Diagnosis of medullary carcinoma of the thyroid (MCT) by calcitonin assay using monoclonal antibodies: criteria for the pentagastrin stimulation test in hereditary MCT.

A new calcitonin (CT) immunoradiometric assay, using anti-11-7 and anti-24-32 CT fragment monoclonal antibodies was evaluated and compared to classical RIA. The sensitivity was 2.5 ng/L, the normal basal level (n = 83) was lower than 10 ng/L, the response to pentagastrin stimulation in control subjects was absent in nine and between 10-30 ng/L in nine others. (mean, 15.4). In patients with renal failure the basal level was increased between 10-52 ng/L. In patients with medullary thyroid carcinoma (MTC; n = 28), the basal level was between 189-28,900 ng/L. A pentagastrin test was performed as screening for familial MTC in eight patients with confirmed MTC at subsequent surgery; the calcitonin peak was equal or greater than 38 ng/L. Large differences exist between CT levels measured by RIA and immunoradiometric assay. The latter method provides a greater sensitivity to pentagastrin test and allows a better identification of microcarcinoma in hereditary cases of MTC.

Synthesis and ligand binding of nortropane derivatives: N-substituted 2beta-carbomethoxy-3beta-(4'-iodophenyl)nortropane and N-(3-iodoprop-(2E)-enyl)-2beta-carbomethoxy-3beta-(3',4'-disubstituted phenyl)nortropane. New high-affinity and selective compounds for the dopamine transporter.

Two novel series of iodinated N-substituted analogs of 2beta-carbomethoxy-3beta-(4'-iodophenyl)tropane (beta-CIT) and N-(3-iodoprop-(2E)-enyl)-2beta-carbomethoxy-3beta-(3',4'-dis ubstituted phenyl)nortropane were synthesized. They were evaluated for their inhibitory properties on dopamine (DA(T)), serotonin (5-HT(T)), and norepinephrine (NE(T)) transporters in rat brain homogenates using [3H]GBR-12935, [3H]paroxetine, and [3H]nisoxetine as specific ligands. All new N-substituted analogs of beta-CIT exhibited higher DAT selectivity over both 5-HT(T) and NE(T) than beta-CIT. Moreover compounds with the N-substituents propynyl (6), crotyl (4), 2-bromoprop-(2E)-enyl (5), and 3-iodoprop-(2E)-enyl (3d) showed similar to higher DA(T) affinities than beta-CIT (respectively 14, 15, 30, and 30 nM vs 27 nM). Compound 3d was found to be the most selective DA(T) agent of this series (5-HTT/DA(T) = 32.0 vs 0.1 for beta-CIT). The N-(3-iodoprop-(2E)-enyl) chain linked to the tropane nitrogen was therefore maintained on the tropane structure, and phenyl substitution was carried out in order to improve DA(T) affinity. K(i) values of N-(3-iodoprop-(2E)-enyl)-2beta-carbomethoxy-3beta-(3',4'-dis ubstituted phenyl)nortropanes revealed that phenyl, 4'-isopropyl, and 4'-n-propyl derivatives weakly inhibited specific binding to DA(T), whereas phenyl substitution with 4'-methyl (3c), 3',4'-dichloro (3b), and 4'-iodo (3d) yielded high-DA(T) reuptake agents with increased DA(T) selectivity compared to beta-CIT. These results demonstrate that the combination of a nitrogen and a phenyl substitution yields compounds with high affinity and selectivity for the dopamine transporter which are usable as SPECT markers for DA neurons.

Bone kinetics of calcium-45 and pyrophosphate labeled with technetium-96: an autoradiographic evaluation.

The uptake of calcium-45 and of pyrophosphate labeled with the long-lived technetium-96 isotope were compared by means of liquid-emulsion microautoradiograms of the epiphyseal plates of 10-week-old rabbits, at 30 min, and 3 and 48 hr after i.v. administration. For both tracers, thin sections confirm the significant role of the blood supply, especially shortly after injection. However, other more specific mechanisms lead to a mixing of the calcium in the mineral mass and to a linear deposition of technetium facing the osteoid surfaces. These findings suggest that the tropism of tin-reduced technetium pyrophosphate is not governed by the mineral pool but rather by exchanges inside a still poorly calcified organic matrix.

Skeletal uptake of pyrophosphate labeled with technetium-95m and technetium-96, as evaluated by autoradiography.

For animal experimentation, the 95m and 96 technetium isotopes offer many advantages over technetium-99m. Their long physical half-lives and the emission of extranuclear electrons of low penetrating power make it possible to obtain autoradiograms of a great precision. The uptake of technetium stannous pyrophosphate by the epiphyseal plate was studied using liquid-emulsion microautoradiography, 3 hr after i.v. injection into 10-week-old rabbits. Microautoradiograms showed a well-defined and rather specific pattern of localization with intense uptake beneath the epiphyseal disk on the extremities of the vascular buds and a lack of accumulation in the cartilage, whether calcified or uncalcified. In the metaphysis, the label was located where new bone was being laid down and also over the cytoplasm of osteoclasts. We deduce from these results that in normal bone the general distribution of this tracer reflects mainly the arrangement of the blood supply, but the specific sites of accumulation are the bone-forming surfaces and the active resorbing osteoclasts.

Therapeutic effectiveness of iodine-131 MIBG metastases of a nonsecreting paraganglioma.

This case report describes the treatment of the bone metastases of a nonfunctioning sympathetic paraganglioma, with [131I]MIBG. After primary tumor excision and unsuccessful external radiotherapy, the patient received three therapeutic doses of [131I]MIBG, resulting in a reduction of the number and volume of metastases, and an improvement of the general condition. At 3 yr following [131I]MIBG therapy, the patient remained in remission. [131I]MIBG appears to be an efficient and safe agent for treating malignant sympathetic paraganglioma.

Luteinizing hormone measurement in polycystic ovary syndrome: a practical approach.

The biological diagnosis of polycystic ovary syndrome (PCO) remains questionable, and a single immunological hLH (ihLH) determination can be misleading. In order better to characterize these patients, we studied hLH pulsatility every 10 min for 4h using a radioimmunoassay and then compared the results with others we obtained with a biological method. Radioimmunological and biological profiles were similar in patients with PCO and in controls. We also studied pulsatility characteristics - frequency and amplitude - and calculated the area under the curve (AUC). There was no significant increase in frequency in our 10 patients with PCO but, as in other studies, increased amplitude of hLH pulses was observed. The most discriminating parameter was the AUC. For practical purposes, we propose that hLH in patients with PCO could be assessed efficiently by taking four samples every 10 min, with computerized calculation of the AUC.

Chronic n-3 polyunsaturated fatty acid diet-deficiency acts on dopamine metabolism in the rat frontal cortex: a microdialysis study.

The effects of alpha-linolenic acid diet deficiency on rat dopaminergic metabolism were investigated in the frontal cortex of male 2-3 month-old rats using the microdialysis method. Increased basal levels of dopamine metabolites were observed in the frontal cortex of awake deficient rats, without modification of dopamine levels. Moreover, using KCl perfusion which releases newly synthesized dopamine, no difference was observed in anaesthetized deficient rats versus control rats. In addition, a decrease in dopamine release was observed in anaesthetized deficient rats versus control rats after tyramine stimulation, which is known to induce release of dopamine from vesicular stores. A working model is proposed which suggests that a chronic n-3 polyunsaturated fatty acids (PUFA) deficiency may lead to modifications in the internalization of dopamine in the storage pool in the frontal cortex.

Correlation of free thyroxine index and thyroxine: thyroxine-binding globulin ratio with the free thyroxine concentration as measured by the thyroxine and thyroxine-binding globulin radioimmunoassays.

The concentration of thyroxine-binding globulin in the serum can now be measured by a simple and specific radioimmunoassay. Triiodothyronine uptake and measurement of total thyroxine have been combined to yield a free thyroxine index which has been found to correlate with the clinical state of the patients. An estimate of the free thyroxine concentration, as measured by the thyroxine and thyroxine-binding globulin radioimmunoassays, provided a good correlation with the free thyroxine index and the thyroxine: thyroxine-binding globulin ratio. However, the thyroxine: thyroxine-binding globulin ratio is inaccurate when thyroxine-binding globulin concentrations are high or low.

Is TISCH a suitable tracer for in vivo study of modifications of the dopamine D-1 receptor?

To detect quantitative modification of dopamine D-1 receptors in vivo, we used [125I]-TISCH in an animal modelin which the striatum was unilaterally lesioned with quinolinic acid. [125I]-TISCH was injected into rats 5 days after the lesion, and the changes in receptor density obtained in vivo were compared to in vitro quantification of dopamine D-1 receptors by binding with either [125I]-TISCH or [3H]-SCH 23390 as a reference ligand. In vivo and in vitro, we obtained the same decrease (-70%) in binding of these ligands in the lesioned striatum. Using an injection of [99mTc]-DTPA to lesioned rats, we also showed the disruption of the blood-brain barrier (BBB) in the lesioned area. Thus, the equivalent decrease observed in vitro and in vivo with [125I]-TISCH confirmed that this molecule would be a valuable tool for exploration of dopamine D-1 receptors by SPECT imaging. Moreover, the fact that the breakdown of the BBB did not interfere with the receptor binding obtained in vivo may be important for future investigations in pathologies with BBB disruption, such as ischemia.

Iododerivative of pargyline: a potential tracer for the exploration of monoamine oxidase sites by SPECT.

Monoamine oxidases are important in the regulation of monoaminergic neurotransmission. An increase in monoamine oxidase B (MAO B) has been observed in some neurodegenerative diseases, and therefore quantification of cerebral MAO B activity by SPECT would be useful for the diagnosis and therapeutic follow-up of these disorders. We have developed an iodinated derivative of pargyline, a selective inhibitor of MAO B, in order to explore this enzyme by SPECT. Stable bromo and iodo derivatives of pargyline were synthesized and chemically characterized. The radioiodinated ligand [125I]-2-iodopargyline was obtained with high specific activity from the bromo precursor by nucleophilic exchange. Affinity and selectivity of 2-iodopargyline were tested in vitro. Biodistribution study of [125I]-2-iodopargyline was performed in rats. Radioiodinated ligand were obtained in a no-carrier-added form. 2-iodopargyline has a higher in vitro affinity for MAO B than pargyline. However, the in vitro selectivity for MAO B was better for pargyline than for 2-iodopargyline. Ex vivo autoradiographic studies and in vivo saturation studies with selective inhibitors of MAO showed that the cerebral biodistribution of [125I]-2-iodopargyline in the rat is consistent with high level binding to MAO B sites in the pineal gland and in the thalamus. In conclusion, 2-iodopargyline preferentially binds in vivo to MAO B sites with high affinity. However, its selectivity for MAO B in rats is not very high, whereas this ligand binds to a lesser extent to MAO A. It will be then of great value to evaluate the specificity of 2-iodopargyline in humans. This new ligand labeled with 123I should therefore be a suitable tool for SPECT exploration of MAO B in the human brain.

Synthesis and characterization of [125I]N-(2-aminoethyl)-4-iodobenzamide as a selective monoamine oxidase B inhibitor.

We described the radiosynthesis of an analog of Ro 16-6491, [125I]N-(2-aminoethyl)-4-iodobenzamide, for SPECT exploration of the monoamine oxidase B (MAO-B) in human brain. The radiolabelling was carried out by nucleophilic exchange of the brominated precursor at solid-state phase in presence of ammonium sulphate. The radiochemical purity of radioiodinated product was higher than 95%. In comparison with Ro 16-6491, the in vitro studies showed a good selectivity of stable N-(2-aminoethyl)-4-iodobenzamide for MAO-B but a slightly lower affinity. Biodistribution studies in the rat showed a high and selective uptake of this compound in the pineal gland 1 h after i.v. injection. The cerebral uptake was low, but the coupling of [125I]N-(2-aminoethyl)-4-iodobenzamide with a lipophilic radical to enhance the passage through the blood-brain barrier can be envisaged.

Synthesis of 4'-iodo-5-methoxy-valerophenone O-(2-aminoethyl)oxime as an agent for exploration of serotoninergic transporter.

The serotonin reuptake process is observed in the central nervous system and in cells derived from the neural crest. It would therefore be of great interest to visualize this reuptake for brain exploration and to visualize the tumors derived from these cells (Apudome). Fluvoxamine has been described as a specific uptake inhibitor for serotonin uptake and we therefore supposed that an iodinated derivative of this compound would be a suitable tracer for this purpose. We had shown by computer-assisted investigation that the trifluoromethyl group of fluvoxamine can be replaced by iodine without changing the steric hindrance of the structure. We therefore expected that this result would allow the development of a new iodinated ligand for human exploration by SPECT which would inhibit for the serotoninergic transporter. This new ligand is 4'-iodo-5-methoxyvalerophenone O-(2-aminoethyl)oxime in its E configuration. In vitro binding studies demonstrated that this iodinated ligand has a weaker affinity for the serotonin uptake sites than fluvoxamine. Steric hindrance is not sufficient to predict affinity, other structural factors such as electronic density and dipole moment must be considered to explain the biological difference between fluvoxamine and its iodinated analog.

Exploration of the dopamine transporter: in vitro and in vivo characterization of a high-affinity and high-specificity iodinated tropane derivative (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-m ethylph enyl)nortropane (PE2I).

For the diagnosis and follow-up of neurodegenerative diseases, many cocaine derivatives have been proposed as radioligands to explore the dopamine transporter. As none of them have all the criteria of specificity and kinetics for human use, we have developed a new derivative, (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methy lphenyl)nortropane (PE2I), which displays promising properties. We report the characterization of PE2I in vitro on rat striatal membranes and in vivo in rats and in monkeys. PE2I had a high affinity (Kd = 0.09 +/- 0.01 nM) and high specificity for the dopamine transporter. In rats we observed a high accumulation in the striatum; by contrast, a very low fixation was measured in the cortex. Moreover, a preinjection of a saturating dose of GBR 12909 prevented the striatal accumulation of PE2I by 74%. These results confirmed the specificity of PE2I for the dopamine transporter. In vivo in monkeys, SPECT studies showed a high accumulation in striatum. Moreover, an equilibrium state was obtained 1 h after injection. PE2I seemed to be the most promising ligand for the dopamine transporter exploration by SPECT using a single-day protocol.

Toxicity, mutagenicity, and behavioral effects of beta-CIT, a ligand for dopamine transporter exploration by SPECT.

The cocaine analog beta-CIT is one of the most used compounds for SPET examination of the dopamine transporter in drug abuse and Parkinson's disease. However, the toxicity of this agent has not yet been studied. We report here acute toxicity, mutagenicity, and effect on locomotor activity of beta-CIT. Acute toxicity experiments were performed in mice and rats. The LD50 values were about 20 mg and 5 mg for mice and rats, respectively. There was no sex difference. The mutagenicity was evaluated using the Ames' test. No mutagenic effect was observed for beta-CIT. Effects on locomotor activity were measured in mice using the open-field test. beta-CIT increased locomotion (+65%) when injected at a dose of 0.312 mg/kg; the maximal increase (+205%) was observed at a dose of 1.25 mg/kg; at higher doses, the effect was decreased slightly. These pharmacological findings are in agreement with an inhibitory effect of beta-CIT at the dopamine transporter. We conclude that with no mutagenic effects and LD50 more than 6 orders of magnitude higher than the routinely used doses in PET or SPET, it can be assumed that beta-CIT can be safely used as a radioligand in humans.

[131I]-TYR3-octreotide: clinical dosimetry and use for internal radiotherapy of metastatic paraganglioma and carcinoid tumors.

Dosimetry and therapeutic application of [(131)I]-Tyr3-octreotide were evaluated in three patients with metastatic paraganglioma and carcinoid tumor. The in vitro stability of [(131)I]-Tyr3-octreotide was verified. Tumor uptake and residence time were between 0.02 and 0.1% and 0.5 to 9.8 h, respectively. The calculated tumor radiation doses were between 0.105 and 0.696 mGy.MBq(-1). No intolerance or adverse effects were observed after the therapeutic doses (3.3-6.6 GBq). A partial tumor response was obtained in one patient and no response occurred in two patients.

PET examination of three potent cocaine derivatives as specific radioligands for the serotonin transporter.

Several positron emission tomography (PET) radioligands based on the aryl tropane structure have been used for studies on monoamine reuptake sites. RTI-364, RTI-330, and RTI-357 (3-beta-(4'-n-propyl-,4'-iso-propyl-, and 4'-iso-propenyl-phenyl)nortropane-2-beta-carboxylic acid methyl ester) are three recently synthesized cocaine analogues with higher affinity for the serotonin (5-HTT) than the dopamine transporter (DAT). Unlabelled RTI-364 and RTI-330 were prepared in a two-step synthesis. The key step was the addition of the appropriate propyl Grignard reagent to anhydroecgonine methyl ester. RTI-357 was prepared in a three-step synthesis with a palladium-catalyzed coupling reaction of beta-CIT and isopropenylzinc bromide as key step. Hydrolysis of the ester functions gave the carboxylic acid analogues of RTI-364, RTI-330, and RTI-357, which were labelled with 11C using [11C]methyl iodide in dimethyl formamide (DMF) and tetrabutylammonium hydroxide (TBAH) as base. All three compounds entered the monkey brain in a high degree (approximately 5-10%). There was a low uptake of [11C]RTI-364 in serotonin-rich brain areas, whereas [11C]RTI-330 and [11C]RTI-357 showed a marked uptake of radioactivity in the thalamus and the brainstem, regions known to contain serotonin transporters. Transient equilibrium was reached at 15 and 40 min for [11C]RTI-330 and [11C]RTI-357, respectively. After pretreatment with citalopram, the ratio of radioactivity in the thalamus and the brainstem to the cerebellum were markedly reduced for [11C]RTI-357 but not for [11C]RTI-330. The results indicate that [11C]RTI-357 is a potential PET radioligand for quantitation of the serotonin reuptake site.

Evolution of dopamine receptors in the rat after neonatal hypoxia-ischemia: autoradiographic studies.

The aim of this work was to follow the evolution of striatal dopamine D1 and D2 receptors after hypoxic-ischemic (H/I) insult in immature rats. SPET imaging of these receptors could be used as an index to assess brain dysfunctions after perinatal H/I without change in cerebral blood flow or neuronal loss. We submitted 1-week-old rat pups to unilateral ligation of the left carotid artery plus 2h exposure to 8% O2. After recovery periods of 1, 2 or 9 weeks, ex vivo and in vitro autoradiographic studies of dopamine receptors were performed on normal appearing brains using specific ligands usable in human SPET imaging. Striatal dopamine D2 receptors tended to decrease bilaterally after one week's recovery. The decrease then reached 40% at 3 weeks of age and at 10 weeks of age the level of receptors had returned to normal values. By contrast, no change in dopamine D1 receptors was seen, whatever the age studied. In conclusion, changes in dopamine D2 receptors could be a valuable index for SPET imaging to evaluate H/I brain damage in the absence of anatomical lesions.

Comparison of two radioiodinated ligands of dopamine D2 receptors in animal models: iodobenzamide and iodoethylspiperone.

Several iodinated compounds have been developed for in vivo exploration of dopamine D2 receptors by SPECT. It is of great value to understand if the same information could be obtained with different radioligands. For this purpose, we compared in vivo properties of two iodinated ligands, iodoethylspiperone (IES) and iodobenzamide (IBZM), using different pharmacological and lesioning treatments in rats. Cerebral biodistribution performed by ex vivo autoradiograms and dissection of brain areas showed that IES and IBZM bound specifically to D2 receptors since a pre-injection of haloperidol prevented accumulation of both ligands. In contrast, when haloperidol was injected after IES or IBZM, only IBZM was displaced from its binding sites. This could be explained partly by a process of dopamine-dependent internalization with IES. The response to striatal quinolinic acid infusion for lesioning post-synaptic neurons was very different for IES and IBZM. In this model a decrease in IBZM accumulation occurred, corresponding to the loss of D2 receptors located on post-synaptic neurons. In contrast, a unexpected increase in IES accumulation was observed on the lesioned side. From these results we concluded that IES and IBZM, two iodinated ligands belonging to different pharmacological families, bound specifically to dopamine D2 receptors. However they have different properties in animal models. Therefore, it appears that IBZM is a more suitable ligand than IES to detect modifications of D2 receptors by in vivo exploration.

An iodinated derivative of moclobemide as potential radioligand for brain MAO-A exploration.

In vivo evaluation of MAO-A would be of great value for the diagnosis and follow-up of therapy of depression. In order to perform this exploration by SPECT, we developed an iodinated derivative of the reversible MAO-A inhibitor, moclobemide. Ro 11-9900 was synthesized and analysed by IR, NMR and HPLC. Radioiodination was carried out by nucleophilic exchange of [125I] on the brominated precursor, and yielded [125I]-Ro 11-9900 with high specific activity. In vitro experiments on rat brain homogenates showed that Ro 11-9900 had poor inhibitory activity on MAO-A, as already described for moclobemide. By contrast, in vivo biodistribution in the rat brain showed that [125I]-Ro 11-990 accumulated in a region corresponding to the localization of locus coeruleus known for its high density of MAO-A. Moreover, preinjection of the irreversible MAO-A inhibitor clorgyline (10 mg.kg-1) prevented accumulation of radioactivity by 40 to 60% and we found that the radioactivity in the brain corresponded exclusively to [125I]-Ro 11-9900 and not to a metabolite. [125I]-Ro 11-9900 was highly accumulated in the pineal gland, both on MAO-A and on MAO-B sites. We concluded that the unmetabolized iodinated derivative of moclobemide, Ro 11-9900, preferentially labeled MAO-A in vivo in the rat brain. This compound would therefore be a potential tracer for evaluation of MAO-A by SPECT.

Iodobenzamide for in vivo exploration of central dopamine receptors: evaluation in animal models of supersensitivity.

Iodobenzamide is a promising agent to investigate D2 receptors by SPECT in living human brain. In this work, we have evaluated this radiolabeled compound in two animal models of D2 receptors supersensitivity. In the first model, rats were treated chronically with haloperidol during three weeks (S.C. injection of 0.5 mg/kg/day). One week after the last day of treatment, they were I.V. injected with 125I-IBZM. In vivo specific binding study showed a 45 percent increase of 125I-IBZM fixation in the striatum of treated rats. In a second step of experiments, animals were unilaterally lesioned by a stereotaxic injection of 6-OHDA in the substantia nigra, 23 days before receiving 125I-IBZM. Autoradiographic analysis of coronal brain sections showed a 38 percent enhancement of 125I-IBZM in vivo binding in the striatum on the lesioned side as compared to the contralateral intact side; this increase occurred in striatal lateral area. These data demonstrate that 125I-IBZM is convenient to detect alterations of dopamine D2 receptors in vivo in the rat. Thus IBZM labelled with 123I can be a very useful imaging agent for the exploration of D2 receptors in pathological situations.