Jeffrey A. Clanton

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [125I]epidepride

The regional distribution of striatal and extrastriatal dopamine D2 receptors in human brain was studied in vitro with (S)-N-[(1-ethyl-2- pyrrolidinyl)methyl]-5-[125I]iodo-2,3-dimethoxybenzamide, [125I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D2 receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D2 receptors (0.17-0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31-0.46 pmol/g tissue). Inhibition of [125I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [125I]epidepride binding was potently inhibited only by dopamine D2 ligands. The present study demonstrates that dopamine D2 receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D2 receptors is comparable to many limbic regions and is considerably higher than in cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmaxs of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50s for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.

The use of GD DTPA as a perfusion agent and marker of blood-brain barrier disruption

To provide contrast enhancement in magnetic resonance imaging, a new class of compounds has been developed, the paramagnetic metal ion chelates. Gadolinium (Gd) DTPA, a prototype of this class, shows a sufficiently high in vivo stability and low toxicity for use in initial clinical trials. This type of agent, designed for rapid clearance by glomerular filtration, allows the assessment on MRI of renal function, alterations in tissue perfusion, myocardial ischemia, and perhaps most significantly disruption of the blood-brain barrier (BBB). Research at Vanderbilt has demonstrated these applications, with particular emphasis in three areas. Tissue perfusion changes, such as those produced by ligation of the arterial blood supply to portions of the spleen and kidney, cannot easily be detected on unenhanced MRI. These acute tissue infarcts can be readily identified following the administration of Gd DTPA. The question of field strength dependence of Gd DTPA has been addressed by experimentation at 0.15, 0.5, and 1.5 tesla. Furthermore, the ability to detect an alteration of the BBB, when present without associated edema, has been demonstrated with the application of control enhancement. The use of contrast agents in MRI will enhance both the sensitivity and specificity of magnetic resonance imaging.

Visualization of extrastriatal dopamine D2 receptors in the human brain

[123I]Epidepride, a potent and selective dopamine D2 radioligand, was administered to a 27 year old normal male volunteer. Single photon tomography revealed that peak striatal uptake occurred at 4 h after injection with a striatal:cerebellar ratio of 7.8 rising to over 100 at 18 h post injection. Uptake above the levels seen in cerebellum was also noted in the thalamus, pituitary, hypothalamus and temporal lobe, particularly medially. Single photon tomography with [123I]epidepride allows visualization of extrastriatal dopamine D2 receptors in man.

Dynamic fluctuations in blood and spleen radioactivity: Splenic contraction and relation to clinical radionuclide volume calculations

Alterations in the blood radioactivity affect ventricular volume calculations using count-based radionuclide ventriculography. To study this phenomenon, the effect of time, posture and supine exercise on blood radioactivity, red blood cell count and splenic radioactivity was evaluated. The red blood cell count, and blood, splanchnic and splenic radioactivity remained stable in five patients studied at rest in the supine position. On standing, blood radioactivity increased 10 +/- 3% (standard error of the mean), and abdominal radioactivity decreased 14.5 +/- 6.5% (both p less than 0.05). In 10 patients, splenic radioactivity decreased after supine exercise by 49 +/- 7%, while blood radioactivity increased 10.5 +/- 1.5% and red blood cell count increased 7.5 +/- 1.5% (all p less than 0.001). Splenic radioactivity increased gradually after exercise and decreased after a second exercise period. In the exercising patients, blood radioactivity increased by 14.5% and correlated with an increase in the red blood cell count (r = 0.57, p = 0.01, 19 samples from 10 patients). Reduction in splenic radioactivity also correlated with the increase in red blood cell count (r = -0.51, p = 0.025). The data demonstrate splenic shrinkage in human beings and an inverse relation between changes in splenic and blood radioactivity. These dynamic fluctuations emphasize the need for simultaneous blood sampling for accurate calculation of left ventricular volume and high-light the importance of regional volume shifts during exercise.

Paramagnetic NMR Contrast Agents: Development and Evaluation

Paramagnetic ions could be theoretically used as NMR contrast agents because of their effect upon T1. However, the toxicity of these ions prevents their application. By the formation of appropriate chemical complexes with these ions, the toxicity of these agents can be substantially reduced while maintaining the paramagnetic effect. Two potential NMR contrast agents, one for oral use and one for intravenous administration, were developed and evaluated both in vitro and in vivo. The effect upon T1 in vitro of these paramagnetic compounds was determined using a JEOL FX-90Q NMR spectrometer. These agents were evaluated in vivo in dogs with a Technicare 0.3 tesla superconducting magnet system and in rabbits with the Aberdeen 0.04 tesla resistive NMR imager. Using calculated T1 NMR images, a nontoxic dose of gadolinium oxalate provided visualization of the gastrointestinal tract. Intravenous administration of chromium EDTA provided enhancement of the kidneys, ureters, and bladder, thereby potentially allowing for the evaluation of renal function with magnetic resonance imaging. Stable paramagnetic complexes can serve as effective, nontoxic, oral and intravenous NMR contrast agents.

High affinity dopamine D2 receptor radioligands. 3. [123I] and [125I]epidepride: In vivo studies in rhesus monkey brain and comparison with in vitro pharmacokinetics in rat brain

Studies of [123I]epidepride uptake in rhesus monkey brain were performed using single photon tomography. Striatal uptake peaked at 0.85% of administered dose/g at 107 min post-injection, then declined slowly to 0.70% of administered dose/g at 6 h. Striatal:posterior brain ratios rose from 2 at 25 min to 6.8 at 105 min, to 15 at 4 h and to 58 at 6.4 h. [123I]Epidepride was displaced by haloperidol (0.1 and 1 mg/kg) with a half-life of washout of 55 min. Little displacement of [123I]epidepride was observed following administration of 1 or 2 mg/kg d-amphetamine, respectively, indicating [123I]epidepride is not easily displaced by endogenous dopamine. In vitro equilibrium binding studies using rat striatum revealed a KD of 46 pM and Bmax of 33 pmol/g tissue at 37 degrees C, while at 25 degrees C the KD was 25 pM and the Bmax 32 pmol/g tissue. In vitro kinetic analysis of association and dissociation curves revealed a half-life for receptor dissociation at 37 degrees C of 15 min and 79-90 min at 25 degrees C. Allowing for the temperature difference, there is good correspondence between in vivo and in vitro dissociation kinetics at 25 degrees C. Increasing in vitro incubation temperature from 25 to 37 degrees C caused a 6-fold increase in the dissociation rate, suggesting that there is a change in binding kinetics at the dopamine D2 receptor at 37 degrees C compared to in vivo binding. The results of this study indicate that [123I]epidepride is an excellent radioligand for SPECT studies of the dopamine D2 receptor in man.

Comparison of CT and MRI brain tumor imaging using a canine glioma model

A canine gliosarcoma model was used to study the effectiveness of magnetic resonance imaging (MRI) with gadolinium contrast enhancement in defining the histologic margins of brain tumors. The effectiveness of this technique was compared to conventional computed tomography (CT) using iodinated contrast enhancement. Cultured canine gliosarcoma cells were injected into the left hemisphere of adult mongrel dogs. The dogs developed brain tumors and progressive clinical signs. Serial MRI with and without gadolinium diethylene triamine penta-acetic acid was compared to serial CT with and without sodium iothalamate obtained on the same days. After the final scans, animals were sacrificed; the brains were removed and processed for routine histopathologic study. All tumors were visualized with contrast-enhanced MRI which proved most sensitive. Gadolinium di-ethylene triamine penta-acetic acid caused bright enhancement of tumors in a distribution that consistently corresponded to areas of pathologically proved tumor infiltration. Gross and microscopic autopsy findings correlated better with MRI than with CT which tended to produce poorer resolution and underrepresent the size of viable tumor. Gadolinium-enhanced MRI is more accurate than unenhanced MRI, unenhanced CT, or enhanced CT in defining the histologic margins of tumors.

Magnetic resonance brain tumor imaging in canine glioma

This study investigates a canine model of experimental brain tumor. Particularly addressed was the usefulness of gadolinium contrast-enhanced MRI for differentiating brain tumor tissue from cerebral edema. Cultured canine glioma cells were injected into the left hemispheres of six adult mongrel dogs. All dogs developed brain tumors. Serum samples drawn prior to and serially after tumor inoculation showed development of antibodies reactive to the tumor. All tumors were visualized with MRI. Contrast-enhanced T1-weighted imaging was the most sensitive with gadolinium producing tumor enhancement due to blood-brain barrier breakdown. Gross and microscopic autopsy findings correlated well with MRIs.

Particulate oral NMR contrast agents

Insoluble paramagnetic compounds in suspension can be used to achieve visualization of the gastrointestinal system on magnetic resonance imaging (NMR). Particulate preparations of these agents decrease the T1 and T2 of solutions to which they are added. Gadolinium oxalate, a prototype of these particulate agents, was evaluated in vitro and in vivo (in rabbits) by NMR imaging. The effect of this compound upon T1 and T2 in vitro was also quantitated by NMR spectroscopy. Opacification of the upper gastrointestinal tract was achieved with gadolinium oxalate following oral administration. The colon was visualized following rectal administration.