Abstract
A mechanism-based pharmacokinetic-pharmacodynamic (PK/PD) model for neuroactive steroids, comprising a separate characterization of 1) the receptor activation process and 2) the stimulus-response relationship, was applied to various nonsteroidal GABAA receptor modulators. The EEG effects of nine prototypical GABAAreceptor modulators (six benzodiazepines, one imidazopyridine, one cyclopyrrolone, and one β-carboline) were determined in rats in conjunction with plasma concentrations. Population PK/PD modeling revealed monophasic concentration-EEG effect relationships with large differences in potency (EC50) and intrinsic activity between the compounds. The data were analyzed on the basis of the mechanism-based PK/PD model for (synthetic) neuroactive steroids on the assumption of a single and unique stimulus-response relationship. The model converged yielding estimates of both the apparent in vivo receptor affinity (KPD) and the in vivo intrinsic efficacy (ePD). The values ofKPD ranged from 0.41 ± 0 ng·ml−1 for bretazenil to 436 ± 72 ng·ml−1 for clobazam and the values forePD from −0.27 ± 0 for methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate to 0.54 ± 0.02 for diazepam. Significant linear correlations were observed between KPD for unbound concentrations and the affinity in an in vitro receptor bioassay (r = 0.93) and between ePD and the GABA-shift in vitro (r = 0.95). The findings of this investigation show that the in vivo effects of nonsteroidal GABAA receptor modulators and (synthetic) neuroactive steroids can be described on the basis of a single unique transducer function. In this paradigm, the nonsteroidal GABAA receptor modulators behave as partial agonists relative to neuroactive steroids.
The pharmacokinetic-pharmacodynamic correlations of benzodiazepines have been the subject of numerous studies in both animals and humans (Danhof and Mandema, 1992; for review, see Laurijssens and Greenblatt, 1996), but the predictive value of the proposed models seems to be limited. To date, there is an increasing interest in the development of mechanism-based PK/PD models because they allow the prediction of drug effects in vivo in a strict, quantitative manner on the basis of results obtained in in vitro test systems. These models not only provide a scientific basis for the prediction of drug effects in humans on the basis of results obtained in animal studies but also allow a mechanistic understanding for observed interindividual variability in drug response (Van der Graaf and Danhof, 1997).
The need for mechanism-based modeling is illustrated by the difficulty of predicting the in vivo intrinsic activity of benzodiazepine receptor partial agonists in humans on the basis of results obtained in preclinical investigations. For example, in humans, the new benzodiazepine Ro 46-2153 behaved as a full agonist, whereas it was selected from preclinical studies based on its partial agonist properties (Goggin et al., 2000).
In mechanism-based PK/PD models that are based on receptor theory, a separation is made between the drug-specific properties and the system-specific properties (Van der Graaf and Danhof, 1997). In previous investigations, the full parametric operational model of agonism (Black and Leff, 1983) has been applied successfully to synthetic opiates (Cox et al., 1998), adenosine A1 agonists (Van der Graaf et al., 1997, 1999), and 5-hydroxytryptamine1A agonists (Zuideveld et al., 2001). A characteristic feature of the operational model of agonism is that it contains a specific expression for the maximum effect that is achievable in the system. However, attempts to develop a mechanism-based PK/PD model for the effects of benzodiazepines have been only partially successful, which can be explained by the fact that in the studies reported to date, no saturation in the stimulus-response relationship has been observed (Tuk et al., 1999). Recently, however, significantly higher EEG effects have been observed for neuroactive steroids compared with benzodiazepines, indicating that with benzodiazepines the system maximum has not been reached (Visser et al., 2002a,b). Similar to benzodiazepines, neuroactive steroids are selective and potent modulators of GABAA receptor function (for review, see Lambert et al., 1995). Benzodiazepines and other ligands for the benzodiazepine site exert their effects through allosteric modulation of the GABAA receptor, thereby enhancing the actions of endogenous GABA (Sigel and Baur, 1988). Neuroactive steroids have dual effects at the GABAA receptor. At nanomolar concentrations neuroactive steroids potentiate the effect of GABA, whereas at micromolar concentrations they can directly activate the GABAA receptor (Cottrell et al., 1987).
Interestingly, all investigated neuroactive steroids (alphaxalone, pregnanolone, ORG 20599, and ORG 21465) exhibited biphasic concentration-effect relationships in vivo. At low concentrations the EEG effect increased from baseline to a maximum value that was the same for each neuroactive steroid and approximately 2 to 3 times higher than the maximum observed for the benzodiazepine displaying the highest intrinsic activity (diazepam). At higher concentrations of neuroactive steroids the effect decreased under the baseline toward isoelectric EEG. Based on these observations, a new mechanism-based PK/PD modeling approach was proposed, which features a parameterized biphasic stimulus response. For alphaxalone the biphasic stimulus-response relationship was successfully characterized by a parabolic function (Visser et al., 2002b). Next, it was shown that a single and unique stimulus-response relationship characterizes the effects of an array of different synthetic neuroactive steroids showing large differences in potency but not in intrinsic efficacy at the GABAA receptor (Visser et al., 2002a).
In the present investigation, the mechanism-based PK/PD model for neuroactive steroids has been applied to the EEG effects of various nonsteroidal GABAA receptor modulators. The EEG effects and the plasma concentrations of six benzodiazepines (diazepam, flunitrazepam, midazolam, clobazam, oxazepam, and bretazenil), an imidazopyridine (zolpidem), a cyclopyrrolone (zopiclone), and a β-carboline (methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, DMCM) were investigated in rats upon 5-min intravenous infusion. In addition, receptor binding studies were performed to obtain in vitro estimates of the affinity and the intrinsic efficacy on the basis of the so-called “GABA-shift” properties (Wood et al., 1983) at the GABAA receptor.
Materials and Methods
Animals and Surgical Procedures.
The protocol of this investigation was approved by the Ethical Committee on Animal Experimentation of Leiden University. Male Wistar rats [289 ± 33 g (mean ± S.D.); Broekman Breeding Facilities, Someren, The Netherlands] were used in this investigation. After surgery, the rats were housed individually in standard plastic cages with a normal 12-h day/night schedule (lights on 7:00 AM) at a temperature of 21°C. The animals had access to standard laboratory chow (RMH-TM; Hope Farms, Woerden, The Netherlands) and acidified water ad libitum.
Nine days before the start of the experiments seven cortical electrodes were implanted into the skull at the locations 11 mm anterior and 2.5 mm lateral (Fl and Fr), 3 mm anterior and 3.5 mm lateral (Cl and Cr), and 3 mm posterior and 2.5 mm lateral (Ol and Or) to lambda, where a reference electrode was placed (Visser et al., 2002b). Stainless steel screws were used as electrodes and connected to a miniature connector, which was insulated and fixed to the skull with dental acrylic cement.
Three days before the start of the experiment, indwelling cannulae were implanted in the right femoral artery for the serial collection of blood samples and in the right jugular vein for drug administration. The cannulae, filled with heparinized 25% polyvinyl pyrrolidone solution, were tunneled subcutaneously to the back of the neck where they were exteriorized and fixed with a rubber ring. The surgical procedures were performed under anesthesia with 0.1 mg · kg−1 i.m. medetomidine hydrochloride (Domitor; Pfizer, Capelle a/d IJssel, The Netherlands) and 1 mg · kg−1 s.c. ketamine base (Ketalar; Parke-Davis, Hoofddorp, The Netherlands). After the first surgery, 4 mg of ampicillin (A.U.V., Cuijk, The Netherlands) was administered to aid recovery.
Drugs and Dosages.
Midazolam, oxazepam and diazepam were purchased from Duchefa Pharma BV (Haarlem, The Netherlands). Clobazam, flunitrazepam, zolpidem, and DMCM were obtained from Sigma-Aldrich BV (Zwijndrecht, The Netherlands). 4-Methyl-1-piperazinecarboxylic acid 6-(5-chloro-2-pyridinyl)-6,7-dihydro-7-oxo-5H-pyrrolo[3,4-β] pyrazin-5-yl ester (zopiclone) was purchased from Tocris Cookson Inc. (Bristol, UK). Bretazenil was kindly donated by F. Hoffmann-La Roche (Basel, Switzerland). Midazolam and zolpidem were dissolved in 250 μl of saline with equimolar hydrochloric acid, and the other compounds were dissolved in 100 μl of dimethylacetamide. Rats were randomly assigned to treatment groups (n = 6–9) that received 9.4 ± 0.3 mg · kg−1 diazepam, 2.2 ± 0.07 mg · kg−1 flunitrazepam, 18.0 ± 0.9 mg · kg−1 clobazam, 4.7 ± 0.1 mg · kg−1 midazolam, 4.0 ± 0.1 mg · kg−1 zolpidem, 8.9 ± 0.1 mg · kg−1 oxazepam, 3.9 ± 0.1 mg · kg−1 zopiclone, 2.9 ± 0.1 mg · kg−1 bretazenil, 0.81 ± 0.03 mg · kg−1 DMCM, or vehicle in a 5-min zero-order infusion.
In Vivo Pharmacological Experiments.
The studies were conducted in accordance with the requirements of national legislation and appropriate guidelines for animal care. All experiments were started between 8:30 and 9:30 AM to exclude influences of circadian rhythms. The rats were placed in a rotating drum to control the level of vigilance, thereby avoiding the interference of sleep patterns. During the experiments, the rats were deprived of food and water. Bipolar EEG leads on the left hemisphere (Fl–Cl) were continuously recorded using a Nihon-Kohden AB-621G bioelectric amplifier (Hoekloos BV, Amsterdam, The Netherlands) and concurrently digitized at a rate of 256 Hz using a CED 1401plus interface (CED, Cambridge, UK). The signal was fed into an 80486 computer (Intel BV, Sassenheim, The Netherlands) and stored on hard disk for off-line analysis. After recording the EEG baseline for 45 min, a zero-order intravenous infusion of one of the compounds was administered to the conscious and freely moving rats using an infusion pump (BAS Bioanalytical Systems Inc., West Lafayette, IN). For each 5-s epoch, quantitative EEG parameters were obtained off-line by Fast Fourier transformation with a user-defined script within the data analysis software package Spike 2, version 4.6 (CED). Amplitudes in the β-frequency band of the EEG (11.5–30 Hz) averaged over 1-min time intervals were used as a measure of drug effect intensity.
Serial arterial blood samples were taken at predefined time points, and the total volume of blood sampled was kept equal to 1.8 ml during each experiment. Bretazenil and clobazam samples were immediately hemolyzed in MilliQ water, to which NaF had been added to stop esterase activity. For the other compounds, the blood samples were heparinized and centrifuged at 5000 rpm for 15 min for plasma collection. Blood and plasma samples were stored at −20°C until high-pressure liquid chromatographic (HPLC) analysis.
HPLC Analysis.
The blood or plasma concentrations of the compounds were determined by a specific HPLC assay with UV detection (slightly modified from Mandema et al., 1991a; Hoogerkamp et al., 1996;Cleton et al., 1999). The procedure was as follows. The blood/plasma samples were diluted with 0.5 ml of 0.1 M NaOH, 50 μl of an internal standard was added and the mixture was extracted with 5 ml of dichloromethane/petroleum ether [45:55 (v/v)]. The mixture was vortexed for 5 min and subsequently centrifuged for 15 min at 4500g. The samples were placed at −20°C to freeze the water phase. The organic phase was transferred to a clean tube and evaporated under reduced pressure at 37°C. The residue was dissolved in 150 μl of mobile phase of which 40 μl was injected into the HPLC system. A mixture of 25 mM phosphate buffer and acetonitrile was used as mobile phase for midazolam [50:50 (v/v), pH 7.5], flunitrazepam [58:42 (v/v), pH 5.0], zolpidem [60:40 (v/v), pH 7.0], clobazam [60:40 (v/v), pH 7.0], and DMCM [60:40 (v/v), pH 7.0]. A mixture of 25 mM acetate buffer and acetonitrile was used as mobile phase for bretazenil [55:45 (v/v), pH 5.0], oxazepam [60:40 (v/v), pH 3.8], zopiclone [56:44 (v/v), pH 5.0], and diazepam [53:47 (v/v), pH 3.8]. The chromatographic system consisted of a M-45 solvent delivery pump, a WISP 717 automatic injector (all of Millipore Corporation, Bedford, MA), a column (150 × 4.6 mm C18, 5 μm; Alltech BV, Breda, The Netherlands) equipped with a hand-packed C18 guard column (20 × 2 mm i.d.), and a spectroflow 757 Kratus UV detector (Spark Holland BV, Emmen, The Netherlands). Bretazenil, clobazam, diazepam, DMCM, flunitrazepam, midazolam, oxazepam, and zolpidem were detected at wavelengths of 235, 222, 227, 278, 218, 222, 230, and 215 nm, respectively. The detector output was recorded using a Shimadzu C-R3A integrator (Shimadzu, Hertogenbosch, The Netherlands). In some cases the analytical procedure was slightly modified (Berrueta et al., 1992). The zopiclone and flunitrazepam plasma samples were diluted with 0.5 ml of 0.2 M borate buffer, pH 8.0, and 0.5 ml of 1 M borate buffer, pH 9.5, respectively. The oxazepam samples were diluted with 0.5 ml of 0.1 M phosphate buffer, pH 5.0, followed by methanol extraction using octadecyl 3-ml solid phase extraction columns (Bakerbond; Baker BV, Deventer, The Netherlands) (Mawa et al., 1996). Zopiclone concentrations were measured using a PerkinElmer LC240 fluorescence detector (Beaconsfield, UK) at wavelengths for excitation of 300 nm and for emission of 470 nm (Foster et al., 1994).
Linear calibration curves were obtained in the range of 0.01 to 10 μg · ml−1 for each compound. Inter- and intraday variability and the extraction recovery were determined using two quality controls (0.3 and 9 μg · ml−1). Limit of quantification for each compound, based on a 50-μl plasma (or 100-μl blood) sample, the inter- and intra-assay variability, and the extraction recovery were for bretazenil 0.025 μg · ml−1, 8, 5, and 85%; for clobazam 0.013 μg · ml−1, 20, 20, and 83%; for diazepam 0.025 μg · ml−1, 9, 8, and 95%; for DMCM 0.01 μg · ml−1, 15, 10, and 80%; for flunitrazepam 0.025 μg · ml−1, 4, 4, and 98%; for midazolam 0.025 μg · ml−1, 6, 6, and 110%; for oxazepam 0.10 μg · ml−1, 5, 5, and 89%; for zolpidem 0.05 μg · ml−1, 10, 9, and 96%; and for zopiclone 0.025 μg · ml−1, 10, 7, and 86%, respectively.
Protein Binding.
Plasma protein binding was determined ex vivo after administration of 5 mg · kg−1(n = 3) and 10 mg · kg−1(n = 3) zopiclone or 1 mg · kg−1 DMCM (n = 6). At two time points 2-ml blood samples were drawn. For the other benzodiazepines the degree of plasma protein binding was determined in vitro at two concentrations (n = 3). Freshly heparinized blood (2 ml) was spiked with the compound and placed in a water bath at 37°C for 30 min. After equilibration, two samples (100 μl) of the spiked blood were hemolyzed with 500 μl of MilliQ water. Subsequently, the tubes were centrifuged for 10 min at 5000 rpm for plasma collection. From each tube, two plasma samples of 50 μl were taken and the remaining plasma was centrifuged at 37°C (15 min, 2000g) using an ultrafiltration device (Centrifree; Millipore Corporation). Two samples of 100- to 400-μl ultrafiltrate were taken. The concentration of drug in the blood, plasma, and ultrafiltrate samples was measured by HPLC, according to the methods described above. The free fraction (fu) was calculated by dividing the free concentration in ultrafiltrate by the total (bound and free) concentration in plasma.
In Vitro Pharmacology.
The receptor binding affinity and the GABA-shift were determined in rat cortex homogenates in vitro. Briefly, the procedures of these assays were as follows: three groups of rats (n = 12–15) were used to obtain three brain homogenates. The rats were sacrificed by decapitation and the whole brain was excised. The homogenates were prepared using the method described by Mandema et al. (1991b), which was slightly modified. The cortex was removed from the whole brain and gently homogenized in 10 volumes of ice-cold “enriched” Tris buffer, pH 7.4, containing 50 mM Tris (Sigma-Aldrich), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2 (all of Baker), in a Potter-Elvehjem homogenizer (10× at 4°C; Braun Potter S, Melsungen, Germany). The homogenates were centrifuged for 20 min at 45,000g at 4°C and the supernatant was discarded. The pellets were resuspended in 10 volumes of the enriched Tris buffer. This procedure was repeated four times. Protein concentration was measured using a DC protein assay (Bio-Rad BV, Veenendaal, The Netherlands). The homogenates (3 mg protein/ml) were stored in 1-ml fractions at −80°C until use.
The receptor binding characteristics (Kd andBmax) of the radioligand [3H]flumazenil (specific activity 87.0 Ci/mmol; New England Nuclear 757, Hoofddorp, The Netherlands) were determined in saturation experiments in all three homogenates. Brain homogenate aliquots containing 1 mg of protein were incubated for 35 min at 37°C with increasing concentrations of the radioligand (0.5–330 nM) in the absence and presence of 50 μM GABA. Subsequently, for each compound, the GABAA receptor binding was determined by displacement of [3H]flumazenil. Cortex homogenate aliquots containing 1 mg of protein were incubated for 35 min at 37°C with increasing concentrations of the compounds and a final assay concentration of 7 nM [3H]flumazenil.
In both the saturation and the displacement experiments the efficacy of the various compounds was evaluated by examining the effect of 50 μM GABA on the receptor binding, the so-called GABA-shift. It has been shown that the ratio of the IC50 values in the absence and in the presence of GABA is a reflection of the agonist efficacy of the compound (Wood et al., 1983). Thus, saturation and displacement experiments were carried out in the absence and presence of 50 μM GABA. In all experiments the nonspecific binding was determined in presence of 20 μM unlabeled flumazenil. In each tube, the final assay volume was 100 μl, including the ethanol concentration necessary to dissolve the compounds, fixed at 0.5% and the NaCl concentration at 1 M, to improve the assay performance. After incubation, the reaction was terminated by filtration through a presoaked GF/B filter (Whatman, Maidstone, UK) under mild suction using a harvester (Brandel Inc., Gaithersburg, MD). The filters were washed three times (or five times in saturation experiments) with 5 ml of ice-cold enriched Tris buffer. The filters were submerged in 5 ml of Emulsifier Safe scintillation fluid (PerkinElmer Life Sciences, Boston, MA) and vortexed. After incubation of 2 h, the radioactivity was measured with a Packard TriCarb 4640 liquid scintillation counter. Each experiment was performed in duplicate.
Pharmacokinetic-Pharmacodynamic Data Analysis.
Pharmacokinetic compartmental analysis was performed by fitting of a two-compartment model to the concentration-time profiles of the compounds by use of the ADVAN3 TRANS4 subroutine within the nonlinear mixed effect modeling software package NONMEM (NONMEM project group, University of California, San Francisco, CA). The NONMEM program is based on a statistical model, which explicitly takes into account both interindividual variability and intraindividual residual error (Schoemaker and Cohen, 1996). The two-compartment model was selected for each compound on the basis of visual inspection of the model fits and the Akaike information criterion (Akaike, 1974). The pharmacokinetic parameters clearance (CL), intercompartmental clearance (Q), and the volumes of distribution of compartments 1 and 2 (V1 andV2) were estimated. The interindividual variability of these parameters was modeled according to the exponential equation:
Individual parameter estimates were used to calculate individual blood/plasma concentrations at the times of the EEG measurements. For each compound, the individual concentration-effect curves thus obtained were fitted simultaneously to the Hill equation:
Mechanism-Based PK/PD Analysis.
Subsequently, the concentration-effect data were analyzed by the recently proposed mechanism-based model for neuroactive steroids. In this model, the effect is thought to be a function of the stimulus induced by the drug-receptor binding (Tuk et al., 1999; Visser et al., 2002a,b). Upon binding to the receptor the drug produces a stimulus that is followed by a cascade of signal-transduction processes leading to the ultimate response (Fig.2; Tuk et al., 1999; Visser et al., 2002a). A unique feature of this model is that the receptor activation process is drug-specific, whereas the stimulus-response relationship is system specific. Thus, the receptor activation can be different for different drugs. The stimulus-response relationship on the other hand is the same, regardless of the drug that is tested.
The characterization of the drug receptor interaction is based on receptor theory adjusted for application to in vivo responses (Kenakin, 1997; Visser et al., 2002a,b). In Fig. 2B is shown that in this approach, the drug-receptor interaction consists of a binding step where drug A binds to the receptor and an initial receptor activation step. The drug binding to the receptor forms a complex [AR]. This complex [AR] produces a stimulus depending on the efficacy (ε). Taking the binding and initial activation together, a concentration-stimulus relationship can be derived. In this model, the interaction with the receptor yields a stimulus S according to the formula:
In the analysis, the parameters determining the shape of the stimulus-response relationship were fixed at the previously obtained values for synthetic neuroactive steroids with the corresponding interindividual variability [A = 9.2 (22%),b = 0.44 (7%), and d = 3.36 (−)], respectively (Visser et al., 2002a,b). To characterize the effects of inverse agonists, negative ePD values must be estimated. For this reason, Sd
was rewritten:
For each GABAA receptor modulator the values ofKPD andePD were estimated. TheePD was estimated relative to alphaxalone for which ePD is 1 (i.e., maximal stimulus). Averaged amplitudes over 40 min of individual EEG recordings before infusion served as input for individual baseline values.
The interindividual variability of KPDwas modeled using an exponential error model, whereas interindividual variability of ePD was modeled using a CCV error model. Similar to the pharmacokinetics, the residual variability in the pharmacodynamics was modeled as a CCV error according to eq. 2. The FOCE method with interaction was used to estimate the population θ, ω2, and ς2. All fitting procedures were performed on an IBM-compatible personal computer (Pentium III, 450 MHz) running under Windows NT 4.0 and Visual-NM 2.2.2. (RDPP, Montpellier, France) with the use of the Microsoft FORTRAN PowerStation 4.0 compiler with NONMEM, version V.
In Vitro Data Analysis.
The specific binding of [3H]flumazenil was calculated by subtracting the nonspecific binding from the total binding measured in the saturation experiments. The [3H]flumazenil concentrations versus the specific binding were fitted using the equation:
The displacement curves were described using the following equation:
Statistical Analysis.
Goodness-of-fit was evaluated on basis of visual inspection of the model fits and the value of the objective function. Model selection was based on the Akaike Information Criterion (Akaike, 1974) and assessment of the parameter correlation. Statistical analysis was performed using one-way analysis of variance (ANOVA) and a Tukey-Kramer multiple comparison test. In case of nonhomogeneity, as determined by Bartlett's test, the nonparametric Kruskal-Wallis test was used. Statistical tests were performed using InStat, version 3.0, for Windows (GraphPad, San Diego, CA). All data are represented as mean ± S.E.M and the significance level was set to α < 0.05.
Results
Pharmacokinetics and Pharmacodynamics.
The concentration-time profiles of the various compounds were best described using a two-compartment pharmacokinetic model. The observed and predicted pharmacokinetic profiles are depicted in Fig.3. The population pharmacokinetic parameter estimates for each GABAA receptor modulator and the corresponding inter- and intraindividual variability are summarized in Table 1. Terminal half-life was 41 ± 2 min for diazepam, 42 ± 4 min for flunitrazepam, 33 ± 2 min for clobazam, 32 ± 1 min for midazolam, 20 ± 1 min for zolpidem, 64 ± 3 min for oxazepam, 69 ± 3 min for zopiclone, 53 ± 9 min for bretazenil, and 20 ± 1 min for DMCM.
For each compound the protein binding was determined. For DMCM, no free fraction could be determined. Except for zopiclone and bretazenil, the blood-plasma ratio was determined for each compound. The fluorescence assay for zopiclone was not suitable for the analysis of blood samples, whereas for bretazenil, no plasma samples could be taken for stability reasons. Protein binding and blood/plasma concentration ratios are summarized in Table 1.
Concentrations were calculated at the time points of effect measurements and the resulting concentration-effect relationships were fitted to the Hill equation (eq. 3), yielding estimates for intrinsic activity (α), potency (EC50), baseline (E0), and slope (nH). The observed concentration-effect relationships for each GABAAreceptor modulator are shown in Fig. 4. The highest intrinsic activity was found for diazepam (10.6 ± 1.2 μV). Bretazenil showed an intrinsic activity of 1.2 ± 0.5 μV, whereas DMCM revealed a negative α of −0.8 ± 0.2 μV as depicted in Table 2. The intrinsic activity for DMCM and bretazenil could only be determined by fixing the individual baseline values, because the variation in baseline between the animals was larger than the intrinsic activity of DMCM and bretazenil itself. However, all individual profiles showed a decrease (DMCM) or increase (bretazenil) in EEG compared with vehicle treatment experiments. Statistical analysis showed that the intrinsic activity of diazepam was different from the intrinsic activity of zolpidem, oxazepam, zopiclone, bretazenil, and DMCM but not from midazolam, flunitrazepam, and clobazam (p < 0.05).
Mechanism-Based PK/PD Modeling.
The concentration-effect relationships were simultaneously fitted to the mechanism-based model wherein the parameters A, b, and d of the stimulus-response relationship fixed to values determined using synthetic neuroactive steroids (Visser et al., 2002a,b). The model was able to successfully describe all individual concentration-effect relationships. The population prediction and individual predictions are shown in Fig. 4. Wide differences were observed inePD andKPD as shown in Table3. The values of theePD ranged from −0.27 (DMCM) to 0.54 (diazepam) and the values of the KPDranged from 0.41 ± 0 ng · ml−1(bretazenil) to 392 ± 76 ng · ml−1(clobazam). For all compounds, the population concentration-stimulus relationship with their standard error ofKPD andePD are shown in Fig.5A. In this graph, the concentration-stimulus relationships of the neuroactive steroids (alphaxalone, pregnanolone, ORG 20599, and ORG 21465) are included (Visser et al., 2002a). Interestingly, it was observed that the GABAA receptor modulators with little difference in their in vivo efficacy, show large differences in intrinsic activity (e.g., zolpidem versus midazolam), whereas bretazenil and DMCM had significant different estimates for in vivo efficacy, but showed marginal EEG effects.
In Fig. 5B, the stimulus-response relationship of the benzodiazepines, imidazopyridine, cyclopyrrolone, and β-carboline are superimposed on the stimulus-response relationship that was found for the neuroactive steroids (Visser et al., 2002a). This analysis shows that benzodiazepines and other allosteric GABAAreceptor modulators differ from neuroactive steroids both with respect to affinity and intrinsic efficacy at the GABAAreceptor.
In Vitro Pharmacology.
In three homogenates, theKd andBmax of [3H]flumazenil were estimated using NONMEM. TheKd andBmax of flumazenil in absence of GABA were 10.6 ± 2.7 nM (51%) and 111 ± 10 fmol · mg−1 protein (18%), respectively. The Kd andBmax were not significantly affected by the presence of 50 μM GABA [10.4 ± 1.2 nM (16%) and 124 ± 5 fmol · mg−1 protein (15%), respectively]. This is consistent with the fact that flumazenil acts as a competitive antagonist at the GABAA receptor with an intrinsic efficacy that is not significantly different from zero.
Subsequently, in displacement studies, for each compound the IC50 for displacement of [3H]flumazenil was measured in the presence and absence of 50 μM GABA. The displacement studies were performed in duplicate in three homogenates. All curves were successfully described by eq. 13. The value for Ki was calculated using eq. 14. The in vitro Ki values and the GABA-shifts are summarized in Table 3. In line with the in vivo estimates of ePD, the GABA-shifts of the compounds do not show large differences except for bretazenil and DMCM.
In Vitro-in Vivo Correlations.
Figure6 shows a plot of the values of theKi determined in vitro in the presence of GABA versus the in vivo KPD. When using total concentrations, a linear relationship was observed between the logarithm of the Ki and theKPD: log(KPD) = 1.15 ± 0.22 · log(Ki ) − 0.003 ± 0.37 (r = 0.85) (A). This relationship was improved by correcting the values of KPD for protein binding (B). The relationship betweenKPD for the free (unbound) concentrations (KPD,unb) andKi was log(KPD,unb) = 1.03 ± 0.12 · log(Ki ) − 0.86 ± 0.02 (r = 0.93). Because no protein binding for DMCM could be determined, the value for KPDof DMCM was not corrected for protein binding. For neuroactive steroids, the in vitro values, representing the IC50 for [35S]t-butylbicyclophosphorothionate inhibition, were taken from Anderson et al. (1997) and the values for the in vivo KPD were fromVisser et al. (2002a).
The relationship between the logarithm of the GABA-shift and estimated in vivo efficacy ePD is shown in Fig.7. The solid line represents the linear relationship that was observed using the data obtained in this investigation: ePD = 1.96 ± 0.19 · log(GABA-shift) − 0.11 ± 0.03 (r = 0.97). The dotted line represents the relationship that includes the GABA-shifts for pregnanolone and ORG 20599 from the literature (McCauley et al., 1995; Van Rijn et al., 1999), which is described by the equation ePD = 1.75 ± 0.21 · log(GABA-shift) − 0.14 ± 0.05 (r = 0.95). On the basis of the information in Fig. 7and the shape of the unique biphasic stimulus-response relationship, it is in principle possible to predict for each compound the maximum EEG effect in vivo on the basis of information on the GABA-shift from the in vitro receptor binding assay. This relationship is complex as is shown in Fig. 8.
Discussion
In the present investigation, the mechanism-based PK/PD model for synthetic neuroactive steroids has been applied to the in vivo EEG effects of six benzodiazepines, an imidazopyridine, a cyclopyrrolone, and a β-carboline. The compounds were selected on the knowledge that they display different intrinsic activity in vivo (Mandema et al., 1991b, 1992). These differences in intrinsic activity (α) and potency were confirmed in the present investigation by the descriptive analysis using the Hill equation. It was shown that benzodiazepines in general have a similar intrinsic activity, although there is a tendency for slightly lower values for flunitrazepam, midazolam, and clobazam relative to diazepam. This difference, however, did not have statistical significance. This is in agreement with several other reports where one of these four benzodiazepines has been reported to have the highest intrinsic activity in vivo (Mandema et al., 1991; Facklam et al., 1992). The nonbenzodiazepines have a lower intrinsic activity. It has been suggested that these compounds have pharmacological profiles different from benzodiazepines due to either differential affinity for different GABAAreceptor subtypes or partial agonistic properties (Depoortere et al., 1986; Ueki et al., 1987). Bretazenil showed a marginal EEG effect, which is in agreement with the results of previous investigations where it was shown that bretazenil has an intrinsic activity that is 20% of the intrinsic activity observed with midazolam (Mandema et al., 1992). DMCM, reported a full inverse agonist (Kemp et al., 1987), showed a small but statistically significant negative EEG effect (Massotti et al., 1985). The potencies (EC50) of the various compounds were in the same range and order as reported previously (Mandema et al., 1991, 1992).
In the analysis with the mechanism-based PK/PD model that was developed for the biphasic concentration-effect relationship neuroactive steroids, it was shown that this model is generally applicable for the description and prediction of the EEG effects of benzodiazepines and other GABAA receptor modulators. An interesting feature of the model is that it allows estimation of the absolute intrinsic efficacy at the GABAA receptor, rather than the intrinsic efficacy relative to other benzodiazepines, because with neuroactive steroids the physiological maximum of the system (i.e., isoelectric EEG) is reached. In addition, the model is able to estimate the intrinsic efficacy of a negative GABAA receptor modulator. The findings of the present analysis show that all investigated compounds behave as partial (or inverse) agonists at the GABAA receptor with intrinsic efficacies varying between −0.27 and 0.54.
The observed and predicted shape of the stimulus-response relationship for benzodiazepines (Fig. 7) is found to be similar to the stimulus-response relationship observed by Tuk et al. (1999) in the sense that no saturation at the higher stimulus intensities is reached. In that investigation, the stimulus-response relationship was characterized as a monotonically increasing function. In the present investigation, it is shown that this stimulus-response relationship coincides with the initial part of the biphasic stimulus-response relationship of synthetic neuroactive steroids. This shows that benzodiazepines and other nonsteroidal GABAAreceptor modulators differ from neuroactive steroids solely with respect to the interaction at the GABAA receptor in the sense that they behave as partial agonists. The present analysis shows that despite the very different in vivo concentration-effect relationships for both classes of compounds the only difference is the degree of maximal receptor activation.
To evaluate the validity of the present modeling, in vitro-in vivo correlations were explored, both with respect to potency and intrinsic efficacy. The binding affinity of the nonsteroidal GABAA receptor modulators was determined in rat brain homogenates in vitro in the presence and absence of GABA (Wood et al., 1983). The binding constant Ki in the presence of 50 μM GABA was taken as the most representative for the in vivo binding affinity. Between the compounds wide differences in in vivo binding affinity (KPD) were observed with values ranging from 0.41 ng · ml−1 for bretazenil to 392 ng · ml−1 for clobazam. Interestingly, the values of the KPD for flunitrazepam, midazolam, oxazepam, and clobazam are remarkably similar to the values reported by Tuk et al. (1999). In the analysis of the in vitro-in vivo correlation for potency both total and free (i.e., unbound) plasma concentrations were considered. Statistically significant linear correlations between the in vitro Ki and the estimated in vivo binding affinity KPDwere observed (Fig. 6). Correction for the free fractions in plasma resulted in a statistically significant increase of the correlation coefficient (p < 0.001), indicating that free plasma concentrations may be the most representative for concentrations at the site of action and the in vivo drug effect. Similar observations have been reported for benzodiazepines (Greenblatt et al., 1983; Arendt et al., 1987; Mandema and Danhof, 1992; Hoogerkamp et al., 1996) and neuroactive steroids (Visser et al., 2002a). For DMCM no free fraction could be determined in the ultrafiltrate despite a sufficiently low detection limit of the assay and might indicate a methodological error (e.g., sticking of DMCM to the membrane of the ultrafiltration device) or that protein binding is not an important factor for the pharmacodynamics of DMCM.
The so-called GABA-shift is a well established parameter to estimate the intrinsic efficacy of benzodiazepines and neuroactive steroids in vitro (Wood et al., 1983). Typically, rather small values of the GABA-shift are observed for benzodiazepines, whereas the values are typically larger for neuroactive steroids. In the present investigations, values for the GABA-shift varying between 0.69 ± 0.2 for DMCM and 1.70 ± 0.4 for midazolam were observed. These values are in the same range as reported by others. It should be realized, however, that considerable differences in the values of the GABA-shift between laboratories have been reported. This parameter may be relatively sensitive to the experimental conditions and the radioligand that has been used in the characterization of the binding range (Wood et al., 1983; Petersen et al., 1984; Kemp et al., 1987; Dawson and Poretski, 1989; Mehta and Shank, 1995; Van Rijn et al., 1999). In the present investigation [3H]flumazenil has been used as a radioligand in the estimation of the GABA-shift for nonsteroidal GABAA receptor modulators. Flumazenil has no intrinsic efficacy itself, which facilitates estimation of the GABA-shift. Estimation of the GABA-shift for neuroactive steroids on the other hand is more complex. Because no direct ligand is available, the use of a ligand with positive or negative intrinsic efficacy is required to assess the GABA-shift, which makes interpretation more difficult. This may explain part of the variability in the values that have been reported.
A highly significant linear correlation was observed between the values of the in vivo intrinsic efficacy parameterePD and the log GABA-shift. In addition, the relationship between the log GABA-shift and the in vivo efficacy enabled the prediction of the “maximal” EEG response (Fig.8). This relationship between the log GABA-shift and predicted intrinsic activity in vivo was found to be nonlinear with a shallow part at low stimulus intensities in both directions and a steeper part at higher stimulus intensities. The relationship between the log GABA-shift and intrinsic activity predicts that compounds with a GABA-shift higher than 2 will show biphasic EEG effects in rats. For pregnanolone, a GABA-shift of 2.3 has been reported (McCauley et al., 1995) and for ORG 20599, a 3.5-fold binding enhancement was observed in the presence of GABA (Van Rijn et al., 1999), which is consistent with our observations. Figure 8 also shows that especially for drugs with a log GABA-shift of around 0.1 to 0.2, a small change in the shape/location of the stimulus-response relationship may result in a large difference in the observed response. These observations might explain why it has been difficult to predict the intensity of the in vivo responses of benzodiazepines (Goggin et al., 2000). In this respect, it is important that the shape and the location of the stimulus-response may differ between species, between individuals of the same species, and even within individual subjects. Such changes can occur as a result of differences/changes in receptor expression.
In conclusion, a mechanism-based PK/PD model has been successfully applied to the effects of benzodiazepines, an imidazopyridine, a cyclopyrrolone, and a β-carboline in vivo. The model was able to separate the drug-receptor interaction and the stimulus-response relationship. It was shown that all investigated compounds are low-efficacy modulators of the GABAA receptor, except for DMCM, which is a negative modulator. The stimulus-response relationship of the benzodiazepines was found similar to the first part of the stimulus-response relationship of neuroactive steroids and it was extended for the description of the response of a negative modulator, yielding a complex nonlinear stimulus-response relationship for the activation of the GABAA receptor in both positive and negative direction. Furthermore, the in vitro-in vivo correlations confirm that the new mechanism-based PK/PD model constitutes a realistic approach to the characterization of the effects of GABAA receptor modulators in vivo.
Acknowledgments
We acknowledge Prof. Dr. Ad IJzerman for the advice in receptor binding studies and Adriaan Cleton for providing the bretazenil data.
Footnotes
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↵1 Current address: AstraZeneca R&D Södertälje, DMPK and BAC, S 15185 Södertälje, Sweden.
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DOI: 10.1124/jpet.102.042341
- Abbreviations:
- PK/PD
- pharmacokinetic/pharmacodynamic
- DMCM
- methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate
- HPLC
- high-pressure liquid chromatography
- CCV
- constant coefficient of variation
- FOCE
- first-order estimation method with interaction
- ORG 21465
- 2β-3α-5α-3-hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione
- ORG 20599
- 2β-3α-5α-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one
- Received July 30, 2002.
- Accepted September 18, 2002.
- The American Society for Pharmacology and Experimental Therapeutics