infusion paradigm for the novel NMDA receptor SPET tracer [123i]CNS 1261

Nuclear Medicine and Biology 31 (2004) 155–164

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A bolus/infusion paradigm for the novel NMDA receptor SPET tracer [123I]CNS 1261 Rodrigo A. Bressana, Kjell Erlandssonb,*, Rachel S. Mulliganb, Roger N. Gunnc, Vincent J. Cunninghamd, Jonathan Owense, Ian D. Cullumb, Peter J. Ellb, Lyn S. Pilowskya,b a

Institute of Psychiatry, De Crespigny Park, Denmark Hill, London, SE5 8AF, UK b Institute of Nuclear Medicine, UCL Medical School, London, UK c McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada d IRSL, Hammersmith Hospital, Du Cane Rd., London, W12 ONN, UK e West of Scotland Radionuclide Dispensary, Western Infirmary, Glasgow, UK Received 18 March 2003; received in revised form 29 July 2003; accepted 2 August 2003

Abstract We have previously performed quantitative kinetic modeling of [123I]CNS 1261, a new SPET ligand for the MK801 intrachannel site of the NMDA receptor. We now report a bolus-infusion protocol, which eliminates the need for arterial blood sampling. Dynamic SPET scanning and venous blood sampling were performed in 7 healthy volunteers. Good agreement was obtained between kinetic and equilibrium analysis. SPET scanning with a bolus-infusion protocol is a valid method to estimate the total volume of distribution for [123I]CNS 1261 in clinical populations. © 2004 Elsevier Inc. All rights reserved. Keywords: NMDA receptors; Dynamic SPET; Kinetic modeling; Constant infusion; Equilibrium analysis; Volume of distribution

1. Introduction The glutamatergic system is the major excitatory neurotransmitter system in the human brain. It has a key role in fast synaptic transmission, neuronal plasticity (e.g. synaptogenesis, excitotoxicity, apoptosis) and higher cognitive function (e.g. learning and memory) [6 –9]. In vivo investigation of NMDA receptor dysfunction in neuropsychiatric disorders has been limited by a lack of useful radioligands [3]. [123I]CNS 1261 (N-(1-napthyl)-N⬘-(3-iodophenyl)-Nmethylguanidine) is a novel SPET probe, binding with high affinity (Kd 4.2 nM) and high specificity to the MK801 site within the NMDA receptor channel [21]. We have previously reported results from bolus studies with [123I]CNS 1261 in healthy volunteers, using quantitative kinetic modeling to estimate the total volume of distribution (VT) [10]. The rank order of receptor binding for different anatomical regions was compatible with that reported by other PCP/ MK-801 NMDA intrachannel site PET ligands, [11C]ket* Corresponding author. Tel.: ⫹44-20-7380 9396; fax: ⫹44-20-7637 0578 E-mail address: [email protected] (K. Erlandsson). 0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0969-8051(03)00154-9

amine [12] and [18F]memantine [1]. It is not yet certain whether these ligands accurately estimate NMDA binding in vivo (for fuller discussion see [10]). Quantitative estimation of neuroreceptor binding parameters by kinetic analysis of bolus studies can be achieved with an input function derived from arterial blood sampling [15,18]. However, arterial cannulation may not be readily tolerated by clinical populations and should be avoided if possible. An alternative experiment design uses a constant infusion of the radiotracer to achieve a prolonged state of equilibrium at the receptor population of interest [4,16]. Administration of an initial bolus of the tracer can be used to reduce the time required to reach equilibrium. SPET studies with constant infusion of the radiotracer can provide an accurate estimation of binding parameters, and can also provide a baseline for pharmacological challenge experiments [17]. We now report on the results of bolus/infusion (B/I) studies with [123I]CNS 1261 in 7 healthy volunteers. VT was estimated based on the ratio of the concentration of radiotracer in tissue and in plasma at equilibrium. Venous samples were used instead of arterial, since our previous bolus studies with [123I]CNS 1261 showed that the two were in

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good agreement from 90 min after injection. This study was constructed to allow for assessment of: i) bolus-infusion versus bolus studies in two groups of healthy volunteers; ii) test-retest values for two individuals; and iii) kinetic versus equilibrium analysis for one subject.

2. Materials and methods 2.1. Subjects The protocol was approved by the South London and Maudsley Trust Ethics Committee. Permission to administer the radiotracer was obtained from the UK Administration of Radioactive Substances Advisory Committee (ARSAC). Written informed consent was obtained from all volunteers after study procedures and possible side effects were explained. Exclusion criteria were: 1) past or present neurological, psychiatric or serious physical illness; 2) use of medications acting at the central nervous system; 3) alcohol or drug dependence or abuse; 4) pregnancy. Seven healthy subjects were included in the study (see Results). Two subjects (subjects 4 and 6) had previously taken part in bolus [123I]CNS 1261 studies (interval between scans: 8 and 10 months, respectively). Healthy volunteers scanned with the B/I protocol were matched to those scanned with the bolus protocol [10] with respect to age, gender, ethnicity, handedness, height, weight, or years of education. 2.2. Tracer 2.2.1. CNS 1261 preparation [123I]CNS 1261 was prepared in a isolated yield of 54% by radioiododestannylation of N-(1-naphthyl)-N⬘-(3-tributylstannylphenyl)-N⬘-methylguanidine hydrochloride, using a method reported previously [10,19]. The product was found to co-elute with reference CNS 1261 and had a radiochemical purity of ⬎99% as shown by analytical HPLC [21]. 2.2.2. Bolus/infusion ratio The principle of a B/I study is to reach a state of equilibrium, where both the tissue and the plasma tracer concentrations are constant over a certain period of time. During this period, the ratio of the tracer concentration in tissue to that in plasma is equal to the total volume of distribution, VT, which is a measure of tracer binding. The role of the constant infusion of the tracer is to reach and maintain a state of equilibrium. The role of the bolus injection of the tracer is to reduce the time required to reach the equilibrium state. The ratio between the activity of the bolus injection [MBq] and the rate of the constant infusion [MBq/min] is defined by the parameter Kbol [min], which can be optimized in order to minimize the time to equilibrium. Our previous studies showed that [123I]CNS 1261 has a

very slow final clearance from plasma following a bolus injection: The tracer concentration in plasma does not go down to zero within a 6 h period p.i., but instead seems to tend towards a low constant level [10]. In this case, it might be expected that it would be possible to reach an equilibrium state after a single bolus injection. However, this did not happen within the 6 h period of our bolus experiments, so a long waiting period between injection and scanning would be required in order to use this approach for quantification of tracer binding, and it would result in poor image quality due to low count rate. Also, a true equilibrium might not be obtained for even longer times, due to inter-individual variations in the plasma clearance rate. Due to the slow plasma clearance, continuous infusion of [123I]CNS 1261 does in general not produce a true equilibrium. Instead, a pseudo-equilibrium is obtained, where both the plasma and the tissue tracer concentration increase slowly with time. We therefore used the following method for determination of the optimal Kbol value, which is a slight modification of the method described by Carson et al. [4], the difference being that we optimized for a constant tissueto-plasma ratio rather than constant tracer concentration. Tissue time-activity curves (TACs) were generated using a 1-tissue compartment (1-TC) model, with rate constants and a mean plasma input function determined from our previous bolus studies. TACs were generated over a period of 6 h, and the Kbol value was optimized for a constant tissue-toplasma ratio after 90 min p.i. The optimization was done using rate constants for a series of brain regions, and the mean Kbol value was obtained as: Kbol ⫽ 120 min. In a state of pseudo-equilibrium, the ratio of tissue to plasma concentrations does not give the true VT value. A larger gradient in the plasma curve will induce a greater bias in the estimated VT. The drawback of optimizing Kbol for a constant tissue-to-plasma ratio is that this does not guard against a sharp monotonically increasing plasma curve. However, using the present optimization method, reasonably constant metabolite corrected plasma curves were obtained. Below we describe a method for correcting the ratio for the slope of the plasma curve so that an unbiased estimate of VT is obtained. We wish to emphasize the fact that the reason for obtaining a pseudo-equilibrium rather than a true one, is related to slow plasma clearance of the tracer and not due to an erroneous Kbol value. 2.3. SPET data 2.3.1. Data acquisition A bolus of 51-62 MBq [123I]CNS 1261 was injected intravenously. Infusion of [123I]CNS 1261 was via a saline drip into an antecubital vein using a volumetric infusion pump (IVAC 572 –Allaris Medical) for 6 hours at a rate of 80 mL/h. The total amount of [123I]CNS 1261 administered was ⬃185 MBq. Dynamic SPET studies were performed using a Prism 3000XP triple-headed scanner equipped with a 153Gd trans-

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2.4. Blood data

Fig. 1. Measured plasma time-activity curves for one healthy volunteer during a bolus/infusion study with [123I]CNS 1261. The solid symbols indicate arterial, and the open ones venous values. Squares and solid lines indicate total activity concentration in plasma, circles and dashed lines indicate metabolite corrected values.

mission source (Philips Medical Systems, Cleveland, OH, USA). The three detectors were fitted with ultra-high resolution low energy fan beam collimators (focal length: 50 cm). Emission, transmission and scatter data were collected as described in [10]. Four fiducial markers, each filled with approximately 0.1 MBq of 123I, were attached to the subject’s head at the level of the orbito-meatal line to allow repositioning of data from different imaging sessions. Data acquisition began immediately after bolus injection and continued up to 6 h p.i. in a series of imaging sessions, the first one for 60 min and the rest for 30 min, interlaced with 30 min breaks. Transmission studies were performed either before injection of the tracer or at the end of the scan, using the simultaneous transmission emission protocol (STEP, Philips Medical Systems, Cleveland, OH, USA). 2.3.2. Data processing Transmission images were reconstructed using an ordered subsets implementation of the convex EM algorithm [14]. Emission images were reconstructed by filtered backprojection with ramp-filter, after scatter correction with the triple energy window method [20]. A 3D Butterworth filter was applied to both emission and transmission images. Attenuation correction was performed with two iterations of the Chang method [5], based on the transmission images. Image realignment was performed based on the centroid positions of the fiducial markers. All image reconstruction, processing and analysis was done using in-house software, mainly written in IDL 5.2 (Interactive Data Language, Research Systems Inc., Boulder, CO, USA). For more details see [10].

2.4.1. Blood sampling The concentration of radioactivity in whole blood and plasma was determined in discrete blood samples taken during the scans, with different sampling schemes for different subjects. i) For all subjects, venous samples (5 mL) were taken at 30 and 60 min p.i., and 1 or 2 samples in each of the following 5 scanning sessions. ii) In addition, for subjects 2 and 4, arterial samples (5 mL) were collected using the same protocol as for venous samples. This was done in order to determine the arterial-to-venous concentration ratio. iii) In addition, for subject 4, fast arterial sampling was performed to allow comparison between equilibrium analysis and kinetic modeling. This fast sampling was done at a rate of 1 sample (1 mL) per 8 s for 5 min from the time of injection, with additional samples taken at 10 and 15 min p.i. 2.4.2. Blood analysis Parent radioligand and radioactive metabolites in plasma were separated and measured by HPLC using the system reported by Mulligan et al. [19]. The HPLC eluent was monitored sequentially for absorbance at 230 nm (Waters 2487) and radioactivity (in-house sodium iodide well detector linked to an analogue voltage output converter). The fraction of unchanged [123I]CNS 1261 was determined by integrating the areas under the HPLC radioactivity curve for each sample. Reference CNS 1261 eluted on this system with a retention time of 14 min. The measured values were fitted to a bi-exponential function, described as follows: ƒ p共t兲 ⫽ a 1e ⫺a2t ⫹ a 3e ⫺a4t

(1)

where fp(t) is the fraction of unchanged parent compound at time t p.i. and a1–a4 are parameters to be determined by non-linear optimization. 2.4.3. Venous-to-arterial correction In bolus studies with [123I]CNS 1261 on healthy volunteers, the arterial and venous time-activity curves (TACs) were in good agreement from 2 h after injection. With the B/I protocol, the arterial-to-venous (A/V) ratio is always ⬎ 1 due to continuous administration of new tracer. When only venous samples are taken, it is necessary to correct for this ratio in the calculation of VT. We calculated the A/V ratio for subjects 2 and 4, and fitted the data to the analytical function: ƒ a␯共t兲 ⫽ b 1e ⫺b2t ⫹ b 3

(2)

where fav(t) is the A/V ratio at time t p.i. and b1– b3 are the parameters to be determined. A separate estimate of the A/V ratio was made using data from our previous bolus studies on two healthy volunteers. For these subjects, the complete arterial and venous TACs had been sampled, including fast initial sampling to mea-

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sure the peak. Using these measured bolus TACs, we simulated TACs corresponding to the present B/I protocol, and calculated the ratio between the arterial and the venous simulated B/I TACs. The average of the four A/V ratio curves was finally determined for use in the VT calculations. The parameters for this curve were: b1⫽0.5258, b2⫽1.343 · 10-2 min-1, b3⫽1.032. 2.4.4. Equilibrium analysis At equilibrium the Michaelis-Menten receptor-ligand equation is satisfied: B⫽(Kd⫹F), where F and B are the concentrations of free and bound ligand, respectively, Bmax is the total concentration of receptor sites, and Kd is the equilibrium dissociation constant [13]. At tracer doses (F⬍⬍Kd), the equation simplifies to: B/F ⫽ Bmax/Kd. The ratio Bmax/Kd is known as the binding potential (BP), and is often used for quantification of receptor binding of neuroreceptor tracers [18]. The total volume of distribution (VT) is defined at equilibrium as the ratio of the concentration of tracer in tissue (free, specifically bound, and non-specifically bound compartments) to the concentration of unchanged tracer in plasma, and is proportional to BP [17]. 2.4.5. Slope correction The principle of a B/I study is to reach an equilibrium state, where both the tissue and the plasma TACs are constant, in which the ratio of the tissue and metabolite corrected arterial plasma activity concentration is equal to the total volume of distribution, VT [4,16]. In the present study this true equilibrium was not obtained due to the slow final plasma clearance of the tracer. Rather a pseudo-equilibrium state was observed in which there was a slow increase in both the tissue and plasma TACs. In this case a simple ratio of the activity concentrations will not give the true VT value. We have derived a correction factor for this effect, as outlined below. A [123I]CNS 1261 B/I plasma TAC can be approximated as follows: C p共t兲 ⫽ c 1 䡠 ␦ 共t兲 ⫹ c 2 ⫹ c 3 䡠 t

(3)

where Cp(t) is the activity concentration of unchanged radiotracer in arterial plasma at time t, ␦(t) is the Dirac deltafunction, and c1, c2 and c3 are constants representing the size of the initial peak, the constant background level, and the slope of the curve in pseudo-equilibrium respectively. We have previously found that the 1-TC model is adequate for describing the kinetics of [123I]CNS 1261 [10]. Combining this model with equation [3], we obtain the following expression for the tissue TAC: C t共t兲 ⫽ K 1 䡠 C p共t兲 䊟 e

再 冉

where Ct( · ) is the activity concentration in tissue, K1 and k2⬙ are first order rate constants, determining the rate of transfer from plasma to tissue and from tissue to plasma respectively, and represents the convolution operator. For large values of t, the tissue-to-plasma ratio can be approximated by:

冉

K 1 c 2 ⫹ c 3 䡠 t ⫺ c 3/k 2⬙ C t共t兲 ⬇ C p共t兲 k 2⬙ c2 ⫹ c3 䡠 t

⫹ c 1 䡠 k 2⬙ ⫺ c 2 ⫹

ƒ s共t兲 ⫽

冊 冎

冊

⫺1

(6)

The correction factor is dependent on the parameters c2 and c3, describing the plasma curve, as well as on the tissue-toplasma transfer rate constant, k2⬙, and the time, t, p.i. In order to use this correction factor in practice, we fitted a straight line to the linear part of the plasma TAC, after correction for metabolites and the arterial-to-venous ratio. The values for the intercept and slope of the fitted line were used for c2 and c3 respectively in equation [6]. The k2⬙ values used for different brain regions were the mean k2⬙ values determined in a group of healthy volunteers scanned previously with a bolus protocol [10]. To validate this correction, we performed simulations based on the 1-TC model and a mean plasma input function, obtained from the bolus studies. A simulated B/I plasma TAC for parent compound is shown is Fig. 2a, together with the fitted straight line. Fig. 1b shows the tissue-to-plasma ratio with and without slope correction. It can be seen that, without correction, an underestimation is made but, with slope correction, the value obtained is very close to the true VT value. The slope correction formula in equation [6] was obtained by disregarding the last term in equation [4]. This approximation might result in a slight overestimation of [123I]CNS 1261 binding or VT at small values of t. To include this term requires knowledge of the constant c1 (the peak size), which can only be obtained with arterial sampling. To investigate the significance of this approximation, we calculated the slope correction factor with and without the last term for subject 4. 2.4.6. Volume of distribution The total volume of distribution (VT) [mL plasma/mL tissue], was calculated as follows: V␶ ⫽

Ct ⫽ (4)

冉

K 1/k 2⬙ 1 ⬇ 1⫺ C t共t兲/C p共t兲 k 2⬙共c 2/c 3 ⫹ t兲

Ct ƒ Cp s

where

c 3 ⫺k2⬙t e k 2⬙

(5)

The true VT value is given by the ratio K1/ k2⬙, and the slope correction factor, fs, is therefore obtained as:

⫺k2⬙t

K1 c3 ⫽ c ⫹ c3 䡠 t ⫺ k 2⬙ 2 k 2⬙

冊

Cm ⫺ Vb 䡠 Cb 1 ⫺ Vb

C p ⫽ C v 䡠 f av 䡠 f p ,

(7)

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Cm is the regional activity concentration measured by SPET, Cb and Cv are the activity concentrations in venous blood and plasma, respectively, fs is the slope correction factor, fav is the arterial-to-venous concentration ratio, fp is the fraction of unchanged parent compound, and Vb is the fractional blood volume in brain tissue. Images from the equilibrium phase of the scans, between 90 and 240 min (90 min of data acquisition), were integrated and transformed into Talairach space using the spatial normalization tool in SPM-99 [11] with a SPET blood flow template. This template is appropriate in this case since [123I]CNS 1261 is widely distributed throughout the brain. Regions of interest (ROIs) were drawn in Talairach space, and used as a standard template for all subjects. The brain regions studied were: cerebellum, frontal cortex, striatum, temporal cortex, thalamus and white matter. For each brain region the VT value was calculated with equation [7] and a Vb value of 5% [18]. The mean and standard deviation (SD) across subjects were calculated for each region. The VT values were compared with those obtained in the bolus studies with the same ROIs by spectral analysis (SA), which was highly correlated with the 1-TC analysis [10]. Data from subject 4 was analyzed with two different methods; kinetic analysis and equilibrium analysis. Timeactivity curves (TACs) were generated for each ROI, and the metabolite-corrected arterial plasma input function was used to fit the 1-TC model to the TACs, and VT values were determined as K1/k2⬙ [10]. Equilibrium VT values were also calculated for the same ROIs using equation [7] and the 90 –240 min data. For white matter, the equilibrium analysis was also done using the 150 –300 min data, in order to investigate the effect of slower equilibration in white matter as compared to gray matter. 2.4.7. Statistical analysis Statistical analyses were implemented using SPSS v.10.0 for windows (SPSS Inc, Chicago, Ill). Student T tests for independent samples were performed to compare age, height, weight and years of education between healthy subjects evaluated in the B/I study and the Bolus study. Chi-square tests were performed to compare gender, ethnicity and handedness. Agreement between outcome measures generated by kinetic and equilibrium analysis was evaluated by Bland-Altman plots [2]. The relationship between the two approaches was explored with correlation analysis.

3. Results 3.1. Demographic characteristics Seven healthy subjects were scanned with the B/I protocol (4 female, 3 male; mean age 29.6 years (SD 6.4), 6 right-handed, 1 left-handed). Four subjects were Cau-

Fig. 2. (a)Simulated plasma time-activity curve for a bolus/infusion study with [123I]CNS 1261. The slope and intercept of the straight line (dashed), fitted to the late part or the curve, are used for calculation of the slope correction factors. (b)Ratio of simulated tissue and plasma time-activity curves without (dashed line) and with (solid line) correction for the slope in the plasma curve. The dotted line indicates the true VT value (K1/ k2“).

casians, 2 were mixed race and one was African. The average time spent in fulltime education was 19 years (SD ⫽ 3.5 years). The average weight was 68 kg (SD ⫽ 12 kg) and height was 170 cm (SD ⫽ 11 cm). The socio-demographic features of the subjects scanned in the B/I study were similar to those of the subjects scanned in the bolus study [10]. The differences between the two groups did not reach statistical significance for age (t ⫽ -0.95, d.f. ⫽12, p ⫽ 0.36), gender (X2 ⫽ 0.29, d.f. ⫽1, p ⫽ 0.59), handedness (X2 ⫽ 0.00, d.f. ⫽1, p ⫽ 1.00), height (t ⫽ 0.19, d.f. ⫽12, p ⫽ 0.85), weight (t⫽ -0.26, d.f. ⫽12, p ⫽ 0.98), ethnicity (X2 ⫽2.20, d.f. ⫽3, p ⫽0.53), or for years spent on full time education (t ⫽ -0.89, d.f. ⫽ 12, p ⫽ 0.39).

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Fig. 3. The fraction of total arterial plasma radioactivity attributed to [123I]CNS 1261 for one healthy volunteer during a bolus/infusion study. The solid line is a fitted bi-exponential curve.

3.2. Blood and plasma activity Fig. 2 shows the total and metabolite corrected plasma TACs measured in arterial and venous plasma for subject 4. The metabolite corrected curves were reasonably constant, and the slight gradient resulted in slope correction factors of 1.12⫾0.08 (average⫾SD across all gray matter regions and all subjects). The fraction of parent compound in arterial plasma is shown in Fig. 3 for the same subject. There was close agreement between the measured arterial and venous parent fraction. Fig. 4 shows the arterial-to-venous activity concentration ratios for subjects 2 and 4, as well as the simulated A/V ratios and the average of the four curves, which was used in the VT calculations.

Fig. 5. (a)Tissue time activity curves obtained during a bolus/infusion study of one healthy volunteer (subject 4) corresponding to temporal cortex (circles). The fitted 1-tissue compartment model curve is also shown (dashed line), as well as the metabolite-corrected arterial plasma curve (solid line). (b)Tissue-to-plasma ratios for the same region as a), after slope correction using the complete (solid line) and the simplified formula (circles).

3.3. Brain tissue time activity and imaging data

Fig. 4. Ratio of arterial to venous activity concentration during bolus/ infusion study with [123I]CNS 1261. Solid lines indicate curves fitted to measured values from two healthy volunteers scanned with the bolus/ infusion protocol. Thin dashed lines indicate results from simulated bolus/ infusion studies, based on data from two bolus studies. The thick dashed line indicates the average of the other four curves.

Figure 5a shows the time activity curve for temporal cortex for subject 4, as well as the 1-TC model fit, and the metabolite corrected arterial plasma curve. Figure 5b shows the ratio of the fitted tissue curve and the plasma curve, including slope correction using the complete and the simplified formula. There is an initial difference between the two curves, but from 90 min p.i. there is practically no difference, and a constant level is obtained, indicating that pseudo-equilibrium has been reached. The period 90 –240 min was chosen for future patient scans. We therefore used data from this time period in the comparisons presented here. The mean equilibrium image for all subjects, integrated over the period 90 –240 min is shown in Fig. 6, including transaxial, coronal and sagittal sections. Figure 7 shows the VT values obtained by equilibrium analysis plotted against those obtained by kinetic modeling

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Fig. 6. Mean VT image of all volunteers (N⫽7). Transaxial (bottom right), coronal (top right) and sagittal (bottom left) sections are shown.

of the B/I data from subject 4, including all investigated regions. There is very good agreement (slope⫽1.00) and good correlation (R2⫽0.96) between the two methods. The graph also includes the 150 –300 min equilibrium value for white matter, which falls closer to the regression line than the 90 –240 min value, since white matter takes longer to reach equilibrium than the other regions. Figure 8a shows the B/I VT values for subjects 4 and 6 plotted against the VT values obtained for the same subjects with the bolus protocol. For subject 6 there was a good agreement (slope⫽1.02) and a good correlation (R2⫽0.93) between the two scans. However, for subject 4, both agreement and correlation were poor (slope⫽0.73, R2⫽0.51). Figure 8b shows the mean VT values, taken over all B/I subjects (n⫽7) for each region, plotted against the corresponding values for the bolus subjects (n⫽7). The graph shows good correlation (R2⫽0.97), but the agreement between the methods is not as good (slope⫽0.93). If B/I subject 4 is excluded, the agreement improves (slope⫽0.99, R2⫽0.96). All the data from Figs. 7, 8a and 8b have been summa-

Fig. 7. Correlation between VT values obtained by equilibrium analysis and those obtained by kinetic modeling, using the 1-tissue compartment model, for one volunteer scanned with the bolus/infusion protocol. The solid symbols represent 90-240 min values for different brain regions, the open symbol represents the 150-300 min white matter value.

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Fig. 9. The difference between the VT values obtained by equilibrium analysis and kinetic analysis (VT,equilibrium-VT,kinetic) plotted against the average of the two: subject 4, B/I data only (diamonds), all regions, 90-240 min data (filled diamonds), white matter only, 150-300 min (open diamond); Subject 4, B/I study vs. bolus study (circles); subject 6, B/I study vs. bolus study (squares); Group mean (triangles).

site within the NMDA receptor channel [21]. Our previous study was done with bolus injection of the tracer and arterial sampling for quantification. Since this protocol is not ideal for patient studies, we have now developed a B/I protocol, which does not require arterial sampling. Issues concerning the methodology and implications for further research are discussed below. 4.1. Blood data analysis

Fig. 8. (a)Correlation between VT values obtained from two healthy volunteers scanned with the bolus protocol and rescanned with the bolus/ infusion protocol (squares: subject 4, diamonds: subject 6). (b)Correlation between mean VT values obtained from two groups of healthy volunteers scanned with the bolus protocol (N⫽7) and with the bolus/infusion protocol (N⫽7). The error bars show standard error values.

There were two differences between the blood data analysis in the B/I studies and our previous bolus studies with [123I]CNS 1261. i) In the bolus studies, we found a good agreement between arterial and venous activity concentra-

rized in a Bland-Altman plot (Fig. 9), showing the difference between the VT values obtained by equilibrium analysis and kinetic analysis plotted against the average of the two. Figure 10 shows the mean VT values and SDs for the different brain regions obtained by kinetic analysis of the bolus studies (n⫽7), as well as those obtained by equilibrium analysis of the B/I studies, excluding subject 4 (n⫽6). There is a good agreement between the mean values obtained with the two methods, but the variability (SD) is larger in the B/I study (average SD⫽32% with B/I vs. 19% with bolus). 4. Discussion [123I]CNS 1261 is a novel ligand for the glutamatergic NMDA receptor, highly specific for binding to the MK-801

Fig. 10. The mean VT values (⫾SD) for different brain regions for all volunteers scanned with the bolus protocol (N⫽7) (dark bars), and with the bolus/infusion protocol (N⫽6) (light bars).

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tion from 90 min p.i. However, with the B/I protocol, the arterial-to-venous ratio is always ⬎ 1 due to continuous administration of new tracer. A correction for this was needed in the VT calculation. ii) In the bolus studies, the time course of the fraction of parent compound in plasma could be described by a mono-exponential-plus-constant function. However, in the B/I case, we found that a biexponential function gave a better fit to the data. 4.2. Equilibrium analysis In the development of the equilibrium analysis method, several approximations were made that related to the bolusto-infusion ratio, the slope correction and the venous-toarterial correction. Here we discuss the possible errors that could result from these approximations. Ideally, Kbol should be optimized for each individual, but this would require a preliminary bolus study for each B/I subject. The use of a fixed Kbol value could introduce some variability in the results, due to differences in equilibration time between subjects, which was estimated as follows. The individual plasma TACs and rate constants for the 7 bolus subjects were used together with the fixed Kbol value in the simulation of B/I TACs for different brain regions. The deviation from equilibrium in the time period 90-240 min p.i. was calculated, and the coefficient of variation (CV) across subjects and regions was 3.5%. The slope correction was based on mean k2⬙ values from the bolus study. To estimate the variability introduced by this procedure, we calculated slope correction factors using both the individual and mean k2⬙ values, based on the simulated B/I plasma curves. The CV of the ratio of the two fs values across subjects and regions for the period 90-240 min p.i. was 3%. The venous-to-arterial correction was based on an average ratio of measured data from 4 different subjects. The mean difference between the measured curves and the average curve in the time period 90-240 min was 2%. 4.3. Equilibrium vs. kinetic analysis The good agreement between VT values obtained with equilibrium analysis and with kinetic modeling in subject 4 (Fig. 7) shows that the two quantification methods yield equivalent results. However, the B/I protocol seems to underestimate the white matter VT value as compared to the bolus protocol. This could be due to slower kinetics in white matter compared to gray matter, due to lower regional blood flow, which leads to a longer equilibration time. This assumption is supported by the fact that the 150-300 min white matter VT value is closer to the regression line than the 90-240 min one (Fig. 7). Two subjects were scanned with both approaches on separate occasions (Fig. 8a). In one case, close agreement was found between the VT values obtained from the two scans. In the other case, however, VT values differed by

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27%. This individual experienced a very brief syncopal attack (simple faint) at 105 min p.i. while sitting up out of the scanner on a short acquisition break during the first bolus scan. The effects of this on central glutamatergic function in humans in vivo are unknown, but it could theoretically provide a physiological explanation for the difference between the bolus and bolus infusion VT values measured in this subject. Yet, there was no difference between the kinetic or equilibrium analysis of this subject’s B/I scan. There were no differences in technical factors associated with either the bolus or bolus infusion scan in this individual compared to the other subjects, and the syncopal attack was unique to this subject in the bolus scanning session. Group comparisons between the two approaches showed a mean discrepancy of 7% when this subject was included in the comparison, which was reduced to 1% when the subject was excluded. The Bland-Altman plot (Fig. 9), which summarizes all the data comparing equilibrium and kinetic analysis, shows that, apart from the white matter values and the one testretest outlier (subject 4), there is no serious systematic bias between the VT values obtained with either approach. There is, however, a larger variability in the B/I data compared to the bolus data, as can be seen in Fig. 10. This is likely in part due to the approximations discussed above, and also to the fact that a smaller amount of SPET data was used in the equilibrium analysis, since no data from the first 90 min p.i. could be utilized.

5. Conclusions We have validated a constant infusion paradigm for the novel NMDA receptor ligand [123I]CNS 1261 in healthy volunteers. A ligand binding to the PCP/MK-801 site of the NMDA ion channel is likely to be a highly relevant research tool applicable to the neuropharmacology of a wide variety of neuropsychiatric disorders involving patients with varying capacity to tolerate interventions during scanning, or long duration scanning protocols. Because there are no reference regions for the NMDA ion channel, full quantification by the bolus method requires knowledge of the arterial plasma input function. Arterial sampling is invasive, and not necessarily generalisable or ethically feasible in clinical settings away from research centers. This new protocol allows more flexibility in data acquisition, and providing venous blood samples are taken during the equilibrium phase, subjects do not have to lie on the scanning bench for both the early and late parts of the scan. The bolus infusion approach is also useful for competitive physiological or pharmacological challenges where the baseline can be obtained first at equilibrium, and then following the challenge in only one scan. This would necessitate a twoscan protocol for the bolus only approach. The present study extends previous work by developing a more patient

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friendly protocol that can easily be undertaken in clinical settings[22].

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Acknowledgments

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We wish to thank the following persons for their invaluable help: DC Costa, G Davies, S Gacinovic and WA Waddington. LSP, KE and RSM were supported by a UK MRC Senior Clinical Fellowship awarded to LSP. RAB was supported by CAPES Foundation (Brazil) and Janssen Pharmaceuticals.

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