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Quantification of D₂-Like Dopamine Receptors in the Human Brain with ¹⁸F-Desmethoxyfallypride

¹ Department of Psychiatry, University of Mainz, Mainz, Germany
² Department of Nuclear Medicine, University of Mainz, Mainz, Germany
³ Institute of Nuclear Chemistry, University of Mainz, Mainz, Germany
⁴ Division of Nuclear Medicine, Department of Radiology, Johns Hopkins Medical Institutions, Baltimore, Maryland

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Substituted benzamides such as ¹¹C-raclopride or ¹²³I-iodobenzamide are selective radiotracers for PET and SPECT imaging of D₂-like dopamine (DA) receptors. ¹⁸F-Desmethoxyfallypride (¹⁸F-DMFP) is a benzamide tracer with the advantage of an ¹⁸F label. We optimized the synthesis and evaluated ¹⁸F-DMFP in PET studies on healthy human volunteers. Methods: The affinity of DMFP for D₂-like DA receptors was characterized in vitro using membrane preparations from rat striatum and the DA receptor ligand ³H-spiperone. PET studies on 10 healthy human volunteers were performed using a whole-body PET scanner after injection of 214 ± 54 MBq (mean ± SD) ¹⁸F-DMFP. Brain images were acquired dynamically over 124 min, and metabolite-corrected plasma activity was used as the input function. Data analysis was performed using several different approaches (compartmental, graphical, equilibrium methods). Results: The mean inhibition constant (K_i) of DMFP was 15 ± 9 nmol/L. In human brain, the striatum-to-cerebellum ratio reached a maximum of about 4 between 60 and 120 min. When specific binding in the striatum was expressed as the difference between binding in the striatum and the cerebellum, it reached a maximum at approximately 60 min after injection and remained almost constant until the end of data acquisition. The ratio of specific striatal to nonspecific cerebellar binding was about 3:1 at 120 min after injection. A small, but significant specific tracer binding could also be detected in the thalamus. Treatment of a schizophrenic patient with a high dose (1,000 mg/d) of another substituted benzamide, amisulpride, resulted in a reduction of specific tracer uptake of about 90% in striatal regions. With regard to measured distribution volumes and binding potentials, there was an excellent agreement between all applied analytic methods. Conclusion: Our study demonstrates that ¹⁸F-DMFP is a highly reliable tracer for PET imaging of D₂-like DA receptors. It offers the major advantage that it can be used independently of an on-site cyclotron within a PET satellite network. Noninvasive analytic methods without blood sampling provide valid measurements of receptor quantities in human striatum. Because of the ¹⁸F label and the favorable imaging properties, ¹⁸F-DMFP could become an efficient substitute for ¹¹C-raclopride in a clinical context.

Key Words: D₂-like dopamine receptors • PET • ¹⁸F-desmethoxyfallypride • caudate nucleus • putamen • thalamus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
The D₂-like dopamine (DA) receptor was the first neuroreceptor to be visualized by PET, with 3-N-¹¹C-methylspiperone (¹¹C-NMSP) as the radiotracer (1–3). Subsequently, ¹¹C-raclopride, a substituted benzamide radioligand, was developed for quantification of D₂-like DA receptors (4). Whereas ¹¹C-NMSP binds not only to D₂-like DA receptors but also to 5-hydroxytryptamine-2A and ₁-receptors to a considerable extent (5,6), ¹¹C-raclopride has the advantages of selective binding to D₂-like DA receptors and a relatively easy tracer kinetic modeling. Substituted benzamide neuroleptics are a group of D₂-like DA receptor antagonists, which share their high affinity and selectivity for those receptors. ¹²³I-Iodobenzamide (¹²³I-IBZM) is an established benzamide tracer for selective labeling of D₂-like DA receptors with SPECT (7). Recently, the benzamide ligands ¹²³I-NCQ 219 (epidepride) (8) and ¹¹C-FLB 457 (9) have been labeled for use with SPECT and PET, respectively, for visualization and quantification of extrastriatal DA receptors.

Substituted benzamides with an ¹⁸F label are currently being developed for broader clinical use. The ¹⁸F label offers the advantage of a longer half-life compared with the ¹¹C label of, for example, ¹¹C-raclopride. This can also allow an increase in time of follow-up examinations after pharmacologic challenges. Two of the most promising ¹⁸F-labeled benzamide radiotracers are ¹⁸F-fallypride (N-[(1-allyl)-2-pyrrolidinyl)methyl]-5-(3-¹⁸F-fluoropropyl)-2,3-di- methoxybenzamide; ¹⁸F-FP) and ¹⁸F-desmethoxyfallypride ((S)-N-[(1-allyl)-2-pyrrolidinyl)methyl]-5-(3-¹⁸F-fluoropro- pyl)-2-methoxybenzamide; ¹⁸F-DMFP), with ¹⁸F-FP having a higher affinity for D₂-like DA receptors (50% inhibitory concentration [IC₅₀], 0.6 nmol/L vs. 15.0 nmol/L) (10). In monkey studies, both tracers have proven to be receptive for amphetamine-induced changes in synaptic DA levels (11,12). Here we report on the first ¹⁸F-DMFP PET studies in humans and our efforts to describe the tracer’s behavior in brain with a variety of different analytic methods.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
¹⁸F-DMFP was synthesized via direct fluorination of a tosylated precursor, ((S)-N-[(1-allyl)-2-pyrrolidinyl)methyl]-5-(3-toluenesulfonyloxy-propyl)-2-methoxybenzamide. It was obtained according to a method described in the literature (10,12). Modifications of the published method led to a much higher yield and simultaneously simplified the purification. Through these modifications it became possible to prepare the precursor of ¹⁸F-DMFP on a larger scale.

The tosylated precursor (5 mg, 10 µmol) was dissolved in 1 mL acetonitrile, treated for 5 min at 65°C with potassium carbonate (5 mg, 36 µmol), and subsequently transferred into a 5-mL vial containing ¹⁸F-fluoride using the method proposed by Hamacher et al. (13). The ¹⁸F-fluoride was delivered from other cyclotron centers with transport periods ranging from 2 to 4 h. The reaction mixture was heated for 20 min at 85°C, diluted with 1 mL phosphoric acid (10%), and separated using high-performance liquid chromatography (HPLC) (250 x 10, RP8, CH₃CN:0.25 mol/L ammonium acetate buffer + 5 mL acetic acid/L 30:70, 5 mL/min). The fraction containing ¹⁸F-DMFP was isolated, diluted with 0.15 mol/L disodium hydrogen phosphate buffer, and adsorbed on a C₁₈ cartridge to remove the HPLC solvent. The column was washed with 2 mL water and eluted with 1 mL ethanol. The elute was diluted with 9 mL of an isotonic NaCl solution and sterilized by filtration. Before injection, quality was controlled, which included determination of chemical and radiochemical purity, specific activity, pH, and absence of pyrogens. The volume of ¹⁸F-DMFP injected as an ethanol:water mixture (1:9) was 5 ± 1 mL.

The specific activity was analyzed by means of the same HPLC using the calibrated fluoro-DMFP ultraviolet intensity. The specific activity at the time of injection ranged from 37 to 834 GBq/µmol (mean ± SD, 295 ± 265 GBq/µmol). Accordingly, the injected tracer mass for all studies was <1 µg.

The binding of DMFP was characterized in vitro using membrane preparations from rat corpus striatum and the DA receptor ligand ³H-spiperone using a published method (14) with slight modifications. Tissue was homogenized in 50 vol of 50 mmol/L Tris-HCl buffer, pH 7.4, containing 0.32 mol/L sucrose. Membranes were prepared by differential centrifugation. The final pellet (40,000g) used for in vitro binding was stored frozen (-20°C).

After thawing, the pellet was resuspended (5 mg original wet weight per mL) in 15 mmol/L Tris-HCl buffer, pH 7.4, containing 120 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl₂, and 1 mmol/L MgCl₂. In the binding assays tubes were filled with 0.9 mL membrane suspension, 50 µL DMFP solution (final concentration, 10–100 nmol/L) or distilled water, 25 µL distilled water, or, for determination of nonspecific binding, 50 µmol/L (+)-butaclamol (final concentration, 1 µmol/L) and ³H-spiroperidol (specific activity, 851 GBq/mmol; Amersham, Braunschweig, Germany). After a 30-min incubation at 30°C, 5 mL Tris buffer were added and the suspension was filtered under vacuum through GF/C filters (Whatman, Ann Arbor, MI) using a 24-manifold filtration apparatus. After 2 rinses of the same buffer, filter-bound radioactivity was determined by liquid scintillation counting. Each determination was run in duplicate.

The study was approved by the local ethics committee in Mainz, Germany, and the German radiation safety authorities. PET studies were performed on 10 healthy male volunteers (age range, 24–44 y; mean age ± SD, 31 ± 6 y), who gave written informed consent to participate in the study. Additionally, to demonstrate blockade of specific ¹⁸F-DMFP binding by DA receptor antagonists, we studied a schizophrenic patient, who was treated with a high dose (1,000 mg/d) of another substituted benzamide, amisulpride, which also binds selectively to D₂ and D₃ DA receptors. This patient had been on an amisulpride monotherapy for 6 wk, and the dose had been kept stable for at least 4 wk. The patient had not been treated with depot neuroleptics for at least 6 mo. Informed consent was also obtained from this patient.

Data Acquisition
Images were acquired on a Siemens ECAT EXACT whole-body PET scanner. The camera has a field of view of 16.2 cm in 47 planes with a plane spacing of 3.375 mm, an axial resolution of 4.6-mm full width at half maximum, and an in-plane resolution of 6.0 mm (resolution in center with scanner in 3-dimensional [3D] mode). Data acquisition comprised of a series of 28 time frames. The scan duration increased progressively from 1 min to 10 min (4 scans of 1 min, 3 scans of 2 min, 3 scans of 3 min, 15 scans of 5 min, 3 scans of 10 min), resulting in a total scanning time of 124 min. A 20-min transmission scan using a ⁶⁸Ge source was obtained before each study for subsequent attenuation correction.

A mean of 214 ± 54 MBq (mean ± SD; range, 157–308 MBq) ¹⁸F-DMFP was injected intravenously as a bolus into a cubital vein over approximately 30 s. Plasma samples for determination of the kinetics of total plasma radioactivity and labeled metabolites were obtained from a radial artery according to the following protocol: continuous sampling for the first 2 min, 1-min intervals up to 5 min after injection, 2-min intervals up to 10 min after injection, 5-min intervals up to 20 min after injection, and 10-min intervals up to 120 min after injection. Blood samples were immediately centrifuged, and plasma radioactivity was counted in a -scintillation spectrometer.

The positioning of the head in the scanner was parallel to the canthomeatal line. In the healthy volunteer, a T1-weighted 3D gradient echo magnetic resonance (MR) scan with 1.5-mm slice thickness and 128 slices was acquired. The MR scan was resliced according to the anterior commissure–posterior commissure (ac–pc) line, which was identified on a midsagittal plane. The PET images were then coregistered with the resliced MR images for accurate anatomic positioning of regions of interest (ROIs) using the Automatic Image Registration (AIR) algorithm (15). Polygonal ROIs were drawn on planes, where the respective regions (cerebellum, thalamus, putamen, caudate nucleus) have maximal areas. The thalamus was chosen as a region for analysis of binding to extrastriatal DA receptors, because this structure is easily identifiable on MR images and its size minimizes possible partial volume effects. Also, the thalamus has been demonstrated to contain a considerable amount of D₂-like DA receptors at least in the rat (16). ROIs were drawn bilaterally and subsequently averaged. For the subsequent analyses, the cerebellum was chosen as a reference region, because it is generally considered to be DA receptor free.

Plasma Metabolite Studies
Blood samples of 2.5 mL for determination of labeled metabolites were withdrawn from the radial artery at the time points 2, 5, 10, 20, 30, 40, 60, 90, and 120 min after injection. The samples were centrifuged for 10 min at 5.5g. One-half milliliter acetonitrile was added to aliquots of 0.5 mL plasma for precipitation, and the resulting mixtures were centrifuged again for 10 min at 5.5g. Aliquots of 10–30 µL of the supernatant were transferred on silica gel 60 plates (Merck, Darmstadt, Germany) for thin-layer chromatography (methanol:ethyl acetate, 1:1). Activity distribution was detected using an Instant Imager (Canberra-Packard, Dreieich, Germany) with R_f values of 0.4–0.5 for ¹⁸F-DMFP and 0–0.1 for polar metabolites.

Data Analysis
Volumes of distribution (VDs) or binding potentials (BPs) were calculated with graphical, compartmental, and equilibrium methods. Here we report on the results obtained with 6 analytic methods.

Kinetic Analysis Invasive (Single- and 2-Tissue-Compartment) Models.
Both single-tissue (3 parameter) and 2-tissue (5 parameter) models were applied to fit the measured data (17–20). For the single-tissue, 2-compartment, 3-parameter model, the tissue concentration C_t is related to the metabolite-corrected plasma concentration C_P according to the equation C_t(t) = K₁C_P(t) exp(-k₂t), and the measured radioactivity concentration within an ROI, C_ROI, is assumed to be equal to C_t + V_PC_P, where K₁ is the unidirectional transport rate constant of tracer across blood–brain barrier from vascular space to tissue (mL/min/mL), k₂ is the rate constant for efflux (1/min), and V_P (mL/mL) is the fractional plasma volume in vascular space. After obtaining the parameters K₁, k₂, and V_P, the VD can be calculated as VD = K₁/k₂. From the VDs determined, BPs for specific brain regions can be calculated as:

The 2-tissue, 3-compartment, 5-parameter model is described by the equations:

Here, the measured radioactivity concentration within an ROI, C_ROI, is assumed to be equal to C_NS+F + C_B + V_PC_P, where K₁ (mL/min/mL) is the transport rate constant from the plasma compartment to the free plus nonspecific binding compartment, k₂ (1/min) is the efflux rate from the free plus nonspecific binding compartment, k₃ (1/min) is the transport rate constant from the free plus nonspecific binding compartment to the specific binding compartment, k₄ (1/min) is the inverse rate constant from the specific binding compartment to the free plus nonspecific bonding compartment, and V_P (mL/mL) is the fractional plasma volume in vascular space. C_NS+F and C_B are the concentrations in the free plus nonspecific compartment and specific bound compartment, respectively. By assuming that K₁/k₂ estimated from the reference tissue cerebellar time–activity curve using single-tissue compartmental analysis is the same as in the target tissue, the model parameters of the 3-compartment model can be estimated by fixing the K₁/k₂ value during nonlinear fitting. The VD and BP values are then estimated as VD = (K₁/k₂)(1 + k₃/k₄) and k₃/k₄ after model fitting, respectively.

Logan Plot with Arterial Blood Sampling.
Plots of ₀^t ROI(t)dt/ROI(t) versus ₀^t C_P(t)dt/ROI(t) (where ROI and C_P are functions of time describing the variation of tissue radioactivity in the ROI and plasma radioactivity, respectively) are generated from the time–activity data in striatal regions, thalamus, cerebellum, and plasma (21). The slope of the linear portion of the plot for the cerebellum represents a = K₁/k₂, and the slope of the linear portion of the plots for the striatal and thalamic regions represents b = K₁/k₂(1 + k₃/k₄), with K₁ being a delivery rate constant and k₂, k₃, and k₄ being first-order kinetic rate constants. The BP k₃/k₄ is then equal to b/a - 1.

Logan Plot with Reference Region.
Logan et al. (22) described a noninvasive graphical method for estimation of the distribution volume ratio, DVR, from which the BP can be calculated by the equation BP = DVR - 1. Under the assumption that C_RF(t)/C_RC(t) becomes reasonably constant for a sufficiently long period of time over the course of the scanning period, knowledge of the transfer constant from tissue to plasma in the reference tissue k'₂ is not necessary, and then the DVR can be estimated from the slope of the linear portion of the plot of ₀^t C_RC(t)dt/C_RC(t) versus ₀^t C_RF(t)dt/C_RF(t) (where C_RC and C_RF are functions of time describing the variation of tissue radioactivity in receptor-containing and receptor-free regions, respectively).

Lammertsma Simplified Reference Tissue Model.
A simplified reference tissue model was applied to estimate BP according to the following equation (23):

where C_RC is the tissue radioligand activity in the receptor-containing ROI, C_RF is the tissue radioligand activity of ¹⁸F-DMFP in the reference tissue (cerebellum), k₂ is the rate constant for transfer from the free compartment to plasma, R₁ is the influx rate constant (K₁) ratio for target region versus reference region, and t is time. The parameters R₁, k₂, and BP (k₃/k₄) were estimated with a nonlinear least-square minimization procedure.

Transient Equilibrium Model.
Under the assumption that tissue radioligand activity in the nondisplaceable compartment is the same in reference and receptor-containing tissues, the tissue radioligand activity bound to receptors C₃(t) equals the difference C_RC(t)–C_RF(t) (24). When C₃(t) is plotted and a curve is fitted to these data, at the peak of this curve, dC₃(t)/dt = 0 (the point of peak equilibrium); thus, C₃(t)/C_RF(t) = BP = k₃/k₄.

Statistical Analysis
Group comparisons were performed by the Mann–Whitney U test. Correlations between the various analytic methods were tested using the Spearman rank order correlation (r_s). The level of significance was set at = 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Chemistry
The synthesis of ¹⁸F-DMFP consists of 2 main steps: the synthesis of (S)-N-allyl-2-aminomethylpyrrolidine and the formation of the tosylated precursor, ((S)-N-[(1-allyl)-2-pyrrolidinyl)methyl]-5-(3-toluensulfonyloxy-propyl)-2- methoxybenzamide. For the synthesis of (S)-N-allyl-2-aminomethylpyrrolidine, a stereo-conservative route first described by Högberg et al. (25) was applied. For this 3-step process the workup has been simplified and yields were improved. The crude product of the first step could be used without further purification as educt for the second step. However, through a higher concentration of sodium dihydrido-bis(2-methoxyethoxo)-aluminate (SDMA) in the third step, much higher yields of (S)-N-allyl-2-aminomethylpyrrolidine could be obtained. Further studies showed that, by using N,N,N',N'-tetramethyl(succinimido)uronium tetrafluoroborate (TSTU) instead of dicyclohexylcarbodiimide (DCC) for coupling the (S)-N-allyl-2-aminomethylpyrrolidine and the 2-methoxy-5-(3-hydroxypropyl)benzoic acid, the product could be purified through an easy extraction system (10). These modifications of the synthesis led to a 4-fold overall yield.

For the optimization of the ¹⁸F fluorination, different reaction parameters such as solvents, reaction temperatures, and phase transfer catalysts were examined. The highest radiochemical yields of 30%, comparable with that reported in the literature (12), were achieved using acetonitrile as the solvent and Kryptofix 2.2.2. (Merck, Schuchardt, Germany) as phase transfer catalyst at a reaction temperature of 85°C and a reaction time of about 20 min. Varying the precursor concentration improved the radiochemical yield only slightly. However, the addition of potassium carbonate (36 µmol/mL) to the precursor (10 µmol/mL) led to drastically increased radiochemical yields of 80% ± 10%.

In Vitro Studies
In vitro binding studies using variable concentrations of ³H-spiperone (0.05–2.5 nmol/L) and 0–50 nmol/L of the macroscopic (^natF) DMFP revealed competitive inhibition. The mean inhibition constant (K_i) of DMFP calculated from 4 independent experiments was 15 ± 9 nmol/L.

PET Studies in Humans
Plasma Metabolite Studies.
The metabolism of ¹⁸F-DMFP was relatively slow. At 120 min after tracer injection, >50% of the injected ligand was found unchanged in arterial plasma (Fig. 1).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Fraction of unchanged 18F-DMFP over scanning time of 124 min (n = 10; mean ± SD).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Typical thin-layer chromatograms for 18F-DMFP and metabolite(s) 10 min (top) and 60 min (bottom) after injection, respectively. 1 = Metabolite; 2 = intact 18F-DMFP.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Typical set of time–activity curves in putamen (), thalamus (), and cerebellum (•) with fitted curves (solid lines) obtained in healthy human volunteer. Dashed lines represent specific binding in putamen (long dashes) and in thalamus (short dashes), when specific binding in these structures is expressed as difference between total binding in structure and cerebellum. From 3-compartment, 5-parameter model, rate constants in putamen in this subject were determined as follows: K1 = 0.405 mL/min/mL; k2 = 0.077/min; k3 = 0.862/min; k4 = 0.338/min.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Time–activity curves in putamen () and cerebellum (•) with fitted curves (solid lines) obtained in schizophrenic patient, who was treated with benzamide antipsychotic amisulpride (1,000 mg/d), which is also highly selective for D2 and D3 DA receptors. Dashed line represents specific binding in putamen, when specific binding in this structure is expressed as difference between total binding in structure and cerebellum. In this patient, scan duration was just 90 min.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Correlations between VDs as determined with 3-compartment model and 2-compartment model (A) as well as between VDs as determined with 3-compartment model and Logan plot with arterial blood sampling (Logan invasive method; B).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Correlations between BPs as determined with 3-compartment model and reference tissue model (A) as well as between BPs as determined with transient equilibrium model and reference tissue model (B).

Regarding the BPs, there was also a very good agreement between all methods (Table 1). BPs in the putamen ranged from 2.19 ± 0.32 (mean ± SD) as determined by the transient equilibrium model to 2.44 ± 0.40 as measured with the compartment kinetic models. In the caudate, the respective values ranged from 1.61 ± 0.34 (Logan plot with reference region) to 1.80 ± 0.41 (2-compartment model). In the thalamus, BPs were determined to be around 0.20 in magnitude, with the Logan graphical methods at the low end and, again, the 2-compartment model revealing the highest value (Table 1).

In general, there was also a very good correlation between the invasive (using the metabolite-corrected arterial blood input function) and the noninvasive (without blood) methods. All applied methods correlated statistically significantly with each other. Figure 6 shows the correlations between representative methods applied to data for the putamen. Results for the other regions are very similar. The strongest correlation was found between the Logan plot with arterial blood sampling and the Lammertsma reference tissue model (r_s = 0.997; P < 0.0001; data not shown). The latter method was also significantly correlated with the gold standard 3-compartment model (r_s = 0.924; P < 0.0001; Fig. 6A). The transient equilibrium model tended to demonstrate weaker correlations with all other analytic methods (with correlation coefficients around 0.70), although the correlations were statistically significant in all cases. Figure 6B shows the correlation between the transient equilibrium model and the reference tissue model (r_s = 0.748; P = 0.013). All other methods were statistically significantly correlated with each other, with correlation coefficients of around 0.90 (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
In this study on human healthy male volunteers we confirmed earlier results from monkey studies demonstrating the selective, high-affinity binding of the benzamide tracer ¹⁸F-DMFP to D₂-like DA receptors (10). ¹⁸F-DMFP belongs to a class of ¹⁸F-labeled substituted benzamide PET radioligands that were initially described by Mukherjee et al. (26). These authors determined an IC₅₀ of 15 nmol/L for ¹⁸F-DMFP at D₂-like DA receptors (using ³H-spiperone) compared with a value of 30 nmol/L for the well-characterized PET radioligand ¹¹C-raclopride (10). The equilibrium dissociation constant K_d for ¹⁸F-DMFP was calculated to be 0.34 nmol/L. In one of their first papers on ¹¹C-raclopride, the Karolinska group reported a K_i of 26 nmol/L (using ³H-spiperone) (27) and a K_d of 1.15 nmol/L. For ¹⁸F-DMFP, we determined a K_i of 15 nmol/L using ³H-spiperone, which is only slightly higher than the K_i reported for ¹¹C-raclopride. Because the K_i is expected to be markedly higher than the K_d when spiperone is used as the competitor (28), our K_i value of 15 nmol/L for ¹⁸F-DMFP is well in line with the K_d of 0.34 nmol/L reported by Mukherjee et al. (10). These available data point to a slightly higher affinity of ¹⁸F-DMFP for D₂-like DA receptors compared with ¹¹C-raclopride.

Corresponding to the minor difference in in vitro affinity between the 2 tracers, ¹⁸F-DMFP differs only slightly from ¹¹C-raclopride with regard to binding indices determined in human brain (19,29). Interestingly, although the BPs obtained with the 2 tracers are very similar in magnitude, VD values measured with ¹⁸F-DMFP are almost 10-fold higher than those from ¹¹C-raclopride studies. The distribution volume represents the blood volume, in which the same amount of tracer is distributed that is allocated to 1 mL tissue (in mL blood/mL tissue). Thus, the distribution volume of a radiotracer in the reference tissue corresponds to the nonspecific binding of the tracer in this tissue, which is generally assumed to be the same as in receptor-rich areas. For ¹¹C-raclopride, Lammertsma et al. (19) report VDs of about 0.40 for the cerebellum and 2.20 for the striatum. With markedly higher VDs in all assessed brain regions, ¹⁸F-DMFP seems to display a higher nonspecific binding than ¹¹C-raclopride, probably because of the higher lipophilicity of the compound caused by the fluorine label. However, according to our studies, this does not influence the calculated BPs derived from these VDs.

Several authors reported the distribution of D₂-like DA receptors in various species using heterogeneous ligands such as sulpride, spiperone, and raclopride (16,30–33). The highest DA receptor densities have been demonstrated in the caudate nucleus and in the putamen, with decreasing numbers in various subcortical structures such as the thalamus and lowest densities—the almost receptor-free cerebellum excluded—in the cortex, especially in layer 5 (16,32). Although we were able to demonstrate that even extrastriatal DA receptors can be quantified to a certain extent—at least in brain regions with higher receptor densities—with this tracer (Fig. 4), the affinity is certainly too low for the quantification of cortical DA receptors. However, the same is true for ¹¹C-raclopride (34). Although attempts have been made to quantify cortical DA receptors with ¹¹C-raclopride or ¹²³I-IBZM, it seems doubtful whether the results obtained with these tracers are truly reliable because the interindividual variability in binding by far exceeds the measured signal (35,36). The quantification of extrastriatal, especially neocortical, DA receptors remains the domain of the high-affinity benzamide ligands ¹¹C-FLB 457 or ¹⁸F-fallypride, the latter being an ¹⁸F-DMFP analog with an affinity for D₂-like DA receptors in the picomolar range (37,38). On the other hand, ¹⁸F-fallypride has the disadvantage of its slow kinetics (because of its high affinity), which requires a data acquisition of at least 3 or even 4 h (39).

Because of the longer half-life of the ¹⁸F label (109.7 vs. 20.4 min), ¹⁸F-DMFP has several advantages over ¹¹C-raclopride. First, ¹⁸F-DMFP can be used independently of an on-site cyclotron, which means in practice that satellite PET centers even hundreds of kilometers away from the manufacturing radiochemistry site are able to administer the ligand for clinical and research applications. Second, ¹⁸F-DMFP allows for more time to follow pharmacologic and behavioral challenges applied in PET studies of the sensitivity of dopaminergic systems in neuropsychiatric disorders. Compared with the established SPECT tracer ¹²³I-IBZM, it demonstrates a markedly higher signal-to-noise ratio. The only true disadvantage of the longer half-life of the fluorine label may be that it cannot be easily administered repeatedly in a short period of time, which may be necessary in pharmacokinetic studies (5). In psychiatric patient populations, studies on D₂ receptor occupancies by antipsychotics and their relationship to doses, plasma levels, and clinical effects and side effects, on the one hand, and the evaluation of pharmacologic and nonpharmacologic challenges on binding of the radioligand could be the domain of ¹⁸F-DMFP. In neurologic samples, the tracer could be an ideal tool for studies of movement disorders.

Another advantage of using an ¹⁸F-derivative is its high specific activity. Whereas maximal values of <100 GBq/µmol are reported for ¹¹C-raclopride, specific activities of >800 GBq/µmol at the time of injection could be obtained with ¹⁸F-DMFP in our study. Specific activities of about 1,000 GBq/µmol could be reached if the ¹⁸F were produced on site.

We have shown that VDs and BPs obtained with ¹⁸F-DMFP are well described by means of various noninvasive analytic methods such as the Logan noninvasive plot (22), the transient equilibrium method (24), or the reference tissue model (23). Thus, for future clinical and research applications it may be possible to use this tracer without arterial blood sampling to obtain quantitative information about the general binding parameters.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 
Our results indicate that ¹⁸F-DMFP is a highly selective PET ligand for quantitative assessment of D₂-like DA receptors, which can be used independently of an on-site cyclotron. Moreover, our comparative analyses demonstrate that noninvasive analytic methods without blood sampling provide valid measures of receptor quantities in human striatum.

	ACKNOWLEDGMENTS

 
The authors thank Ralf Schirrmacher, Sabine Höhnemann, Stephan Maus, and Wilhelm Hamkens from the Institute of Nuclear Chemistry for performing the radiochemical syntheses and the metabolite studies and Heike Armbrust from the Department of Nuclear Medicine for assistance in performing the PET studies. The authors also thank Peter Stoeter and his staff from the Institute for Neuroradiology for performing the MRI studies and Professor Hakan Håll from the Karolinska Institute, Stockholm, Sweden, for discussion of the K_i value of ¹⁸F-DMFP in comparison with ¹¹C-raclopride. Part of this work was supported by the Deutsche Forschungsgemeinschaft (grant Ba 1011/2-1), by Merck KgaA, Darmstadt, Germany, and by the National Institutes of Health (grants R01 DA11080, R01 AA1283, K24 NS389279, RO1 MH4282, and RO1 DA 094821).

	FOOTNOTES

 
Received Apr. 8, 2002; revision accepted Jul. 26, 2002.

For correspondence or reprints contact: Gerhard Gründer, MD, Department of Psychiatry, University of Mainz, Untere Zahlbacher Strasse 8, 55131 Mainz, Germany.

E-mail: gruender{at}mail.uni-mainz.de

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION REFERENCES

 

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