Division of Pharmacology, Leiden/Amsterdam Center for Drug
Research, Leiden University, Leiden, The Netherlands (S.A.G.V.,
W.W.F.T.G., M.D.); Pfizer Global Research and Development, Discovery
Biology, Kent, United Kingdom (P.H.v.d.G.); and Mathematical Institute,
Leiden University, Leiden, The Netherlands (L.A.P.)
The objective of the present investigation was to characterize the in
vivo EEG effects of (synthetic) neuroactive steroids on the basis of a
recently proposed mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model. After intravenous administration, the time course of the
EEG effect of pregnanolone,
2
-3
-5-3-hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione (ORG 21465),
2-3-5-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one (ORG 20599), and alphaxalone was determined in conjunction with plasma concentrations in rats. For each neuroactive steroid the PK/PD
correlation was described on the basis of a two-compartment pharmacokinetic model with an effect compartment to account for hysteresis. The observed concentration EEG effect relationships were
biphasic and characterized with a mechanism-based pharmacodynamic model, which is based on a separation between the receptor activation process and the stimulus-response relationship. A single unique biphasic stimulus-response relationship could be identified for all
neuroactive steroids, which was successfully described by a parabolic
function. The receptor activation process was described by a hyperbolic
function. Estimates for the maximum activation (ePD) were similar for the different
neuroactive steroids but values of the potency estimate
(KPD) ranged from 157 ± 16 ng · ml
1 for pregnanolone, 221 ± 83 ng · ml1 for ORG 20599, and 483 ± 42 ng · ml1 for alphaxalone to 1619 ± 208 ng · ml1 for ORG 21465. A statistically significant
correlation was observed between the in vivo potency and the
IC50 in an in vitro
[35S]t-butylbicyclophosphorothionate
binding assay (r = 0.91). It is concluded that the
new PK/PD model constitutes a new mechanism-based approach to the
quantification of the effects of (synthetic) neuroactive steroids in
vivo effects. The results show that the neuroactive steroids differ in
potency but not in intrinsic efficacy at the GABAA receptor
in vivo.
 |
Introduction |
Neuroactive
steroids have long been known to produce anesthesia (Seyle, 1942
), but
the clinical development of neuroactive steroid anesthetics has been
hampered by side effects that are in part related to the pharmaceutical
formulation required for the intravenous administration (Anderson et
al., 1997; Sear, 1998). At present, there is a renewed interest in the
efficacy of neuroactive steroids for the management of epilepsy,
anxiety, insomnia, migraine, drug dependence, depression, stress, and
premenstrual syndrome (Gasior et al., 1997; Rupprecht and Holsboer,
1999). An important characteristic in this respect is the intrinsic
efficacy at the GABAA receptor in vivo. It
is known that both synthetic and endogenous neuroactive steroids are
selective and potent modulators of GABAA receptor
function, which are devoid of effects through activation of
glucocorticoid and/or mineralocorticoid receptors upon acute administration (Paul and Purdy, 1992; for reviews, see Lambert et al.,
1995; Rupprecht and Holsboer, 1999). Electrophysiological studies have
demonstrated that neurosteroids have dual effects at the
GABAA receptor upon binding. At nanomolar
concentrations neuroactive steroids potentiate GABA-evoked currents,
whereas at micromolar concentrations and in the absence of applied
GABA, they can directly elicit membrane currents through activation of
GABAA receptors (Harrison and Simmonds, 1984;
Cottrell et al., 1987).
In vitro it has been shown that synthetic neuroactive steroids and
other GABAA receptor modulators can differ in
intrinsic efficacy, covering the entire spectrum from full agonists to
inverse agonists (Sieghart, 1995; Anderson et al., 1997). It is well
established that intrinsic efficacy is a major determinant of
pharmacological actions in vivo (Kenakin, 1999). This underscores the
importance of estimation of the intrinsic efficacy in vivo.
In recent years, quantitative EEG parameters have often been used
as pharmacodynamic endpoint for the characterization of PK/PD
relationships of drugs acting at the GABAA
receptor and the µ-opioid receptor (Danhof and Mandema, 1992; Cox et
al., 1999). In addition, important progress has been made in the
development of a new class of mechanism-based PK/PD models that use
concepts from receptor theory (Van der Graaf and Danhof, 1997). A
specific feature of these models is a separation between the
characterization of the drug-receptor interaction on one hand and the
stimulus-response relationship on the other hand. In the mean time,
such mechanism-based PK/PD models have been successfully developed for
drugs such as benzodiazepines (Tuk et al., 1999), synthetic opiates
(Cox et al., 1998a), adenosine A1 agonists (Van
der Graaf et al., 1999), and
5-hydroxytryptamine1A agonists (Zuideveld
et al., 2001). It has been shown that these mechanism-based PK/PD
models constitute an excellent approach to the estimation of the in
vivo intrinsic efficacy.
Recently, we have proposed a novel mechanism-based PK/PD modeling
approach to describe the biphasic concentration-EEG effect relationship
of the synthetic neuroactive steroid alphaxalone (Visser et al., 2002).
In this approach, the initial receptor activation process is described
by a monophasic and saturable function, whereas the stimulus-response
function has a biphasic shape. In the model, the receptor activation
was described on the basis of a hyperbolic function and the biphasic
transducer on the basis of a parabolic function. In this manner, it has
been possible to identify the biphasic stimulus-response relationship of alphaxalone.
An important question is, however, whether the biphasic
stimulus-response relationship is only specific for alphaxalone or whether it applies to neuroactive steroids in general. In this respect,
it is important that the stimulus-response relationship should be
specific for the functioning of the biological system in vivo and
independent of the administered drug. A second important question is
whether quantitative differences in the EEG between neuroactive
steroids are caused by differences in potency (i.e., affinity),
intrinsic efficacy, or a combination of both.
In this investigation, we have characterized the in vivo PK/PD
relationships of a series of neuroactive steroids (i.e., alphaxalone, ORG 20599, ORG 21465, and pregnanolone) with known differences in
affinity at the GABAA receptor (Anderson et al.,
1997) to determine whether a unique stimulus-response relationship can
be identified for neuroactive steroids in general. A second objective
was to estimate the in vivo potency and intrinsic efficacy at the
GABAA receptor.
| |
Materials and Methods |
Animals and Surgical Procedures.
The protocol of this
investigation was approved by the Committee on Animal Experimentation
of Leiden University (Leiden University, The Netherlands). In this
investigation, groups of six to eight male Wistar rats with a mean body
weight of 297 ± 3 g (mean ± S.D., n = 42) were used (Charles River BV, Zeist, The Netherlands). 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 as described previously (Visser et al.,
2002). Briefly, the electrodes were placed 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. A reference electrode was placed
on lambda. 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. The surgical procedures were performed
under anesthesia with 0.1 mg · kg1 i.m.
medetomidine hydrochloride (Domitor; Pfizer, Capelle a/d IJssel, The
Netherlands) and 1 mg · kg1 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.
Three days before the start of the experiment indwelling cannulae were
implanted in the right femoral artery for the serial collection of
arterial blood samples and in the right jugular vein for drug
administration. The cannulae were filled with heparinized 25% (g/v)
polyvinylpyrrolidone in saline (Brocacef, Maarssen, The Netherlands)
and tunneled subcutaneously to the back of the neck where they were
exteriorized and fixed with a rubber ring.
Drugs and Dosages.
2-3-5-3-Hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione
(ORG 21465) and
2-3-5-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one (ORG 20599) were kindly donated by Organon Laboratories Ltd.
(Newhouse, Scotland) (Fig. 1).
Pregnanolone (5-pregnan-3-ol-20-one) and alphaxalone
(5-pregnan-3-ol-11,20-dione) were purchased from Sigma-Aldrich BV
(Zwijndrecht, The Netherlands) (Fig. 1). Infusion solutions were
prepared in DMSO (Baker, Deventer, The Netherlands). Per rat, 100 µl
of the infusion solution was administered. ORG 21465 was administered
in a dose of 8.7 ± 0.2 mg · kg1 in
5 min (n = 6). Pregnanolone was administered in two
dosages of 4.0 mg · kg1
(n = 7) and 9.8 mg · kg1
(n = 6) in 5 min. ORG 20599 was administered in two
dosages of 21 mg · kg1
(n = 8) in 5 min and 24 mg · kg1 (n = 6) in 15 min.
Alphaxalone was administered in a dose of 4.8 mg · kg1 (n = 8), which was group C
in a previous investigation (details in Visser et al., 2002). Control
experiments with administration of the vehicle DMSO were also included.
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Fig. 1.
Chemical structures of pregnanolone (A), ORG 21465 (B), alphaxalone (C), and ORG 20599 (D), which is a methane sulfonate
salt (MeSO3H).
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Pharmacokinetic-Pharmacodynamic 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. Two bipolar EEG leads (Cl
Ol) and
(CrOr) 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 1401plus interface (CED,
Cambridge, UK). The digitized signal was fed into a Pentium III
computer and stored on hard disk for off-line analysis. EEG baseline
was recorded for 45 min. Thereafter, the neuroactive steroids were
administered in a zero order intravenous infusion to the conscious and
freely moving rats using an infusion pump (Bioanalytical Systems, West
Lafayette, IN). The EEG recordings lasted until 250 min after the end
of the infusion. 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 for Windows,
version 3.18 (CED). Amplitudes in the -frequency band of the EEG
(11.5-30 Hz) averaged over 25-s time intervals were used as a measure
of drug-effect intensity. Serial arterial blood samples were collected
at predefined time intervals in heparinized tubes and centrifuged at
5000 rpm for 15 min for plasma collection. Total volume of redrawn
blood samples was kept equal to 2.1 ml during each experiment. Plasma samples were stored at 20°C until drug analysis.
Drug Analysis.
Pregnanolone, ORG 21465, and alphaxalone
plasma concentrations were determined by HPLC using the derivatization
and fluorescence detection method described previously (Visser et al.,
2000, 2002), which was slightly modified for ORG 21465. Briefly, to 50 µl of plasma, 50 µl of the internal standard (3 µg · ml1 pregnenolone dissolved in acetonitrile) was
added. Subsequently, 200 µl of acetonitrile was added to precipitate
plasma proteins. After centrifugation, the supernatant was transferred
to a clean tube, and 50 µl of 2 M NaOH and 25 µl of dansyl
hydrazine solution (20 mg in methanol acidified with 40 µl of
sulfuric acid) were added. The mixtures were stored in a dark place at
room temperature for 20 h. Subsequently, 500 µl of 1 M NaOH
(pregnanolone) or 500 µl of 0.2 M NaOH-glycine buffer pH 11 (ORG
21465) and 5 ml of dichloromethane were added, and the mixture was
vortexed for 5 min. The phase system was centrifuged for 15 min at
4500g and the organic phase was transferred to a clean tube
and evaporated under reduced pressure on a vortex vacuum evaporator
(Buchler Instruments, Fort Lee, NJ) at 37°C. The residue was
dissolved in 100 µl of mobile phase, of which a volume of 50 µl was
injected into the HPLC system. The HPLC system consisted of a
Spectroflow 400 solvent delivery system (Applied Biosystems, Ramsey,
NJ), a 712 Autosampler (Waters, Milford, MA) and a PerkinElmer LC240 fluorescence detector (PerkinElmer Ltd., Beaconsfield, UK).
Chromatography was performed on a C18 3-µm
cartridge column (100 × 4.6-mm i.d.; Chrompack, Bergen op Zoom,
The Netherlands) equipped with a guard column. The mobile phase
consisted of a mixture of 25 mM acetate buffer, pH 3.7, and
acetonitrile (40:60, v/v, for pregnanolone; 45:55, v/v, for
alphaxalone) or 17 mM phosphate buffer, pH 7.2, and acetonitrile
(56:44, v/v, for ORG 21465). Flow rate was 1 ml · min1. Fluorescence detection occurred at an
excitation wavelength of 332 nm and an emission wavelength of 516 nm.
Data acquisition and processing was performed using a Chromatopac C-R3A
reporting integrator (Shimadzu, Kyoto Japan). Using 50 µl of plasma,
the limit of quantification was 0.01 µg · ml1 for pregnanolone, alphaxalone, and ORG
21465. Linear calibration curves were obtained in the range 0.025 to 25 µg · ml1 (r > 0.995, n = 11) for pregnanolone, in the range 0.025 to 20 µg · ml1 (r > 0.997, n = 9) for ORG 21465 and in the range 0.025 to 10 µg · ml1 (r > 0.990, n = 17) for alphaxalone. For pregnanolone the
intra-assay coefficients of variation at 0.5 and 5.0 µg · ml1 were 9 and 12% (n = 15),
for ORG 21465 the intra-assay coefficients of variation at 0.1 and 1.0 µg · ml1 were 12 and 6%
(n = 5), and for alphaxalone the intra-assay
coefficients of variation for 0.25 and 2.5 µg · ml1 were 6 and 8% (n = 10).
The interassay coefficients of variation were 10 and 12%
(n = 29) for pregnanolone, 16 and 5%
(n = 23) for ORG 21465, and 16 and 12%
(n = 28) for alphaxalone, respectively.
ORG 20599 plasma concentrations were determined using HPLC with mass
spectrometry detection. To the plasma samples, 50 µl of 1.5 µg
· ml1 ORG 21465 as internal standard and 500 µl of 0.1 M acetate buffer, pH 4, were added. Extraction was
performed with a mixture of 5 ml of petroleum/ether (40:60) and
dichloromethane (55:45, 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 100 µl of mobile phase of
which 50 µl was injected into the liquid chromatography system with
mass spectrometry detection. The mobile phase consisted of a mixture of
methanol and water (80:20, v/v) and 1% (v/v) acetic acid. The system
consisted of a Broma solvent delivery pump, set at a flow rate of 1.0 ml · min1 (LKB, Uppsala, Sweden); a
717plus Autosampler (Waters); and a triple state
quadropole (T.S.Q.) 700 mass spectrometer (Thermo Finnigan, San Jose,
CA). Positive electrospray was used as ionization method with a sheath
flow of 1 µl · min1 with
MeOH/H2O (80:20, v/v) and 1% (v/v) acetic acid.
Chromatography was performed on a lichroma Rosil
NH2 column (156 × 4.6-mm i.d.; Alltech
Associates, Deerfield, IL). Components of [M + H] at 438.5 and
446.5 Da were quantified. Run-time was 5 min. The limit of quantification was 0.05 µg · ml1.
Linear calibration curves were obtained in the range of 0.1 to 10 µg · ml1 (r > 0.976, n = 5). The recovery of ORG 20599 was 90% at
concentrations of 0.25 to 5 µg · ml1.
The intra-assay coefficients of variation at 0.5 and 5.0 µg · ml1 were 7 and 6% (n = 5),
whereas the interassay coefficients of variation were 13 and 11%
(n = 18), respectively.
Protein Binding.
Plasma protein binding was determined in
vivo after administration of 5 mg · kg1
pregnanolone and 10 mg · kg1 ORG 21465. For each dose three rats were used. The protein binding for alphaxalone
has been determined previously (Visser et al., 2002) At two time points
after the administration of the neuroactive steroids 2-ml blood samples
were drawn and collected in heparinized glass tubes. After the second
sample the rats were directly sacrificed.
The tubes were centrifuged for 10 min at 5000 rpm to collect plasma.
From each tube, two plasma samples of 50 µl were taken and the
remaining plasma was centrifuged at 37°C (15 min, 2000 relative
centrifugal field) using an ultrafiltration device (Centrifree; Millipore Corporation, Bedford, MA). Two samples of 100 to 400 µl of
ultrafiltrate were taken.
Plasma protein binding of ORG 20599 was determined in vitro by
adding 5, 15, 30, and 50 µg of ORG 20599 to 2 ml of plasma (n = 3/concentration). After equilibration of the
mixtures for 30 min at 37°C, two plasma samples of 50 µl were taken
from each tube, and the remaining plasma was centrifuged at 37°C
using the ultrafiltrate device. Subsequently, two samples of 100 to 400 µl of ultrafiltrate were taken. After sample preparation, all plasma
and ultrafiltrate samples were analyzed. The free fraction (fu) was calculated by dividing the free
concentration in ultrafiltrate by the total (bound and free)
concentration in plasma.
Stability of ORG 20599 and ORG 21465 in Biological Fluids.
The stability of ORG 20599 and ORG 21465 in biological fluids was
studied in an ex vivo experiment. Fresh rat blood was obtained by
decapitation. ORG 20599 and ORG 21465 were added to 2 ml of plasma and
2 ml of blood. The decline of the concentration ORG 20599 and ORG 21465 was studied at 37 and 0°C. At fixed time points (0-30 min) samples
of 100 µl were taken and stored at 20°C until analysis at the
same day. In another experiment, ORG 20599 was added to 3 ml of blood
for determination of the blood-plasma concentration ratio. Samples of
200 µl were taken and split. One sample was immediately hemolyzed and
the second was heparinized for plasma collection. All samples were
immediately assayed as described above.
Pharmacokinetic Data Analysis.
In a population approach, the
neuroactive steroid plasma concentration-time profiles of all
individual rats in the different treatment groups were fitted
simultaneously while explicitly taking into account both
intraindividual variability in the model parameters as well as
interindividual variability. A two-compartment model was selected for
all compounds on the basis of the Akaike information criterion (Akaike,
1974). The concentration-time courses were modeled according to the
following equations:
|
(1)
|
|
(2)
|
in which Cp and
Ct represent the concentration of the
neuroactive steroid in the compartments 1 and 2, respectively. The input = R0 for t < =
T and input = 0 for t > T,
where R0 and T are the zero
order infusion rate and the duration of infusion. In these equations CL
is the clearance, Q is the intracompartmental clearance, and
V1 and
V2 are the volumes of distribution of
compartments 1 and 2.
The interindividual variability of these parameters was modeled
according to an exponential equation:
|
(3)
|
where 
is the population estimate for parameter P,
Pi is the individual estimate, and
i the random deviation of
Pi from P. The values of
i are assumed to be independently normally
distributed with mean zero and variance 
2.
For Q interindividual variability was fixed at zero. The
residual error in the plasma drug concentration was characterized by a constant coefficient of variation error model:
|
(4)
|
where Cpij represents the
jth plasma concentration for the
ith individual predicted by the model.
Cmij represents the predicted concentration, and
ij accounts for the residual deviation of the
model-predicted value from the observed concentration. The value for
was assumed to be independently normally distributed with mean zero
and variance 
2.
The model was implemented in the ADVAN6 subroutine in NONMEM (version
V, NONMEM project group, University of California, San Francisco, CA).
The first-order conditional estimation method (first-order conditional
estimation interaction) was used to estimate the population ,
2, and 2. From
individual Bayesian post hoc parameter estimates, CL, Q, V1,
V2,
Vdss, and half-lives were calculated
following standard procedures (Gibaldi and Perrier, 1982).
Subsequently, the individual Bayesian post hoc pharmacokinetic
parameter estimates were used to calculate the individual neuroactive steroid plasma concentrations at the time points of EEG measurements. Hysteresis was characterized on the basis of a hypothetical
effect-compartment model. In the effect compartment approach it is
assumed that the rate of onset and offset of effect is governed by the
rate of drug distribution to and from a hypothetical "effect site"
(Sheiner et al., 1979). Under this interpretation the effect
compartment model is linked to the plasma compartment by a first-order
equilibration rate constant (k1e) and
with a first-order rate constant for drug loss
(keo). The rate of change of the drug
concentration in the effect compartment can then be expressed by the
following differential equation:
|
(5)
|
where Cp represents the plasma
concentration and Ce the effect-site
concentration (see eqs. 1 and 2). Under the assumption that in
equilibrium the effect site concentration equals the plasma concentration, this equation can be simplified to the following:
|
(6)
|
The keo was calculated
nonparametrically using the program keo-obj.exe (S. J. Shafer,
Palo Alto VA Medical Center, Stanford University, Palo Alto, CA). In
the subsequent PK/PD analysis, the individual pharmacokinetic
parameters (CL, Q, V1, and
V2) and the
keo were fixed at the estimated
values, and effect-site concentrations were calculated at the time
points of the pharmacodynamic measurements.
Mechanism-Based Pharmacodynamic Analysis.
Concentration-effect data from the four neuroactive steroids
alphaxalone, ORG 20599, ORG 21465, and pregnanolone served as input for
the subsequent pharmacodynamic analysis. The data were analyzed by a
recently proposed mechanism-based model in which the effect considered
a function of a stimulus resulting from the drug-receptor binding (Tuk
et al., 1999; Visser et al., 2002). In this theory, the drug at the
effect site produces, upon binding to the receptor, a stimulus that is
followed by a cascade of signal transduction processes, leading to the
ultimate response (Fig. 2).
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Fig. 2.
Proposed mechanism-based PK/PD model consists of two
parts. The first part consists of a model for drug-receptor interaction
(eq. 10), which is a hyperbolic function of the effect-site
concentration producing a stimulus. KPD is
the concentration producing the half-maximal stimulus and
ePD is the maximal stimulus. The second part
consists of a biphasic stimulus-response model, which is represented by
a parabolic function (eq. 11). E0 is
baseline response, the top of the stimulus-response relationship is
located at the value Etop and is obtained at
the value b1/d, and the slope of the
parabolic function is determined by a. Exponent
d > 1 produces the asymmetry of the parabola.
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The definition of a drug-mediated response in terms of the classical
occupation theory, as proposed by Stephenson and Furchgott (Kenakin,
1997), considers the drug effect to be the result of the interaction
with a specific receptor followed by a stimulus to the biological
system. Thus, the interaction with the receptor yields a stimulus
S according to the following formula:
![S=<FR><NU>ϵ · [R<SUB><UP>t</UP></SUB>]<UP>·</UP>C</NU><DE>C+K<SUP>−</SUP><SUB>A</SUB></DE></FR>](/content/vol303/issue2/fulltext/616/img007.gif)
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(7)
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where S is a function of the concentration
(C), the parameter is a constant that
measures the capacity of a drug to initiate a stimulus from one
receptor and is a strictly drug-related parameter, [Rt] is the total number of
receptors, and KA is the equilibrium dissociation constant. Subsequently this stimulus is propagated into
the ultimate effect (E); its relation to the stimulus is given by an unknown function f:
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(8)
|
Receptor mediated drug responses in any given tissue depend,
therefore, on 1) two quantities determined by the drug: the intrinsic
efficacy and the equilibrium dissociation constant KA; and 2) two quantities determined
by the tissue: the constant [Rt] and
function f. Two adjustments to this general model have to be
made to apply it to in vivo systems. First, the total amount of
receptors cannot easily be measured in vivo, thereby allowing only the
product of and [Rt]
to be estimated. This can be defined as follows:
![e<SUB><UP>PD</UP></SUB>= ϵ· [R<SUB><UP>t</UP></SUB>]](/content/vol303/issue2/fulltext/616/img009.gif)
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(9)
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where ePD is the in vivo
efficacy. Second, the maximal stimulus achieved by the drug must be set
to 1 (ePD = 1), to allow an
independent estimation of f and
ePD of other compounds. The relationship between effect-site-drug concentration and effect is thus
characterized by the following equation:
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(10)
|
where KPD is the in vivo
potency and ePD the in vivo relative
efficacy. In this investigation KPD is
defined as in vivo potency instead of in vivo affinity, because for
compounds exerting the maximal effect, differences in efficacy cannot
be assessed, and because efficacy has no upper limit in principle,
differences in efficacy are indistinguishable from differences in
affinity when the efficacy is high (Colquhoun, 1998). In the analysis
of low-efficacy compounds versus the drug reaching the highest
stimulus, KPD can reflect the in vivo affinity.
Previously, we have shown that the neuroactive steroid alphaxalone
showed biphasic EEG effects, which reached at high concentrations isoelectric EEG. This implied a physiological maximum of the stimulus (i.e., the maximal stimulus is observed at an EEG effect of 0 µV;
Visser et al., 2002). Therefore, the value of
ePD of alphaxalone was fixed at 1 in
eq. 10. In the current investigation, pregnanolone also revealed
isoelectric EEG at the highest dosage. Although ORG 20599 and ORG 21465 did not reach the isoelectric EEG, it cannot be excluded that higher
dosages of ORG 20599 and ORG 21465 also give maximal EEG effects (i.e.,
isoelectric EEG).
The relationship f between the initial stimulus
(S) and the observed EEG effect was parameterized on the
basis of a parabolic function:
|
(11)
|
where Etop represents the top
of the parabola, a is a constant reflecting the slopes of
the parabola. b1/d is the stimulus for
which the top of the parabola (i.e., the maximal effect,
Etop) is reached, and the exponent
d results in an asymmetry of the parabola (Fig. 2). When no
drug is present the EEG is equal to its baseline value
(E0). Equation 11 then reduces to the
following:
|
(12)
|
Substituting eq. 12 in eq. 11 and rearranging yields the
following:
|
(13)
|
In the present analysis, the concentration-effect relationships
of the different neuroactive steroids were fitted simultaneously to
identify the drug-receptor interaction and the stimulus-response relationship. The KPD and the
ePD (relative to alphaxalone) were estimated for each neuroactive steroid, whereas the parameters a and d were estimated for the whole population.
Averaged amplitudes over 40 min of individual EEG recordings before
infusion served as input for individual baseline values
(E0).
In the previous analysis (Visser et al., 2002), it was shown that the
EEG effect dropped below baseline values and reached isoelectric EEG at
maximal stimulus of alphaxalone. Because in the present investigation
the effects did not drop under the baseline value except for the
highest dose of pregnanolone, parameter b could not be
identified. Therefore, b was fixed at the value obtained in
the previous analysis [0.44 ± 0.1 (7%); Visser et al., 2002]. This did not influence the estimation of exponent d. In the
previous analysis of the stimulus-effect relationship of alphaxalone it seemed impossible to estimate the value of parameter d. The
only a priori information about the value of d was that
d > 1 was used to describe the observed asymmetry of
the parabola. Based on numerical evaluation that revealed that the
value of d was likely to be between 2 and 4, d
was fixed at 3 (Visser et al., 2002). In the present analysis, due to
the availability of information on a series of different neuroactive
steroids, it became possible to estimate the value of d in
the simultaneous analysis of all neuroactive steroids.
It was observed that low baseline EEG
(E0) corresponded with low values for
the visually determined Etop. The
relationship between E0 and
Etop is given in eq. 12. The variation
in the Etop was not fully explained by
the variation in E0 when parameters a and b are kept constant. Parameter b
determines the location of the Etop,
whereas parameter a determines the height of the parabola.
Therefore, it was investigated whether parameter a could be
estimated in a fixed relationship to the baseline
(E0) in vivo:
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(14)
|
where A represents a constant that amplifies
variation in baseline into parameter a.
The interindividual variability in the pharmacodynamic parameter
KPD was modeled according to the
exponential eq. 3 and for the parameter a according to a
constant coefficient of variation error:
|
(15)
|
Similar to the pharmacokinetic analysis, the residual
variability in the pharmacodynamics was modeled as a coefficient of variation error according to eq. 4. The first-order estimation method
was used to estimate the population , 2,
and 2.
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 parameter correlation. Statistical
analysis was performed using one-way analysis of variance and a
Tukey-Kramer multiple comparison test. In the case of nonhomogeneity,
as determined by Bartlett's test, the nonparametric Kruskall-Wallis
test was used. Statistical tests were performed using InStat version
3.0 for Windows (GraphPad Software, San Diego, CA). All data are
represented as mean ± S.E., and p < 0.05 was
considered significant.
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Results |
Pharmacokinetics.
Fig. 3 shows
the observed, the individual, and population-predicted plasma
concentration-time profiles and the averaged EEG effect-time profiles
for 8.7 mg · kg1 ORG 21465 (A), 21 and
24 mg · kg1 ORG 20599 (B and C), 4.0 and
9.8 mg · kg1 pregnanolone (D and E), and
4.8 mg · kg1 alphaxalone (F). For each
neuroactive steroid, the individual pharmacokinetic profiles were best
fitted to a two-compartment model. For alphaxalone, CL and Q
were described as a function of body weight, which was described in
detail previously (Visser et al., 2002). The population pharmacokinetic
parameter estimates for each neuroactive steroid and the corresponding
inter- and intraindividual variability are summarized in Table
1. The values of the distribution and
elimination half-lives were 0.1 ± 0.01 and 22 ± 2 min
(n = 6) for ORG 21465, 0.6 ± 0.1 and 33 ± 2 min (n = 15) for pregnanolone, 2.0 ± 0.8 and
51 ± 1 min (n = 15) for ORG 20599, and 0.7 ± 0.01 and 10.8 ± 1 min (n = 8) for alphaxalone, respectively. No differences in the individual post hoc pharmacokinetic predictions were observed between the two dosages of pregnanolone and
ORG 20599, except for the estimate of
V1 for ORG 20599. No differences were
observed between the groups of ORG 20599 for volume of distribution at
steady state (Vdss). The clearance of ORG 20599 was 5 times faster than for the other neuroactive steroids and the volume of distribution two times larger. This was associated with a larger inter- and intraindividual variability.
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Fig. 3.
Observed (dotted line and circles) and predicted
(thin line, individual; thick line, population) concentration-time
profiles (left y-axis, on a logarithmic scale) and
averaged (mean ± S.E.) effect-time profiles (right
y-axis) for 8.7 mg · kg1 in 5 min
of ORG 21465 (A, n = 6); 21 mg · kg1 in 5 min (B, n = 8) and 24 mg · kg1 in 15 min (C, n = 6)
of ORG 20599; 4.0 mg · kg1 in 5 min (D,
n = 8) and 9.8 mg · kg1 in 5 min (E, n = 6) of pregnanolone; and 4.8 mg · kg1 alphaxalone (F, n = 8).
Alphaxalone is taken from Visser et al. (2002).
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TABLE 1
Population pharmacokinetic parameter estimates for CL, Q,
V1, and V2 with the corresponding
interindividual coefficient of variation (%) and 95% confidence
interval
Intraindividual residual variation was 18% for ORG 21465, 30% for
pregnanolone, 49% for ORG 20599, and 23% for alphaxalone,
respectively.
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The stability of 40 µg of ORG 20599 and 40 µg of ORG 21465 was
studied ex vivo in 2 ml of both blood and plasma at temperatures of 37 and 0°C. ORG 21465 seemed to be stable in blood and plasma at 37 and
0°C. In contrast, the concentration of ORG 20599 rapidly declined in
blood at 37°C, but not in plasma, where it seemed to be stable during
at least 30 min at 37°C (data not shown). The blood-plasma
concentration ratio of ORG 20599 was determined by adding 50 µg to 3 ml of blood. Each time point, samples of 200 µl were taken and split.
One sample was immediately hemolyzed and the second was heparinized for
plasma collection. At 37°C, ORG 20599 rapidly declined in blood,
whereas the concentration of ORG 20599 remained 100% at 0°C.
Blood/plasma concentration ratio was 0.73 ± 0.15 (mean ± S.D., n = 10) at 37°C and 0.92 ± 0.04 (mean ± S.D., n = 10) at 0°C.
The free fraction in plasma was 2.5 ± 0.5% for ORG 21465 (mean ± S.E., n = 11), 3.0 ± 0.8% for
pregnanolone (mean ± S.E., n = 10), and 3.2 ± 0.3% (n = 18) for alphaxalone. Plasma protein binding of ORG 20599 was determined ex vivo in plasma. No
concentrations ORG 20599 were measured in the ultrafiltrate despite
large volumes (400 µl). The free fraction in plasma was less than 2%
(n = 12) when taking the detection limit into account.
Pharmacodynamics and Hysteresis.
The EEG effects of the
neuroactive steroids, expressed as absolute amplitude in a 11.5- to
30-Hz band versus time, revealed a biphasic pattern as shown in Fig. 3.
Upon start of the infusion, the amplitude immediately increased,
followed by a partial decrease. After termination of the infusion, the
effect increased again to the same height and then gradually returned
to baseline. The partial decrease in amplitude was correlated to a
state of unconsciousness of the rats and the decrease of the amplitude
was deeper with higher dosages. For the highest dose of pregnanolone,
isoelectric EEG was reached during this partial decrease. Not all rats
in the treatment group of 24 mg · kg1
ORG 20599 showed a biphasic EEG effect, presumably as a result of the
lower infusion rate and as a consequence, the lower maximal plasma
concentrations (Cmax). Duration of the
effect (from the start of infusion until the return to baseline values
of the effect) was 100 min for 8.7 mg · kg1 ORG 21465, 35 and 40 min for 21 and 24 mg · kg1 ORG 20599, and 130 and 200 min
for 4.0 and 9.8 mg · kg1 pregnanolone,
respectively. For 24 mg · kg1 ORG 20599 and 9.8 mg · kg1 pregnanolone the
observed values of the baseline EEG were significantly lower than for
the other groups (Table 3). In control experiments, the vehicle DMSO
did not affect the EEG amplitudes (data not shown).
The individual pharmacokinetic parameter estimates were used to
calculate the plasma concentrations at the time points of the
individual effect measurements. The derived plasma concentration-effect relationships were biphasic and showed hysteresis. For each neuroactive steroid a representative plasma concentration-EEG effect profile is
shown in Fig. 4. The individual dose is
depicted in the graphs. Nonparametric hysteresis minimization yielded
estimates for keo of 0.53 ± 0.1 min1 (mean ± S.E., n = 6)
for ORG 21465, 0.17 ± 0.03 min1
(mean ± S.E., n = 14) for pregnanolone, and
0.26 ± 0.06 min1 (mean ± S.E.,
n = 14) for ORG 20599, corresponding to
keo half-lives of 1.3, 4.1, and 2.6 min, respectively.
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Fig. 4.
Representative individual profiles for the plasma
concentration versus amplitude in -frequency range for four
neuroactive steroids. For each neuroactive steroid, hysteresis and a
biphasic EEG effect with an increase in amplitude at low concentrations
and a decrease at high concentrations are observed. The individual dose
is depicted in the graph. The x-axis represents the
plasma concentration (nanograms per milliliter) for each neuroactive
steroid on a logarithmic scale, and the y-axis
represents the effect on the amplitude in -frequency range.
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Mechanism-Based Pharmacodynamic Modeling.
The mechanism-based
pharmacodynamic model was fitted to the biphasic effect-site
concentration-effect relationships of all individual rats
simultaneously. The individual profiles for all dosages could be
successfully described using the mechanism-based pharmacodynamic model,
yielding population estimates for the parameters KPD and
ePD for each neuroactive steroid and
estimates for the system parameters a and d.
Figure 5 shows the observed and predicted effect-site concentration versus EEG-effect relationship for
representative rats of each neuroactive steroid (for the same rats as
in Fig. 4). The population pharmacodynamic parameter estimates are
shown in Table 2. The
concentration-stimulus relationship (A) and the stimulus-effect
relationship (B) for the representative rats are depicted in Fig.
6. It shows that the
concentration-stimulus relationships of the neuroactive steroids differ
in the potency (i.e., the value of
KPD) but not in efficacy (i.e., the
value of ePD), because the estimates
of ePD of the various neuroactive
steroids were not different from 1.
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Fig. 5.
Observed (circles) and predicted (line) effect-site
concentration versus EEG effect for the same representative rats as
depicted in Fig. 3. The x-axis represents the effect
site concentration (nanograms per milliliter) for each neuroactive
steroid on a logarithmic scale and the y-axis represents
the effect on the amplitude in -frequency range.
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TABLE 2
Averaged (mean ± S.E.) Bayesian post hoc parameter estimates for
each neuroactive steroid for KPD,
ePD, a, and d with the
corresponding interindividual coefficient of variation in parentheses
and 95% confidence interval
The value for ePD was relative to the
ePD of alphaxalone, which was fixed at 1. The
intraindividual residual variation was 19%.
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Fig. 6.
A, drug-receptor interaction. The relationship
between effect-site concentration and stimulus for neuroactive steroids
for the representative individuals rats. Effect site concentration
(nanograms per milliliter) is depicted on the x-axis on
a logarithmic scale, and the stimulus is depicted on the
y-axis. B, stimulus-effect relationship. The biphasic
stimulus-effect relationship as described by the parabolic function for
steroids for the representative individual rats. Dots represent the
observed amplitudes. The lines represent the best-fitted
stimulus-effect relationship for each neuroactive steroid.
Intraindividual variability is 16% for all neuroactive steroids.
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A unique stimulus-effect relationship was observed for all neuroactive
steroids. Population estimates for a and d were
103 ± 25 (52%) and 3.36 ± 0.7 (), respectively. A
large interindividual variability (52%) was found for parameter
a. For pregnanolone (9.8 mg · kg1), this parameter was significantly lower
than for the other groups. It was investigated whether this large
variability could be explained by the observed differences in baseline
EEG effect (E0) between the groups. In
this respect, it is of interest that the calculated Etop and the baseline EEG effect
(E0) was lower for both 24 mg · kg1 ORG 20599 and 9.8 mg · kg1 pregnanolone compared with the other
treatment groups (Table 3). The
relationship between the baseline (E0)
and the predicted Etop and the
visually determined top of the effect (measured
Etop) are depicted in Fig.
7B. For comparison, all individual
observed and predicted stimulus-response profiles are shown in Fig. 7A. The regression coefficient shows that the
Etop is approximately 3 times
E0. The measured values for
Etop are slightly higher due to the
imperfection of visual determination. The ratio of a and E0 for each dosing group is ~10.
When parameter a is estimated as a function of the baseline,
following the model in eq. 14, the population estimate of A
is 9.2 ± 0.4 (22%), indicating that a difference in baseline of
1 µV is almost 10 times amplified in the value for a.
Individual estimates for A (ratio a and
E0) and the ratio
Etop and
E0, are similar for each neuroactive
steroid (Table 3).
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TABLE 3
Averaged values for baseline EEG (E0) and the
predicted maximal increase of the EEG (Etop),
parameter a, the ratio between a and
E0, and the ratio between E0 and
Etop (mean ± S.E., n = 6-8
for each group)
Etop is calculated from the post hoc estimation of
a, b, and E0 for each individual and each
dose of neuroactive steroid. For two dosages E0,
a, and Etop are significantly lower, but
the ratio between E0 and a or
Etop is constant. Population estimate of
A: the ratio a/E0 was 9.2 ± 0.4 (22%).
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Fig. 7.
A, biphasic observed and predicted stimulus-effect
relationship as described by the parabolic function for steroids for
all individuals. Dots represent the observed amplitudes. The thin lines
represent the best-fitted stimulus effect relationship for each
individual. The relationship between baseline and
Etop is illustrated with the broken lines.
B, baseline (E0) versus the predicted
Etop after the mechanism-based PK/PD
analysis and the visually determined (measured)
Etop derived from the individual graphs of
the neuroactive steroids. Linear regression: predicted
Etop = 2.9 · E0 (r = 0.80) is
consistent over a range of 5 to 15 µV and in agreement with the
experimental values [measured Etop = 3.2 · E0 (r = 0.79)].
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Discussion |
Pharmacokinetics.
The pharmacokinetics of pregnanolone,
alphaxalone, and ORG 21465 was successfully described by a
two-compartment model with linear elimination. The various neuroactive
steroids exhibit similar pharmacokinetic characteristics, such as a
very short distribution half-life and an elimination half-life between
20 and 50 min. The value of the clearance is around ~20 ml · min1, which is equal to the rat liver blood
flow, suggesting hepatic elimination with a high extraction ratio
(Sear, 1996; Visser et al., 2000, 2002). The volume of distribution at
steady state is larger for pregnanolone (~1.8 l · kg1) than for alphaxalone and ORG 21465 (~0.8
l · kg1). Interestingly, the
pharmacokinetics in rats shows great similarity to that in humans. In
humans, a clearance of ~25 ml min1
kg1 and a volume of distribution at steady
state between 0.7 and 2.5 l · kg1 have
been reported for pregnanolone, alphaxalone, and ORG 21465 (Hering et
al., 1996; Sear, 1996; Sneyd et al., 1997a,b).
To date, no pharmacokinetic information about ORG 20599 has been
reported. In the present investigation, it was shown that the clearance
of ORG 20599 is more than 5 times faster and that the volume of
distribution was ~2 times larger compared with the other steroids.
The fast clearance of ORG 20599, which is much higher than the hepatic
blood flow and the large volume of distribution might be explained by
the rapid decline of ORG 20599 in blood due to fast metabolism in
tissues other than the liver. The results of the ex vivo studies
suggest that components of whole blood (e.g., red blood cells) might be
an important factor in metabolism of ORG 20599, because the
concentration of ORG 20599 declined rapidly in whole blood at 37°C
and not in plasma, in contrast to ORG 21465, which was stable at 37°C
in both blood and plasma. A possible explanation for this difference is
the presence of an electron-drawing chloro-substitution in ORG 20599, which could make the keto-group more reactive and vulnerable to
metabolism by keto-reductase in comparison with ORG 21465. The
instability of ORG 20599 in blood has not been a confounding factor in
the estimation of the pharmacokinetic parameters estimates, because ORG
20599 is stable at 0°C. In these in vivo experiments, it required maximally 20 s to take the blood sample and to put it on ice to stop the breakdown of ORG 20599. It was shown that in the in vitro experiments the breakdown of ORG 20599 was less than 5% within 20 s.
The plasma protein binding for each neuroactive steroid was at least
~97%, and to our knowledge no values have been reported previously
for these compounds. The protein binding of ORG 20599 was determined in
plasma. Even though, no ORG 20599 could be found in the ultrafiltrate,
despite high plasma concentrations.
Values for keo half-life were between
1 and 5 min for each compound. The value of
keo half-life for pregnanolone was in
the same range as reported for human volunteers (Hering et al., 1996).
Mechanism-Based Pharmacokinetic-Pharmacodynamic
Modeling.
The recently proposed mechanism-based pharmacodynamic
model was successfully applied in this investigation. It was shown that the neuroactive steroids differ in their in vivo potency and not in
their in vivo efficacy. The biphasic stimulus-effect relationship f was fully characterized on the basis of a parabolic
function and was similar for each neuroactive steroid. An important
aspect of the present analysis is that an estimate of the exponent
d could be obtained on the basis of a simultaneous analysis
of the data from different neuroactive steroids. In the previous
analysis (Visser et al., 2002), when only data of alphaxalone were
considered, the value had to be fixed to 3, which seemed to be an
appropriate approximation at that time. Interestingly, the population
estimate of d (3.36 ± 0.7) is very close to the
previously assumed value of 3, confirming the validity of the approach.
In the present investigation a fixed relationship (a = A · E0, with A
= 9.2) was found between the baseline values
(E0) and values of a, and
thereby explaining the large interindividual variation of a
(and thus in Etop). This confirms that
the parameters of the stimulus-response relationship are not
drug-related but system-related parameters (Fig. 7). The maximal EEG
effect (Etop) that can be observed in
vivo, before the EEG starts to decrease, is ~3 times the baseline.
This relationship is important for future investigations where
variation in baseline is observed. In this investigation, the uniform
and unique shape of the stimulus-effect relationship, which is
determined for the all the neuroactive steroids studied so far,
indicates that this relationship is indeed a unique system related
property, reflecting the generation of the response upon
GABAA receptor activation.
The neuroactive steroids differed in their in vivo potency
(KPD) and not in their in vivo
efficacy. Because for high-efficacy compounds differences in efficacy
are indistinguishable from differences in affinity, the
KPD is herein defined as the in vivo
potency. It was shown that neuroactive steroids all have a relative
intrinsic efficacy that was not different from the value of the
ePD of alphaxalone. This is in agreement
with the observations that all these neuroactive steroids were able to
induce anesthesia in a similar way. Furthermore, in in vitro
investigations on the modulation of human recombinant GABAA receptors in oocytes, ORG 20599, and
alphaxalone differed only little in the maximal enhancement and potency
(Hill-Venning et al., 1996). It seems reasonable to assume that the
efficacy of neuroactive steroids cannot be higher in vivo, because the isoelectric EEG at higher dosages indicates that the physiological limit has been reached. Furthermore, anesthetics such as propofol and
pentobarbital have similar EEG patterns (Mandema and Danhof, 1990; Cox
et al., 1998b). In this investigation, pregnanolone was the most potent
neuroactive steroid, followed by ORG 20599, alphaxalone, and ORG 21465. It is of interest to compare the values of the
KPD obtained in the present in vivo
investigation to the values obtained in vitro. Anderson et al. (1997)
have studied the inhibition of [35S]TBPS
binding of the neuroactive steroids in vitro. In Fig.
8, these in vitro
IC50 estimates in the in vitro receptor assays are correlated to the in vivo KPD. The
order of the in vivo potency was similar to the ranking in the in vitro
IC50 of inhibition of
[35S]TBPS binding with the values for in vivo
potency. Interestingly, the rank order of the potency in vitro and in
vivo is similar. Estimates for in vivo
KPD are 2 to 6 times higher than the
values for the in vitro IC50. There are several
mechanisms that might explain such a difference. One possible factor is
the role of protein binding as a determinant in the pharmacodynamics of
the neuroactive steroids. For benzodiazepines, it is well known that effects are correlated to the unbound concentrations (Mandema et al.,
1991; Hoogerkamp et al., 1996). After correction for the degree of
protein binding, values of the
KPD,unbound are obtained that are in
the same potency order but 8 to 10 times lower than the in vitro
IC50 (Fig. 9). This
might suggest a large receptor reserve, which is not uncommon for
high-efficacy agonists in vivo (Cox et al., 1998a). In this respect,
however, also the allosteric modulation of the
GABAA receptor by GABA needs to be considered. The in vitro experiments were performed in the presence of 0.6 µM
GABA, which enhances binding and thereby lowers the
IC50 in the in vitro investigations. At present,
it is unknown to what extent such an effect occurs also in vivo. To
date, no selective radioligand has been reported for the neuroactive
steroid binding site; therefore, the affinity of an allosteric ligand
cannot be determined directly. Recently, van Rijn et al. (1999) have
described a method to assess the allosteric interactions between the
binding of GABA and GABAergic anesthetics at the
GABAA receptor in vitro using molecular modeling.
It was shown that ORG 20599 had an in vitro
Kd of 214 ng · ml1 and that the presence of GABA enhances the
affinity. This in vitro Kd of ORG
20599 is remarkably similar to the in vivo
KPD.
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Fig. 8.
Linear correlation (solid line) is observed between
the in vitro IC50 versus the in vivo
KPD (closed circles, log
KPD = 1.13 · log
IC50 0.32, r = 0.91) and versus
the KPD,unbound (open circles, log
KPD,unbound = 1.15 · log
IC50 1.28, r = 0.89) of four
neuroactive steroids. The dotted line represents the line of unity. The
in vitro data were taken from Anderson et al. (1997).
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Fig. 9.
Simulation of the EEG effects of compounds with
varying efficacy (0.3, 0.5, 0.7, 0.9, and 1) predicts that compounds
having a ePD lower than 0.7 have monophasic
EEG effects in contrast with compounds with a
ePD close to 1, which have biphasic EEG
effects.
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The mechanism-based PK/PD model for neuroactive steroids presented in
this investigation could be used for the prediction of the
concentration-effect relationships of other modulators of the
GABAA receptor. In Fig. 9, a simulation is shown
for compounds varying in efficacy. It is predicted that monophasic
concentration EEG effects profiles are produced by compounds that have
a relative efficacy lower than 0.7. This shows that the biphasic effect
contains specific information on the intrinsic efficacy of the drug
under investigation. In this respect, it is of interest that
benzodiazepines typically produce monophasic concentration-effect
relationships (Mandema et al., 1991, 1992). In theory, according to the
present mechanism-based pharmacodynamic model, this could be explained by the fact that benzodiazepines behave partial agonists (i.e., relative intrinsic efficacy <0.7) relative to synthetic neurosteroids.
In conclusion, a novel mechanism-based model has been successfully
applied to characterize the biphasic concentration-effect relationships
of neuroactive steroids. It was shown that the biphasic stimulus-effect
relationship has a uniform and unique shape, which was similar for each
neuroactive steroid. The exponent d and the relationship
between baseline and Etop could be
identified. All investigated neuroactive steroids are high-efficacy
modulators and differ only in their potency. The endogenous
neurosteroid pregnanolone was found to be the most potent neuroactive
steroid. Interestingly, this novel mechanism-based model predicts
monophasic concentration-EEG effects for compounds with an
ePD lower than 0.7.
We thank Erica Tukker for excellent technical assistance and
Bertil Hofte for the mass spectrometry analysis. ORG 20599 and ORG
21465 were kindly donated by Dr. Hamilton (Organon Laboratories Ltd.).
PK/PD, pharmacokinetic/pharmacodynamic;
DMSO, dimethyl sulfoxide;
HPLC, high-performance liquid chromatography;
TBPS, t-butylbicyclophosphorothionate;
ORG 21465, 2-3-5-3-hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione;
ORG 20599, 2-3-5-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one.
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Abstract
Introduction
