European Journal of Pharmacology, 213 (1992) 107-115

1117

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52324

Central benzodiazepine receptors in human brain: estimation of regional Bmax and K D values with positron emission tomography P a s c a l e A b a d i e , J e a n C l a u d e B a r o n , J e a n C l a u d e B i s s e r b e , J e a n P h i l i p p e B o u l e n g e r , P a t r i c e Rioux, J e a n M a r c e l T r a v ~ r e , L o u i s a BarrY, M a r i e C h r i s t i n e P e t i t - T a b o u 6 a n d E d o u a r d Z a r i f i a n Inserm U. 320, Cyc~ron, CEA DSV-DPTE, Centre F. Baclesse, Centre Psychiatrique Esquirol, Unit'ersity of Caen, C'aen, France Received 8 August 1091, revised MS received 6 December 1991, accepted 10 December 1991

Studies of central benzodiazcpinc reccptors in thc human brain in vivo are now possible using positron emission tomography (PET) and [llC]flumazenil. With the aim of measuring Bm~ and K d in brain regions, we used a two-injection [llC]flumazenil (at high and low specific radioactivity, respectively) pseudo-equilibrium paradigm to evaluate, in seven unmedicated healthy volunteers, the relative merits of three 'reference' structures (pons, hemispheric white matter and corpus callosum) in which the free radioligand concentration in brain tissue was estimated 15-40 rain after i.v. injection of the radioligand. By mcans of high-resolution PET, the Bm~x and Kj were calculated for each subject in 18 gray matter structures, based on a two-point Scatchard plot. We found that the use of the corpus callosum as reference often resulted in spurious Bm;~x and K s values. The pons was the best reference structure because it provided satisfactory B,,,~,xvalues (closest to in vitro data) and most consistent K d values, and was the region easiest to sample on PET images. The pattern of regional Bmax was consistent with that expected from in vitro studies, with values highest in the cerebral cortex, intermediate in the cerebellum, and lowest in the striatum and the thalamus. The K d values were uniform among regions and were consistent with earlier in vitro and in vivo data. This work documents the feasibility of estimating Bm~x and K d of central bcnzodiazepinc receptors in multiple brain regions for clinical research. Benzodiazepine receptors; Flumazenil; PET (positron emission tomography)

1. Introduction

Benzodiazepines are known to exert their effects on the central nervous system through the so-called central benzodiazepine receptors (M6hler and Okada, 1977; Squires and Braestrup, 1977), which are structurally and functionally associated with the GABAergic system. Studies of the central benzodiazepine receptors in the human brain in vivo are now possible using positron emission t o m o g r a p h y ( P E T ) and [~1C] flumazenil, and detailed reports on blood and brain regional pharmacokinetics of this radioligand have been published previously (for review: Abadie and Baron, 1990). 'Pseudo-equilibrium' quantitative methods have been developed to measure the density (Bm~x) and affinity (K 0) of central benzodiazepine receptors in the human brain with [t~C]flumazenil (Pappata et al., 1988; Persson et ai., 1989; lyo et al., 1991); all these methods require the estimation of free radioligand concentra-

Correspondence to: J.C. Baron, Inserm U. 320, BP 5027, 14021 Caen cedex, France. Tel. 33.31.47.02.00, fax. 33.31.47.02.22.

tion (F) in brain tissue, thereby allowing the measurement of specifically bound ligand (B) in gray matter structures of interest by subtraction of free radioligand concentration from total radioligand concentration (T). However, the application of these methods to clinical investigations is logistically cumbersome because several P E T sessions are required for each subject for the repeated administration of [ltc]flumazenil at different specific radioactivities. To investigate epileptic patients, Savic et al. (1988, 1990) used a simplified approach that required only two injections of the radioligand at widely different specific radioactivities. This method is derived from the original 4 - 5 injection paradigm developed by Persson et al. (1989), who pioneered the use of a reference structure assumed to be devoid of central benzodiazepine receptors (pons or hemispheric white matter) to estimate the p a r a m c t c r F. In the present study, our aim was to investigate systematically the validity and feasibility of Savic's approach in unmedicated healthy volunteers, with two main objectives: (1) the optimal selection of a reference structure, in terms of anatomical definition and reliability of regional Bmax and K d values. To this end,

108

we explored three different spheric white matter and systematic quantitation of gray matter areas with the P E T camera.

structures: the pons, hemicorpus caliosum; (2) the Bmax and K d in multiple help of a high-resolution

2. Material and methods

2.1. Subjects The study protocol was approved by the regional Ethics Committee of Caen. After giving their informed consent, seven healthy volunteers (five women and two men) participated in the project. Their mean weight was 64.5 kg and their mean age was 36.4 years (range: 22-52 years). Exclusion criteria were: (1) present or past systemic, neurologic or mental disorder (according to DSM3 R criteria), (2) history of previous head injury, (3) drug, alcohol, caffeine or tobacco dependence, and (4) current pregnancy. None had ever been treated with antipsychotic or antidepressant drugs. Occasional use of benzodiazepines at low doses for hypnotic purposes was not a cause of exclusion from the protocol, but, if present, was discontinued at least one month before P E T scanning. On the morning of the P E T study, screening for the presence of benzodiazepines in plasma by gas phase chromatography was carried out and reported as negative in each subject. X-Ray computerized tomography (CT) scans of the brain were also normal in all subjects. Standard biological tests, performed immediately before the P E T study, indicated that the renal, hepatic and hemostatic functions were normal in each subject. No drug was permitted throughout the study day. On the evening before the P E T examination, alcohol intake was prohibited; coffee was to be avoided on the morning of the P E T examination.

2.2. Radiochemistry Flumazenil (Ro15 1788) was labeled by reaction of [it C]methyl iodide on the desmethyl precursor (Mazi~re et al., 1984). The radiochemical yield was around 55% with an overall synthesis time of 45 min. [1~C]Ro15 1788 was isolated on a semi-preparative H P L C column (/x-porasil, 7.8 mm x 30 cm, Waters, France) with a mobile phase of c h l o r o f o r m - e t h a n o l - w a t e r - e t h y l amine (99 : 0.96 : 0.02 : 0.02 v / v / v / v ) and detection took place at 254 nm. The collected fraction was evaporated to dryness, dissolved in physiological saline and run through a Millipore filter (0.22 /zm, Waters, France). The average specific radioactivity obtained at the end of synthesis was about 750 mCi/~tmol (about 28 G B q / # m o l ) , but showed some variability. Two succes-

sivc injections were given out at 2 h intervals: the first one (mean + S.D.: 9.23 + 2 mCi) with high specific radioactivity (mean + S.D.: 317 + 282 m C i / i z m o l ) and the second one (mean + S.D.: 9.33 + 0.63 mCi) with low specific radioactivity ( m c a n + S.D.: 1.75 + 0.25 mCi//zmol). The two successive radiosyntheses were carried out in different lead shielded hoods. With the second injection of [11C]flumazenil, unlabelcd flumazenil was coinjected at a fixed dose of 0.025 m g / k g . This dose was bascd on previously published data from partial saturation studies (Pappata ct al., 1988; Savic et al.. 1988). The specific radioactivity in the first (high specific radioactivity) study was more difficult to control, due to reasons inherent to P E T radiochemistry as well as to the occasional delay between the end of synthesis and injection into the subject.

2.3. Camera We used a time-of-flight T r v 0 3 model P E T camera (LETI, Grenoble) which acquires seven slices simultaneously (four direct and three crossed slices). The axial and lateral rcsolutions are 9 mm and 5.5 mm. respectively, with 3 mm undetected void between slices. Before emission data acquisition, a transmission scan was made with an external source of 68Ge-68Ga for attenuation correction.

2. 4. Procedure A 1 mm radius catheter was inserted into the radial artery under local anesthesia (xylocaine 1%, 1 ml) after a negative Allen's test. After the positioning procedure (see below) and the transmission scan, an intravenous slow bolus injection of [llC]flumazenil of high specific radioactivity was given while the subject was lying at rest, with ears unblocked. P E T data acquisition started at the end of injection and lasted 60 min, at which time the subject was allowed to get up and have lunch. About 1 h later and after the repositioning procedure (see below), the low specific radioactivity radioligand was administered as a slow infusion over about 4 min. Using this infusion schedule, we did not observe undesirable systemic or behavioral effects attributable to the active compound. The second period of P E T data acquisition also lasted 60 min. Arterial blood gases, pulse rate and arterial blood pressure were determined after about 5, 30 and 60 min and were stable in all cases.

2.5. Positioning In this study, the brain structures of interest were defined in relation to Talairach's stereotaxic atlas (Talairach et al., 1967) by means of a CT scan obtained according to the same references as PET. The proce-

109

dure of positioning used for both PET and CT scanning was derived from that of Fox et al. (1985). This method also ensures an accurate repositioning during the second P E T study, thus allowing the subject to walk around between the first and second PET scanning sessions, but allowing sampling of the same brain structures in both studies. The procedure used is based on external osseous landmarks, the glabella-inion (GI) line. This line is chosen by refercnce to the A C - P C (anterior commissure-posterior commissure) line, which is the reference axis of Talairach ct al. (1967), because the A C - P C line has consistent relationships with the GI line (Fox et al., 1985). The latter is defined on a lateral skull X-ray obtained once the subject is lying supine on the PET or C.I" couch with the head immobilized in a Laitinen stereotaxic frame. This noninvasive and painless frame not only serves to immobilize the head but also serves as a reference to focus the X-ray source and to calculate the coordinatcs of the couch and the tilting of the detector gantry, taking into account the distortion due to point source of X-rays. Seven corresponding PET and CT slices were obtained parallel, and centred at the following levels, with respect to the GI line: - 4 , +8, +20, +32, +44, +56, and +68 ram, assumed to correspond to levels - 2 5 , - 1 3 , - 1 , +11, +23, +35 and +47 mm relative to the A C - P C line, respectively.

2.6. Blood pharmacokinetics of [ IIC]flumazenil During each of the two PET scanning procedures, a total of 18 arterial blood samples were collected to determine the timc course of total radioactivity concentration in whole blood (uncorrectcd for metabolites), as well as that of unchanged [~lC]flumazenil, using a validated extraction procedure (Barr6 et al., 1991). The unlabeled desmethyl metabolite [Ro15 5528] was also assayed using an HPLC method on a single arterial blood sample obtained during the second PET study. The results of these assays are reported elsewhere (Debruyne et al., 1991).

2. 7. Quantification method We used a 'pseudo-equilibrium' approach derived from that used by Savic et al. (1988). The assumptions underlying this method are developed in the Appendix. Briefly, the radioactivity concentration measured in a reference structure is assumed to represent F (free [~lC]flumazenil brain concentration), non-specific binding being considered negligible (Brown and Martin, 1984; Goeders and Kuhar, 1985; Potier et ai., 1988; Pappata et ai., 1988). Specifically bound radioligand (B) in a given structure can be calculated according to the equation: B = T - F , where T represents the total radiotracer concentration, assuming F is uniform in all

brain structures (Pappata et al., 1988; Persson et al., 1989). The time interval to measure T and F used by Savic et al. (1988) was between 15 and 36 min after tracer injection. That study as well as earlier studies by Pappata et al. (1988) had demonstrated that the B / F ratio is essentially stable during this time interval. In our study, the value F was estimated from a PET image summated over 15-40 min (see below), in order to optimize accuracy in the measurement of low levels of radioactivity in the reference structures; this accuracy would be compromised in images with shorter summation times (unpublished data from our laboratory). The value T in gray matter structures of interest was obtained for the same time interval (15-40 min) but the tracer concentration was averaged from five images, each summatcd over 5 min acquisition time, because counting statistics are more favorable in these high uptake brain areas. The value B was then calculated according to the above equation, and was expressed in molar units (nM) by normalization with the specific radioactivity value of each study. Using this procedure, we could establish B and B / F separately for each of the 18 structures of interest (see below) in both the high and low specific radioactivity studies in each subject and using each of the three reference structures (i.e., pons, white matter and corpus callosum). Bin, x and K d (both expressed in nM units) were obtained for each brain structure in each subject according to the Scatchard representation, using a two-point determination.

2.8. PET reconstruction From the data acquired from time 0 (injection) to 60 min (end of study), and using the list-mode procedure, several image frames were reconstructed in a standardized scheme: 0-8 min: 'early frame' (whcre [lIC] flumazenil distribution and uptake is assumed to be influcnced by perfusion). 8-60 min: 'late frame' (assumed to bc essentially independent of perfusion); 15-40 min frame: 'static frame', (see above); 'kinetic' 5 min frames from 0 to 60 min (N = 12). The images obtained were expressed in quantitative terms of nCi/pixci, corrected for attenuation, scatter, and physical decay of [ I I C] during acquisition.

2. 9. Definition of structures of interest Eleven discrete brain areas were selected for analysis of binding parameters: cerebellum, medial frontal cortex, cingulate gyrus, medial occipital cortex, lateral frontal cortex (right and left), lateral temporal cortex (right and left), mesial temporal cortex (right and left), occipital cortex (right and left), parietal cortex (right and left), striatum (head of caudate and lenticular nucleus right and left) and thalamus (right and left).

110

Based on corresponding CT scan cuts obtained in the same subject, the various cortical areas and the cerebellum were sampled by means of circular regions of interest. These were defined on the 'late' image frame (8-60 rain) with respect to an computer generated iso-contour set at 50% of maximal pixel value in the image, such that the isocontour delineates the cortical surface. The regions of interest were set tangentially to this iso-contour in such a way as to sample the gray matter optimally (i.e. to limit the partial volume effect over the brain surface). The subcortical areas were defined on the 'early' static image (0-8 min) because these structures with low central benzodiazepine receptors density are better delineated during initial tracer distribution; the regions of interest were centered over the subcortical structures with the aid of the CT Scan. For all areas a region of interest radius of 9 mm was selected as a compromise between the lateral resolution of the P E T camera (5.5 ram) and the minimum sample volume which allows an acceptable signalto-noise ratio, and hence an adequate accuracy in the determination of the radioactivity time course, taking into account the count rate characteristics of typical [lIC]flumazenii studies (unpublished data from our laboratory). For further analysis the data obtained from the regions of interest were averaged for each structure of interest (total number of regions of interest per structure: 3, 6, 3, 6, 11, 8, 2, 7, 5, 2 and 1 for the cerebellum, medial frontal, cingulate gyrus, medial occipital, lateral frontal, lateral temporal, mesial temporal, occipital, parietal, striatum and thalamus, respectively, defined on one to four planes). In some subjects, the images containing specific brain structures were not available, or the structure was not adequately sampled, resulting in missing regional values (cingulate gyrus, parietal cortex, thalamus and striatum in two subjects).

2.10. Definition of reference structures Three different reference structures were investigated to determine the p a r a m e t e r F: (1) basis pontis; (2) hemispheric white matter (defined in the corona radiata); and (3) splenium of corpus callosum, referred to in the text as 'ports', 'white matter' and 'corpus callosum', respectively. The regions of interest were defined on the 'latc' framc (8-60 min) using a single circular region of interest for pons and white matter, and an irregular region for corpus callosum. Region of interest placement was determined manually with the help of CT scan cuts and corresponding Talairach atlas levels. The region of interest for the pons was positioned on slice G I - 4 mm, using a fixed radius of 13 mm. The region of interest for white matter (radius 10 mm) was placed in the center of the corona radiata (slice: GI +

44 or 56 mm) where radioactivity was lowest, avoiding the lateral ventricles (as identified on the corresponding CT scan cut) as far as possible. The region of interest for the corpus callosum was even more difficult to define; it consisted of a crescent-shaped region of interest based on CT scan morphology, drawn to match the section of the splcnium of the corpus callosum on planc GI + 20 or 32 mm. Its shape and area (3.14 + 0.2 cm 2) were standardized but slightly adjusted to the individual subject's anatomy. Because of technical problems, the P E T cuts containing the pons and white matter were inadequate or missing in one and two subjects, respectively.

2.11. Statistical analysis A set of repeated measures A N O V A s with two factors (reference structures and brain areas) was carried out for the parameters B,,~x and K d, using the B M D P - 2 V program. We used the following tests: (1) sphericity tests for repeated measures applied to orthogonal components; and (2) F tests for interaction and between-groups comparisons. In case sphericity and interactions tests were not significant but the between-group tests for Bmax and K d showed stastistical significance, post-hoe A N O V A s comparing pairs of reference structures were performed. In addition, linear regrcssion analysis and correlation measures with testing of null hypothesis were carried out for each of the variables (B .... and K d) and the injected amount of flumazenil (or its log transform) in the high spccifie radioactivity study.

3. Results

The table shows the mean ( + 1 S.D.) Bmax and K a values calculated in the various brain areas for each subject individually and according to each reference structure (pons, white matter or corpus callosum). This table indicates the occasional occurrence of negative Bm~x or K a values, which were discarded from region averaging. These spurious results concerned mainly subcortical regions (striatum and thalamus), and only on one occasion included the cerebellum and the mesial temporal cortex: of 19 such negative values, 18 occurred when the corpus callosum was used as reference structure, and one with white matter as reference.

3.1. B,,ax z'alues 3.1.1. Global statistical analysis The A N O V A revealed a significant reference structure effect (P = 0.02), indicating the presence of significant differences between mean Bm~x values obtained using the three reference structures. There was no

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3.3. Variability of results

statistically significant i n t e r a c t i o n , i n d i c a t i n g that all three r e f e r e n c e s t r u c t u r e s resulted in the same p a t t e r n of regional Bmax values. T h e highest values were obt a i n e d for the medial occipital, medial frontal a n d cingulatc cortices, a n d lowest values for the s t r i a t u m a n d t h a l a m u s ; i n t e r m e d i a t e values were f o u n d in the o t h e r cortical areas a n d in the c e r e b e l l u m .

T a b l e 1 shows that the S.D.s for Bin, X of cortical areas were smallest for data o b t a i n e d with white matter as reference. With respect to K d values, the smallest S.D.s were t b u n d with the pons as reference. As for the s t r i a t u m a n d thalamus, the variance of data was large for N)th Bm~~ a n d Ka values, especially when the corpus callosum was used as reference structure (which also r e p e a t e d l y yielded negative values, scc above).

3.1.2. Post-hoe analysis T h e r e was a significant difference (P < 0 . 0 1 ) between the Bmax values o b t a i n e d with white m a t t e r as r e f e r e n c e structure a n d those o b t a i n e d with the pons. No significant difference was f o u n d either b e t w e e n the pons and corpus callosum, or b e t w e e n white m a t t e r a n d corpus callosum. T h e w i t h i n - g r o u p A N O V A d e m o n s t r a t e d a significant region effect (P = 0.001), which is c o n s i s t e n t with the k n o w n h e t e r o g e n e i t y of c e n t r a l b e n z o d i a z e p i n e receptors density in the h u m a n brain.

4. Discussion As a whole, o u r study d e m o n s t r a t e s the feasibility of e s t i m a t i n g the density a n d affinity of central b e n z o d i a z e p i n c receptors in various cortical a n d non-cortical structures of individual h u m a n subjects in vivo, using a relatively simple p r o c e d u r e . T h r e e specific points reg a r d i n g the results of this study will bc discussed: (1) the issue of the reference structure; (2) the results of the regional data analysis: a n d (3) the validity of the q u a n t i t a t i v e a p p r o a c h used.

3.2. K a ralues T h e A N O V A indicatcd n e i t h e r a significant reference s t r u c t u r e effect nor a significant i n t e r a c t i o n . T h e s e results indicate no statistical difference in m e a n regional K d values a n d no difference in p a t t e r n of regional K,, values o b t a i n e d using the t h r e e r e f e r e n c e structures. T h e w i t h i n - g r o u p A N O V A d e m o n s t r a t e d no significant regional effect, indicating a lack of significant h e t e r o g e n e i t y of K d a m o n g b r a i n regions.

4. I. Reference structure A l t h o u g h all three r e f e r e n c e s t r u c t u r e s resulted in the same p a t t e r n of Bmax values with u n i f o r m i t y of K d values a m o n g the various b r a i n regions analyzed, there

TABLE 1 Quantitative positron emission tomography in healthy w)lunteers; regional brain B,,,~ and Kd determination using three different references structures. Reference structures White matter

Pons

Corpus callosum

N

Bm~x (nM)*

K o(nM) *

N

Bm¢,x(nM) *

Kd (nM)*

N

B.... (nM)*

Ka (nM)*

Medialfrontalcortex Cingulategyrus Medialoccipitalcortex Lateral frontal cortex (right) Lateral frontal cortex (left) Lateral temporal cortex (right) Lateral teml~ral cortex (left) Mesial temporal cortex (right) Mesial temporal cortex (left) Occipital cortex (right) Occipital cortex (left) Parietal cortex (right) Parietal cortex (left)

5 5 5 5 5 5 5 5 5 5 5 5 5

63+ g 78.+17 87+ 7 55-+ 8 56_+ 6 57.+10 57_+12 40_+ 7 31.+11 64+_10 62-+ 4 58-+ 5 56_+ 6

114-_ 14.+ 14+ I1-+ 11_411-+ 11-+ 10-+ 7.+ 13-+ 12-+ 11± I1±

6 4 6 6 6 6 6 6 6 6 6 4 4

78+_13 92.+_18 103+15 66-+14 72..+ 9 67_+17 76_+11 55-+12 49_+14 80_+17 83+13 75-+ 9 75-+ 4

12+ 2 16.+ 4 14-+ 2 lOt 4 13+ 2 11_+ 3 13-+ 3 12-+ 2 10_+ 2 14:1:2 14-+ 3 12_+ 2 14+ 3

7 7 7 7 7 7 7 7 6 7 7 5 5

68.+28 02t39 96+31 55-+26 63-+30 53_+27 66-+38 41-+26 45_+23 71_+32 60-+28 61+_25 62-+311

22.+ 33.+ 27+ 21).+ 24-+ 19_+ 24+ 16_+ 20+ 26+_ 22+_ 22_+ 25±

Striatum(right) Striatum(left) Thalamus(right) Thalamus(left) Cerebellum

5 5 4 5 5

16± 9 21+_16 23.+12 12+_ 8 40+_11)

8-+ 6 9.+12 15+_13 a 7± 6 13+_ 6

5 5 5 5 6

35_+10 44+_18 35-+18 25+_15 55+_11

10+ 3 12.+ 4 19+_14 9_+ 5 14t 2

4 5 3 3 6

12+10 ~ 13+_ 8 ~' 65+69 ~ 10± 5 c 45+_39"

16+_ 24 h 8.+ 4" 208+320 c 14.+ 12 ~ 28..+ 27 ~'

4 6 7 4 4 6 3 5 2 7 6 5 4

* Mean + S.D. ~,.b.~One, two or three negative values obtained, respectively, which were discarded from averaging.

10 18 8 10 12 9 15 I1 I0" 11) 11 II 14

112 was a statistically significant difference between Bmax values obtained with white matter and the pons as reference structures. In addition, the use of the corpus callosum resulted in spuriously high variance for both Bmax and K,~ values, while the latter were much larger than expected from previous in vitro and in vivo data (lor review: Abadie and Baron, 1990). In addition, negative values for K d or B .... were frequently observed for the striatum and thalamus, and occasionally for the cerebellum and mesial temporal cortex as well. These problems with the corpus callosum presumably result from both partial volume effects in P E T imaging (with spilling over of radioactivity from ncighbouring gray matter) and difficulties encountered in the region of interest positioning procedure (see Methods). They indicate that this structure does not reliably reflect the p a r a m e t e r F and is thus unsuitable for central benzodiazepine receptors quantitation in vivo. Although both white matter and the pons resulted in satisfactory results, the former would theoretically appear preferable because of its total lack of central benzodiazcpine receptors in vitro (for review: Abadie and Baron, 1990; Miillcr, 1987). Nevertheless, several lines of arguments speak in favor of the pons as a reference structure: (1) the higher B,,,,~ values for both cortical and non-cortical areas obtained with the pons were closer to in vitro human data (Sicghart et al., 1985; Trifiletti et al., 1987; Ferrarcse et al., 1989); (2) the variance of K d values was much smaller with the pons than with white matter as reference; and (3) the region of interest positioning procedure was much easier for the pons than for white matter, because avoiding the lateral ventricle in region of interest positioning is a quite difficult and subjective operation. Although, to the best of our knowledge, data for the density of central benzodiazepine receptors in the human pons as measured in vitro with [3H]RoI5 1788 are not available, [~C]flumazenil P E T data suggest this structure constitutes an acceptable index of the p a r a m e t e r F (Persson et al., 1989).

4.2. Regional analysis Regardless of the reference structure used, the pattern of regional Bin, ~ values obtained was consistent with earlier measurements made on human brain samples in vitro with [3H]Ro15 1788 (Sieghart et al., 1985; Trifiletti et al., 1987; Ferrarese et al., 1989). Thus, the highest values were obtained in neocortical areas (especially the medial occipital and medial frontal areas), intermediate values were found in cerebellar and mesial temporal cortices, and lowest values in the thalamus and striatum. However, as already noted, the use of the pons as reference structure resulted in Bm~x values closcst to in vitro data. Previous P E T studies

with [lIC]flumazenii in groups of control subjects have reported only neocortical BmaX values (Persson et al., 1989; Savic et al., 1988, 1989). The range of Bm~Xvalues for various neocortical areas observed in this study is quite consistent with that reported for whole neocortex by Persson et al. (1989), who used a cumbersome, 4 - 5 [llC]flumazenil injection paradigm. Our mean Bm~~ values are also consistent with those reported for several neocortical areas by Savic et al. (1988, 1989), but the coefficients of variation tabulated in their publications are considerably wider than those found here despite the use of the same quantification procedure and reference structure (white matter). We are the first to attempt to estimate the density of central benzodiazepine receptors in the striatum and thalamus in humans in vivo. With the pons as reference structure, the mean Bmax values obtained were consistent with in vitro human data (Miiller, 1987). However, the coefficients of variation were much higher for subcortical nuclei than for neocortex, presumably because the twofold lower receptor density in the former notably reduces the signal-to-noise ratio in P E T imaging. With respect to K d values, one salient finding of this study is that the values obtained are higher than those measured in vitro with [3H]RoI5 1788 (for review: Abadie and Baron, 1990). However, in the in vivo approach they are measured at physiological conditions of temperature and cellular environment. It has been well documented that in vitro K d values for [H]RoI5 1788 are temperature-dependent, and a recent study on human brain tissue reported K d values of about 10 nM when measured at 37°C (Kopp ct al., 1990): our findings are entirely consistent with this recent report. They are also consistent with previous [llC]flumazenil P E T studies that used a 4-5-injection paradigm (Persson et al., 1989). However, using the two-point Scatchard method with white matter as reference, Savic et al. (1988, 1989) reported very high K d values, in the range of 24-27 nM, about twice as high as our corresponding data. We cannot provide an adequate explanation for these discrepant findings, except perhaps the higher spatial resolution of the P E T camera used here. We also investigated each methodological step in a systematic fashion to estimate the p a r a m e t e r F. Finally, the K d values obtained with either the ports or white matter as reference structure were essentially uniform among the various gray matter structures, in agreement with in vitro data (for review: Abadie and Baron, 1990). However, very large coefficients of variation for K d values were occasionally obtained for subcortical nuclei, as well as one negative K d values for the thalamus (using white matter as reference), indicating the limited reliability of our method to investigate areas with low density of central benzodiazepinc receptors.

113

4.3. Validity of the quantitation paradigm Although our methodology provided satisfactory values for B ~ and K j in brain regions when the pons was used as reference structure, several issues regarding the quantitation paradigm used need to be addressed. First, we chose the 'equilibrium', rather than thc 'dynamic', approach to measure Bmax and K,~ bccause the latter, which is based on complex compartmental modelling, requires two administrations of [ltC]flumazenil, and is also based on several underlying assumptions. Only preliminary findings in a single human subject using a non-validated model have been published in full (Biomqvist et ai., 1990). In this subject, the results obtained with the dynamic approach were very close to those found with the equilibrium measuremcnt (two-point Scatchard procedure). Only future work will tell whether this dynamic approach is applicable to large series of subjects in clinical research paradigms. Second, a state of strict pharmacological equilibrium is unlikely to occur in vivo because the free ligand concentration in brain depends on that present in plasma, where ligand is normally subject to biological removal (see Appendix). Based on previous extensive investigations on the time course of the ratio B / F in various brain regions in humans after intravenous injection of [l~C]flumazcnil (Pappata et al., 1988; Persson et al., 1989), Savic et al. (1988) selected a period of 'pseudo-equilibrum' where this ratio is essentially stable, i.e. from 15 to 40 min post-injection, to carry out the quantitation procedurc (scc Appendix for details). In a diffcrent PET paradigm with [~tC]raclopride to measure D z receptors in the striatum using a similar two-point Scatchard approach, Farde et al. (1990) introduced the notion of 'transient equilibrium', which is reached in vivo when the time derivative of B is zero, i.e. at the peak of specific binding kinetics. However, our experience of PET kinetic studies with [~C] flumazenil in standard doses (around 10 mCi) indicates that the ~C count rates in reference structures become so low in rapid PET image frames as to result in considerable errors in estimates of Bm~~ and K j with the Farde paradigm. Our choice to measure Bm~x over an extended time interval ensured that F was estimated with statistical confidence and also allowed comparison with the carlier findings of Savic et al. (1988). Finally, the specific radioactivity of the first [~lC]flumazenil study (high specific radioactivity) was inevitably variable from subject to subject, for both radiochemical and PET procedure timing reasons (see Methods). To investigate whether this variability could have affected the determination of Bm~x and K,~ using the two-point Scatchard procedure, we looked for correlations between measured Bmax and K d values on one hand and the specific radioactivity (or its log

transform) on the other hand, using Pearson's linear regressions. There was no statistically significant correlation, whatever the brain area considered, indicating the independence of our findings on exact specific radioactivity. Regarding thc second [~C]flumazenil study, in which the specific radioactivity was controlled (see Methods), the latter was selected so as to obtain a binding inhibition of about 80%, based on the data published by Pappata et al. (1988). The two-point Scatchard method requires that the two measurements are made at the two ends of receptor occupancy (Farde et al., 1990). Our findings further establish the reliability of this simple methodology. The present study indicates the feasibility of estimating Bm~~ and K d of central benzodiazcpine receptors in multiple cortical and cerebellar regions, using a procedure readily applicable to clinical rcscarch.

Acknowledgements We wish to thank C. Lc Po~c and the cyclotron staff, F. Gourand and D. Brault for preparation of [~tC]flumazenil, V. Beaudouin for PET data analysis, D. Debruyne for benzodiazepine screening in plasma, P. Allain, D. Luet and S. Steinville for establishing the patient positioning procedure, and J. Druart, M. Huguet, B. David and M.T. Baptiste for secretarial assistance.

5. Appendix 5.I. Underlying assumptions for quantification of cent. al benzodiazepine receptors" in brain regions Several assumptions support the quantitation method used in this study

5.1.1. Radioligand [nIC]Flumazenil is regarded as the radioligand of choice for the in vivo quantitation of central benzodiazepine receptors (Mazib~re et al., 1983; Hantraye et al., 1984; Samson et al., 1985; Abadie and Baron, 1990; Brouillet et al., 1990). Flumazenil has been shown to bind solely to central benzodiazepine receptors in vitro (Mohler and Richards, 1981). Both in vitro and in vivo, including in the h u m a n brain, Scatchard plots and Hill coefficients close to one have demonstrated binding to a single class of receptors (MiJller, 1987; Pappata et al., 1988; Persson et al., 19891, allowing the application of the two-point Scatchard procedure (a method initially developed for PET studies of D 2 receptors, Farde et al., 19901 to [~lC]flumazenil (Savic et al., 1988, 199(I).

5.1.2. Brain radioacticity consists only of the parent radioligand and not of labeled metabolites Thirty min after in vivo administration of L"H]Rol5 1788 in rats, only pure radioligand was recovered in brain tissue (Inoue et al., 1985). In humans, virtually the only circulating 11C-labeled metabolite of flumazenil is its acid derivative, Ro15 3890 (ttalldin ct al., 1988, Swahn et al., 1989; Barr6 et al., 19911. Studies with intravenous administration of [InC}Rol5 389(I in m a n have demonstrated that this metabolite has virtually no access to brain tissue (Swahn et al., 19891. Although it cannot be totally excluded that minute amounts of

114 [liC]Rol5 3890 are formed within brain tissue, it can be reasonably assumed that brain radioactivity measured by PET after [i iC]flumazeniI injection is entirely or quasi-exclusively in the parent form (Swahn et al., 1989).

5.1.3. Non-specific binding (NS) in brain is negligible In agreement with in vitro binding and autoradiographic studies, in vivo studies have shown that NS was well below 10% of total binding in brain membranes of rat after intravenous injection of [3tl]flumazcnil, and could be neglected for quantitative and autoradiographic studies (Brown and Martin, 1984; Goeders and Kuhar, 1985). In vivo PET studies with [iiC]flumazeni I have demonstrated in both baboons and humans that the displaceable fraction in cortical areas was about 90% of total [itC]flumazeni I 1(I-2(I min after injection (Pappata et al., 1988; Shinotoh et al., 1986; Brouillet ct al., 1990). The remaining 10% must thus represent thc sum of F and NS, indicating that the latter can be assumed to be negligible for practical proposes.

5.1.4. Pharmacok~gical equilibrium In PET studies of specific binding in vivo, true pharmacological equilibrium is unlikely to bc reached because of the elimination and disposition of thc externally administered radioligand. True equilibrium would exist only if the concentration of specifically bound radioligand were stable, indicating no net flux in the association/ dissociation reaction (Young et al.. 1986). In human PET studies with[ I IC]flumazcnil, Pappata et al. (1988)showed that the B / F ratio rose slowly for about 15-20 min and was essentially stable until 60 min; in most regions, however, this ratio slightly declined after 411 rain. Persson et al. (19891, using pons or corona radiata to determine F, found that the B / F ratio for the neocortex rose until about 25 min and was essentially stable thereafter, but with a larger variability aftcr 40 rain. Savic et al. (1988. 1990) documented thc stability of the B / F ratio for the parietal cortex as being between 18 and 54 rain. Thus, a "pseudo-equilibrium" interval bctween 15 and 40 min has been assumed to be an acceptable time period for determination of F and B (Savic et al.. 1988).

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