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Pharmacokinetic-Pharmacodynamic Modeling of theElectroencephalogram Effect of Synthetic Opioids in the Rat:

Correlation with the Interaction at the Mu-Opioid Receptor

EUGE ` NE H. COX, THOMAS KERBUSCH, PIETER H. VAN DER GRAAF and MEINDERT DANHOF

Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, University of Leiden, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden, The Netherlands

Accepted for publication November 13, 1997 This paper is available online at http://www.jpet.org

ABSTRACT

The purpose of our investigation was to characterize the rela-tionships between the pharmacodynamics of synthetic opioids in vivo and the interaction at the mu-opioid receptor. The phar-macokinetics and pharmacodynamics were determined in vivoafter a single i.v. infusion of 3.14 mg/kg alfentanil (A), 0.15mg/kg fentanyl (F) or 0.030 mg/kg sufentanil (S) in rats. Ampli-tudes in the 0.5 to 4.5 Hz frequency band of the electroenceph-alogram (EEG) was used as pharmacodynamic endpoint. TheEEG effect intensity was related to the (free) concentration inblood (A) or in a hypothetical effect compartment (F, S) on basisof the sigmoidal Emax pharmacodynamic model. The interactionat the mu-opioid receptor was determined in vitro on basis ofthe displacement of [3H]-naloxone binding in washed rat brainmembranes. The value of the sodium shift was used as ameasure of in vitro intrinsic efficacy. For the EEG effect the in

vivo potencies based on free drug concentrations (EC50,u ) were4.62 Ϯ 0.66 ng/ml (A), 0.69 Ϯ 0.05 ng/ml (F) and 0.29 Ϯ 0.06ng/ml (S). In the receptor binding studies the affinities at the mu-opioid receptor ( K

I ) were 47.4 Ϯ 6.6 nM (A), 8.6 Ϯ 4.1 nM (F)

and 2.8 Ϯ 0.2 nM (S). For each opioid the ratio between EC50,u

and K I

was the same with a value of 0.23–0.25, indicating theexistence of receptor reserve for the EEG effect. The intrinsicactivity (Emax ) of the three opioids in vivo was similar with valuesof 111 Ϯ 10 V (A), 89 Ϯ 11 V (F) and 104 Ϯ 4 V (S)However, the values of the sodium shift varied between 2.8 (S)and 19.1 (A). Further analysis of the in vivo pharmacodynamicdata on basis of an operational model of agonism providedevidence for a large receptor reserve, which explains why com-pounds with different values of the sodium shift all behave asfull agonists in vivo.

Synthetic opioids are frequently used in clinical analgesiaand anesthesia. Among these opioids important differencesin pharmacokinetics and pharmacodynamics exist and thishas important implications for the clinical application (Sha-fer and Varvel, 1991). Currently, a number of new syntheticopioids with more favorable pharmacokinetic and pharmaco-dynamic properties relative to existing opioids is under development (James, 1994; Rosow, 1993).

In recent years considerable progress has been made in theunderstanding of opioid receptor pharmacology. Differentopioid receptor subtypes have been identified, and it has beendemonstrated that opioids may exhibit selectivity with re-gard to the binding at the various subtypes (Pasternak,1993). Important differences in the signal transduction path-ways between the various receptor subtypes have been ob-served (Schiller et al., 1992). Furthermore several com-

pounds have been shown to act as partial agonists, indicatingthat there may be differences in intrinsic efficacy betweenopioids (Miller et al., 1986; Kajiwara et al., 1986; Berzeteigur-

ske et al., 1995). In several studies the functional role of thedifferent opioid receptor subtypes has been determined (Pas-ternak, 1993). At present mu-opioid receptors are thought to

be responsible for most of the analgesic effects (Mathhes et

al., 1996) and some of the other effects such as respiratory

depression and sedation (Ling et al., 1983). In clinical inves-

tigations the effects of opioids are typically quantified on thebasis of rating scales (Shannon et al., 1995). Recently quan-

titative EEG monitoring has been used successfully to char-acterize the time course of the pharmacological actions inman after i.v. administration of anesthetic doses. By appli-

cation of pharmacokinetic-pharmacodynamic modeling itwas demonstrated that the value of certain EEG parameterscan be directly related to the concentration at a hypotheticalReceived for publication July 21, 1997.

ABBREVIATIONS: PK/PD, modeling-pharmacokinetic-pharmacodynamic modeling; EEG, electroencephalogram; CNS, central nervous system

SPF, specific pathogen free; I-E ratio, inspiration expiration ratio; pCO2, partial CO2 pressure; pO2, partial O2 pressure; GC, gas chromatography

ID, internal diameter; HPLC, high pressure liquid chromatography; E0, no drug effect; Emax, maximum drug effect; EC50, concentration resulting

in 50% of the maximum drug effect; ANOVA, analysis of variance; Cl, total body clearance; Vd,ss, volume of distribution at steady-state; t1/2,␣n

half-life associated with the nth exponential phase; RIA, radioimmunoassay, fu, fraction of drug concentration unbound in the body.

0022-3565/98/2843-1095$03.00/0THE JOURNAL OF PHARMACOLOGY AND E XPERIMENTAL THERAPEUTICS Vol. 284, No. 3Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A

JPET 284:1095–1103, 1998

1095



effect site on basis of the sigmoidal Emax pharmacodynamicmodel (Scott et al., 1985; 1991; Lemmens et al., 1994; Egan et

al., 1996). In this way major differences in potency (i.e., EC50)among the opioids were observed. Furthermore, clear differ-ences in the onset of the effect were observed which could becharacterized on the basis of the rate constant keo. The EEGeffect was finally shown to occur in a clinically relevantconcentration range with regard to the anesthetic effect

(Ebling et al., 1990).Despite the important progress that has been made in both

the receptor pharmacology and the clinical pharmacology of opioids, thus far no attempts have been reported to relate thetwo to each other. In other words the quantitative relation-ships between receptor binding and pharmacological effectintensity have not been explored in vivo. This is importantbecause the pharmacological actions of a drug are related tothe receptor pharmacology in a rather complex fashion. Thepotency and the intrinsic activity of a drug in vivo are notonly dependent on drug-related properties such as affinity toand the efficacy at the mu-opioid receptor but also on tissuerelated factors such as the efficiency of the receptor-effector

coupling and on homeostatic mechanisms that may be oper-ative (Haynes, 1988; Johnson and Fleming, 1989). Recentlymechanism based models to characterize the pharmacody-namics of drugs in vivo have been proposed (Tuk et al., 1995; Van der Graaf et al., 1997). In these models the interaction atthe receptor has been separated from the multitude of pos-treceptor events. This separation of drug-specific effect fromthe tissue specific stimulus-effect propagation is of value toexplore and understand changes in pharmacodynamics,caused by factors such as aging, tolerance, disease states anddrug interactions and may furthermore be helpful in thedesign of new compounds.

The purpose of our investigation was to quantitativelyexplore the relationships between receptor binding and phar-macological effect of synthetic opioids in vivo. QuantitativeEEG parameters were used as a pharmacodynamic endpoint,as this provides a continuous and realistic measure of theirpharmacologic effect (Cox et al., 1997). The in vivo pharma-codynamic data were analyzed on basis of an operationalmodel of agonism (Black and Leff, 1983) to examine therelationships with the information obtained in the in vitro

receptor binding assays.

Methods

Chemicals. Alfentanil hydrochloride, fentanyl citrate, sufentanilcitrate and the internal standard R38527 were donated by JanssenPharmaceutica BV (Beerse, Belgium). Midazolam was donated byHoffmann-LaRoche (Basel, Switzerland). Vecuronium bromide wasobtained from Organon Technika BV (Boxtel, The Netherlands).

Experimental animals. Male SPF rats of Wistar descent with abody weight between 250 to 300 g were used in the experiments(Brookman BV, Someren, The Netherlands). The rats were housedindividually in plastic cages at constant temperature of 21°C and acontrolled light-dark cycle (lights on: 7.00 A .M. to 7.00 P.M.). Food(Standard Laboratory Rat Mouse and Hamster Diets, RMH-TM,Hope Farms, Woerden, The Netherlands) and tap water were avail-able ad libitum. One week before the EEG experiment the rats hadseven cortical EEG electrodes implanted under fentanyl/fluanisoneanaesthesia (Hypnorm, Janssen Pharmaceutica BV, Beerse, Bel-gium) as described before (Mandema and Danhof, 1990). One day

before the experiment, four permanent cannulas were implanted:

one in the femoral artery, two in the left jugular vein and one in thefemoral vein. The cannulas in the left jugular vein and the femoral

vein were used for the administration of alfentanil, midazolam and vecuronium respectively. The cannula in the femoral artery was usedfor the collection of blood samples. The protocol of the study wasapproved by the Committee on Animal Experimentation of LeidenUniversity.

Pharmacokinetic-pharmacodynamic experiments. Thepharmacokinetics and pharmacodynamics of the opioids were deter-

mined after i.v. administration. Rats were randomly assigned to fourdifferent treatment groups of seven to eight rats. Alfentanil (3.14mg/kg in 40 min), fentanyl (0.15 mg/kg in 20 min) and sufentanil(0.030 mg/kg in 40 min) were administered at a constant rate. Agroup of control animals received an infusion of 1.5 ml saline for 40min. To determine the pharmacokinetics of the opioids arterial bloodsamples (20–1000 l) were serially collected at fixed time intervalsover a period during and after the infusion. Alfentanil, fentanyl andsufentanil blood samples were immediately hemolyzed with 0.5 ml ofdeionized water and stored at Ϫ20°C until analysis.

Two bipolar EEG leads (Cl-Ol and Cr-Or) were continuously re-corded using a Nihon-Kohden AB-621G Bioelectric Amplifier (Hoek-loos, Amsterdam, The Netherlands) and concurrently digitized at arate of 210 Hz using a CED 1401plus interface (CED, Cambridge

UK). The digitized signal was fed into a 80486 computer (Inteb BV,Sassenheim, The Netherlands) and stored on hard-disk for off-lineanalysis. For each 5-sec epoch, quantitative EEG parameters wereobtained off-line by fast Fourier analysis using a user-defined pro-gram within the data analysis software package Spike 2, version4.60 (CED, Cambridge, UK). Reduction of EEG data was performedby averaging spectral parameter values over predetermined timeintervals.

To prevent opioid-induced seizures which are typically observedupon the administration of these high anesthetic doses, rats receiveda continuous infusion of midazolam at a rate of 5.5 mg/kg/hr (Cox et

al., 1997). To reach steady-state rapidly, midazolam was adminis-tered according to a Wagner infusion scheme, with an initial infusionrate at three times the steady-state infusion rate for 16 min (Wagner1974). The midazolam infusion was started 30 min before the ad-

ministration of the opioid. In each of the rats midazolam was able tosuppress seizure activity completely.

During and after the infusion of the opioids, severe respiratorydepression and muscle rigidity occurred. Rats were artificially ven-tilated with air using an Amsterdam Infant Ventilator, model MK3(Hoekloos, Amsterdam, The Netherlands) through a custom made

ventilation mask. The ventilation settings were: ventilation fre-quency 62 beats/min, I-E ratio 1:2 and air supply flow rate 0.7 to 1.0l/min. When muscle rigidity appeared, the rat received an i.v. bolusinjection of 0.15 mg vecuronium bromide and artificial ventilationwas started. Administration of vecuronium in a dose of 0.10 mg wasrepeated each time muscle rigidity reappeared (usually every 5 min)until spontaneous respiratory activity returned and muscle rigiditydid not reappear. To determine whether artificial ventilation wasadequate arterial pH, -pCO2 and -pO2 values were monitored usinga Corning 178 Blood Gas Analyzer (Ciba Corning, Houten, TheNetherlands).

During the experiments, body temperature was stabilized be-tween 37.5 and 38.5°C with the aid of Delta Phase Isothermal Heat-ing Pads (Braintree, Braintree, MA) and ventilation with air pre-heated to 32°C. Body temperature was monitored using a YSI TeleThermometer (Yellow Springs Instrument Corporation Inc, YellowSprings, OH).

Determination of the cerebrospinal fluid over total blood

concentration ratio. The relative free fraction of the opioids in thebrain was determined on the basis of the cerebrospinal fluid/totalblood concentration ratio in steady-state. For these experimentsthree groups of seven to eight male Wistar rats were used. Thecannulation procedure was as described above. The rats received a

steady-state infusion of midazolam as described above. Instanta-

1096 Cox et al. Vol. 284



neous pseudo steady-state concentrations of the opioids were ob-tained using a computer controlled infusion device. The STANPUMPprogram (Shafer and Gregg, 1992) was used to clamp blood opioidconcentrations at the respective EC50 value, as determined by thepharmacokinetic-pharmacodynamic experiment. Before the start of the opioid infusion, a midazolam infusion was initiated as describedabove. During the opioid infusion, rats were artificially ventilatedand muscle rigidity was managed as described above. Adequatenessof artificial ventilation was monitored by measuring arterial pH-,

-pCO2- and pO2 levels. Thirty minutes after the start of the opioidinfusion, two arterial blood samples of 100 l were drawn. Simulta-neously, a CSF sample was obtained by cisternal puncture. Bloodand CSF samples were hemolyzed with 0.5 ml deionized water andstored at Ϫ20°C until analysis.

Drug assays. Blood and CSF concentrations of alfentanil weredetermined by gas chromatography with nitrogen-phosphorus detec-tion as described previously (Cox et al., 1997). The intra- and inter-assay variability was generally less than 5% and the lower limit of quantitation was 1 ng/ml for a 0.1-ml sample.

Blood and CSF concentrations of sufentanil were determined us-ing a slightly modified RIA method, as described by Woestenborghs

et al. (1994). Hemolyzed blood samples were alkalinized with 1 ml 0.1N NaOH and subsequently extracted with 5 ml of n-heptane/isoamy-

lalcohol (98.5:1.5, v:v). The organic phase was transferred to a cleantube and subsequently evaporated to dryness under reduced pres-sure. The residue was then reconstituted in 50 l methanol and 0.5ml 2% phosphate buffered bovine serum albumin (BSA) solution.3H-sufentanil (activity approximately 25,000 dpm) and antiserumwere added followed by 2 hr of incubation. Unbound ligand was thenprecipitated by incubation with 200 l 2% dextran-charcoal suspen-sion for 1 hr. Supernatant was transferred to scintillation vialscontaining 4 ml of Packard UltimaGold scintillation fluid (Packard,Meriden, CT). Finally ␤-activity was measured for 4 min using aPackard Tri-Carb 1500 liquid scintillation analyzer (Packard, Down-ers Grove, IL). Calibration curves were obtained in duplicate in therange of 0.040 to 10 ng/tube using a blank standard for nonspecificbinding and a zero standard for the determination of B0. Activity wasexpressed as percent binding relative to B0. Relative binding was

used to construct a linear logit-log plot. Binding ability (B0) wasapproximately 33%. Corresponding binding levels were 82 and 10%for 0.040 and 2.0 ng per assay tube, respectively. With the extractionof a blood sample of 1 ml at the end of the experiment sufentanilconcentrations of 0.040 ng/ml could be measured. Intraassay vari-ability was 35 and 14% for 0.040 and 2.0 ng per assay tube, respec-tively.

For the determination of fentanyl concentrations in blood and CSFessentially the same RIA procedure was used as for sufentanil.Binding ability (B0) was approximately 25%. Corresponding binding levels were 89 and 20% for 0.040 and 2.0 ng per assay tube, respec-tively. With the extraction of a sample of 1 ml at the end of theexperiment fentanyl concentrations of 0.040 ng/ml could be mea-sured. Intra assay variability was 37 and 4% for 0.040 and 2.0 ng perassay tube, respectively.

The blood concentrations of midazolam were determined by HPLCwith UV detection as previously described (Mandema et al., 1991a).The intra- and interassay variation was less than 6%.

Receptor binding. Because in our investigation, the emphasis ison in vitro/in vivo correlations in the pharmacodynamics of opiates,the receptor binding characteristics were determined in brain homo-genates rather than in cells transfected with mu-opioid receptor.Brain homogenates were prepared according to the method of Lohse

et al. (1984). Briefly, rat brain (minus cerebellum) was homogenizedin 10 volumes 50 mM Tris-HCl buffer (pH ϭ 7.4) at 25°C. Thesuspension was centrifuged at 5000 ϫ g for 20 min and the pellet waswashed three times with Tris-HCl.

The protein concentration in the homogenate was 2 mg/ml, asdetermined with the Pierce Micro BCA assay (Pierce, Rockford, IL).

Opioid receptor binding was determined by displacement of [3

H]-

naloxone (New England Nuclear-719, specific activity 57.5 Ci/mmolNew England Nuclear, Boston, MA) at 25°C. Brain homogenatealiquots of 100 l were incubated with 2.5 nM [3H]-naloxone at

various concentrations of the opioids. After 30 min of incubation thesamples were filtered through a presoaked glass fiber filter (What-man GF/B) and eluted six times using 3 ml 50 mM Tris-HCl buffer of4°C under reduced pressure. The filters were submerged in 3.5 mlPackard UltimaGold scintillation fluid and radioactivity was mea-sured for 4 min by a Hewlett-Packard Tri-Carb 1500 liquid scintil-

lation counter.In this binding assay the agonistic character of the opioids was

evaluated by examining the effect of a high concentration of sodium(100 mM) on the receptor binding affinity. It has been shown that theshift in K i value is a reflection of the agonist efficacy of the ligand(Pert and Snyder, 1974). The sodium shift was expressed as the ratioof the K i value in the presence, and in the absence of 100 mM NaClrespectively. The receptor binding characteristics in the presence ofsodium were determined by replacement of 100 l 50 mM Tris-HClbuffer with 100 l 400 mM NaCl solution in the incubation mixture.

The receptor binding characteristics of the radioligand [3H]-nal-oxone ( K d and Bmax) were determined in a saturation experimentunder similar conditions as described above, using various concen-trations of the ligand up to 20 nM. Nonspecific binding was deter-

mined by calculating the binding of

3

H-naloxone in the presence of10Ϫ4 M fentanyl. Free radioligand concentrations were calculated bysubtracting the nonspecific binding from the concentrations in theincubation mixture. In the displacement studies the concentration of[3H]-naloxone was equivalent to the K d value in the saturationexperiment. In both displacement and saturation experiments bind-ing was determined in triplicate for each concentration.

Data analysis. The pharmacokinetics and the pharmacokineticsof the opioids were determined in each individual rat. The bloodconcentration time profiles were described by a poly-exponentiaequation:

C t ϭ iϭ1

nCi

i ⅐ T 1 Ϫ e

Ϫi ⅐ t t Ͻ T (1A)

C t ϭ iϭ1

nCi

i ⅐ T 1 Ϫ e

Ϫi ⅐ T ⅐ e

Ϫi ⅐ tϪT t Ն T

(1B)

where C(t) is the blood concentration of opioid at time t, T is theduration of the infusion and Ci and i are the coefficients and theexponents of the equation, respectively. Different models were inves-tigated and tested according to the Akaike Information Criterion(Akaike, 1974). Various pharmacokinetic parameters were calculated from the coefficients and exponents of the fitted functions bystandard methods (Gibaldi and Perrier, 1982).

The sigmoidal Emax pharmacodynamic model was used to describe

the relationship between opioid concentration and EEG effect:

EC ϭ E0ϩ

Emax ⅐ Cn

EC 50 ϩ Cn (2)

in which EC is the EEG effect at opioid blood concentration c, E0 isthe no-drug effect, Emax is the maximum effect, EC50 is the opioidconcentration at 50% of the maximum effect and n is the Hill factorreflecting the sigmoidicity of the curve. When hysteresis was ob-served in the blood concentration-EEG effect relationship, hysteresiswas minimized parametrically by postulating a hypothetical effectcompartment in which the concentration is determined by a mono-exponential model (Sheiner et al., 1979; Danhof and Mandema, 1994

C e t ϭ Cb t e

Ϫ Keo ⅐ t

(3)

1998 PK/PD Modeling of Opioids in Rats 1097



where Ce(t) and Cb(t) are the opioid concentration in the effect andthe blood compartment, respectively, at time t, denotes convolutionand Keo is the exponent of the unit impulse disposition function forthe effect compartment. The sigmoidal Emax pharmacodynamicmodel (equation 2) was used to relate the EEG effect to effect com-partment concentration.

The concentration-EEG effect relationship of the three opioidswere also simulated according to the operational model of agonism asproposed by Black and Leff (1983):

EC

Em

ϭ

n

⅐ Cn

K A ϩ Cnϩ

n⅐ C

n (4)

where EC is the effect at the free ligand concentration C, Em is themaximum effect in the system, is the efficacy parameter, defined asthe ratio of [R0], the total concentration of available receptors andK E, the concentration of occupied receptors that elicits half-maximaleffect, n the slope factor of the transduction function and K A is theagonist equilibrium/dissociation constant. The receptor binding characteristics of the radioligand [3H]-naloxone were determined byfitting the following equation to the data from the saturation exper-iment:

B ϭ Bmax C f

K d ϩ C f

(5)

where B is the number of receptors occupied, Bmax is the totalnumber of specific binding sites, K d is the ligand concentration atwhich 50% of the receptors is occupied and C f is the free ligandconcentration. IC50 values for the three opioids were determined byfitting the following equation to the data from the displacementstudy:

B ϭ B0 IC 50

IC 50 ϩ Cd

(6)

In which B0 is the specific binding of the radioligand in the absenceof displacer and Cd is the concentration of displacer added. The K

value for the three opioids were calculated from the IC50 valuesaccording to the Cheng-Prusoff equation:

K I ϭ

IC50

1 ϩ L*/ K *d(7)

where IC50 is the opioid concentration that displaces 50% of 3H-

naloxone, L* is the concentration of 3H-naloxone and K *d is theequilibrium dissociation constant of 3H-naloxone as determined inthe saturation experiment.

The pharmacokinetic and pharmacodynamic data were analyzedusing the nonlinear least-squares program Siphar version 3.0 (SimedSA, Creteil, France). Pharmacokinetic and pharmacodynamic parameter estimates for the different opioids were statistically evaluated using a one-way ANOVA. In case of non-homogeneity, as deter-mined by Bartlet’s test, the nonparametric Kruskal-Wallis test wasused. A significance level of 5% was selected. The receptor bindingdata were analyzed using the software package GRAPH PRISM,

version 2.0 (GraphPad, San Diego, CA).

ResultsThe changes in the EEG effect (amplitude in the 0.5–4.5

Hz frequency band of the EEG power spectrum) and drugconcentrations in the blood as a function of time in fourrepresentative rats of the treatment groups are shown infigure 1. During i.v. infusion of the opioids a rapid andpronounced increase in the amplitude of the 0.5 to 4.5 Hzfrequency band of the EEG was observed for all drugs. Theeffect reached a maximum and maintained that level forsome time after termination of the infusion. The effect thengradually returned to preinfusion values. The pharmacoki-

Fig. 1. Blood concentration (F)and EEG effect (—) vs. time pro-files on i.v. infusion of alfentanil(3.14 mg/kg in 40 min), sufentanil(0.030 mg/kg in 40 min), fentanyl(0.15 mg/kg in 20 min) or physio-

logical saline (0.8 ml in 40 min).The solid bars represent the dura-tion of the infusion. The dashedline through the concentrationpoints represents the best fit of data according to the poly-expo-nential equation.




netics of all opioids were most adequately described using abi-exponential equation. The averaged pharmacokinetic pa-rameters calculated for each opioid in the individual rats aresummarized in table 1. The clearance of sufentanil was abouttwice the value obtained for alfentanil. The volumes of dis-tribution at steady-state ranged from 0.75 liter/kg for alfen-tanil to 5.53 liter/kg for sufentanil. The terminal half-life foralfentanil was considerably smaller than for fentanyl and

sufentanil.For alfentanil the EEG effect could be related directly to

the opioid blood concentrations. For fentanyl and sufentanilprofound hysteresis was observed between blood concentra-tions and EEG effect (fig. 2). After minimization of hystere-sis, the concentration-EEG effect relationship of the threeopioids could be described satisfactorily on basis of the sig-moidal Emax pharmacodynamic model (equation 2). In figure3 the concentration-EEG effect relationship is shown for thesame three individual rats from which the data are shown infigure 1. The averaged pharmacodynamic parameter esti-mates obtained from the individual rats are represented intable 2. Significant differences were observed for the EC50

values of the opioids. Sufentanil (1.43 ng/ml) was the mostpotent opioid for the EEG effect, and alfentanil (289 ng/ml)was the least potent. No significant differences in Emax, E0

and Hill factor were observed for the three opioids.From the start of the opioid infusion midazolam concentra-

tions were constant in all treatment groups with an averageconcentration of 992 Ϯ 318 ng/ml (mean Ϯ S.D., n ϭ 80).

The determination of the ratio of cerebrospinal fluid/totalblood concentrations under steady-state conditions revealedsignificant differences in the relative free fraction of theopioids in the brain (table 1). The relative free fraction of alfentanil in the brain was considerably lower than those forfentanyl and sufentanil. In general, the measured blood con-centrations were in agreement with the concentration levelsto which the STANPUMP program was instructed to target,confirming the adequacy of the pharmacokinetic parameterestimates obtained from the pharmacokinetic-pharmacody-namic experiment.

The receptor binding parameters of [3H]-naloxone were K dϭ 2.1 Ϯ 0.5 nM and Bmax ϭ 154 Ϯ 28 fmol/mg. The displace-ment studies with [3H]-naloxone revealed significant differ-ences in in vitro receptor affinity between the different opi-oids (table 3). Sufentanil showed highest affinity for themu-opioid receptor ( K I ϭ 2.8 nM), while alfentanil showedthe lowest affinity ( K i ϭ 47.4 nM). In the presence of 100 mMNaCl, K i shifted to higher values for all opioids. This sodium-shift, expressed in the ratio of the two K i values, ranged from

2.8 (sufentanil) to 19.1 (alfentanil). In contrast, the receptorbinding for the opioid antagonist [3H]-naloxone was not sig-nificantly affected: K d ϭ 1.8 Ϯ 0.3 nM and Bmax ϭ 216 Ϯ 16pmol/mg protein. A high correlation (r Ͼ 0.999) was observed

between in vitro mu-opioid receptor affinity ( K i) and in vivo

potency (EC50,u) as can be seen from the constant ratio ofthese two values for each opioid (table 3).

The relation between the in vitro and in vivo results wasfurther investigated by simulations with the operationalmodel of agonism (see “Methods”). Figure 4a shows that themodel could simulate the concentration-effect curves of alfen-tanil, fentanyl and sufentanil simultaneously by keeping theagonist-independent parameters (Em and n) constant andassuming that the operational affinity (K A ) for each agonistwas identical to the K i value estimated in vitro and that the values for each agonist were identical to the sodium shiftmeasured for each agonist multiplied by a constant, agonist-independent factor (4.3). On the basis of the model simula-tions it can also be understood why all three opioid agonistsbehaved as full agonists despite the considerable differences

in sodium shifts. The simulations in figure 4b show that, due

TABLE 1

Pharmacokinetic parameter estimates and CSF/total bloodconcentration ratio of three opioids in the rat (mean Ϯ S.E.)

Drug nCl

(ml/min/kg) Vd,ss

(liter/kg)t1/2,n(min)

CSF

Total blood(%)

Alfentanil 8 37 Ϯ 4 0.75 Ϯ 0.10 25 Ϯ 2 1.6 Ϯ 0.1Fentanyl 8 42 Ϯ 5 2.91 Ϯ 0.39 73 Ϯ 4 6.8 Ϯ 1.2Sufentanil 7 77 Ϯ 8 5.53 Ϯ 0.76 79 Ϯ 5 19.9 Ϯ 9.5

Fig. 2. EEG effect vs. opioid blood concentration profiles of the opioidsFor fentanyl and sufentanil a profound hysteresis loop is observedwhereas for alfentanil hysteresis is absent.




to the high receptor reserve in the system, even compoundswith a sodium shift close to unity (i.e., compounds that wouldbehave as almost “silent” agonists in vitro) would still beexpected to behave as full agonists in vivo.

Discussion

The interaction of 4-anilidopiperidine opioids, such as fen-tanyl, sufentanil and alfentanil, with the central mu-opioidreceptor resulting in analgesic action has been characterizedin a number of pharmacological evaluations (Niemeegers et

al., 1976; Janssens et al., 1986). Although numerous studieshave shown that these opioids interact with the central mu-opioid receptor, the knowledge of the quantitative character-istics of this pharmacological interaction is surprisingly in-complete. Major attention has been focused on receptoraffinity whereas the systematic investigation of the agonisticcharacter (potency and intrinsic activity), both in vivo and in

vitro, has been rather limited. To our knowledge the compar-ative intrinsic activities in vitro of alfentanil, fentanyl andsufentanil have only recently been reported by James et al.

(1991) using the guinea pig ileum assay. Insight in the fun-damental pharmacology can provide a suitable basis for thedevelopment of new synthetic opioids with both pharmacoki-netically and pharmacodynamically superior characteristicsfor use in clinical anesthesia (James, 1994). Also the system-atic investigation of factors that affect the pharmacodynam-ics of these opioids, such as the development of functionaltolerance and the pharmacodynamic interaction with otherdrugs, will be possible in a mechanistic way. Integrated phar-macokinetic-pharmacodynamic modeling is therefore a use-

ful approach to characterize the in vivo pharmacodynamics of synthetic opioids. This approach, in conjunction with in vitro

receptor binding characteristics may reveal relationshipsthat provide insight in the underlying fundamental pharma-cology of these opioid effects.

Quantitative EEG monitoring has been proposed as aneffect measure that is continuous, sensitive, objective andreproducible. As such it possesses the most important char-acteristics of the ideal pharmacodynamic parameter neededfor pharmacokinetic-pharmacodynamic modeling. Also, theEEG effect occurs in a clinically relevant concentration rangeencountered in anesthesia and can be obtained in both ani-mals (Cox et al., 1997) and man (Scott et al., 1985, 1991).

In our study the concentration-EEG effect relationship of

the three opioids could be characterized on the basis of thesigmoidal Emax pharmacodynamic model. This resulted inestimates of in vivo intrinsic activity (Emax) and potency(EC50) as shown in table 2. An important question is to whatextent the values of the pharmacodynamic parameters havebeen affected by the fact that the rats had also received asingle dose of fentanyl during the surgical implantation ofthe EEG electrodes, 1 wk before the experiment. At present

this question cannot be answered. However, an importantfact is that the pretreatment has been identical in all ratsThis means that a comparison of the pharmacodynamics ofthe three different opioids is indeed justified. No differencesin Emax were observed between the three opioids, indicatinga similar intrinsic activity for the compounds. Similar intrin-sic activities for the three opioids have also been observed inthe guinea pig ileum assay (James et al., 1991). Comparisonof the obtained EC50 values, however, resulted in differencesin in vivo potency based on total blood concentrations. Sufen-tanil was approximately 10 and 200 times more potent thanfentanyl and alfentanil, respectively.

Differences in the relative free fraction of the opioids in the

brain were corrected for by the determination of the CSF overtotal blood concentration ratio. To obtain equilibrium be-tween the concentration in the blood and in the brain, thesteady-state concentration of the opioid in the blood wasmaintained for 30 min before this ratio was determined. Thistime appears to be sufficient to reach equilibrium of the drugconcentrations in cerebrospinal fluid and blood, since the values of t1/2,keo range between 0 and 4.2 min for the differentopioids that have been studied (table 2).

As can be seen from table 1, considerable differences wereobserved in the relative free fraction of the opioids in thebrain, ranging from 1.6% for alfentanil to approximately 20%for sufentanil. These differences indicate the need to correctEC50 values for differences in relative free fraction of theopioids in the brain to reliably compare the in vivo potency ofthe different drugs. After this correction, differences in in

vivo potency between the three opioids were still observedSufentanil showed the highest potency of the three opioidsand alfentanil the lowest potency. When these in vivo poten-cies were compared with in vitro binding affinity constantsfor the central mu-opioid receptor, as determined by [3H]-naloxone displacement studies, a high correlation betweenthe two parameters was obtained (table 3). Although [3H]-naloxone is not particularly selective for mu-opioid receptorsthe synthetic opiates that were studied are known to the veryselective in the concentration ranges tested. The findingsfrom our investigation justify therefore the conclusion that

the EEG effect of synthetic opioids results from their directreversible interaction with the central mu-opioid receptorThis is further supported by the fact that the K I values for thethree opioids obtained from the displacement studies were inthe similar concentration ranges as obtained previously (Ley-sen et al., 1983; Ilien et al., 1988; Clark et al., 1988; Maguire et al., 1992; Hustveit, 1994). Similar correlations between in

vivo potency and in vitro receptor affinity have also beenreported for the CNS effects of benzodiazepines (Mandema et

al., 1991b) and for the cardiovascular effects of adenosineagonists (Mathot, 1995). Interestingly, the EC50,u values ob-tained for the three opioids in the EEG model are essentiallysimilar to the respective IC50 values obtained in the guinea

pig ileum assay (James et al., 1991). It has been shown that

Fig. 3. EEG effect vs. effect site concentration plots of alfentanil (}),fentanyl (E) and sufentanil (F) in three individual rats. The solid linesrepresent the best fit through the EEG data according to the sigmoidalEmax pharmacodynamic model.




the effect of opioids on the guinea pig ileum is mediatedthrough the mu2-opioid receptor subtype (Gintzler and Pas-ternak, 1983). It is possible that the EEG effects of theseopioids are also mediated through mu2-opioid subtype recep-tors in the CNS. It requires additional investigations to con-firm this.

For each ligand the EC50,u value was about 4-fold lower

than the K I value in the absence of NaCl. Thus, a maximumpharmacological response of the opioids is already observedwhen the available opioid receptors are not fully occupied,indicating the existence of a receptor reserve. It cannot be

excluded that the opioids have different intrinsic efficaciesbut that this is masked by a considerable receptor reserve. Ithas been shown that the K I value for opioids increases in thepresence of sodium ions, whereas the K I values for antago-nists are unaffected or even decreased in the presence ofsodium (Pert and Snyder, 1974). Sodium shifts among opi-oids agonists, with values ranging from 2 to 140, have beenreported by Kosterlitz and Leslie (1978). The value of thesodium shift may therefore be a measure of intrinsic efficacyof the ligand. From table 3 it can be seen that in the presenceof 100 mM NaCl the K I values for the opioids increasedsignificantly. This depressant effect differed widely with val-ues between 2.8 and 19.1 for sufentanil and alfentanil, re-

spectively. The value of 2.2 for the sodium shift of sufentanilin our study is in agreement with the value reported byLeysen et al. (1983). It is of interest to investigate if thesodium shifts of the opioid ligands in combination with the in

vivo pharmacodynamic estimates of the opioids obtainedfrom the EEG model would be able to predict the efficacy ofeach ligand. The concentration-EEG effect data were there-fore simulated according to the operational model for phar-macological agonism as proposed by Black and Leff (1983). As can be seen from figure 4a, this resulted in concentration-effect relationships for the opioids that adequately describedthe concentration-effect relationship obtained with the sig-moidal Emax pharmacodynamic model. The simulations

showed that the sodium shift measured in vitro provided anaccurate prediction of the expression of efficacy in vivo, thatis could be expressed as the product of sodium shift and anagonist-independent constant. In the operational model ofagonism, is given by the ratio of the total receptor concen-tration ([R0]) and the midpoint location of the transducerfunction (K E) which relates agonist-occupied receptor concen-tration to pharmacological effect (Black and Leff, 1983). Be-cause [R0] is specific for the tissue and therefore ligandindependent, differences in are likely to reflect differencesin K E values between the opioid ligands. Furthermore, themodel predicts that on the basis of the sodium shift it will notbe possible to detect a ligand that will behave as a partial

agonist in vivo, because a sodium shift close to unity will still

Fig. 4. a, Operational model of agonism simulations of the self-nor-malised concentration-EEG effect relationships of the three opioids. Thesolid lines were simulated using equation 4 with the values for Em (100%)and n (2.3) held constant for all three agonists. The affinity (K A ) valueswere set to the K I values estimated in the presence of NaCl (table 3) andthe efficacy parameter ( ) of each agonist was expressed as a constant

(4.3) times the sodium shift (table 3). The dashed lines were simulatedwith the Emax model (equation 2) using the parameter values estimatedfrom the experimental data (table 2). b, Operational model of agonistsimulations showing the expected concentration-EEG effect relationshipson the basis of radioligand binding studies for compounds with constantaffinity and different sodium shifts. The lines were simulated using equation 4 with the following parameter values: Em ϭ 100%, n ϭ 2.3, K A ϭ 20 nM and ϭ 4.3 ϫ sodium shift.

TABLE 2

Pharmacodynamic parameter estimates of three opioids in the rat (mean Ϯ S.E.)

Drug E0 ( V) Emax ( V) EC50 (ng/ml) N t1/2,keo(min)

Alfentanil 53 Ϯ 4 111 Ϯ 10 289 Ϯ 41 1.7 Ϯ 0.2Fentanyl 48 Ϯ 3 89 Ϯ 11 10.1 Ϯ 0.8 2.6 Ϯ 0.3 2.2 Ϯ 0.2Sufentanil 72 Ϯ 4 104 Ϯ 4 1.43 Ϯ 0.30 2.4 Ϯ 0.4 4.2 Ϯ 0.6

TABLE 3

Relationship between in vivo potency (EC50,u) and in vitro receptor affinity (K I) for the three synthetic opioids

Drug K I,ϪNa (nM) K I,ϩNa (nM) Na-Shifta EC50,u(nM) EC50,u /K I,ϪNa

Alfentanil 47.4 Ϯ 6.6 906 Ϯ 115 19.1 11.3 0.23Fentanyl 8.6 Ϯ 4.1 114 Ϯ 17 13.3 2.02 0.25Sufentanil 2.8 Ϯ 0.2 7.9 Ϯ 1.6 2.8 0.70 0.24

a Expressed as the ratio of the K I value in the presence and the absence of 100 mM NaCl, respectively.




result in the expression of maximal possible intrinsic activity(fig. 4b).

Another interesting issue is the relationship between ourfindings in rats and the pharmacological properties in hu-mans. In our study, profound hysteresis was observed forboth fentanyl and sufentanil. To derive the effect-site concen-tration-EEG effect relationship, the hysteresis was mini-mized successfully using a parametric approach as described

by Sheiner et al. (1979).Concentration-EEG effect relationships of synthetic opi-

oids as obtained in our study have also been obtained inhumans (Scott et al., 1985, 1991). Interestingly, the EC50

values obtained for each opioid was in the same concentra-tion range in both rats and humans. Also the three opioidsshowed equal intrinsic activity (Emax) in both species. Unfor-tunately, no free drug concentrations were obtained in thestudy by Scott et al. (1985, 1991) and therefore no directcomparisons could be made between the EC50,u value of thetwo species. Interestingly, the relative magnitude of hyster-esis of the three opioids observed in rats in the present studyare essentially similar to those observed in humans. In hu-

mans fentanyl and sufentanil show remarkable hysteresis inthe plasma concentration-EEG effect relationship (t1/2,keo is6.6 and 6.2 min, respectively), whereas hysteresis for alfen-tanil is substantially smaller, resulting in a t1/2,keo of 1.1 min(Scott et al., 1985, 1991). The similarities suggest that iden-tical physicochemical and/or physiological processes are in- volved in the hysteresis phenomenon in both species.

The pharmacokinetic parameter estimates showed signifi-cant differences between the three opioids. The systemicclearance was the highest for sufentanil (77 ml/min/kg) ap-proaching total hepatic blood flow (Flaim et al., 1984) and thelowest clearance was observed for alfentanil. However, alfen-tanil portrayed the lowest volume of distribution at steady-

state, and sufentanil showed the largest volume of distribu-tion. Slightly different values for total body clearance and volume of distribution at steady-state for alfentanil and fen-tanyl were observed, however, by Bjorkman et al. (1993).These differences may be explained by differences in ratstrains used.

From the results of this study it can be concluded thatthere is a close correlation between the pharmacodynamics of synthetic opioids at the mu-opioid receptor in vitro and theeffect on the amplitudes of the 0.5–4.5 Hz frequency band of the EEG in vivo. Mechanism-based modeling of the pharma-codynamics of synthetic opioids may therefore be a usefulapproach in the design of new compounds, and provides the

scientific basis for the extrapolation from preclinical to clin-ical investigations of the pharmacodynamics of opioids.

Acknowledgments

The authors are grateful for the technical assistance of EricaTukker and Mariska Langemeijer with the animal surgery and of Jacobien von Frijtag Drabbe Kunzel with the receptor binding stud-ies. We also thank Dr. Ad P. IJzerman and Professor Douwe D.Breimer for critically reading the manuscript. The STANPUMP pro-gram was kindly provided by Dr. S. L. Shafer, Department of Anes-thesia, Stanford University, Palo Alto, CA. The generous donation of the opioids by Janssen Pharmaceutica (Beersse, Belgium) and of midazolam by Hoffman LaRoche (Basel, Switzerland) is highly ap-

preciated.

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