Using Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (LC/MS/MS) steroid profiles were made of samples of the cortex and plasma. Samples were prepared for assay by dichloromethane/ethanol extraction essentially as previously described (Karssen et al., 2001). The brain samples (weighing about 350 mg) were homogenised in 2 ml 0.1 M perchloric acid with a Potter-Elvehjem tissue homogeniser (10 times up and down, 1000 rpm). To check for differences in recovery 100 ng of dexamethasone was added to each sample. The homogenates were transferred with a 4 ml wash of dichloromethane (DCM) to screw-cappedglass tubes. After adding an extra 4 ml DCM the tubes were shaken on a horizontal reciprocating shaker for 30 minutes and subsequently centrifuged at 1000x g at 4°C for 10 minutes. The DCM layer was transferred to a clean coned tube and rinsed with 1 ml water, centrifuged at 700x g for 10 minutes. Then, the DCM-layer was transferred to a long tube and evaporated to dryness under nitrogen. To maximise the amount transferred, the extracts were redissolved in 750 µl ethanol and after transferring to an eppendorf, evaporated again. The final extracts were resuspended
in 100 µl 25% methanol and centrifuged at 13000 rpm for 5 minutes. To avoid potential dissimilarities between different extraction methods, 250 µl plasma samples were extracted in the same way.
The LC/MS/MS assays were performed on a Triple Stage Quadrupole mass spectrometer (ThermoFinnigan TSQ-Quantum, San Jose, USA) with an atmospheric pressure chemical ionisation interface. A modification of the method of Van der Hoeven et al. (1997) was used. The analysis was performed in positive ionisation mode using selective reaction monitoring of C-1073, its three main metabolites, corticosterone and dexamethasone. The [M+H]+ precursor ions were fragmented using argon as collision gas. The m/z ratios of the most abundant product ions were alternately scanned. The ion-source temperature and the nebulisation heater were kept at 200°C and 400°C, respectively. The voltages on the corona needle and on the electron-multiplier were set at 10 µA. Each experiment, a new standard series was made in 25% methanol with concentrations ranging from 1-500 ng/ml of all steroids. Dexamethasone (1µg/ml) was used as an internal standard. A Surveyor LC System (ThermoFinnigan) was used to inject 20 µl of the standard or extraction samples. A gradient of methanol-water (containing 1 g/l acetic acid) changing from 50/50% to 90/10% at a flow rate of 500 µl/min separated the steroids on an ADS C18 column. All samples were measured in duplo. The detection limit of this assay was 1-5 ng/ml for each steroid.
Steroid concentrations were calculated from a standard plot of area under the curve versus concentration. The standard curves usually displayed an r2 of more than 0.95. Presented data are corrected for recovery of dexamethasone, which was in the order of 25-50%.
Transepithelial transport and inhibition studies
In order to examine the inhibitory action of RU486 on Pgp-mediated cortisol transport we used monolayers of the porcine kidney epithelial cell-line LLC-PK1, and LLC-PK1 cells stably transfected with cDNA of the human MDR1 gene encoding P-glycoprotein (LLC- PK1:MDR1) (Schinkel et al., 1995) as previously described (Karssen et al., 2001).
During culturing and experiments complete medium, which consisted of DMEM supplied with HEPES (25 mM) and glucose (4.5 g/l) and supplemented with penicillin (100.000 U/l), streptomycin (100 mg/l), L-glutamine (2 mM) and 10% (v/v) foetal calf serum, was used. The cells were seeded on microporous polycarbonate membrane filters (0.4 µM pore size, 12 mm diameter; Costar, USA) at a density of 120*103 cells/cm2. Two hours before the start of the experiment, the medium was replaced with 800 µl fresh medium at both the apical and basal side of the monolayer. One hour later 8 µl of a 100x stock of GR antagonists or vehicle (ethanol) was added at the apical side. RU486 was added alone at a final concentration of 10 or 100 µM. In a separate experiment a mix of RU486 and metabolites at therapeutically relevant concentrations was added. At the start of the experiment (t=0), 8 µl of a 100x stock of
3H-cortisol (Amersham Pharmacia Biotech, UK; specific activity 63 Ci/mmol) in ethanol
(final concentration 15 nM) was added in triplicate at the apical or basal side respectively. To measure the transepithelial transport from the apical to the basal side or from basal to the
apical side, the appearance of radioactivity in the compartment opposite that to which activity was added, was examined. Over the four hours of study 75 µl samples were taken once every hour from this compartment. These samples were counted in a Tricarb ß-counter together with eight µl samples of the 100x stock. From the other compartment 75 µl aliquots were removed each time to ascertain an undisturbed action of Pgp. Basal to apical and apical to basal transport is presented as percentage of total activity added at the beginning of the experiment. Transepithelial electrical resistance was measured before and after the experiments to check the integrity of the monolayers (Gaillard and De Boer, 2000).
Statistical analysis
Data were evaluated by Student’s t-test. The results of the monolayer experiments were analysed by Repeated Measures ANOVA. Significance was taken at p < 0.05.
Resu lts
The steroid profiles of rat brain and plasma made with LC/MS/MS show that three hours after the last treatment with 16 mg/kg the C-1073 levels were low in both brain and plasma (figure 1). Brain levels were 19.7±13 ng/gr tissue, whereas levels in plasma were 12.6±5.5 ng/ml. There was, however, quite a large variability in C-1073 levels between animals. In addition, plasma levels did not correlate with brain levels. The concentrations of the three metabolites were below the detection limit, except for RU42633 levels, which were very low but detectable (data not shown).
Corticosterone levels in brain at the same time point late in the afternoon were about 8-10% of plasma levels in both treatment groups (figure 1). Due to the low number of animals no significant differences in corticosterone levels in blood plasma or in brain were found between
FIGURE 1. Steroid levels at 3 hours after last administration of 16 mg/ml C-1073; levels of C-1073 were in the same range in both brain and plasma (10-20 ng/ml). Brain levels of corticosterone were 10-12 times lower than plasma levels in both groups. N=2-4, shown is mean+SEM. Note the difference in scale.
Corticosterone C-1073 Steroids in plasma 0 100 200 300 400 500 VEH group C-1073 ng/ml Steroids in brain 0 10 20 30 40 50 VEH group C-1073 ng/g tissue
vehicle and C-1073 treated animals. There was a strong significant correlation between corticosterone plasma and brain levels (figure 2).
Extremely high brain and plasma levels of C-1073 were found in animals treated with a high dose of 50 mg/kg at one hour after the last administration in the morning (figure 3).
After oral treatment….?? (Experiment in progress)
C-1073 treatment did neither affect adrenal and thymus weight nor body weight after any treatment schedule.
FIGURE 2. Representative example of the strong correlation between corticosterone brain and plasma levels. At 3 hours after the last administration of C-1073 (8 or16 mg/kg) or vehicle brain and plasma levels correlated significantly (r2=0.92; p<0.005).
Correlation corticosterone plasma and brain levels
0 10 20 30 40 50 60 0 100 200 300 400 500 600
corticosterone plasma levels [ng/ml]
brain levels [ng/gr] Corticosterone C-1073 Steroids in plasma 0 200 400 600 800 1000 1200 1400 1600 1800 VEH group C-1073 ng/ml Steroids in brain 0 500 1000 1500 2000 2500 3000 3500 VEH group C-1073 ng/g tissue
FIGURE 3. At one hour after the last administration of 50 mg/kg C-1073, C-1073 were extremely high in both brain and plasma. For unknown methodological reasons false positive results were obtained in vehicle treated animals. Although less strong, again a significant correlation was found between corticosterone brain and plasma levels (not shown).