Effects of atropine treatment on in vitro and in vivo binding of 4-[125I]-dexetimide to central and myocardial muscarinic receptors

Upregulation of muscarinic cholinergic receptors (mAChR) after chronic atropine treatment has been described previously. The present study was designed to evaluate 4-iodine-125 dexetimide as an agent to determine changes in the number of mAChR. Rats were injected subcutaneously with atropine (500 mg/kg) either once or chronically, once daily for 10 days, and sacrificed 24 h later. In vitro binding assays with 4-[125I]-dexetimide showed significant increases in the number of mAChR in cerebra (21%) and ventricles (45%) after chronic atropine treatment but not after acute treatment. The affinity of binding to cerebral and ventricular mAChR declined after acute and chronic atropine treatment. In vivo studies were carried out involving intravenous injection of 4-[125I]-dexetimide 24 h after atropine treatment. Binding was markedly reduced in the brain and heart. Upregulation of mAChR, as seen in in vitro studies, could not be observed because of the remaining atropine. Occupancy of mAChR by atropine persisted as long as 7 days after one dose. The results of these studies indicate that 4-[125I]-dexetimide binding reflects the effects of atropine on central and peripheral muscarinic cholinergic receptors in vitro and in vivo.     

In vitro and in vivo characterization of 4-[125I]iododexetimide binding to muscarinic cholinergic receptors in the rat heart

4-[125I]iododexetimide binding to muscarinic cholinergic receptors (mAChR) was evaluated in the rat heart. 4-[125I]iododexetimide displayed high in vitro affinity (Kd = 14.0 nM) for rat myocardial mAChR. In vivo, there was high accumulation of 4-[125I]iododexetimide in the rat atrium and ventricle which could be blocked by approximately 60% by preinjection of atropine. In contrast, accumulation of the radiolabeled stereoisomer, 4-[125I]iodolevetimide, was 63% lower than 4-[125I]iodolevetimide and was not blocked by atropine. The blood clearance of 4-[125I]iododexetimide was rapid, providing heart-to-blood ratios of up to 14:1; however, heart-to-lung and heart-to-liver ratios were below unity. The data indicate that 4-[125I]iododexetimide binds potently to rat mAChR. However, since nonspecific binding is relatively high, it is not clear whether iododexetimide labeled with 123I will be useful in SPECT imaging studies of myocardial mAChR. Further studies in humans are indicated.     

Effect of partial volume correction on muscarinic cholinergic receptor imaging with single-photon emission tomography in patients with temporal lobe epilepsy

Animal experiments and preliminary results in humans have indicated alterations of hippocampal muscarinic acetylcholine receptors (mAChR) in temporal lobe epilepsy. Patients with temporal lobe epilepsy often present with a reduction in hippocampal volume. The aim of this study was to investigate the influence of hippocampal atrophy on the quantification of mAChR with single photon emission tomography (SPET) in patients with temporal lobe epilepsy. Cerebral uptake of the muscarinic cholinergic antagonist [¹²³I]4-iododexetimide (IDex) was investigated by SPET in patients suffering from temporal lobe epilepsy of unilateral (n=6) or predominantly unilateral (n=1) onset. Regions of interest were drawn on co-registered magnetic resonance images. Hippocampal volume was determined in these regions and was used to correct the SPET results for partial volume effects. A ratio of hippocampal IDex binding on the affected side to that on the unaffected side was used to detect changes in muscarinic cholinergic receptor density. Before partial volume correction a decrease in hippocampal IDex binding on the focus side was found in each patient. After partial volume no convincing differences remained. Our results indicate that the reduction in hippocampal IDex binding in patients with epilepsy is due to a decrease in hippocampal volume rather than to a decrease in receptor concentration.     

Iodine-123 labelled Z-(R,R)-IQNP: a potential radioligand for visualization of M1 and M2 muscarinic acetylcholine receptors in Alzheimer’s disease

Z-(R)-1-Azabicyclo[2.2.2]oct-3-yl (R)-α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (Z-IQNP) has high affinity to the M₁ and M₂ muscarinic acetylcholine receptor (mAChR) subtypes according to previous in vitro and in vivo studies in rats. In the present study iodine-123 labelled Z-IQNP was prepared for in vivo single-photon emission tomography (SPET) studies in cynomolgus monkeys. SPET studies with Z-[¹²³I]IQNP demonstrated high accumulation in monkey brain (>5% of injected dose at 70 min p.i.) and marked accumulation in brain regions such as the thalamus, the neocortex, the striatum and the cerebellum. Pretreatment with the non-selective mAChR antagonist scopolamine (0.2 mg/kg) inhibited Z-[¹²³I]IQNP binding in all these regions. The percentage of unchanged Z-[¹²³I]IQNP measured in plasma was less than 10% at 10 min after injection, which may be due to rapid hydrolysis, as has been demonstrated previously with the E-isomer of IQNP. Z-[¹²³I]IQNP showed higher uptake in M₂-rich regions, compared with previously obtained results with E-[¹²³I]IQNP. In conclusion, the radioactivity distribution from Z-[¹²³I]IQNP in monkey brain indicates that Z-[¹²³I]IQNP binds to the M₁- and M₂-rich areas and provides a high signal for specific binding, and is thus a potential ligand for mAChR imaging with SPET. Received 18 February and in revised form 15 July 1999     

Decreased ipsilateral [123I]iododexetimide binding to cortical muscarinic receptors in unilaterally 6-hydroxydopamine lesioned rats

IntroductionDysfunction of the cholinergic neurotransmitter system is present in Parkinson’s disease, Parkinson’s disease related dementia and dementia with Lewy bodies, and is thought to contribute to cognitive deficits in these patients. In vivo imaging of the cholinergic system in these diseases may be of value to monitor central cholinergic disturbances and to select cases in which treatment with cholinesterase inhibitors could be beneficial. The muscarinic receptor tracer [¹²³I]iododexetimide, predominantly reflecting M₁ receptor binding, may be an appropriate tool for imaging of the cholinergic system by means of SPECT. In this study, we used [¹²³I]iododexetimide to study the effects of a 6-hydroxydopamine lesion (an animal model of Parkinson’s disease) on the muscarinic receptor availability in the rat brain.MethodsRats (n = 5) were injected in vivo at 10–13 days after a confirmed unilateral 6-hydroxydopamine lesion. Muscarinic receptor availability was measured bilaterally in multiple brain areas on storage phosphor images by region of interest analysis.ResultsAutoradiography revealed a consistent and statistically significant lower [¹²³I]iododexetimide binding in all examined neocortical areas on the ipsilateral side of the lesion as compared to the contralateral side. In hippocampal and subcortical areas, such asymmetry was not detected.ConclusionsThis study suggests that evaluation of muscarinic receptor availability in dopamine depleted brains using [¹²³I]iododexetimide is feasible. We conclude that 6-hydroxydopamine lesions induce a decrease of neocortical muscarinic receptor availability. We hypothesize that this arises from down regulation of muscarinic postsynaptic M₁ receptors due to hyperactivation of the cortical cholinergic system in response to dopamine depletion.Advances in knowledgeIn rats, dopamine depletion provokes a decrease in neocortical muscarinic receptor availability, which is evaluable by [¹²³I]iododexetimide imaging.Implications for patient careThis study may further underline the role of a dysregulated muscarinic system in patients with Lewy body disorders.     

Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP

We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[⁷⁶Br]BrQNP,E(-,-)-[⁷⁶Br]BrQNP andE(-,+)-[⁷⁶Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [⁷⁶Br]BrNH₄ and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M₂ selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M₁ selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., theE(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively.Z(-,-)-[⁷⁶Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for theE(-,+)-[⁷⁶Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors.Z(-,-)-[⁷⁶Br]BrQNP, which is displaced in higher amounts from M₂ mAChR-enriched structures, reveals an M₂ affinity. The two isomers having the (-,-)-configuration are potential probes for investigating central muscarinic receptors. The absolute configuration on the acetate chiral centre influences their muscarinic subtype selectivity and thecis-trans isomerism of the vinyl moiety affects their specific fixation.     

Influence of acetylcholine on binding of 4-[i]iododexetimide to muscarinic brain receptors

The distribution of nicotinic and muscarinic cholinergic receptors in the human brain in vivo has been successfully characterized using radiolabeled tracers and emission tomography. The effect of acetylcholine release into the synaptic cleft on receptor binding of these tracers has not yet been investigated. The present study examined the influence of acetylcholine on binding of 4-[¹²⁵I]iododexetimide to muscarinic cholinergic receptors of porcine brain synaptosomes in vitro. 4-Iododexetimide is a subtype-unspecific muscarinic receptor antagonist with high affinity. Acetylcholine competed with 4-[¹²⁵I]iododexetimide in a dose-dependent manner. A concentration of 500 μM acetylcholine inhibited 50% of total specific 4-[¹²⁵I]iododexetimide binding to synaptosomes when both substances were given simultaneously. An 800 μM acetylcholine solution reduced total specific 4-[¹²⁵I]iododexetimide binding by about 35%, when acetylcholine was given 60 min after incubation of synaptosomes with 4-[¹²⁵I]iododexetimide. Variations in the synaptic acetylcholine concentration might influence muscarinic cholinergic receptor imaging in vivo using 4-[¹²³I]iododexetimide. Conversely, 4-[¹²³I]iododexetimide might be an appropriate molecule to investigate alterations of acetylcholine release into the synaptic cleft in vivo using single photon emission computed tomography.     

Synthesis and evaluation of radioiodinated phenoxyquinazoline and benzylaminoquinazoline derivatives as new EGF receptor tyrosine kinase imaging ligands for tumor diagnosis using SPECT

Objective

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, the radioiodinated 4-(3-iodoanilino)-6,7-diethoxyquinazoline ([¹²⁵I]m-IPQ) has been reported to possess good characteristics as a tumor imaging agent; however, it was also found to have low in vivo stability. To improve the in vivo stability, m-IPQ derivatives, 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) and 4-(3-iodobenzylamino)-6,7-diethoxyquinazoline (BAY) were designed and synthesized, and the biological studies of [¹²⁵I]PHY and [¹²⁵I]BAY were performed to evaluate these new ligands as in vivo tumor diagnosis agents.

Methods

PHY and BAY were synthesized according to previous reports. The EGFR-TK inhibitory potency of these new compounds was measured and compared to other EGFR-TK inhibitors. Radiolabeled [¹²⁵I]PHY and [¹²⁵I]BAY were synthesized by an iododestannylation reaction. Biodistribution studies of these radioligands were conducted in normal mice and tumor-bearing mice. Furthermore, selectivity and binding characteristics of [¹²⁵I]PHY were analyzed by in vitro blocking studies and a binding assay.

Results

The new derivatives were found to have high inhibitory potency against EGFR-TK (PHY: IC₅₀?=?12.7?±?7.2?nM, BAY: IC₅₀?=?51.0?±?8.9?nM). [¹²⁵I]PHY and [¹²⁵I]BAY were conveniently synthesized from tributylstannyl precursors. In in vivo biodistribution studies, [¹²⁵I]PHY and [¹²⁵I]BAY were observed to have lower uptake in the stomach, an indication of deiodination, than [¹²⁵I]m-IPQ. Moreover, [¹²⁵I]PHY showed high uptake and prolonged retention in tumors and low accumulation in blood and muscle tissue resulting in a good tumor-to-blood ratio (0.94?C1.50) and tumor-to-muscle ratio (1.02?C1.95). The EGFR-TK selectivity of [¹²⁵I]PHY was confirmed by pretreatment experiments with specific EGFR-TK inhibitors.

Conclusion

New radioiodinated quinazoline derivatives were synthesized, which were found to have improved in vivo stability. In particular, [¹²⁵I]PHY showed higher tumor accumulation than the other ligands which was indicative of selective binding to EGFR-TK. These desirable characteristics for [¹²⁵I]PHY suggest that the ¹²³I-labeled counterpart, [¹²³I]PHY, could be a possible candidate for cancer diagnosis radiopharmaceutical.     

Iodo-QNB cortical binding and brain perfusion: effects of a cholinergic basal forebrain lesion in the rat

This study deals with the question of whether in vivo application of [125I]iodo-quinuclidinyl-benzilate (QNB) is able to demonstrate changes in cortical muscarinic receptor density induced by a cholinergic immunolesion of the rat basal forebrain cholinergic system, and whether the potential effects on IQNB distribution in vivo are also associated with effects on regional cerebral perfusion. Immunolesioned and control animals were injected with (R,S) [125]iodo-QNB and with [99mTc]-d,l-hexamethylpropyleneamine oxime (HMPAO). The cerebral distribution of both tracers was imaged using double tracer autoradiography. Impaired cholinergic transmission was paralleled by a 10-15% increase of [125I]iodo-QNB binding in the regions of cortex and hippocampus. The local cerebral blood flow remained unchanged after cholinergic lesion.     

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion

To clarify whether muscarinic acetylcholine receptor (mAChR) binding can be a viable muscarinic neuronal marker which provides therapeutic information different from perfusional information in global brain, we evaluated the discrepancy between the distribution of cerebral blood flow (CBF), mAChR and its live subtypes of messenger ribonucleic acid (mRNA) in the acute (n=9) and chronic (n=8) phases of a middle cerebral artery (MCA) occlusion model and in sham-operated controls (n=6). In the acute phase, regional CBF was markedly reduced in the MCA territory, whereas mAChR was not reduced and the mRNA was reduced only slightly. In the chronic phase, mAChR was reduced markedly in the infarcted lesion and the mRNA was also reduced. The mAChR was slightly reduced in the ipsilateral substantia nigra and pouline nucleus because of remote effects; however, regional CBF in the substantia nigra was slightly increased and did not change in the pontine nucleus. The discrepancy between CBF and mAChR was clarified, and the tendency toward a reduction in mRNA in the acute ischaemic region without a reduction in mAChR suggested the presence of cholinergic neurons which were viable but hypometabolic. It is concluded that mAChR imaging may be of value for the assessment of the viable cholinergic neuron density in vivo.     

Development and evaluation of muscarinic cholinergic receptor ligands n-[c]ethyl-4-piperidyl benzilate and n-[c]propyl-4-piperidyl benzilate: a pet study in comparison with n-[c]methyl-4-piperidyl benzilate in the conscious monkey brain

The muscarinic cholinergic receptor ligands N-[¹¹C]ethyl-4-piperidyl benzilate (4-EPB) and N-[¹¹C]propyl-4-piperidyl benzilate (4-PPB) were developed and evaluated in comparison with N-[¹¹C]methyl-4-piperidyl benzilate (4-MPB) in the conscious monkey brain using positron emission tomography (PET). Time-activity curves of [¹¹C]4-EPB, unlike [¹¹C]4-MPB, showed peaks within 91 min in regions rich in muscarinic receptors. [¹¹C]4-PPB showed no specific binding even in the regions rich in these receptors. These observation demonstrated that increases in [¹¹C]alkyl chain length could alter the kinetic properties of receptor ligands for PET.     

Radioiodination and preclinical evaluation of 4-benzyl-1-(3-[<Superscript>125</Superscript>I]-iodobenzylsulfonyl)piperidine as a breast tumor imaging tracer in mouse

Objective

4-Benzyl-1-(3-iodobenzylsulfonyl)piperidine, 4-B-IBSP, has shown high-binding affinity to both sigma (σ) receptors in our previous work. In current study, radiolabeling and preclinical evaluation of 4-benzyl-1-(3-[¹²⁵I]-iodobenzylsulfonyl)piperidine, 4-B-[¹²⁵I]IBSP, in human ductal breast carcinoma (T47D) cells and in breast adenocarcinoma-bearing BALB/c mice are described.

Methods

Radioiodination of this new σ ligand was performed by a palladium-catalyzed stannylation approach followed by oxidative iododestannylation reaction using Iodo-Gen. Competition-binding assays for binding of 4-B-[¹²⁵I]IBSP to guinea pig brain membranes and to T47D cells were performed with known σ ligands. The selectivity and binding characteristics (B _max and K _d) were analyzed. In vitro stability and in vivo blood metabolism studies were also evaluated. Moreover, biodistribution studies were performed in normal and into the tumor-bearing mice at interval time points post-injection (p.i.). Both in vitro and in vivo blockade experiments were done in the presence of the σ receptors blocking agents.

Results

Radioiodinated ligand was obtained in high yield and high specific activity. The σ inhibition constants (K _i, nM) for 4-(3-iodobenzyl)-1-(benzylsulfonyl)piperazine (4-IBBSPz), (+)-pentazocine, haloperidol, DTG, and 4-B-IBSP were 1.37?±?0.19, 3.90?±?0.77, 2.69?±?0.33, 30.62?±?2.01, and 0.61?±?0.05, respectively. 4-B-[¹²⁵I]IBSP bound to σ receptor sites preferably to very high-affinity binding sites on T47D cells. The radioligand showed acceptable in vitro and in vivo stabilities in the blood pool. However, in vivo biodistribution studies in normal Swiss albino mice revealed fast clearance of 4-B-[¹²⁵I]IBSP from blood and the other normal organs. Biodistribution experiments of 4-B-[¹²⁵I]IBSP in breast adenocarcinoma tumor-bearing BALB/c mice showed a relatively high tumor uptake at 30 min p.i. (4.13?±?0.95) that reaches to 1.57?±?0.24 even after 240 min p.i. A pre-injection of 4-B-IBSP and haloperidol with 4-B-[¹²⁵I]IBSP resulted in 36–57% decrease in activity in the tumor, liver, and brain at 60 min p.i.

Conclusions

The high affinity of 4-B-[¹²⁵I]IBSP to σ receptor-binding sites, its relatively high uptake, and preferential retention in the tumor as well as an increasing trend observed in the tumor to blood and in the tumor to muscle ratios suggests that an iodine-123 labeled counterpart, 4-B-[¹²³I]IBSP, would be a promising σ radioligand for pursuing further studies to assess its potential for breast tumors imaging with SPECT.

     

A useful EGFR-TK ligand for tumor diagnosis with SPECT: development of radioiodinated 6-(3-morpholinopropoxy)-7-ethoxy-4-(3′-iodophenoxy)quinazoline

Objective

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, radioiodinated 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) was reported to possess good characteristics as a tumor imaging agent. We have explored the feasibility of developing tumor diagnosis ligands superior to radioiodinated PHY.

Methods

New phenoxyquinazoline derivatives were designed with various side chains introduced to the 6th position of PHY. The IC₅₀ values of the new derivatives to interrupt EGFR-TK phosphorylation were evaluated and compared to well-known EGFR-TK inhibitors. Tumor uptake studies of the new ¹²⁵I-labeled derivatives were conducted with A431 tumor-bearing mice. Selectivity and binding characteristics were analyzed by in vitro blocking studies and a binding assay. Furthermore, SPECT/CT scans were performed using A431 tumor-bearing mice.

Results

Six quinazoline derivatives were designed and synthesized, and among these, 6a–d were found to have relatively high EGFR-TK inhibitory potency. In tumor uptake studies, [¹²⁵I]6a ([¹²⁵I]PYK) was found to have the highest tumor uptake and longest retention in tumors. In contrast, [¹²⁵I]PYK was rapidly cleared from peripheral tissues, resulting in a high tumor-to-tissue ratio 24 h after injection. Moreover, the EGFR-TK selectivity of [¹²⁵I]PYK was confirmed by pretreatment experiments with specific EGFR-TK inhibitors. Furthermore, [¹²⁵I]PYK provided clear SPECT images of tumors.

Conclusions

Radioiodinated PYK, one of the newly synthesized quinazoline derivatives, was found to be a desirable ligand for EGFR-TK SPECT imaging. [¹²⁵I]PYK showed high tumor accumulation and selective EGFR-TK binding and also succeeded in delivering high contrast imaging of tumors. These favorable characteristics of [¹²⁵I]PYK suggest that the ¹²³I-labeled counterpart, [¹²³I]PYK, would have great potential for diagnostic SPECT tumor imaging.     

Demonstration of a reduction in muscarinic receptor binding in early Alzheimer's disease using iodine-123 dexetimide single-photon emission tomography

Decreased muscarinic receptor binding has been suggested in single-photon emission tomography (SPET) studies of Alzheimer's disease. However, it remains unclear whether these changes are present in mildly demented patients, and the role of cortical atrophy in receptor binding assessment has not been investigated. We studied muscarinic receptor binding normalized to neostriatum with SPET using [¹²³I]4-iododexetimide in five mildly affected patients with probable Alzheimer's disease and in five age-matched control subjects. Region of interest (ROI) analysis was performed in a consensus procedure blind to clinical diagnosis using matched magnetic resonance (MRI) images. Cortical atrophy was assessed by calculating percentages of cerebrospinal fluid in each ROI. An observer study with three observers was conducted to validate this method. Alzheimer patients showed statistically significantly less [¹²³I]4-iododexetimide binding in left temporal and right temporo-parietal cortex compared with controls, independent of age, sex and cortical atrophy. Mean intea-observer variability was 3.6% and inter-observer results showed consistent differences in [¹²³I]4-iododexetimide binding between observers. However, differences between patients and controls were comparable among observers and statistically significant in the same regions as in the consensus procedure. Using an MRI-SPET matching technique, we conclude that [¹²³I]4-iododexetimide binding is reduced in patients with mild probable. Alzheimer's disease in areas of temporal and temporoparietal cortex.     

A 4-methyl-substituted <Emphasis Type="Italic">meta</Emphasis>-iodobenzylguanidine analogue with prolonged retention in human neuroblastoma cells

Purpose As a part of our efforts to develop a meta-iodobenzylguanidine (MIBG) analogue with improved characteristics for the diagnosis and treatment of neuroendocrine tumours, 3-[¹³¹I]iodo-4-methyl-benzylguanidine ([¹³¹I]MeIBG) has been developed. The purpose of this study was to evaluate [¹³¹I]MeIBG in vitro using the uptake-1 positive SK-N-SH neuroblastoma cell line and in vivo in normal mice and mice bearing human neuroblastoma xenografts.Methods The ability of SK-N-SH human neuroblastoma cells to retain [¹³¹I]MeIBG in vitro over a period of 4 days, in comparison to [¹²⁵I]MIBG, was determined by a paired-label assay. Paired-label biodistributions of [¹³¹I]MeIBG and [¹²⁵I]MIBG were performed in normal mice as well as in athymic mice bearing SK-N-SH and IMR-32 human neuroblastoma xenografts.Results Retention of [¹³¹I]MeIBG by SK-N-SH cells in vitro was increased by factors of 1.2, 1.5, 2.0, 2.5 and 3.1 compared with [¹²⁵I]MIBG at 8, 24, 48, 72 and 96 h, respectively. In normal mice, the uptake of [¹³¹I]MeIBG in the heart was similar to that of [¹²⁵I]MIBG at 1 and 4 h; in contrast, myocardial uptake of [¹³¹I]MeIBG was 1.6-fold higher than that of [¹²⁵I]MIBG (p<0.05) at 24 h. When mice were pre-treated with the uptake-1 inhibitor desipramine (DMI), the heart uptake of both tracers was reduced to about half that in untreated controls at 1 h post injection (p<0.05). The hepatic uptake of [¹³¹I]MeIBG was two- to threefold lower than that of [¹²⁵I]MIBG. On the other hand, blood levels of [¹³¹I]MeIBG were substantially higher (up to sixfold), especially at early time points. Uptake of [¹³¹I]MeIBG in heart and tumour at 1 h in the murine SK-N-SH model was specific and comparable to that of [¹²⁵I]MIBG. However, [¹³¹I]MeIBG uptake was 1.6- to 1.7-fold lower than that of [¹²⁵I]MIBG over 4–48 h. While the uptake of both tracers in IMR32 xenografts was similar, it was not uptake-1 mediated.Conclusion Introduction of a methyl group at the 4-position of MIBG seems to be advantageous in terms of higher tumour retention in vitro and lower hepatic uptake in vivo. However, the slower blood clearance of MeIBG may be problematic for some applications.     

Synthesis, rodent biodistribution, dosimetry, metabolism, and monkey images of carbon-11-labeled (+)-2 alpha-tropanyl benzilate: a central muscarinic receptor imaging agent.

Muscarinic cholinergic receptors (mAChR) are abundant in the brain, and the mAChR system mediates many aspects of brain function. There is evidence of alterations in muscarinic binding in degenerative brain disorders. A muscarinic receptor radioligand, carbon-11-(+)-2 alpha-tropanyl benzilate ([11C]TRB), has been prepared through N-[11C]methylation of N-desmethyl TRB, and evaluated in rodents and primates. Full body biodistribution in rats has been determined and the expected human dosimetry calculated. Comparisons with [11C]scopolamine in rats showed 2-6 times greater brain uptake of [11C] TRB. Highly specific and saturable binding of [11C]TRB in the striatum and cortex was demonstrated by greater than 85% blockade of uptake following QNB or scopolamine pretreatment. Striatum/cerebellum ratios in mice at 60 min exceeded 12.6. TLC analysis of rat tissues showed the absence of 11C-metabolites in brain and heart, and a rapid solid phase C-18 Sep-Pak method found that unmetabolized plasma [11C]TRB in monkeys fell from 81% at 5 min to 48% at 80 min. Finally, brains of living primates have been imaged using PET and [11C]TRB; regional localization was consistent with muscarinic receptor distribution. These results represent intermediate steps in the development of [11C]TRB for quantification of central muscarinic receptors in man.     

Iodine-123N- methyl-4-iododexetimide: a new radioligand for single-photon emission tomographic imaging of myocardial muscarinic receptors

Cardiac muscarinic receptor ligands suitable for positron emission tomography have previously been characterised. Attempts to develop radioligands of these receptors suitable for single-photon emission tomographic (SPET) imaging have not been successful due to high lung retention and high non-specific binding of previously investigated potential tracers. The purpose of this study was to evaluate the biodistribution and in vivo imaging characteristics of a new radiopharmaceutical, [¹²³I]N-methyl-4-iododexetimide. Biodistribution studies performed in rats showed high cardiac uptake (2.4% ID/g) 10 min after injection with a heart to lung activity ratio of 5:1. Specificity and stereo selectivity of cardiac binding were demonstrated using blocking experiments in rats. Dynamic imaging studies in anaesthetised greyhounds demonstrated rapid and high myocardial uptake and low lung binding with stable heart to lung activity ratios of >2.5:1 between 10 and 30 min, making SPET imaging feasible. Administration of an excess of an unlabelled muscarinic antagonist, methyl-quinuclidinyl benzylate rapidly displaced myocardial activity to background levels and the pharmacologically inactive enantiomer, [¹²³I]N-methyl-4-iodolevetimide, had no detectable cardiac uptake, indicating specific and stereoselective muscarinic receptor binding. SPET revealed higher activity in the inferior than in the anterior wall, this being consistent with previously described regional variation of cardiac parasympathetic innervation. [¹²³I]N-methyl-4-iododexetimide shows promise as an imaging agent for muscarinic receptor distribution in the heart and may be helpful in evaluating diverse cardiac diseases associated with altered muscarinic receptor function, including heart failure and diabetic heart disease.     

Characterization of FlipIDAM as a SERT-selective SPECT imaging agent

IntroductionBiological evaluation of [¹²⁵I]FlipIDAM (2-((2-((dimethylamino)methyl)-4-iodophenyl)thio)phenyl)methanol ([¹²⁵I]4), a new single-photon emission computed tomography (SPECT) radioligand for imaging the serotonin transporter (SERT) which displayed improved in vivo kinetics for mapping SERT binding sites in the brain.MethodsIn vitro binding studies of [¹²⁵I]4 were performed with membrane homogenates of LLC-PK1 cells stably transfected and overexpressing one of the monoamine transporter (SERT, DAT or NET) and rat cortical homogenates. Biodistribution and ex vivo autoradiography studies were carried out in rats. In vivo competition experiments were evaluated to determine the SERT selectivity of [¹²⁵I]4 vs. [¹²⁵I]IDAM ([¹²⁵I]1).ResultsIn vitro binding studies of 4 showed excellent binding affinity (K_i,SERT = 0.90 ± 0.05 nM) and excellent selectivity over the other monoamine transporters (100 fold and > 4000 fold for NET and DAT respectively). Scatchard analysis of saturation binding of [¹²⁵I]4 to rat cortical homogenates gave a K_d value of 0.5 ± 0.09 nM and a B_max value of 801.4 ± 58.08 fmol/mg protein. The biodistribution study showed rapid high brain uptake (3.09 ± 0.11% dose/organ at 2 min) and a good target to non-target ratio (hypothalamus to cerebellum) at 30 min (2.62) compared to [¹²⁵I]1 (2.19). Ex vivo autoradiography showed that FlipIDAM localizes in accordance with SERT distribution patterns in the brain. In vivo and ex vivo competition experiments with specific and non-specific SERT compounds also showed that [¹²⁵I]4 binds specifically to SERT rich regions.ConclusionsThe biological evaluation of [¹²⁵I]4 demonstrates that [¹²³I]4 would be a good candidate for SPECT imaging of SERT.     

SPET imaging of central muscarinic acetylcholine receptors with iodine-123 labelled E-IQNP and Z-IQNP

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP) is a muscarinic acetylcholine receptor (mAChR) antagonist and the racemic ligand contains eight stereoisomers. In a single-photon emission tomography (SPET) study in monkeys we recently confirmed that [123I]E-(R,R)-IQNP ([123I]E-IQNP) is a radioligand with modest selectivity for the M1 and M4 subtypes, whereas [123I]Z-(R,R)-IQNP ([123I]Z-IQNP) is non-subtype selective. In the present SPET study, E- and Z-IQNP were examined in human subjects. SPET examination was performed on three male subjects after i.v. injection of [123I]E-IQNP and in another three after i.v. injection of [123I]Z-IQNP. The binding potential (BP) for [123I]E-IQNP was calculated using several quantitative approaches with the cerebellum as a reference region. High-performance liquid chromatography was used to measure radioligand metabolism in plasma. Following [123I]E-IQNP, the radioactivity was high in the neocortex and striatum, intermediate in the thalamus and low in the pons and cerebellum, which is consistent with the rank order for the regional density of M1 and M4 subtypes in vitro. For all regions, peak equilibrium was identified within the 48-h data acquisition. The simplified reference tissue approach using SPET data from 0 to 48 h was the most reliable in this limited series of subjects. Following injection of [123I]Z-IQNP, radioactivity was high in the neocortex and striatum, intermediate in the thalamus and pons and low in the cerebellum, which is in agreement with the density of M1, M2 and M4 subtypes as measured in vitro. Quantitative analyses provided indirect support for specific M2 binding of Z-IQNP in the cerebellum. The high selectivity of [123I]E-IQNP for M1 and M4 receptors allowed the use of cerebellum as a reference region devoid of specific binding, and may be advantageous for applied clinical studies of M1 and M4 receptors binding in man. [123I]Z-IQNP has potential for exploration of M2 receptor binding in the cerebellum.     

FlipADAM: a potential new SPECT imaging agent for the serotonin transporter

IntroductionSingle photon emission computed tomography (SPECT) imaging of the serotonin transporter (SERT) in the brain is a useful tool for examining normal physiological functions and disease states involving the serotonergic system. The goal of this study was to develop an improved SPECT radiotracer with faster kinetics than the current leading SPECT tracer, [¹²³I]ADAM, for selective SERT imaging.MethodsThe in vitro binding affinities of (2-(2′-((dimethylamino)methyl)-4′-iodophenylthio)benzenamine) (FlipADAM) (1c), were determined using Hampshire pig kidney cells stably overexpressing the serotonin, norepinephrine (NET) or dopamine transporter (DAT). Localization of [¹²⁵I]FlipADAM (1c) was evaluated through biodistribution and autoradiography in male Sprague Dawley rats, and the specificity of binding was assessed by injecting selective SERT or NET inhibitors prior to [¹²⁵I]FlipADAM (1c).ResultsFlipADAM (1c) displayed a high binding affinity for SERT (K_i=1.0 nM) and good selectivity over NET and DAT binding (43-fold and 257-fold, respectively). [¹²⁵I]FlipADAM (1c) successfully penetrated the blood brain barrier, as evidenced by the brain uptake at 2 min (1.75% dose/g). [¹²⁵I]FlipADAM(1c) also had a good target to non-target (hypothalamus/cerebellum) ratio of 3.35 at 60 min post-injection. In autoradiography studies, [¹²⁵I]FlipADAM (1c) showed selective localization in SERT-rich brain regions such as the thalamic nuclei, amygdala, dorsal raphe nuclei and other areas.Conclusion[¹²⁵I]FlipADAM (1c) exhibited faster clearance from the brain and time to binding equilibrium when compared to [¹²⁵I]2-(2′-((dimethylamino)methyl)-phenylthio)-5-iodophenylamine [¹²⁵I]ADAM (1b) and a higher target to non-target ratio when compared to [¹²⁵I]5-iodo-2-(2′-((dimethylamino)methyl)-phenylthio)benzyl alcohol [¹²⁵I]IDAM (1a). Therefore, [¹²³I]FlipADAM (1c) may be an improved SPECT tracer for imaging SERT.