Alpha-1-acid glycoprotein directly affects the pharmacokinetics and the analgesic effect of methadone in the rat beyond protein binding

Alpha-1-Acid Glycoprotein Directly Affects thePharmacokinetics and the Analgesic Effect ofMethadone in the Rat Beyond Protein Binding

MONICA RODRIGUEZ, IGNACIO ORTEGA, ITZIAR SOENGAS, NEREA LEAL, ELENA SUAREZ,ROSARIO CALVO, JOHN C. LUKAS

Department of Pharmacology, Faculty of Medicine, University of the Basque Country, Leioa s/n, Vizcaya 48940, Spain

Received 9 February 2004; revised 31 May 2004; accepted 6 June 2004

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20171

ABSTRACT: Methadone is a basic drug highly bound to a1-acid glycoprotein (AGP), aplasma protein that increases in several pathological situations. Our aims were toevaluate the processes (pharmacokinetics—PK and/or pharmacodynamics—PD) asso-ciated with changes of methadone analgesia under conditions of increased AGP, andwhether these changes are dependent on binding, secondary to a pathology, or directlyattributable to AGP. AGP levels, in rats, were increased by two different methods: (a)experimental inflammation with turpentine oil (TP), and (b) by directly infusing theprotein (exo-AGP). Both had a corresponding control group. Tail-flick analgesia and PKwere evaluated after methadone dose (0.35 mg/kg i.v.). Bicompartmental PK parametersas well as interanimal and assay variabilities were estimated using NONMEM. Therelationship betweenCpand analgesic effect (PD) was analyzed with WINNONLIN. AGPlevels in both pretreated groups (TP and exo-AGP) were significantly increased, and theunbound fraction (fu) was decreased, compared to controls. All PK parameters were lowerin the pretreated groups, but in exo-AGP the difference was maintained even whencorrected by fu. Paradoxically, also in exo-AGP the analgesic effect was practicallynonexistent, although the unbound Cp remained high, possibly associated to a change inthe PD. AGP appears responsible for alterations in both PK and PD, beyond proteinbinding and inflammatory processes. � 2004 Wiley-Liss, Inc. and the American Pharmacists

Association J Pharm Sci 93:2836–2850, 2004

Keywords: alpha-1-acid glycoprotein; protein binding; methadone; population phar-macokinetics; analgesic effect; pharmacokinetics/pharmacodynamics; NONMEM

INTRODUCTION

It is known that patients to be treated withmethadone, for example, patients with cancer, orabstinence syndrome, typically also have abnor-mally high levels of a-1-acid-glycoprotein (AGP), aplasma protein of the reactive phase.1,2 Given

that methadone is a basic pH drug, preferablytransported in plasma by AGP, binding to thisprotein is increased in the mentioned pathologies,and it was suggested that binding to AGP may bepartly responsible for the observed variability inthe therapeutic response.3,4 The PK-related sour-ces of variability in methadone have been recentlyreviewed, reaffirming the role of binding to AGP.5

Nevertheless, AGP may be involved in the altera-tions to the therapeutic response, and not solelythrough binding.

In previous work in the animal model ofmorphine abstinence, it was observed that theAGP levels as well as binding to methadone were

2836 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 11, NOVEMBER 2004

John C. Lukas’s present address is Laboratory of Biophar-maceutics and Pharmacokinetics, Department of Pharmacy,University of Athens, Athens, Greece.

Correspondence to: Rosario Calvo (Telephone: 34 946012761; Fax: 34 94 4800128; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 93, 2836–2850 (2004)� 2004 Wiley-Liss, Inc. and the American Pharmacists Association

increased and the analgesic effect was decreas-ed. In contrast, in the abstinent animals, themethadone total concentration (Cp) and, to a lesserextent also the unbound, were higher than inthe controls.6 The lack of relationship betweenconcentrations and the effect was explainedthrough an integrated pharmacokinetic/pharma-codynamic (PK/PD) analysis. Nevertheless, be-cause that work dealt with an abstinence animalmodel, the mechanism involved could be attribu-ted either to crosstolerance, to a change in proteinbinding, or independently to the increased levelsof AGP. The same occurs with other methodsof increase of AGP. For example, experimentalinflammation may alter the PK and PD per seof drugs via processes independent of proteinbinding,7 an aspect that has not yet been clarified.

Our aims here were to evaluate the PK and PDof intravenous (i.v.) methadone in rats with highlevels of AGP following two scenaria: (a) classicalexperimental inflammation by administration ofturpentine oil, and (b) direct infusion of a-1-acid-glycoprotein thus excluding the possible influenceof inflammation. High-dose AGP infusion shouldlargely amplify the effects of AGP on drug binding,and therefore provide a useful method of study-ing the contribution of this protein in drug PK andPD alterations. This method would also helpclarify which of the PK, or the PD, best reflectsthe changes in the effect observations, and there-fore help understand the observed variability inmethadone response in the clinic.

Particularly, for sparse or point-per-animalstudies, such as the present, population modelingis appropriate.8 Here, the analysis of the observa-tions was performed with a mixed effects popula-tion method.

MATERIALS AND METHODS

Drugs and Reagents

Methadone (racemic mixture) chlorhydrate wassupplied by Alcaliber (Madrid, Spain). 14C-metha-done (specific activity 30 nCi/mmol and 98.8%of purity) was purchased from Amersham LifeScience (Barcelona, Spain). Turpentine oil(‘‘purissimum’’ grade) was obtained from Panreac(Barcelona, Spain). Human AGP was generouslysupplied by Hamosan Laboratories (Viena,Austria). Benzhexol chlorhydrate (dl-trihexyphe-nidyl) was obtained from Sigma (Madrid, Spain).BiosolTM (Biodegradable Tissue Solubilizer) and

BioscintTM (Liquid Scintillation Solution) werepurchased from National Diagnostics (Atlanta,GA). All the remaining reactants and solventswere of analytical grade.

Animals and Surgical Procedures

Male Sprague-Dawley rats were supplied by theUniversity of the Basque Country. The experi-mental protocol was approved by the Committeeon Animal Experimentation of the University ofthe Basque Country. Animals were randomly dis-tributed into groups of six, and were kept under acontrolled temperature of 208C and humidity of70% with a normal 12-h light/dark cycle (8:00 a.m.to 8:00 p.m.) for 1 week before any experimentwas initiated. Food (standard laboratory rat,mouse, and hamster diets, Panlab, Barcelona,Spain) and water were available ad libitum. Theday before the experiment, animals were fastedovernight.

Animals were divided in four grand experimen-tal sets for the in vivo study: turpentine oil (TP)(n¼ 40), human AGP infusion group (exo-AGP)(n¼ 30) each with its own control group (CTP,n¼ 20 and Cexo-AGP, n¼ 20, respectively). Theformer received an injection of 0.5 mL of turpen-tine oil subcutaneously (s.c.) in the rear leg of theanimal 48 h before the experiments while itscontrol received 0.5 mL of saline.9 The latter wasinfused i.v. with a dissolution of human AGP,while its control received the same volume ofsaline. A dose of 2 g/kg was calculated to increasethe serum AGP concentration 10- to 20-fold basedon a volume of distribution equal to the extra-cellular fluid volume and a normal rat serum AGPconcentration of 5 to 10 g/L. Rats received humanAGP as a 250 mg/mL solution (total volume 1.0–1.3 mL) in 0.45% saline, which required gentleagitation for 2 h at 48C, infused over a 30-minperiod via a jugular vein cannulae. It has been de-monstrated that this dose of human AGP producedno physiological changes that could complicateinterpretation of its effects on drug PK or PD.10

Two distinct groups of rats were used for time-effect evolution and Cp measurements becausea destructive sampling, one-point-per-animaldesign was used to measure serum methadoneconcentrations.

The time course of the analgesic effect wasevaluated in male Sprague-Dawley rats (n¼ 46;179–260 g) after i.v. administration of 0.35 mg/kgmethadone to all. Each animal had a completeeffect evolution profile measured.

ALPHA-1-ACID AND ANALGESIC EFFECT OF METHADONE 2837

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 11, NOVEMBER 2004

Also, a second group of Sprague-Dawley rats(n¼ 64; 182–272 g) received i.v. methadone(0.35 mg/kg) to assess the time course of metha-done Cp (PK). The drug was administered at thesame time of the day to eliminate circadian vari-ations in the PK parameters.

The day before the experiment, rats were lightlyanesthetized with ether, and a polyethylenecatheter (i.d. 0.3 mm, 10 cm length; Vygon,France) was implanted in the right carotid arteryfor blood sample collection. Another catheter wasinserted into the right jugular vein for the i.v.administration of methadone in all groups ofanimals, and for the infusion of the AGP solutionin the corresponding group. All catheters werefilled with a solution containing NaCl 0.9% and1% heparin (50 IU/mL, Roger Lab, Spain). Thecatheters were tunnelled under the skin of theanimal and externalized on the dorsal surface ofthe neck. After surgery, rats remained underfasting conditions but with ad libitum access towater (for 24 h).

Measurement of the Methadone AnalgesicEffect Temporal Evolution

The analgesic effect of methadone was measuredat 0, 2.5, 5, 10, 15, 30, 60, 90, 120, and 180 minafter i.v. administration of 0.35 mg/kg methadonewith the tail-flick as the antinociceptive endpoint.11 Animals responded to a heat stimulusby moving their tail away from the focus of thestimulus, thereby exposing a photocell locatedunder the tail. The reaction time was automa-tically recorded. The intensity of the heat wasadjusted so that the basal measurements were of2–5 s; animals with longer baseline latenciesthan 5 s were excluded. A maximal cutoff time of10 s was used to prevent tissue damage. Metha-done analgesic effect was expressed as percentageof the maximum possible (observed) response(MPR %) and was calculated as,

MPR %¼ test latency � baseline latency

cutoff time � baseline latency

� �� 100:

The analgesia experimental set consisted of ratsfor CTP (n¼ 10), Cexo-AGP (n¼ 10), TP (n¼ 10), andexo-AGP (n¼ 16). Weight ranges were 182–243 g(for both control groups), 183–260 g, and 179–224 g, respectively. The AUCE0–180 was assessedusing the trapezoidal method aided by Graph Pad(version 3.0, Graph Pad Software Inc, San Diego,CA).

Pharmacokinetic Assay

Rats of all groups were administered 0.35 mg/kgi.v. of methadone in 30 s dissolved in salinesolution. A single arterial blood sample at eachtime point (2.5 mL per rat) was drawn at 1, 2,3, 4, 5, 10, 15, 30, 60, 90, and 120 min aftermethadone administration. Exceptionally, somerats corresponding to the 1-min time point couldbe used for extraction of a point at 90 or 120 min.Blood was placed in heparinized tubes, centri-fuged at 2500 rpm for 15 min at 378C, andthe plasma kept frozen at �208C until assay ofmethadone. This set was divided into CTP (n¼ 10),Cexo-AGP (n¼ 10), TP (n¼ 30), and exo-AGP(n¼ 14). Weight ranges were 183–288 g (for bothcontrol groups), 207–272 g, and 183–234 g,respectively.

HPLC Methadone Analysis

Plasma methadone levels were determined byhigh-performance liquid chromatography (HPLC)according to a known method with minor modi-fications.12 Briefly, the HPLC equipment includedan HPLC pump (type 422, Kontron Instru-ments, Bilbao, Spain) and an ultraviolet detector(Waters 486, Waters Corp, Milford, MA). Theseparation was performed in a 5-mm silica column(Apex I, 25� 0.45 cm, Teknochroma, Barcelona,Spain) and a Corasil Type II silica precolumn(Teknochroma, Barcelona, Spain). Methadoneand the internal standard (benzhexol) were de-termined by a UV detector at a wavelength of215 nm.

One milliliter of plasma was mixed with 10 mLofa solution containing the internal standard (1mg/mL) and 0.5 mL of sodium carbonate buffer (1 M,pH 10) to which 5 mL of n-buthyl chloridesaturated in water were added, for the extractionof methadone. The mixture was mechanicallyshaken for 15 min and then centrifuged at4000 rpm at 48C for 10 min. The n-butyl chlorideupper layer was vacuum aspirated and placed in a5-mL glass tube and evaporated at 508C (AES1000, Speed Vac Concentrator, Savant, Spain).The solid residue was dissolved in 110 mL ofmethanol, and 100 mL were injected into thechromatograph system. The mobile phase wasmethanol/1,2-dichloroethane/isopropanol/ammon-ium perchloride aqueous solution (100 g/L) in theproportion 90.5/5/4/0.5 V/V, at a flow rate of 2 mL/min. The sensitivity of the analytical procedure is5 ng/mL of methadone.

2838 RODRIGUEZ ET AL.


Disposition of Methadone in the Brain

Rats were randomly distributed in three groups ofsix rats each (n¼ 18): control, TP, and exo-AGP.The rats were sacrificed by decapitation underlight ether anesthesia at minute 1 (n¼ 9; threeeach) and minute 5 (n¼ 9; three each) after0.35 mg/kg i.v. drug administration, time at whichequilibrium between plasma and brain concen-trations has been reached.6

Methadone brain levels were measured in eachgroup using an already described experimentalprotocol with minor modifications.6 Briefly, thebrains were rapidly removed and 50 mg of brain(cortex) was dissected. Tissue samples (50 mg)were mixed with 1 mL of BiosolTM Tissue Solu-bilizer, and were incubated for 1.5 h (time neededfor the complete tissue digestion) at 508C, and10 mL of BioscintTM Liquid Scintillation Solutionwas added when the solution was at room tem-perature. Radiolabeled methadone concentra-tions were measured by scintillation countingusing a Packard Tricarb 2200 CA liquid scintil-lation analyser (Packard Instrument, Meriden,CT).

Protein Binding In Vitro Studies

An aqueous solution of 14C-methadone (14C-M)(10 mL) was added to 990 mL of plasma from con-trol rats (n¼ 10), TP rats (n¼ 10), and exo-AGPrats (n¼ 10) to obtain a final concentration of70 ng/mL (CT) (analgesic concentration in rats).Samples were mixed and incubated at 378C for10 min. Aliquots of all samples (1 mL) were thentransferred to Amicon Micropartition Units(MPS-1). The devices contain a membrane filterof controlled porosity with a cutoff molecularweight of 10,000 Daltons that retains plasmaprotein and allows free drug in solution to passthrough. The MPS-1 were centrifuged at 3000 rpmfor 8 min at 378C, in a procedure considered asa reliable and relatively easy system to use forseparating protein-free from protein-boundligand for several drug families.13 The free con-centration obtained as ultrafiltrate (Cu) was mea-sured by scintillation spectrometry with aPackard Tricarb 2200 CA. Samples (100 mL) weremixed with 10 mL of BioscintTM Liquid Scintilla-tion Solution, and radioactivity (cpm) wascounted in the spectrometer for 5 min. With eachseries of samples, vials containing aliquots of theultrafiltrate and known quantities of the labeledcompound in the range 0.5 to 250 ng/mL, were

used as standards. The percent fraction ofunbound methadone (fu) was determined as:

% Unbound ¼ Cu

CT� 100

The above procedure was developed accordingto a validated method.14

Protein Levels

Albumin and AGP levels were measured in allcontrol and pretreated rats (TP and exo-AGP).100 mL of plasma were used to evaluate AGPlevels based on a fluorimetric determinationmethod of AGP in plasma with quinaldine red.15

Albumin reagent BCG (bromocresol green) wasused for the quantitative, colorimetric determina-tion of albumin in 10 mL of plasma at 628 nm. Theapplied procedure is a modification of a previouslypublished method.16

Pharmacokinetic Data Analysis

The PK was evaluated in separate control rats(n¼ 20; m¼ 23 time points), and TP (n¼ 30;m¼ 37), and exo-AGP (n¼ 14; m¼ 17) rats, afterthe same dose of methadone i.v. (0.35 mg/kg).

The nonlinear mixed effects method as imple-mented in NONMEM (nonlinear mixed effectmodeling, NONMEM Project Group, Universityof California at San Francisco, CA) was used forfitting compartmental pharmacokinetic and ap-propriate statistical models to the observationsto obtain estimates of the typical parametersand of the interanimal and assay variances.Alternative models were compared based on theNONMEM objective function (minus twice thelog �likelihood, �2LLD), the Akaike criterion forp parameters (AIC¼�2LLDþ 2p) and the visualinspection of plots of the residuals. The �2LLD isdistributed as a x2-like variable and a difference of7.7 in �2LLD between models differing in a singleparameter is significant at the p< 0.005 level andwas used for comparisons here.

Pharmacokinetics

Mono, bi, and tricompartmental pharmacokineticmodels were tested for fit to the data in all groups.The present was a single time point per animaldesign (with very few exceptions of two pointsper rat), the ith rat’s concentration at the jthtime point was represented as, Cpij ¼ gðyi; tijÞ þ eijwhere Cpij are the elements of the matrix of



concentrations in plasma and g(yi,tij) is the bicom-partmental structural model for parameter vec-tors yi¼ (V1, CL, V2, Q)i, one for each rat, withvariances oy (y� 1 vector). Both y and oy are fixedeffects and tij is the matrix of time elements.The residual ‘‘noise’’ matrix elements, eij weremodeled as normally independently and identi-cally distributed with mean E[eij]¼ 0 and var-iance Var[eij]¼se

2 g(yi, tij)2, for heteroscedastic

errors, where se is a fixed effect coefficient ofvariation to be determined. This structure wasselected after inspection of residuals and pre-liminary runs with a fitted exponent for se thatwas closer to 2 than to 0 for the controls (indicatinga proportional model) and was 0 for the TP andexo-AGP groups (for an additive error model). Thefirst order conditional estimation (FOCE) withinteraction (for proportional error models, controlanimals), and FOCE (for TP and exo-AGP rats)was employed.

Preliminary tests of the parameter distribu-tion with Bayesian estimates based on literaturevalues showed normal-like distributions for V1,V2, CL, and Q. Additive statistical models for theparameter variability consistently failed due tonegative values in the process of estimation, so alognormal model for the parameters was used forthe random effect elements,

yi ¼ y� eh yi

where �yy is the typical population value for eachparameter, and h_y is the random effect for theparameter. This models results in variance esti-mates, oij, that are coefficients of variation (CV)for the parameters.

NONMEM obtains estimates of the PK and PDparameter distribution for the population thatcan then be used as Bayesian priors for individua-lization of the PK parameters (empirical Bayesparameters). To obtain the unbound PK para-meters, individual PK parameters from eachgroup were divided by the corresponding unboundfraction.

Pharmacodynamic Data Analysis

The PD model corresponding to the relationshipbetween the Cp and analgesic effect, measuredin each rat, was selected using the nonlinearregression package WINNONLIN 3.0 (Pharsight,Palo Alto, CA). Model selection was based on theAkaike Information Criterion (AIC) and analysisof the weighted residuals, as well as the standarderror of the parameter estimates.

Due to the negative analgesia values obtained inthe exo-AGP group (when analgesia was expressedas MPR %), the PD analysis was carried out withanalgesia expressed as reaction time (in seconds)and PD models with baseline effect were evalu-ated.

Simple Emax and sigmoid Emax models weretested for fit to the data in all groups described bythe following equation:

E ¼ E0 þ ðEmax � E0ÞC

Cþ EC50

E ¼ E0 þ ðEmax � E0ÞgCg

Cg þ ECg50

where E is the model effect, E0 the basal effect(effect in the absence of analgesic drug), Emax themaximum effect (10 s), Cp is the concentration atthe time of the response observation, EC50 is theconcentration in plasma at half the maximumeffect, and g (the Hill exponent) is related to thenumber of binding sites per receptor molecule forthe drug and results in the sigmoidicity of thecurve.

Statistical Analysis

Observations are reported as mean� standarderror of the mean (SEM). Two-tailed unpairedStudent’s t was used to check for significant dif-ferences between the pretreated groups with theircorresponding controls. Independent variableswere statistically compared between groups usinga single-factor analysis of variance (ANOVA) withBonferroni post hoc test. Tests with p< 0.05 wereconsidered statistically significant.

RESULTS

As shown in Table 1, AGP levels in both pre-treated groups (TP, exo-AGP) were significantlyincreased compared to the control animals. In theexo-AGP group, the levels of AGP increased signi-ficantly when compared both to the correspondingcontrols and also to the TP group. There was alsoa slight decrease, compared to CTP, in the levels ofalbumin in the TP group. The unbound fractiondecreased in the TP and exo-AGP groups com-pared to the common control for fu. There were nosignificant differences (p> 0.05) in the free frac-tion between the two pretreated groups, TP andexo-AGP.



Figure 1 shows the temporal evolution ofthe analgesic effect (expressed as MPR %) after0.35 mg/kg bolus of i.v. methadone in the threegroups of animals. The two control groups were notdifferent in their effect or Cp evolution at all timepoints (p> 0.05) and thus for subsequent presen-tation were fused into a single control group. Both

TP and exo-AGP showed, at all times, a reducedand significant degree of analgesia, compared tocontrols (p< 0.05). Statistically significant differ-ences were observed between both pretreatedgroups at 2.5, 5, and 15 min after methadonei.v. administration (p< 0.05). Therefore, there isa change in the global analgesic effect seen as a

Table 1. AGP, Albumin Levels in All Groups of Rats

CTP TP Cexo-AGP exo-AGP

AGP (g/L) 0.52 (0.05) 1.56 (0.08)a 0.54 (0.06) 6.98 (0.63)b,d

Albumin (g/L) 34.07 (1.24) 26.36 (0.58)a 35.76 (0.83) 28.08 (1.66)Control TP exo-AGP

fu (%) 21.96 (1.54) 10.95 (0.46)c e 9.09 (0.73)c

(Turpentine Inflammation, TP; AGP infusion, exo-AGP; with corresponding controls, CTP andCexo-AGP), shown as mean (SEM). Unbound fraction in the control, TP and exo-AGP groups,obtained in vitro, is also listed.

ap< 0.05.bp<0.001.cp<0.0001 when compared with the corresponding control group.dp<0.001 when compared with the TP group.eA single group (n¼10) was used to obtain the fu in vitro for the control rats.

Figure 1. Observed time course of analgesic effect (MPR%) after i.v. methadone(0.35 mg/kg) in the control group (n¼ 20, for all time points; open diamonds with solidline), in the TP group (n¼ 10, for all time points; open squares with dashed line), and in theexo-AGP group (n¼ 17, for all time points; open circles with dotted line). Barsare standard errors of the mean (SEM). Marked time points showed statisticaldifferences: 1p< 0.05 when compared with control animals, 2p< 0.05 when comparedwith TP animals. In the insert is overall magnitude of antinociception as reflected bythe AUCE0–180 (mean�SEM) in control (solid fill), TP (horizontal lines), and exo-AGP (dotted fill) rats after 0.35 mg/kg i.v. methadone. Each column represents themean�SEM. Significant differences were found when comparing with the control group(***p< 0.0001).



reduction in the area under the effect–time curve(AUCE0–180), from 5284� 414 min in the controls,to 2904� 444 min in the TP group (p< 0.001) andto 1831� 252 min in the exo-AGP group (p< 0.001)(see insert in Fig. 1).

Disposition of Methadone in the Brain

No statistical differences were found in metha-done brain levels in the three groups of animalsat minute 1 after drug i.v. administration. Thus,the controls group showed brain concentrationof 628.75� 6.63 ng/g of tissue similar to thoseobtained in the TP group of 565.10� 23.44 ng/gand in the exo-AGP group of 409.00� 75.89 ng/g(p> 0.05). Nevertheless, at 5 min the differencewere significant in the exo-AGP group comparedto the controls (295.99� 28.06 versus 630.48�27.15 for exo-AGP versus control rats, repectively;p< 0.05) but not in TP (465.50� 79.14 versus630.48� 27.15; p> 0.05).

Pharmacokinetics

Plasma concentrations of methadone versus timewere best described, in control and pretreated(both TP and exo-AGP) animals, by a semiphy-siological bicompartmental model described byfollowing PK parameters: central volume ofdistribution (V1), peripheral volume of distribu-tion (V2), intercompartmental clearance (Q), andsystemic clearance (CL).

Initially, the three groups were analyzed as asingle population but the PK was different amongthem as seen in theCp–time profiles. The observedCp at the first time point was 86.36� 8.89 ng/mLin controls, 183.69� 19.36 ng/mL in TP, and971.62� 63.25 ng/mL in the exo-AGP groups.Therefore, populations were studied separately.

The model fits for methadone concentrations inthe three groups, control, TP and exo-AGP, areshown in Figure 2 and estimated parametersare in Table 2. Population prior distributionswere obtained for each group. In the exo-AGPrats the data was very sparse (n¼ 14, m¼ 17) andthe interindividual variability in V1 and Q wasiteratively obtained, based on objective functionreduction, and then fixed to 30% (CV%). V2 had noestimate of variability. This process did not affectthe typical population value estimates.

In the TP group there was a significant reduc-tion in V1, CL, and Q compared with the controlgroup (p< 0.05, p< 0.001, and p< 0.05, respec-tively). In the exo-AGP group, the elevated Cpcorresponded to a low V1 and reduced CL. In

addition, the increase in the levels of AGP (or ofbinding) caused a sharp reduction in the periph-eral distribution of methadone as reflected in V2,and Q, when compared to the controls. In thisgroup, all changes were statistically significantwith respect to the controls and to the TP.

The weight-corrected parameters maintainedsignificant differences between the groups andconsequent covariate analysis with the weightshowed no influence in all groups (data not shown).

To obtain the unbound PK parameters (Table 3)for each rat the empirical Bayes parameters weredivided by the unbound fraction, particular toeach group, and the mean and standard errorsobtained. The unbound PK parameters of the TPgroup were corrected towards those of the controlgroup (although slightly higher). In contrast, forthe exo-AGP group, the parameters remainedvisibly lower than those in the controls or in theTP animals.

Simulating the temporal evolution of the un-bound concentrations in each group, with theircorresponding unbound parameters, it was ob-served how the profiles were almost identical inthe TP and the control groups (Fig. 3). However, inthe exo-AGP group the unbound concentrationsof methadone were significantly higher than inthe other two groups. Nevertheless, the analgesiceffect in this group of rats was practically nonexistent.

Pharmacodynamics

Plasma concentrations were related to the analge-sic effect (PD). Due to the negative analgesiavalues obtained in the exo-AGP group (whenanalgesia was expressed as MPR %), the PDanalysis was carried out with analgesia expressedas reaction time and PD models with baselineeffect were evaluated.

Figure 4 shows the best fit for the PD modelwhen methadone Cp versus effect were used incontrol (a) and TP (b) groups. PD parameters forboth groups are shown in Table 4. In the controlgroup, a sigmoid Emax model was fitted to the totalCp versus effect observations, reaching an Emax of8.57 s and an EC50 of 27.05 ng/mL. The curve forpretreated rats is shifted to the right indicatingthat a higher concentration is needed for thisgroup to reach the same effect as the control. In theTP group, the sigmoid Emax model also best des-cribed the observations, but the Emax was solely6.32 s, and the EC50 was almost twice that of thecontrols (57.49 ng/mL).



The parameters corresponding to the effectversus unbound concentration from the best PDmodel fits are listed in Table 5. The Emax remainslower in TP; however, the unbound EC50 is nowsimilar in both control and TP groups.

In the exo-AGP group, due to the complete lackof relationship between the Cp (total or unbound)and the effect, no model could be adjusted to theobservations. To compare the three groups, the

analgesic effect was simulated in the control groupand the TP group up to an unbound concentrationof 70 ng/mL (that is the concentration reachedin the exo-AGP group) (Fig. 5). At that unboundCp of methadone, the effect reached in the con-trols was close to 100% (9.5 s). However, in theexo-AGP group and for the same unbound Cp ofmethadone, the effect reached was 3.2 s (similar tothe baseline).

Figure 2. Population model fit and plasma concentration (Cp) in (A) the control group(open diamonds), (B) the TP group (open squares), and (C) the exo-AGP group (opencircles) after i.v. administration of 0.35 mg/kg of methadone. Predicted initial con-centrations are also shown.



DISCUSSION

Most of the work studying the influence of ele-vated AGP on the effect of drugs has usedmethods involving the prior administration ofsubstances that modify the expression, at liverlevel, of this protein.17,18 In some studies, knownenzymatic inducers have been used to increaseAGP levels.19

Some authors have used experimental inflam-mation models, of which the most common is thesubcutaneous injection of turpentine oil in therear leg of the animal.20 After 48 h, this method

produces an increase in AGP to near three timesthe base levels. Others have employed transgenicmice with elevated AGP levels. Nevertheless, noneof these methods has proven entirely appropriatefor establishing the role proper of alterations inAGP on the PK/PD of analgesic drugs.

AGP and Analgesic Effect of Methadone

In the present study, the classical experimentalinflammation model was employed for comparisonachieving a significant increase in the levelsof AGP. There was also a slight decrease in the

Figure 2. (Continued)

Table 2. Typical Population PK Parameter Mean Estimates (y) with Standard Errors of the Estimates (SEE) fromthe NONMEM FOCE Method in the Three Groups of Rats

Parameter

Control TP exo-AGP

y o CV % y o CV % y o CV %

CL (L/min) 0.024 (0.002) 19.13 0.017 (0.001)b 19.47 0.006 (0.001)c,f 50.00V1 (L) 0.681 (0.126) 31.62g 0.393 (0.034)a 21.52 0.061 (0.006)b,f 31.62g

Q (L/min) 0.113 (0.040) 31.62g 0.038 (0.008)a 79.49 0.010 (0.002)a,d 31.62g

V2 (L) 0.934 (0.299) 31.62g 0.578 (0.085) 31.62{ 0.071 (0.019)a,e —s (CV %) 16.97 (0.002) — — — — —s (SD) — — 1.95 (2.53) — 9.54 (8.57) —

Interanimal variability (o) is expressed as coefficient of variation (CV %). The interassay variability (s) is reported asCV % for theproportional error model (control) and as standard deviation for the additive error model (TP and exo-AGP).

ap<0.05.bp<0.001.cp<0.0001.dp< 0.05; when comparing TP with exo-AGP.ep<0.001; when comparing TP with exo-AGP.fp<0.0001; when comparing TP with exo-AGP.gFixed.



levels of albumin in this group, as has beendescribed earlier.21 But given that methadone ismainly bound to AGP the final result was anincrease in protein binding, or a twofold decreasein the unbound fraction. In parallel, there was achange in the global analgesic effect seen as areduction, to almost half, in the area under theeffect–time curve (AUCE0–180).

There are several studies that use an experi-mental inflammation model to assess the influenceof changes in binding to the effect and also the PKof drugs.22,23 In a classic reference the influence ofelevated AGP (in TP rats) in the effect of four b-blockers is evaluated.24 It was observed how thepharmacological effect of propranolol and oxpre-nolol decreases drastically in animals with experi-mental inflammation. The effect of atenolol andmetoprolol, with low binding to AGP, did not showdifferences among animal groups. Nevertheless,the same authors in an earlier study had observedthat the plasma concentrations of propranololwere significantly higher in experimental inflam-mation (due to a decrease in plasma clearance).Although this is not mentioned explicitly in the

study, there was a lack of relation between totalCpand the effect of the drug.

In inflammation, metabolic cytochrome(CYP3A4)25 and P-glycoprotein26 expression, inrats, is affected. Consequently, with this inflam-mation model it is not possible to establish thedirect role of AGP, or of binding, on both the PK or

Table 3. Unbound Mean Empirical PK Parametersof Methadone [Mean, (SEM)] in the Three Groups ofRats

Controls TP exo-AGP

CLu (L/min) 0.106 (0.002) 0.150 (0.003) 0.069 (0.008)Vlu (L) 2.982 (0.096) 3.522 (0.062) 0.674 (0.002)Qu (L/min) 0.491 (0.012) 0.390 (0.042) 0.109 (0.033)V2u (L) 4.074 (0.113) 5.435 (0.159) 0.791 (—)

Figure 3. Simulated temporal evolution of the un-bound Cp for the control group (solid line), the TP group(dashed line), and the exo-AGP group (dotted line).

Figure 4. (A) Best pharmacodynamic model fit for thetotal methadone plasma concentration versus effect(expressed as reaction time in seconds) for the controlgroup (solid line). Observations are also included (opendiamonds). (B) Best pharmacodynamic model fit for thetotal methadone Cp versus effect for the TP group(dashed line). Mean observations are plotted (opensquares).

Table 4. Pharmacodynamic Parameters as Mean(SE) of Methadone in Control and TP Rats

Parameters Control TP

Emax (s) 8.57 (0.14) 6.32 (0.62)EC50 (ng/mL) 27.05 (0.67) 57.49 (14.29)E0 (s) 4.15 (0.12) 2.88 (0.24)g 10.77 (2.58) 1.88 (0.74)

The SE are single fit SEs of the parameter estimate and notused for significance comparison.



the drug effect. Alternative methods have beendeveloped instead. One approach consists of eval-uating the possible influence of AGP in vitro in theeffect of certain drugs. Most of such studies havebeen performed in cellular cultures infected withHIV to assess the direct role of AGP on the effect ofantiretroviral drugs. A proportional reduction wasobserved in the antiretroviral activity when theculture was spiked to elevated levels of AGP.27,28

In a later study,29 it was demonstrated that raisedlevels of AGP reduced the intracellular concentra-tion of antiretroviral drugs. This confirmed earlierobservations in HIV patients with elevated AGPlevels who showed a reduced and highly variableresponse to amprenavir.30

New in vivo models for AGP level increase werealso developed, for example, transgenic mice withoverinduction of the gene that codes for AGPexpression (ORM1).31 The modified mice had up toan eightfold increase, over nominal levels, of AGP.Its influence was studied on the antidepressanteffect of fluoxetine (assessed by the forced swim-

ming test). After intraperitoneal administration ofthe drug, a reduction in the volume of distributionwas observed, but none in the unbound fractioncompared to that in the controls. The concentra-tions in the brain were less in the transgenic mice,and so was the antidepressant effect. Neverthe-less, it has recently been observed that the use oftransgenic animals is not always appropriate,particularly with analgesia.32

Another model, used in some studies,10,33 con-sists of direct infusion of human AGP in solution,without need for provoking inflammation. Thismodel was also employed here. As expected, thelevels of AGP increased significantly in this group,almost 13-fold compared to the corresponding con-trols and also fivefold compared to the TP group.

In the exo-AGP group, the in vitro unboundfraction (fu) of methadone was significantlyreduced, almost twice compared with the controls.Curiously, the fu was similar between the twopretreated groups, TP and exo-AGP, besides thesignificant difference in AGP levels. This appearsto be due, at least in part, to the different degree ofbinding between humans and rats of AGP (recallthat the exo-AGP group received human AGP).The binding of methadone in the rat is higher thanin humans, and therefore, the observed differencebetween the two groups with increased AGP isreduced.5,34 Other factors, suchs as the known factthat purified protein binds with lesser affinity todrugs, can further contribute in explaining thatobservation.

In the exo-AGP group, the analgesic effect wassignificantly reduced when compared to the con-trols. The pretreated animals did not reach, at anytime, analgesia superior to 2%, while the controlsafter the same dose never went below 13% after0.35 mg/kg methadone in both groups. This wasnot completely explained by changes in access ofmethadone to the brain in neither of the pretreatedgroups (no significant change in TP and nearly50% reduction in exo-AGP).

To establish the mechanism involved in thechange of the pharmacological effect in bothpretreated groups, a PK study was performed bothwith total and unbound drug concentrations.Stereoselectivity in the PK of methadone hasbeen widely observed, principally in metabolismand binding. However, in this study we have per-formed the PK and PD with racemic methadonebecause the majority of the existing studies eval-uating the influence of protein binding are withdl-methadone, and also because it is the form ofthe drug typically administered in the clinic.5,35,36

Table 5. Unbound Pharmacodynamic Parameters ofMethadone and [Mean (SE)] in Control and TP Rats

UnboundParameters Control TP

Emax (s) 8.88 (0.23) 6.69 (0.32)EC50 (ng/mL) 5.23 (0.48) 5.68 (1.49)E0 (s) 4.20 (0.24) 2.28 (0.17)g 7.24 (2.43) —

The SE are single fit SEs of the parameter estimate and notused for significance comparison.

Figure 5. Relationship between unbound methadoneconcentration and effect in the control (solid line) and TP(dashed line) groups (simulated data up to 70 ng/mLof free drug) and in the exo-AGP group (dotted line)(observed data).



AGP and Pharmacokinetics of Methadone

A lack of relationship was observed betweenplasma levels and analgesic effect in both pre-treated groups (TP and exo-AGP versus control)when the Cp–time and effect–time evolutionprofiles of all groups were compared. The entireCp–time profile in both pretreated groups wassuperior to the controls; however, the correspond-ing effect—time was inferior. At this point it wasdecided that a PK/PD analysis was necessary.

Mixed effects population models are appro-priate for studying sparse data (less than thenumber of parameters in the model) in multiplesubjects as was the case here. A semiphysiologicalbicompartmental model best described the Cp–time observations in all groups. The mean popu-lation parameters for the controls were similar tothose reported previously.6

Initially, the three groups were analyzed as asingle population but the PK was different amongthem, apparently due to the AGP, at this initialstep introduced as a covariate, and the populationswere studied separately. The differences in the PKparameters among populations were maintainedeven after correction by weight.

In the TP group there was a reduction in V1,CL,and Q when compared with the control group. Theresults are reasonable for methadone, a drug withelevated distribution and clearance that wouldhave been affected by a change in binding. Theyalso explain the elevated (total) Cp after the samedose with controls. In the exo-AGP, the elevatedCp were also explained by the reduced PK para-meters. The difference in the TP group vanisheswhen the unbound parameters are compared, oreven show a slight increase. Specifically, the smallincrease observed in CLu appears to contrast withevidence that IL-6 secreted in the inflammatoryprocess reduces the expression of isoforms ofCYP450. Nevertheless, researching the issue, wehave found contradicting evidence in the literatureregarding the net effect of inflammation on clear-ance of drugs mainly due to P-glycoprotein mod-ulation.26 Therefore, the net influence in the caseof methadone was not predictable. Regarding Vu,the small increase in this parameter could beexplained by the known fact that the inflammationproduces an alteration in the microvasculaturefacilitating the intrinsic diffusion of the molecules.In any case, these changes in the unbound para-meters are quantitatively of little significance inrelation to the controls, as is finally appreciated inthe almost complete superposition of the unbound

concentration time courses of TP and controls(Fig. 3). However, this is not so for exo-AGP. In thisgroup, the unbound PK parameters remainedclearly lower than those in the controls or in theTP. Therefore, intrinsic alterations due to AGP,beyond binding, are possibly involved.

Similar AGP-dependent changes in the un-bound parameters have also been observed inother drugs. In a study with saquinavir37 in trans-genic mice a significant drop was observed in thevolume of distribution of the unbound drug, and itwas concluded that AGP blocks distribution bycausing a change in the net charge of the vascularendothelium. In another study,33 the administra-tion of AGP produced an in vitro reduction of thedrug UCN-01 capture by hepatocytes. The pre-sence of elevated AGP reduces the intrinsic clear-ance of some drugs. The causes are unclear,although it appears that AGP affects cellularmitosis and interacts with cellular surfaces.38 It isstill not known whether AGP affects the enzymesystem or causes changes in diffusion acrossbiological barriers.

The reduction in the CLu and Vu in exo-AGPlead to the increased unbound concentrationsof methadone in this group. Nevertheless, theanalgesic effect in this group of rats was stillpractically non existent.

A similar observation was made in a studywith prazosin in rats,39 where in the presenceof elevated concentrations of AGP the relationbetween free drug concentrations and effect wasaltered. Apparently, AGP decreased the sensi-bility to prazosin. The same authors also studiedother a-antagonists (tiadazosin) in the aorta in therabbit. They observed that at equal unbound drugconcentrations, the activity of the a-antagonistwas much less in the presence of AGP that in itsabsence.7

In our study, because the PK alone did notexplain the changes observed in the effect, otherprocesses that could alter the dose–effect relationwere considered and, particularly, the concentra-tion–effect relationship (PD).

AGP and Pharmacodynamics of Methadone

The consequences of the increase in the concen-trations of AGP on the PD have been studied onrare occasions. For example, for drugs such aspropranolol,24 penbutolol,1 and disopyramide.40

In these studies the pharmacological responsecorrelated better with the unbound drug concen-tration than with the total; therefore, in most



cases, alteration is observed in the EC50 but not inunbound EC50.

In the present study, in the TP group, the EC50

was almost twice that of the controls showing alower value of Emax. The change in the EC50 wascorrected by the fu, but not the Emax. The fact thatthe Emax was lower could be attributed to changesat receptor level, as has been suggested in otherresearch.7,39 In a recent study the PK and PDof propranolol was evaluated after oral admi-nistration in rats with experimental arthritis(chronic inflammation).41 An increase of the Cpwas observed, but it was accompanied by adecrease in the effect. Additionally, the unboundAUC was also higher in the inflammation group.It was suggested that the inflammation coulddownregulate the b-adrenergic receptor, a reduc-tion in the potency/efficacy of the receptor proper.With sotalol, there were no changes in the PK ofrats under inflammation but there was a reductionin the effect.42 The two studies point to theinfluence of nitrous oxide (NO) as well as TNF onreceptor regulation, both secreted during the in-flammatory process.

It has also been demonstrated in vitro thatneuronal NO is involved in the destabilization of mreceptors in neurons of the locus coeruleus in therat.43 Further, in vivo, the administration of NOsynthesis inhibitors attenuates the developmentof tolerance induced by chronic treatment withmorphine.44 The above experiments with NOappear to point to a role of the oxide in the receptor,particularly the m receptor, deactivation, and thuscould also be involved in the reduction of efficacyobserved in the TP group here.

The change in PD was larger in the exo-AGPgroup. Due to the complete lack of relationshipbetween the Cp (total or unbound) and the effect,no model could be adjusted to the observations.

To compare the groups, the analgesic effect wassimulated in the control group up to an unboundconcentration of 70 ng/mL (that is, the concentra-tion reached in the exo-AGP group). At thatunbound Cp of methadone, the effect reached inthe controls was close to 100% (9.5 s). However, inthe exo-AGP group and for the same unboundCp ofmethadone, the effect reached was 3.2 s (similar tothe baseline).

The integrated PK/PD point of view verifies thedirect influence of AGP on the effect of methadone.AGP could alter the pharmacological activitythrough mechanisms different to processes relatedto the binding to plasma protein, such as changesin the interaction of the drug with the receptor, as

has been commented for prazosin,7 or changes inthe distribution to the effect site, such as in thecase of fluoxetin.31 These drugs, as in our case,showed absence of correlation between unbounddrug concentrations and the effect. In thesestudies there were no changes in the unboundconcentration, but there were differences in thePD parameters when the AGP was elevated.The mechanism involved has not been clarified.The fact of having employed racemic methadonedoes not obscure this outcome because it isthe enantiomer with no analgesic effect capacity(S-methadone) that presents the most bindingto AGP.5

Regarding prazosin, an interaction of AGPwith the a-receptor has been suggested, in sucha manner that the drug cannot block the effect ofthe a-adrenergic agonists, and would thus explainthe alteration in its antagonistic activity.39

In the present study, the relative differencesamong the three groups observed in the timecourse of the analgesic effect in the rat, can bepartly attributed to alterations in the PK, speci-fically changes in the ubound fraction in TP.However, they are also due to changes in the PDparameters (mainly Emax), possibly related to aninteraction of AGP with the receptor similarly aswith prazosin.

In conclusion, the population PK analysishas allowed the detection of differences in thePK parameters between the three populations.Nevertheless, the PK did not completely ex-plain the changes in the observed effect. ThePD analysis suggests a role of the protein inthe effect of methadone, principally based on theresults of the exo-AGP group. The benefit ofintegrated PK/PD studies is clear for complexinteractions such that of AGP with the methadonePK and PD.

ACKNOWLEDGMENTS

Support was from a Basque Government scholar-ship (MR), a UPSA Pain Institute scholarship(I.O.), the Gangoiti Foundation scholarship (I.S.),and University of the Basque Country scholarship(N.L.). The work was also supported in part byEuropean Commission Grant HPMF-CT-2002-01922 (J.L.). We are thankful to Mr. HerwigReichl for his generously supplying human AGPfrom Hamosan Laboratories. We are also thankfulto Mr. Jokin Lodos for his careful animal surgicalprocedures.



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Documents

Alpha-1-acid glycoprotein directly affects the pharmacokinetics and the analgesic effect of methadone in the rat beyond protein binding