Sumalee Chumpradit

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain

Binding characteristics of a novel radioiodinated ligand, [125I]R(+)trans-7-hydroxy-2-(N-n-propylN-3′-iodo-2′-propenyl) aminotetralin (125I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [125I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (Kd = 0.42 nM). A majority (70%) of the sites labeled by [125I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [125I]R(+)trans-7-OH-PIPAT to 5-HT1A receptors, with a Kd value of 1.4 nM and a Bmax value of 210 fmol/mg protein, was observed. Binding of [125I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D 2 and 5-HT1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [125I]R(+)trans-7-OH-PIPAT. [125I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D 3 receptors.

4-Iodotomoxetine: A novel ligand for serotonin uptake sites

The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compounds high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.

Characterization of radioiodinated TISCH : a high-affinity and selective ligand for mapping CNS D1 dopamine receptor

Abstract: In developing CNS D1 dopamine receptor‐imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7‐chloro‐8‐hydroxy‐1‐(3′‐iodophenyl)‐3‐methyl‐2,3,4,5‐tetrahydro‐1 H‐3‐benzazepine], an analog of SCH 23390 (7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)‐[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(–) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD= 0.21 ± 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 > (±)‐TISCH > (+)‐butaclamol = (±)‐FISCH [7‐chloro‐8‐hydroxy‐1‐(4′‐iodophenyl)‐3‐methyl‐2,3,4,5‐tetrahydro‐1 H‐3‐benzazepine] ≥ WB4101 = spiperone > dopamine, serotonin, (±)‐propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)‐[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)‐TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine‐123, it may be suitable for noninvasive imaging in humans.

Characterization of a novel iodinated ligand, IPMPP, for human dopamine D4 receptors expressed in CHO cells.

A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.