• Abstract
  

• Introduction
  

• Materials and Methods
  

• Results
  

• Discussion
  

• Acknowledgments
  

• References
  

ABSTRACT

The cocaine analog 2b-carbomethoxy-3b-(4-fluorophenyl)-n-(1-iodoprop-1-en-3-yl) nortropane ([¹²³I] IACFT) labeled dopamine transporters in brain striatum. Preliminary single photon emission computed tomography (SPECT) studies with [¹²³I] IACFT were performed in two healthy volunteers. Serial SPECT images were acquired over 2 hrs. Arterial blood samples were collected in parallel with imaging, and plasma radioactivity was analyzed chromatographically to derive metabolite corrected arterial input functions. Binding potential (BP, B'_max / K_D) for striatal (Str) dopamine transporter (DAT) sites was measured by 2 methods using the occipital cortex (Occ) as reference. In the first method, the time-activity data was analyzed by the linear graphical method developed for reversible receptor ligands by Logan et al. For the second method, the expression (Str_TAC - Occ_TAC) was fitted to a gamma variate function and the maximum was used to estimate BP (Farde, et al.). Plasma analysis indicated that [¹²³I]-IACFT is rapidly converted to polar metabolites. The imaging results showed that [¹²³I]-IACFT accumulates rapidly and selectively in the striatum and yields excellent quality images within one hour after injection. DAT selectivity of [¹²³I]-IACFT was indicated by absence of tracer accumulation in 5-HT transporter rich regions of the hypothalamus and mid-brain. Binding potential measured by the 2 methods were remarkably similar; subject 1: method 1- 1.81, method 2-1.88, subject 2: method 1 - 3.00, method 2 - 3.04. These results demonstrate that [¹²³I]-IACFT is a promising SPECT ligand for imaging and quantification of DAT sites in the human brain.

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INTRODUCTION

Parkinson's disease (PD) is a neurodegenerative condition characterized clinically by tremor at rest, rigidity, bradykinesia and postural instability. The primary pathological features of PD are degeneration of striatal dopamine (DA) neurons and their terminals in the caudate and putamen and 80-99% reductions in striatal concentrations of DA and its transporter (1,2,3,4). Currently, the most effective treatment for PD is dopamine replacement with L-DOPA or receptor agonists. However, as the disease progresses, these therapies become less effective in relieving symptoms and drug side-effects become prominent. Thus, in recent years, research has been directed to the development of alternative medical and surgical approaches to treatment such as: neuroprotective agents (5), fetal cell transplants (6) and pallidotomy (7,8). Since these therapies are designed to slow disease progression or permanently correct the deficit in striatal DA, there is a compelling need for an accurate, precise and non-invasive procedure for monitoring disease activity.

Unfortunately, conventional imaging techniques such as CT and MRI are not useful for detecting early disease or monitoring subtle changes in disease activity. Furthermore, since DA neurons make a small contribution to overall striatal metabolism, PET tracers of glucose utilization and SPECT tracers of blood flow have not been effective for detecting decreases in striatal DA innervation before the >80% reduction that is associated with clinical symptoms (9,10). In contrast, positron emission tomography (PET) studies with [¹⁸F]-6-fluoro DOPA have demonstrated that early asymptomatic disease can be detected at 50-60% loss (11). Despite the success of this tracer, it is currently recognized that radiolableled phenyltropanes targeted to the DA transporter (DAT) are superior imaging agents. Recently, several of these compounds have been shown to be promising PET ligands; [¹¹C]-CFT (WIN 35,428) (12, 13, 14, 15), [¹¹C]-b-CIT (16, 17, 18) and [¹¹C]-CPT (WIN 35,065) (19).

Although PET is a precise and accurate noninvasive method for quantification of DA terminal density and clinical monitoring of PD patients, the expense and complexity of the technique limits general applicability at this time. Clearly, a radiopharmaceutical suitable for single photon emission computed tomography (SPECT) would have significant advantages. Recently, 2b-carbomethoxy-3b-(4-iodophenyl)tropane (b-CIT or RTI-55) was applied for this purpose (20, 21, 22, 23). This ligand has high affinity for the dopamine transporter in the striatum (IC₅₀ = 1.08) (24) and ex vivo autoradiography with [¹²⁵I]-b-CIT have demonstrated high levels of accumulation in DA terminals of monkey brain. However, several lines of evidence suggest that this ligand may not be optimal for SPECT studies of DAT sites: 1. Autoradiographic and SPECT studies have demonstrated that like cocaine, b-CIT concentrates in both DA and 5-HT rich regions of the brain (20-26), 2. A delay of 24 hours between injection and imaging is required for quantitation of DAT sites in the human striatum (27, 28), and 3. Radiolabeled lipophilic metabolites of [¹²³I] b-CIT may complicate quantitation of DAT density (29).

Recently we prepared the E isomer of [¹²³I]-2b-carbomethoxy-3b-(4-fluorophenyl)-N-(1-iodoprop-1-en-3-yl)nortropane (E-IACFT as designated as Altropane^TM) as an alternative ligand for SPECT imaging of DAT sites (30). Based on in vitro binding studies with [³H]-CFT and [³H]-citalopram (a high affinity and selective ligand for 5-HT transporter sites (31), E-IACFT was shown to have high affinity (IC₅₀=6.62+0.78 nmol) and selectivity (DA/5-HT=25:1) for DAT sites. In addition, autoradiographic and SPECT studies in monkeys demonstrated rapid and extremely high levels of accumulation in the striatum and minimal activity in other brain regions (30, 32, 33).

In the current study, we evaluated the SPECT imaging characteristics and transporter selectivity of [¹²³I]-IACFT in healthy human volunteers. In addition, methods for quantification of DAT sites were developed.

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MATERIALS AND METHODS

Preparation of [¹²³I]-IACFT
[¹²³I]-IACFT was prepared from the corresponding tributylstannyl precursor by iodination with [¹²³I] (Nordion International, Ltd., Vancouver, B.C., Canada) using tert-BuCOOH as the oxidizing agent as described previously (33). With this procedure [¹²³I]-IACFT was obtained in an average radiochemical yield of 65+5.8% (mean + SD), radiochemical purity of >98% and specific activity of ~5,000 mCi/mmole. To confirm that the product was free of pyrogens, the Limulus amebolysate (LAL) test was performed prior to injection. Sterility was verified after injection.

Safety considerations
The results of acute toxicity studies in rats and rabbits and radiation dosimetry measurements in rats, the procedures for ligand synthesis and formulation and process control protocols were submitted to the Food and Drug Administration (FDA) as an investigator sponsored IND application. After approval of the application, whole-body planar imaging studies were performed in healthy human subjects. Based on these data, MIRDOSE calculations indicated that approximately 5-10 mCi of [¹²³I] IACFT can be administered without delivering a radiation burden in excess of 20 mGy to any organ (unpublished results).

Human subjects
Two healthy subjects (one male; age 38 years and one female; age 27 years) participated in these studies. The inclusion criteria were: 1. absence of any current medical condition and 2. absence of present or past history of neuropsychiatric illnesses or substance abuse. Physical examination, ECG, and routine blood and urine tests were performed before injection of [¹²³I] IACFT and were repeated 24 hrs and 1 week later. All subjects received 0.6 g of potassium iodide (SSKI solution) daily for 7 days beginning 48 h prior to radiopharmaceutical injection.

The imaging protocol was approved by the committees on human studies, pharmacy and radioisotopes of the Massachusetts General Hospital. All subjects gave written informed consent prior to participation in the study.

SPECT Imaging
Images were acquired with a Siemens MultiSPECT 2 gamma camera equipped with fan-beam collimators. The primary imaging parameters of this device are intrinsic resolution of 4.6 mm (x and y) full width at half maximum (FWHM), and a sensitivity of ~236 cps/mCi. Images were acquired over 360^o (60 projection / head, 128 x 128 matrix) in the continuous imaging mode and were reconstructed using a conventional filtered back-projection algorithm to an in-plane resolution of 10 mm FWHM. Attenuation correction was performed using the Chang algorithm (34). The SPECT camera was cross-calibrated with a well scintillation counter by comparing the camera response from a uniform distribution of an [¹²³I] solution in a 15 cm cylindrical phantom with the response of the well counter to an aliquot of the same solution.

Prior to imaging, a venous catheter was placed in an anticubital vein for radiopharmaceutical administration and a radial arterial catheter was placed in the opposite wrist for blood sampling. The subjects were positioned supine on the imaging bed of the SPECT camera with their arms extended out of the field of view and their heads immobilized with individually fabricated head holders (Tru Scan Image Inc, Annapolis MD). Approximately 10 mCi of [¹²³I] IACFT were injected intravenously over 60 seconds and serial SPECT images were acquired. Dynamic SPECT image collection was started at the end of the infusion in 2 min. acquisitions for the first hour and 5 min. acquisitions for the second hour. Arterial blood samples (1 ml) were collected at 20 sec. intervals for the first 5 min., 1.0 min. intervals for the next 15 min. and 5.0 min. intervals for the remainder of the study. At 1, 5, 10, 30, 60 and 90 min., 5 ml blood samples were obtained for [¹²³I] IACFT metabolite analysis.

Analysis of [¹²³I]-IACFT metabolites in blood
The arterial blood samples were centrifuged (5,000 x g for 5 min.) and total plasma radioactivity was measured in aliquots. The remainder of the plasma was analyzed by chromatography on C₁₈ Sep-paks that were activated with 10 mls of methanol and washed with 10 mls of phosphate buffer (pH 7.4). Twenty mls of each plasma sample was diluted with 5 mls of phosphate buffer (pH 7.4) and applied to the Sep-paks. The cartridges were washed with two additional 5 ml volumes of buffer, followed by 5 ml of methanol and [¹²³I] radioactivity was measured in each fraction, including the cartridge. Further details of this procedure have been reported elsewhere (33).

 

Image analysis
The SPECT slices with highest striatal activity or in which the cerebellum was visualized were summed and five regions of interest (ROI's) were constructed. In the striatal planes, ROI's were placed on the right and left striatum, frontal cortex and occipital pole. Activities from the right and left striatum were averaged. A single ROI was placed on the cerebellum. Average ROI activities (cpm/cc) were decay corrected to the time of injection and expressed as mCi/cc using a calibration factor of 0.00338 mCi/cpm. Corrections for partial volume effects or scattered fraction were not performed.

Kinetic modeling
Figure 1 illustrates the kinetic model that was used to analyze the [¹²³I] IACFT SPECT data. In this model, the rate constants K₁ ( ml min^-1 g^-1) and k₂ (min^-1) define ligand transport into and out of the free compartment in tissue, and k₃ (k_on B'_max, min^-1) and k₄ (k_off, min^-1) represent binding to and dissociation from DA transporters. For occipital cortex, k₃ and k₄ were assumed to be zero and this tissue was used as a reference to calculate K₁ and k₂. The vascular volume fraction in cortex and striatum, V_p, was fixed at a previously determined value of 4.5% and binding potential for [¹²³I] IACFT interaction with DAT sites was calculated as the ratio k₃ / k₄ = B'_max / K_D (35) by two methods.

 

Figure 1: Compartmental models describing the receptor kinetics of [123I] IACFT in human brain. A 3 compartment model (upper) was used to analyze the striatal time-activity data and a 2 compartment model (lower) was used to fit the occipital cortical data.

In the first method, the time-activity data was analyzed by the linear graphical method developed for reversible receptor ligands by Logan et al. (36). Briefly, the integrated tissue activity from time zero to T corrected for intravascular tracer with a fixed V_p and normalized to tissue activity at time T was plotted against the metabolite corrected integrated plasma time activity data which was also normalized to tissue activity at time T. This plot becomes linear when pseudo-equilibrium is reached and assuming that nonspecific binding is negligible the asymptotic slope for the striatal data equals K₁ / k₂ [1 + k₃ / k₄]. In contrast, since it can be assumed that DA transporters are not present in occipital cortex, k₃ and k₄ are negligible and the asymptotic slope becomes K₁ / k₂ . Thus the measured ratio of slopes depends only on k₃ / k₄.

The second method of analysis is an adaptation of one of the procedures that has been used to quantify [¹¹C] raclopride binding to DA D₂ receptors (37). Briefly, by assuming that non specific binding is negligible in striatum and occipital cortex, the striatal TAC (Str_TAC) represents the kinetic behavior of specifically bound plus free ligand, whereas the occipital cortex TAC (Occ_TAC) represents the kinetic behavior of free ligand. Under these assumptions, the function, (Str_TAC - Occ_TAC) defines the time dependence of bound tracer. Fitting this curve to a gamma variate function (Atⁿe^-mt) and division of the maximum by the value of Occ_TAC at the same time yields and equilibrium estimate of k₃ / k₄.

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RESULTS

Neither subjects experienced any subjective or objective adverse affects associated with injection of [¹²³I] IACFT. In both subjects, ECG's and blood chemistry values were normal at baseline and after drug treatment.

Figure 2 shows representative time activity curves for striatum, occipital cortex, frontal cortex and cerebellum of one of the healthy volunteers. These data indicate that accumulation of tracer in the striatum is rapid, reaches a maximum by approximately 10-15 min. after injection and decreases nearly to background levels by 2 hrs. In contrast, tracer accumulation in occipital and frontal cortex and cerebellum is lower and decreases much more rapidly. Figure 3 shows high count density images (summation of data acquired from 30 to 45 min. after injection) displayed in trans-axial, sagittal and coronal projections. From these data, it is clear that there is a high concentration of radiopharmaceutical in the striatum with minimal accumulation in other areas of the brain. In particular, lack of accumulation in the thalamus, hypothalamus or midbrain, regions that are rich in 5-HT transporters, supports the specificity of this tracer for DAT sites.

 

Figure 2: Representative time-activity curves for: striatum (average), occipital cortex, frontal cortex and cerebellum expressed as nCi/ml.

 

Figure 3: Trans-axial, sagittal and coronal SPECT images of the brain of a normal subject at 30 to 45 min. after injection of 10 mCi of [123I] IACFT.

Chromatographic profiles of radioactivity in plasma collected at various times after injection of [¹²³I]-IACFT indicated that during the first minute after injection, nearly all of the radioactivity co-eluted with intact [¹²³I]-IACFT. At the later times, significant concentrations of hydrophilic metabolites were detected and by 60 min. after injection almost no intact [¹²³I]-IACFT remained. Figure 4 shows the time dependence of total plasma radioactivity and the concentration of intact [¹²³I]-IACFT in plasma. These data demonstrate that the concentration of intact ligand decreases rapidly and approaches zero by 60 minutes after injection.

 

Figure 4: Time dependence of total plasma radioactivity and intact [123I]-IACFT.

Figure 5 illustrates representative examples of Logan plots for occipital cortex and striatum derived from SPECT data acquired after injection of [¹²³I]-IACFT. For occipital cortex the slope was interpreted in terms of the 2 compartment model and for striatal data the slope was interpreted in terms of the 3 compartment model. In both subjects, the plots were linear for both tissues by approximately 10 min. after injection. Thus, asymptotic slopes were calculated from the data acquired between 10 and 120 min. after injection. In both subjects, the slope was greater for the striatum compared with the occipital cortex. Since fitting of the occipital cortex TAC to a 3 compartment model indicated that k₅ / k₆ is negligible, the slope of the Logan plot was K₁ / k₂. Thus the slope ratio yielded a value of 1.81 for the binding potential. For the second subject, the binding potential was 3.00.

 

Figure 5: Representative Logan plots for striatum and occipital cortex derived from SPECT data acquired after injection of 10 mCi of [123I] IACFT.

Figure 6 shows a plot of (Str_TAC - Occ_TAC) and the corresponding gamma variate fit (171.6t^0.562e^-0.0265t) for one of the subjects. From the maximums of the fitted curves for the two subjects, the binding potential was determined to be 1.83 and 3.04, in excellent agreement with the results of Logan plot analysis.

 

Figure 6: Plot of (StrTAC - OccTAC) and the corresponding gamma variate fit for one of the subjects.

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DISCUSSION

The results of this study establish that [¹²³I]-IACFT accumulates rapidly and selectively in the striatum of healthy humans and yields excellent quality high contrast SPECT images within one hour after injection. The DAT selectivity of [¹²³I]-IACFT is clearly evidenced by the lack of accumulation of radioactivity in 5-HT transporter rich regions of the hypothalamus and mid-brain. These pharmacokinetic properties of [¹²³I]-IACFT are much different from the in vivo behavior of [¹²³I]-b-CIT. In contrast to [¹²³I]-IACFT, striatal accumulation of [¹²³I]-b-CIT is much slower and does not reach steady state until >24 hrs after injection. During this period, two half-lives of the radioligand are lost. There is significant accumulation of [¹²³I]-b-CIT in hypothalamus and mid-brain. In addition, [¹²³I]-b-CIT is metabolized to lipophilic products which may cross the blood brain barrier and confound quantification whereas [¹²³I]-IACFT has only polar metabolites. From a clinical perspective, these characteristics of [¹²³I]-IACFT lead to several distinct advantages, including: 1. a complete quantitative study of dopamine transporter kinetics can be completed within 2 hrs.; 2. measurements can be performed with lower radiopharmaceutical doses; 3. pre- and post interventional studies can be performed in a single imaging session; 4. quantification of dopamine transporter is not influenced by binding of the probe to serotonin transporter sites; 5. the absence of striatal accumulation of radiolabeled metabolites increases the likelihood of precise measurements.

In general, the pharmacokinetics of [¹²³I]-IACFT closely approximate the behavior of reversible receptor ligands such as [¹¹C] raclopride (37). This similarity, in binding kinetics suggests that the approaches that have been developed for analyzing the kinetics of reversible ligands can be applied to [¹²³I]-IACFT. These methods range from multiple injection protocols with ligand preparations of varying specific activities which can be used to derive individual estimates of K_D and B'_max (38) to graphical techniques which yield values for binding potential (B'_max /K_D).

Although only two healthy volunteers were imaged in the current study, the similarity of the results obtained with the two methods of analysis are very encouraging. Furthermore, the high degree of linearity of the Logan plots lend support to the hypothesis that [¹²³I]-IACFT is a reversible ligand. For research studies, either method of analysis should yield reliable results. However, from a clinical perspective, the second method has the distinct advantages of not requiring arterial blood sampling or metabolite analysis. The very low level of slow non specific binding with this tracer (k₅ and k₆ ~ 0), clearly support the utility of the second method.

In conclusion, this preliminary study demonstrates that [¹²³I]-IACFT is a promising SPECT ligand for imaging and quantification of DAT sites in the human brain. In the future, this technique is likely to be of considerable value for detecting early PD, probing the fundamental pathophysiology of the disease, and for monitoring the effects of new and novel medical and surgical therapies.

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ACKNOWLEDGMENTS

We thank the following agencies for their support: NS30556, DA06303, DA09462, RR00168 and Boston Life Sciences, Inc.

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