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Model-Based Methods for Measuring Binding Potential in SPECT Imaging, Study notes of Neurology

A study that aimed to develop model-based methods for measuring the binding potential (BP) of iomazenil to benzodiazepine receptors in the human brain using SPECT imaging. The study compares equilibrium methods and discusses the importance of distinguishing between peak-up and transient equilibrium methods. It also provides information on the yield of iomazenil preparations and the estimation of the optimal bolus-to-infusion ratio for constant infusion. The document also includes data on the peripheral clearance parameters in single-bolus experiments and the estimation of the optimal bolus-to-infusion ratio for reaching equilibrium rapidly in all ROIs.

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bg1
been used as a PETradiotracer forvisualization and quan
tification ofbenzodiazepine receptors in humans (Z12-16).
Recently, an iodinated analog of fluinazenil, iomazenil (Ro
16-0154), has been introduced as a SPECT radiotracer (17).
SPECT studies in nonhuman primates (18) and healthy
human subjects (19) have shown that [‘@IJiomazenilhad a
high brain uptake (iO%—i2%of the injected dose). About
90% of this brain activity is displaceable and therefore,
associated with specific binding to benzodiazepine recep
tors.
Analysis of SPECT neuroreceptor imaging studies have
been typically restricted to empirical, semiquantitative
methods such as the ratio of activities in regions of interest
(RO!s) or the RO! washout rates. Since these empirical
measures do not control for factors such as peripheral
clearance, binding to plasma proteins and cerebral blood
flow, the ability of these measures to provide quantitative
information about the receptors under study is question
able (20). The goal of the studies reported here was to
develop model-based methods for SPECT measurementof
[‘@!]iomazenilbinding to benzodiazepine receptors in the
humanbrain.
Model-based methods for in vivo quantification of neu
roreceptors can be broadly divided into kinetic and equi
librium methods. Kinetic methods yield quantitative inior
mation aboutthe receptors by estimatingthe rate constants
which characterize the transferbetween plasma, brainand
receptor compartments (21). Equilibrium methods derive
this information by analyzing the activity distribution at
equilibrium, i.e., when the receptor-ligand association and
dissociation rates are equal (22). With both approaches,
the outcome measure of experiments performed at tracer
doses is a unitless number, the binding potential (BP),
which equals the product of the receptor density (Bm@,
nM) and affinity (i/Ks, @J@1)(21). The BP is also the
equilibrium distribution volume of the receptor compart
ment, i.e., the ratio of specific binding in the brain-to
Iodine-123-iomazenilbindingto benzoduazepinereceptorsin hu
manbrainwasmeasuredwithSPECTusingkineticandequilib
nummethods.Mthods: Inthe kineticexperiments(n = 6),
regionalthne-activitycurvesaftera singlebolusinjec@onof the
tracerwere fit to a three-compartmentmodelto provideesti
matesoftherateconstantsK1to k. Thebindingpotential(equal
to the productof the receptordensityandaffinity)was derived
fromthe rateconstants.In theequilibriummethod(n = 8), the
tracerbolusinjectionwasfollowedby a constanttracerinfusion
toinducea sustalnedequilibriumstate.Theregionalequilibrium
volumeof distributionwascalculatedastheratiooftheregional
brainCOnCentrabOn-ID-thefreeparenttracersteady-stateplasma
concentration.Inthreeexperiments,a receptor-saturatingdose
offlumazenil was injectedfor direct measurementofthe nondis
placeablecompartmentdistributionvolume.Results: The Id
neticandequilibriummethodresuftswereingoodagreementin
all regions investigated. Iodine-125-iomazenilbinding potential
measuredin vitroin 12 postmortemsampleswasfound to be
consistent wfth SPECT in @vomeasurements. Conclusion:
Thesestudiesdemonstratedthe fea@brIftyof quantificationof
receptorbindingwithSPECT.
KeyWords:SPECT;brain;benzodlazeplnereceptors;10-
dlno-123-Iomazsnll
J Nuci Md 1994; 35:228-238
terations of central benzodiazepine receptors have
been described in several neuropsychiatricconditions, in
cluding epilepsy (1—3),Alzheimer's disease (4,5), Hunting
ton's chorea (6—8)and schizophrenia (9—11).Carbon-li
flumazenil (Ro 15-1788), a benzodiazepine antagonist, has
ReceivedJul.6, 1993;revisionaxepled Nov.4, 1993.
Forcorrespondenceand reprirdscon@ As@ssaAbi-Dar@wn,MD,De@ of
Psychiatry,Y@eUniversityand West HavenVA MedicalCerter/116A2,950
CaIT;@bstAve.,West Haven,CT06516.
228 TheJournalof NudearMedicine•Vol.35•No.2 •February1994
SPECT Measurement of Benzodiazepine
Receptors in Human Brain with
Iodine-123-Iomazenil: Kinetic and
Equilibrium Paradigms
Anissa Abi-Dargham, Marc Laruelle, John Seibyl, Zachary Rattner, Ronald M. BaldWin, Sami S. Zoghbi,
Yolanda Zea-Ponce, J. Douglas Bremner, Thomas M. Hyde, Dennis S. Charney, Paul B. Hoffer and
RobertB. !nnis
DepartmenL@ ofPsychiatiy and Diagnostic RadiOlOgy, Yale UniVeTSÜySchool ofMedicine, New Haven, Connecticut and
West Haven VA Medical Center, West Haven@Connecticut; Clinical Brain Disorders Branch, IRP, NIMH Neuroscience
Center at St. Elizabeths, Washington, DC
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pf4
pf5
pf8
pf9
pfa

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Download Model-Based Methods for Measuring Binding Potential in SPECT Imaging and more Study notes Neurology in PDF only on Docsity!

been used as a PETradiotracer for visualization and quan

tification ofbenzodiazepine receptors in humans (Z12-16).

Recently, an iodinated analog of fluinazenil, iomazenil (Ro

16-0154), has been introduced as a SPECT radiotracer (17).

SPECT studies in nonhuman primates (18) and healthy

human subjects (19) have shown that [‘@IJiomazenilhad a

high brain uptake (iO%—i2%of the injected dose). About

90% of this brain activity is displaceable and therefore,

associated with specific binding to benzodiazepine recep

tors.

Analysis of SPECT neuroreceptor imaging studies have

been typically restricted to empirical, semiquantitative

methods such as the ratio of activities in regions of interest

(RO!s) or the RO! washout rates. Since these empirical

measures do not control for factors such as peripheral

clearance, binding to plasma proteins and cerebral blood

flow, the ability of these measures to provide quantitative

information about the receptors under study is question

able (20). The goal of the studies reported here was to

develop model-based methods for SPECT measurement of

[‘@!]iomazenilbinding to benzodiazepine receptors in the

human brain.

Model-based methods for in vivo quantification of neu

roreceptors can be broadly divided into kinetic and equi

librium methods. Kinetic methods yield quantitative inior

mation about the receptorsby estimatingthe rate constants

which characterize the transferbetween plasma, brainand

receptor compartments (21). Equilibrium methods derive this information by analyzing the activity distribution at

equilibrium, i.e., when the receptor-ligand association and

dissociation rates are equal (22). With both approaches,

the outcome measure of experiments performed at tracer

doses is a unitless number, the binding potential (BP),

which equals the product of the receptor density (Bm@,

nM) and affinity (i/Ks, @J@1)(21). The BP is also the

equilibrium distribution volume of the receptor compart

ment, i.e., the ratio of specific binding in the brain-to

Iodine-123-iomazenilbindingto benzoduazepinereceptorsin hu

manbrainwasmeasuredwithSPECTusingkineticandequilib

nummethods.Mthods: In the kineticexperiments(n = 6),

regionalthne-activitycurvesaftera singlebolusinjec@onof the

tracerwere fit to a three-compartmentmodelto provideesti

matesofthe rateconstantsK1to k. Thebindingpotential(equal

to the productof the receptordensityand affinity)was derived

fromthe rateconstants.In the equilibriummethod(n = 8),the

tracerbolusinjectionwasfollowedby a constanttracerinfusion

toinduceasustalnedequilibriumstate.Theregionalequilibrium

volumeof distributionwascalculatedasthe ratioof the regional

brainCOnCentrabOn-ID-thefreeparenttracersteady-stateplasma

concentration.Inthreeexperiments,a receptor-saturatingdose

offlumazenil was injectedfor direct measurementofthe nondis

placeablecompartmentdistributionvolume.Results: The Id

neticandequilibriummethodresuftswereingoodagreementin

all regions investigated. Iodine-125-iomazenilbinding potential

measuredin vitroin 12 postmortemsampleswasfoundto be

consistent wfth SPECT in @vomeasurements. Conclusion:

Thesestudiesdemonstratedthe fea@brIftyof quantificationof

receptorbindingwithSPECT.

KeyWords:SPECT;brain;benzodlazeplnereceptors;10-

dlno-123-Iomazsnll

J Nuci Md 1994; 35:228-

terations of central benzodiazepine receptors have

been described in several neuropsychiatric conditions, in

cluding epilepsy (1—3),Alzheimer's disease (4,5), Hunting

ton's chorea (6—8)and schizophrenia (9—11).Carbon-li

flumazenil (Ro 15-1788),a benzodiazepine antagonist, has

ReceivedJul. 6, 1993;revisionaxepled Nov.4, 1993. Forcorrespondenceand reprirdscon@ As@ssaAbi-Dar@wn,MD,De@ of Psychiatry,Y@eUniversityand West Haven VA MedicalCerter/116A2, CaIT;@bstAve.,West Haven,CT06516.

228 TheJournalofNudearMedicine•Vol.35 •No.2 •February^1994

SPECT Measurement of Benzodiazepine

Receptors in Human Brain with

Iodine-123-Iomazenil: Kinetic and

Equilibrium Paradigms

Anissa Abi-Dargham, Marc Laruelle, John Seibyl, Zachary Rattner, Ronald M. BaldWin, Sami S. Zoghbi,

Yolanda Zea-Ponce, J. Douglas Bremner, Thomas M. Hyde, Dennis S. Charney, Paul B. Hoffer and

RobertB. !nnis

DepartmenL@ ofPsychiatiy and Diagnostic RadiOlOgy, Yale UniVeTSÜySchool ofMedicine, New Haven, Connecticut and

West Haven VA Medical Center, West Haven@Connecticut; Clinical Brain Disorders Branch, IRP, NIMH Neuroscience

Center at St. Elizabeths, Washington, DC

yieldof the [@!Jiomazenilpreparationsaveraged66.4%±8.2%

(with these and subsequent data expressed as mean ±s.d., n = 14) and the radiochemical purity averaged 97.6% ±1.7% (n = 14).The specific activity was determined by comparh@gthe UV absorbance

of thelabeledpmductwitha standardcurvegeneratedfromknown

concentrations of nonradioactive iomazenil. The specific activity of E'23!liomazenilin these preparations was found to be greater than 5000 Ci/mmole and may be estimated to be on the order of 180,

CL'mmole(37).Sterilitywas assuredby lackof bacterialgrowthin

twomediaandapyrogenicitywasconfirmedusingthelimulusame

bocyte lysate (LAL)test. The specific activity of [1@I]iomazenilwas estimated by UV detection to be 1090 Ci/mmole on the day of

labelin@

@ He&thy

Fourteen healthy subjects (age 26 ±6 yr. weight 75 ±7 kg, 11 males and 2 females) were recruited for these studies. Inclusion

criteria were: (1) absence of current medicalconditionsand (2)

absence of neumpsychiatric illness, alcohol or substance use. A physical examination, EKG and routine blood and urine tests were performed in the screening procedure. All subjects gave written informed consent. Protocols were approved by the insti tutional human investigation committee. Subjects received potas sium iodide (SSK! solution 0.6 g in the 24 hr period prior to imaging).

Data Acquisition

SPECF datawere acquiredwith the multislice brain-dedicated

CERASPECF camera (Digital Scintigraphics,Waltham, MA)

(38). The transaxial and axial resolution in air are 7.7 and 5.9 mm

FWHM,respectively(39).Inwater,theresolutionis 10—12mmin

thethreeplanes.

Four fiducialmarkersfilled with 10 j@Ciof [@Tc]sodium

pertechnetatewere attachedon both sides of the subjects's head

at the level of the cantho-meatalline to control positioningof the

head in the gantrybefore tracerinjectionand to identifythe

cantho-meatal plane during image analysis.

Singlebolus experimentswere performedin six subjects(age

23 ±0.7yr,weight78 ±11kg).Iodine-123-iomazenil(12.8±4.

mCi)was injectedas a single bolus over 30 sec. Scans were

acquired in continuous mode every 2 mm for 145 ±5 mm. Arterial blood samples (1—2ml) were collected every 20 sec for the first 2

mis with a peristalticpump(Harvard2501-001,SouthNatick,

MA)andthenmanuallyat3, 4, 6, 8, 12,16,20,30,45and60mm.

After the first hour, samples were drawn every 30 mis until the

endof theexperiment.

Constant infusion experiments without flumazenil displace ment were performedin five subjects (age 26 ±9 yr, weight 75 ±

4 kg).Thedosewasdividedinabolusof3.89 ±0.39mCifollowed

by aconstantinfusionof 1.08±0.12mCi/hr(IMEDpump,Jemini

PC-i, San Diego, CA) given over 450 miii, resultingin a bolus-to

infusionratioof3.61 ±0.27hr.Scanswereacquiredincontinuous

modeevery 5 ruinfrom250 to 450 ruinpostinjection.In three

subjects, data were also acquired from 0 to 150mis postinjection.

Arterialsampleswere collectedin a pattern similarto the single

bolus experiments in the first three experiments, and every 15 mm starting 2 to 4 hr postradiotracer injection in the last five experi

ments.

Humazenildisplacementduring[‘@I]iomazenilconstantlain

sion was performedin threesubjects(age 27 ±2.6 yr. weight

75 ±12 kg). The injection and acquisition protocols were similar to the previous constant infusion studies (bolus 4.1 ±0.54 mCi, infusion 1.12 ±0.16 mCi/hr resulting in a bolus-to-iniusion ratio of

plasma level of free parent tracer at equilibrium.Benzodi

azepine receptors have been measured with [11C]flumazenil

and PET with kinetic (1423,24) and various “equilibrium―

methods (2,25-28).

Among equilibrium methods used to measure [“C]flu

mazenil BP, it is important to distinguish the “peakup

take―equilibriummethod (2) and the “transient―equiib

rium method (25,27,29). “Peakequilibrium― methods measure the BP at peak uptake of the specific binding, usually obtained by subtracting the activity in the white

matter (2) or the pons (2428) from the activity in the RO!.

At peak uptake, the BP is calculated as the ratio of activity

in the ROl-to-activity in a reference region. The difficulty

of this method is the proper identificationof peak uptake

for ligands such as [1@I]iomazenilwhich exhibit a pro

tracted plateau phase (19). The “transient―equilibrium

method, also called quasi-equilibrium(27) or pseudo-equi

librium (25), refers to the measure of the BP when the

specific-to-nonspecific ratio or the specific binding-to

plasma tracer concentration ratio becomes constant over

time (i.e., when both are decreasing at the same rate).

Carson et a!. (30) proposed the term “transient―equilib

rium to describe these conditions. As opposed to the peak

uptakeequilibriummethod, the transientequilibriummeth

ods do not satisfy the Michaelis Menten equilibriumequa

tion and can result in an overestimation of the BP (30). To overcome the technical and theoretical difficulties associ ated with the use of equilibrium methods after single bolus

injection of the tracer, we implemented in humans the

constant infusion/sustained equilibrium method (30—32)

that we previously described in baboons (33—35).

In the study reported here, benzodiazepine receptor BP

was measured in 14 healthy subjects with [‘@!Jiomazenil

and SPECT using both a kinetic (i.e., bolus only) and a

sustained equilibrium (i.e., bolus plus constant infusion)

paradigm. Plasma clearance of the tracer estimated from

the single bolus experiments was used to design the tracer

administration protocol of the bolus plus constant infusion

experiments. A direct measure of the nonspecific compart

ment was obtained in three of the eight constant infusion

experiments by injecting a receptor saturating dose of flu

mazenil (0.2 mg/kg) at the end of the study. In addition, the

accuracy of SPECT measurements was confirmed with

[‘@!]iomazenilbinding studies to postmortem human brain

samples.

METhODS

Radlolab.llng Iodine-123-iomazenil and [‘@!Jiomazenilwere prepared by io dodestsnnylation of ethyl 7-(thbuty@tannyl)-5,6-dihydro-5-meth yI-6-oxo-4H-hnidazo[1,5.a][i,4jbenzodiazepthe-3-carboxylate with

chloramine-Tin methanolicaceticacidat 120°C(36).Radiolabeled

productswere purifiedby reversed-phaseHPLC(C-18column,

3.9x 300mm,55%CH3OH/H20,0.7mI/rain;R@9.2mm),formu

latedin 5%ethanolin normalsalinecontaining0.1mML-ascorbic

acid, pH 5-6, and filtered through a O.2-@membrane filter into a sterile 10-mI serum vial or sterile 10-mi syringe. The radiochemical

SPECTMeasurementofBenzodiazepineReceptors•Abr-Darghametal. 229

@@ K1 = FE = F(1—e (ml. g ‘. miii Eq.

15k3 k2 K1/V2f1(min@ 1),Eq.

16k4=k0ff(miii@'),Eq.17 kcn@mJ2(mm ‘),Eq.

FiGURE1. Thethree-comparb@nentmodelusedforbothkinetic

and equilibriumanalyses.C, = arterialplasmaconcentration,me

taboiftecorre@ed;C2= tra@erconcentrationinIntr@erebralnondis

@abIecompartment(freeandnonspecificbinding);C, = specif

Icallyboundconcentration;K, to k, = fractionalrateconstants

describingthekineticsoftracertransferbetweencompartments.

compartment (f') is constant over time. The vascular activity present within an RO! was estimated assuming a blood volume equal to 5% of the ROI volume (21) and subtracted from the total

RO! activity prior to analysis.The activity concentrationin the

RO! at time t, C@01(t),is the sum of the concentrations in the

second and third compartments:

CRo@(t)= C,Jt)+ C@(t). Eq. 7

The equilibriumvolume of distributionof compartmenti is the ratio of the tracer concentration in compartment i to the free tracer concentration in the arterial plasma at equilibrium, i.e.,

when no net transferbetweenthe plasmaand compartmenti

exists:

vi = @a.

f1C

V2is theequilibriumvolumeofdistributionofthe nondisplaceable

compartment and V3 is the equilibrium distribution volume of the specifically bound compartment. The total tissue equilibrium vol. ume of distribution,V.@.,is the sum of both compartments:

CR0!

VT j@ = V2+ V3.

Passive diffusion is assumed to be the mechanism of transfer of the tracer across the blood-brainbarrier(BBB). When the non displaceable compartment is at equilibrium with the plasma, the free concentration is assumed to be equal on both sides of the BBB,

f1C5= f2C@, Eq.

which yields the inverse relationship between f2 and V2:

f2= i/V2.

Kinetic Anc4ySiS. The tracer concentration over time in each compartment is given by:

dC@(t)

.—@;.—=K1C@(t)—k2C@(t)—k3C@(t)+k@C@(t),Eq.

dC@(t)

—@i--=k3C@(t)—k@C@(t), Eq. 13

where F is the regionalblood flow (ml •g'. min1); E the

unidirectional extraction fraction; PS the permeability surface area productof the tracer (ml •.g'. @i@@');and k@,and k.@the association and dissociation rate constants for ligand-receptor binding. Equation 15 can be derived from Equation 12 by setting k3, k4 and the derivates to zero and multiplyingboth sides by f1.

Attracerdoses,Equations12and13haveconstantcoefficients

and can be solved analytically (43,44), and BP can be derived from the rate constants using Equations 15, 16 and 17:

BP Bmax koaBmax k3 K1k3 i — KD 1(01! k42k2k41 Eq.

Equilibnum Ana@YSiS.At equilibrium, the rate of association and dissociation of the tracer-receptor complex are equal:

Thus,

@ @f2C@B@= Eq. 19

Bmax C3 C

BP-@=@=@-@, Eq.

1@ 8 which demonstrates that BP is equal to the equilibrium volume of £.d@1. distribution of the bound compartment (V3, Equation 8).

At equilibrium,VTwas calculatedwithEquation9. For con

stant infusion experiments followed by an injection of a saturating

dose of flumazenil,V2was calculatedas the ratio of the nondis

placeableactivity-to-theplasma-freeparentcompound(from15to

45miiipostflumazenilinjection)andBP(V@)wascalculatedasthe

differencebetweenVTandV2(Equation9).

Eq. 9 Cwve-Fitting Plvcedwe. Rate constants for arterialclearance and brain uptake ofthe tracerwere estimated by nonlinear regres sion using a Levenberg-Marquartleast-squaresminimizationpro cedure (45) implemented in MATLAB (The Math Works, !nc., South Natick, MA) on a Macintosh Quadra 950. The number of exponential terms used to describe the plasma clearance was

determinedby the F-test (46) and the AIC criteria (47). The

standard error of the parameters was given by the diagonal of the covanance matrix (48) and expressed as percent of the parame

ters (coefficientof variation,%CV).Thus, %CVindicatesthe

identifiabilityofthe parameterby the least-squaresprocedureand

should not be confused with the Standard deviation (s.d.) of the Eq. ii distributionof the parameteramong subjects.

inVitror@'iiom.@anIIBk@dingExpsrlm.nts Twelve brains were collected from the District of Columbia Medical Examiner's Office. The brains were examined by a fo rensic pathologistor a neuropathologistfor the detection of gross abnormalities and were cut into i-cm thick coronal slices. Sam

pleawere dissectedon ice and immediatelyfrozenat -70°Cuntil

assayed. Inclusion criteria were absence of gross pathological

abnormalitieson examinationof the brain and absenceof neuro

psychiatricdisordersas noted by reviewof the medicalrecords.

wherethe kineticparametersK1to k, aredefinedas follows: Ages varied from 22 to 87 yr (50 ±22 yr). Brains from four

SPECTMeasurementofBenzodlazeplneReceptors•Abr-Darghamatal. 231

SubjectV@CLno.Ofter/kg)(lfter/hr/kg)

VboI initial volume of distribution In plasma CL = P@riP'@' dEW

ance;andf1= freefractionInplasma.

females and eight males were used. Time between death and freezingof tissue was 24 ±10 hr. Preservationtime at —80°Cwas 76 ±12 mo.

Onthedayof theassay,brainsampleswereweighed,thawed

and homogenizedwith a Polytron (setting6 for 10 5cc) in 1/40wet weight in mg/vol in ml (wAr)of buffer(25 mM KH2PO4'150 mM NaCl, pH 7.4). After homogenization, tissues were centrifuged (20,000g, 4°C,10min). The supernatantwas discardedandpellets were resuspended and recentrifuged. This procedure was re

peated twice. Incubation(22°C,45 mm)was initiatedby the suc

cessive additionof 100 @tlof [‘@!]iomazenil,100 @dof bufferor

unlabeled iomazenil and 800 @dof tissue solution. A final tissue dilution of 1/2,400 w/v was selected so that total binding was between 5% and 10% of total ligand concentration. Incubation was terminated by rapid filtration though OF/B ifiters on a 48- channelCell Harvester (Brandel,Gaithersburg,MD). Filterswere rapidly washed three times with 5 ml of ice-cold buffer and counted in a COBRA 5010 gamma counter (Packard, Menden, CT) with an efficiency of 80%. Saturationexperiments (n = 12) were performedby the isotopic dilution method (“cold―satura lion) on the occipital cortex from each brain using one concentra tion of [‘@!]iomazethl(0.02 nM) and 15 concentrations of unla beled iomazenil ranging from 10_14 to 106 M. Saturation experiments were analyzed by weighted nonlinear regression analysis using the programLIGAND (NIH, Bethesda, MD) (49).

RESULTS

SingleBolusEXperimentS After single bolus injection of [‘@!]iomazenil,the six subjects exhibited similar kinetics of plasma clearance of

the parentcompound (Table 1). In all cases, a sum of three

exponentials provided a statistically significant improve ment in the fit as compared to a two-exponential fit. A four-exponential fit provided a statistically significant im

provement of the fit in only two of six subjects. Therefore,

a three-exponential model was chosen (Fig. 2A). !mtial

volume of distributionwas 0.44 ±0.14 liter/kg and clear

ance was 7.4 ±1.1 liter/hr/kg.The average A3(0.013 ±0.

min') corresponded to a terminalhalf-life of 97 mm. The

free fraction ofthe parent compound was between 20% and 30% of the total parent compound (f1 = 0.23 ±0.02). The occipital region showed the highest and latest up take, peaking at about 30 mm (Fig. 2B). Other investigated

40 80 120 160

i

C.)

C 900

F

@ 300

80

TiME(mln)

160

FiGURE2. (A@Arterialtlme-actMtycurve of free parent

[1@lJiomazenilfollowinga bolusInjectionof 12.5mCIIn a 22-yr-old male.Valueswerefittoatriexponentlalmodel(solIdlIne)toderive

the peripheralclearance(Cl = 8.5 lIter/hr/kg).(B)Regionalbrain

time-actMtycurvesfromthesamesubjectasInpanelA Measured

values (drcles,trianglesand squares) were fitto a three-comport

mentmodel(solIdlines).BPvalueswere272,174and58 in the

occipitalcortex,frontalcortexandcerebellum,respectivaly.

RO!s peaked earlier and at lower values (in decreasing

**order temporal > frontal > striatum, thalamus, cerebellum

pons). RO! time-activity curves were fit to the three** compartment model (Fig. 2B). The iteration procedure converged for all regions, providing estimates of the re

gional rate constants (K1 to k@)and values for the outcome

measures (‘@2@f2, BP and VT, Table 2).

K1 rangedfrom 0.385 nil. g@ •@Ji@@1(pons) to 0.546 ml

. g'. min' (occipital), with an average value for all

regions of 0.467 ml. g@. piJ1@1.‘flij@parameter was

reasonably identified in the frontal, occipital, temporal and

cerebellarregions (%CVbetween 5%and 6%)but was less

well identified in the striatum (%CV ±s.d.; 16% ±9%),

thalamus (%CV ±s.d.; 18% ±15%) and pons (%CV ±

s.d.; 19% ±7%). The rate constant k2 showed a large

variation across regions, from 0.094 ±0.118 min1 (tem

poral) to 0.300 ±0.214 (pons)with a mean regional value of

0.151 ±0.150 min', and was poorly identified in all re

gions, with %CV ranging from 26.6% ±8.9% in the thal

amus to 127% ±159% in the striatum. The value of k

varied from 0.175 ±0.095 min' (occipital) to 0.104 ± 0.037 min@' (cerebellum) but was as poorly identifiable as

TABLE 1

Peripheral Clearance Parametersin Singie-BolusExperiments

10.296.80.2220.528.20.2330.487.30.2340.418.50.2350.295.60.2760.648.40.21Mean±s.d.0.

±0.147.4 ±1.10.23 ±0.

232 TheJournalof NuclearMedicine•Vol.35 •No.2 •February 1994

A 1000

iii C.) @ £

z

@ 10

Ef 2

B 1500

Parameters Frontal Occiplal Temporal Striatum Thalamus Cerebellum Pore

Valuesaremean±s.d.of sixexperiments.

mctoi@,= transferrateconstants;BP= bindingpotentialasdertvedfromkineticanalysis;@2= freefr@IonInthenondispleceablecompartment

V2- nondlspIaceai@Ieequilbiumvolumeofdl@rIbutIon;VT= t@aItissueequiblumvolumeofdistribution;and% NSB= percentnonspecific

t@ndIng

TABLE 2

KineticAnalyaisof Single BolusExperiments

mc@@jn@1)0.4630.5460.4950.4460.5340.3980.385±(ml. g

s.d.0.2190.1670.2220.2000.3550.1710.172k (m1n1)0.1560.1360.0940.1230.1430.1060.300± s.d.0.1940.0980.1180.1340.2120.0800.214k3(mln-1)0.1450.1750.1080.1340.1180.1040.114±

s.d.0.1540.0950.0610.0800.1330.0370.064k (m1n1)0.0170.0140.0170.0340.0290.0180.024± s.d.0.0050.0040.0060.0270.0100.0040.008BP161.00240.00189.0081.0078.00101.0030.00±

s.d.15.0041.003.0012.0027.0023.0011.00f20.070.050.040.050.050.060.17±

s.d.0.090.030.030.030.040.030.09V237.0031.0054.0027.0041.0020.008.00±

.004.00VT196.00271.00242.00108.00119.00121.0037.00±s.d.25.0030.0054.0019.0030.001 1

.0011.00%s.d.16.0029.0055.00180034. .0019.0024.0033.0016.0022.00± NSB18.001 1 s.d.12.0011.0015.0013.0020.006.0013.

InVitroraswom@eniIBindingin Postmortem HumanBrain

In the occipital cortex, [‘@!JiomazemlBm@was 162±

nM and KD was 0.59 ±0.14 n.M (n = 12). These values

corresponded to a BP of 287 ±95. No significantcorrela

tion was observed between age and [‘@!]iomazenilB@,, or

KD.

DISCUSSION

These studies demonstrated the feasibility of measuring

benzodiazepine receptors regional binding potential with

SPECF. Two methods were tested: kinetic analysis of a

single bolus injection and equilibrium analysis ofbolus plus

constant infusion. With the exception of the pons (where

the low level of counts may have contributed to a higher

level of noise), both methods gave similar regional results.

For example, the occipital BP was 240 ±41 (n = 5) and 248

±53 (n = 3) for kinetic and equilibrium experiments, respectively, and veiy close to the in vitro homogenate

binding value of 287 ±95.

The three-compartment kinetic analysis applied here

was comparable to the kinetic analysis developed for re

versible PET tracers such as [‘1Cjflumazenil(16,24). As

previously observed with PET, the rate constants K1 to k@

were poorly identified (high standard errors due to covari

ance) when unconstrained kinetic analysis was applied

(30,44). Furthermore, the values of the kinetic parameters wereinconsistentwiththeirphysiologicalinterpretation. Regional differences in K1 and k2 should reflect regional

differences in blood flow. Their ratio, V2 should reflect the

ments. Therefore, in subsequent experiments, arterial

plasma samples and scan datawere acquiredonly from 250

to 450 min.

To quantifythe stability of the plasma and brainuptake,

a linear regression was performedon plasmavalues (last 4

hr, mean number of points = 12) and on RO! values (last 2

hr of the experiment, mean numberof points = 22). !n the

plasma, the mean change was 2.5% ±2.1%/hr. With the

exception of the pons, all RO!s showed changes of less

than 5%/hr.

RegionalVTwas measuredby calculatingthe ratioof the

averaged RO! activity and the free unmetabolized plasma

activity over the last 2 hr (Table 3).

Bolus Plus Infusion Plus Dlsplacsmsnt Experimsnts

Flumazenil injection was well tolerated by all three sub

jects with no side effects reported at the dose of 0.2 mg/kg.

Flumazenil induced 88% displacement of the tracer in the

occipital, 87% in the temporal, 86% in the frontal, 84% in

the thalamic and cerebellar, 81%in the striataland 60%in

the pontine RO!s (n = 3). Plasma tracer activity exhibited

a slight (11% ±3%)and transient (5—15mm) increase after

flumazenilinjection. At the time ofthe measurementof V2,

the plasma tracer concentration was back to baseline. Re

gional V2 varied from 34 (occipital) to 23 (pons, Striatum

and thalamus; Table 3 and Fig. 6). The value of f2, calcu

lated as 1/V2, varied from 0.043 (pons) to 0.030 (occipital

and temporal).An importantobservation was the presence

of 60% displaceable binding in the pons, indicating that the

pons cannot be used as an area devoid of benzodiazepine

receptors sites.

234 TheJournalof NucisarMedicine•Vol.35 •No.2 •February

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CEREBELLAR I 00

TIME (mm)

TiME (mln)

FiGURE4. (A)Simulatedplasmafree[1@l)momazenilconcenta

tionaftera bolusinjectionof3.4 mCIfollowedbya constantInfusion

of 1 mCI/hrfor420 mm.Thesoftdlinerepresentstheprojected

time-ectivitycurvein a 70-kgsubjectwith [1@IJiomazenilclearance

of553lfter/hr(= averageclearancemeasuredInsixsubjects).The

dottedline(.... ) andthe brokenline(———)areprojectedactMtles insubjectsw@,slow(398Ilter/hr)andhigh(708llter/hr)clearance, respectivaly.In all cases,Steady-Stateis achlevedat 300 mm.Sim ulatlonswere generatedusingthe followingparameters:V@ = @ lfterf01 = 1.0;f@= 0.11;f@= 0.026;A1= 1.18m1n1; 0. min1; A3 = 0.013 mlrr1, 0.007 min@ and 0.022 mWr1 In mean, sk@wand fast clearancescenarios,respectively.(B) Simulatedvol umes of distribution(regionalactivity/freeplasma activity)in the occlpftalcortex and cerebellum.In both regions and In all three clearancescenarios,the volumeof distributionreechedan equ@Ib

numvalue(‘IT)at 360 mm.Parametersusedforthissimulation

were:f1 = 0.231; K, = 0.546ml. mmn@. g1; k@= 0.136m1n@;k@ = 0.175 min1 (ooclpltaJ) and 0.104 min1 (cerebellum), k, = 0.0141 m1n1; V2 = 17; BR = 215 (occipital),91 (cerebellum).

nonspecific distribution volume and is not expected to vaiy

much between regions. In fact, V2. as derived from kinetic

analysis (Table 2), showed more regional variation than V

as measured directly by flumazenil displacement (Table 3).

According to the model, k3 is the parameter that includes

the receptor density (B@, Equation 16), leading theoret

ically to a correlation between VT and k3. No such corre

lation was observed (Table 2). The molecular dissociation

constant, k4 (k@) is expected to be similar across regions,

which was not the case (Table 2). Thus, the rate constants

did not reflect the physiological processes ascribedto them

by the model. However, the regional distribution of the

outcome measure VT was better identified and in accor

FIGURE5. (A)Arterialtime-activitycurveoffreeunmetabolized (1@qioru@i after a bolus of 3.68 ma followed by a constant mnfuslonof 1.12 m@libr(B/I ratio of 3.68 hr) in a 21-yr-oldmale. Valueswere fit to a tilexponentlalmodel (solidlIne)to derivethe peripheralclearance(CI = 6.7 liter/hr/kg).(B) RegionalAOl time activitycurves.Total AOl activityat equllibdum(averageof the last 180 mW@)was 0.61 @CWrnlin the occipital cortax@0.54 @CWmlin the temporalcortex and 0.19 @&CIfmlIn the cerebellum.The average leveloffree parentradlotrecerIn plasma(2.2nCl/mI)duringthe last

2hrwasassumedtobeequaltothefreeredictrecerinthebrain.The

ratiooftotal brainactMtyto thefreegaveVTvaluesof 276,242and 89 Inoccipital, temporal and cerebellar ROIs, respectively.

dance with the known distribution of BDZ receptors in

human brain (50—52).

A similar situation is observed in plasma. Whereas it is

unclear ha defined physiological process is associated with

each exponential, the overall outcome measure, the clear

ance of the compound from the plasma, is adequately de

scribed by a three-exponential model. Similarly, the total

tissue volume of distribution is a well defined outcome

measure.

Theidentifiabilityandphysiologicalmeaningof therate

constants can be improved by constraining the regression

process to values obtained in a region devoid of receptors

(16,44,53).K1,k2ortheirratiocanbederivedfromthese

regions and used in fittingreceptor-richregions. This strat

egy could not be implemented with [‘@I]iomazemldue to

the absence of a region devoid of receptors. Flumazenil

displacement (n = 3) clearly showed that 60% of the activ ity measured in the pons was displaceable. This displace

SPECTMeasurementof Benzod,azepneReceptors•Ab@Darghamat al. 235

6. No arterial sampling. At steady-state, the arterial and

venous plasma concentrations equilibrate, allowing

the measurement of tracer C@ concentration from

one venous blood sample, which further facilitates

the experimental procedure.

Several disadvantagesofthe constant infusiontechnique

must be noted. Although scanning time is reduced, the

total experimental time is increased from 120 miii to 500

min. There is a potential for pump failure and finally, if

subject peripheralclearance is extremely fast or slow, equi

librium may not be reached by the time of scan acquisition.

Low specific activity [‘@I}iomazenilconstant infusion

experiments were used in primates to derive in vivo

[‘@I]iomazenilK@and Bm@(33,35). Derivation of these

parameters with kinetic analysis is complex since the

model becomes nonlinear at significant levels of receptor

occupancy. In contrast, Scatchard analysis can easily be

performed on equilibriumdata. We are in the process of

extending these low specific activity experiments to hu

mans.

Assuming an in vivo K@of 0.59 nM (value measured in

primates with low specific activity SPECT experiments)

(33), the occipital BP value of 244 reported here would

correspond to a Bm,, of 141 nM. This value is close to the

Bm@ measured in vitro in 12 subjects (Bm@ of 162 ± 52 nM). In vitro and in vivo values were thus consistent. In conclusion, both kinetic and equilibrium methods pro

vide adequate measures of regional total equilibriumdis

tribution volume of [@I]iomazenil. Following establish

ment of equilibrium tracer conditions, the injection of

receptor-saturating doses of flumazenil directly measures

the equilibriumdistributionvolume of the nonspecific com

partment. The reproducibility of each method is currently

being tested.

In comparison to PET, quantitation of SPECT activity is

less well developed and more vulnerable to scatter and

attenuation. In lightofthese limitations,the SPECT results

reported here compare favorably with PET measurements

of benzodiazepine receptors in terms of absolute values

and identifiabilityof parameters. The decreased cost and

wider availabilityof SPECF technology may facilitate the

applicationof these research methods to clinical studies.

ACKNOWLEDGMENTS

TheauthorsthankWalterHunkeler,PhD(Hoffman-LaRoche,

Basel, Switzerland) for providing samples of flumazenil and iomazenil; E.O. Smith, 0. Wisnlewski and Quinn Ramsby for theirexcellent technical assistance in collecting and analyzingthe in vivo data; and S. Giddings for performing the in vitro experi

ments.

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238 The Journal of Nuclear Medicrne•Vol. 35 •No. 2 •February 1994