Battino D , Bonizzoni E , Craig J , Lindhout D , Sabers A , Perucca E , Vajda F . Dose-dependent risk of malformations with antiepileptic drugs: an analysis of data from the EURAP epilepsy and pregnancy registry . Lancet Neurol 2011 ; 10 ( 7 ): 609 – 617 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - The anti-epileptic drug lamotrigine inhibits the CYP17A1 lyase reaction in vitro
JO - Biology of Reproduction
DO - 10.1093/biolre/ioy098
DA - 2018-04-26
UR - https://www.deepdyve.com/lp/oxford-university-press/the-anti-epileptic-drug-lamotrigine-inhibits-the-cyp17a1-lyase-LQL0iaKLxh
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -