123I]Epidepride neuroimaging of dopamine D2/D3 receptor in chronic MK-801-induced rat schizophrenia model

Purpose[¹²³I]Epidepride is a radio-tracer with very high affinity for dopamine D₂/D₃ receptors in brain. The importance of alteration in dopamine D₂/D₃ receptor binding condition has been wildly verified in schizophrenia. In the present study we set up a rat schizophrenia model by chronic injection of a non-competitive NMDA receptor antagonist, MK-801, to examine if [¹²³I]epidepride could be used to evaluate the alterations of dopamine D₂/D₃ receptor binding condition in specific brain regions.MethodRats were given repeated injection of MK-801 (dissolved in saline, 0.3 mg/kg) or saline for 1 month. Afterwards, total distance traveled (cm) and social interaction changes were recorded. Radiochemical purity of [¹²³I]epidepride was analyzed by Radio-Thin-Layer Chromatography (chloroform: methanol, 9:1, v/v) and [¹²³I]epidepride neuroimages were obtained by ex vivo autoradiography and small animal SPECT/CT. Data obtained were then analyzed to determine the changes of specific binding ratio.ResultChronic MK-801 treatment for a month caused significantly increased local motor activity and induced an inhibition of social interaction. As shown in [¹²³I]epidepride ex vivo autoradiographs, MK-801 induced a decrease of specific binding ratio in the striatum (24.01%), hypothalamus (35.43%), midbrain (41.73%) and substantia nigra (37.93%). In addition, [¹²³I]epidepride small animal SPECT/CT neuroimaging was performed in the striatum and midbrain. There were statistically significant decreases in specific binding ratio in both the striatum (P < .01) and midbrain (P < .05) between the saline and MK-801 group.ConclusionThese results suggest that [¹²³I]epidepride is a useful radio-tracer to reveal the alterations of dopamine D₂/D₃ receptor binding in a rat schizophrenia model and is also helpful to evaluate therapeutic effects of schizophrenia in the future.     

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.     

In vivo evaluation of [123I]mZIENT as a SPECT radioligand for the serotonin transporter

IntroductionIn vivo imaging of the serotonin transporter continues to be a valuable tool in drug development and in monitoring diseases that alter serotonergic function. The purposes of this study were to: 1) evaluate the test/retest reproducibility of [¹²³I] 2β-Carbomethoxy-3β-(3′-((Z)-2-iodoethenyl)phenyl)nortropane ([¹²³I]mZIENT); and 2) to assess displacement of [¹²³I]mZIENT following administration of SERT specific drugs.MethodsSix female baboons (Papio anubis) were scanned following i.v. administration of [¹²³I]mZIENT. The regional binding potential (BP_nd) was determined using a simplified reference tissue model, with the cerebellum used as a reference region. The test/retest reproducibility of BP_nd was determined following repeated injection of [¹²³I]mZIENT on a different day. To assess the displacement of [¹²³I]mZIENT from SERT, citalopram (0.01–5 mg/kg) or sertraline (0.01–0.5 mg/kg) was given as iv bolus at ~ 4 h following administration of [¹²³I]mZIENT.ResultsThe test/retest variability of BP_nd was less than 10% for all SERT-rich brain regions. Estimates of ED50 for displacement of [¹²³I]mZIENT in SERT-rich regions were consistent with previous reports for the [¹¹C] analog of [¹²³I]mZIENT. Both citalopram and sertraline displaced [¹²³I]mZIENT from SERT in a dose-dependent manner, with maximal observed displacements of greater than 80% in the diencephalon and greater than 75% in brainstem for both citalopram and sertraline.Conclusions[¹²³I] mZIENT demonstrates good test–retest reproducibility; and initial displacement studies suggest that this compound is highly selective for SERT. Overall, this radioligand has favorable characteristics for use in drug development studies and/or longitudinal studies interrogating SERT.     

Dopamine D2/D3 receptor binding of [123I]epidepride in risperidone-treatment chronic MK-801-induced rat schizophrenia model using nanoSPECT/CT neuroimaging

IntroductionEpidepride is a compound with an affinity in picomolar range for D₂/D₃ receptors. The aim of this work was designed to investigate the diagnostic possibility of [¹²³I]epidepride imaging platform for risperidone-treatment chronic MK-801-induced rat schizophrenia model.MethodsRats received repeated administration of MK-801 (dissolved in saline, i.p., 0.3 mg/kg/day) or saline for 4 weeks. After 1-week administration of MK-801, rats in MK-801 + risperidone group received risperidone (0.5 mg/kg/day) intraperitoneally 15 min prior to MK-801 administration for the rest of 3-week treatment. We obtained serial [¹²³I]epidepride neuroimages from nanoSPECT/CT and evaluated the alteration of specific binding in striatum and midbrain.ResultsRisperidone reversed chronic MK-801-induced decrease in social interaction duration. IHC and ELISA analysis showed consistent results that chronic MK-801 treatment significantly decreased striatal and midbrain D₂R expression but repeated risperidone administration reversed the effect of MK-801 treatment. In addition, [¹²³I]epidepride nanoSPECT/CT neuroimaging revealed that low specific [¹²³I]epidepride binding ratios caused by MK-801 in striatum and midbrain were statistically alleviated after 1- and 2-week risperidone administration, respectively.ConclusionsWe established a rat schizophrenia model by chronic MK-801 administration for 4 weeks. [¹²³I]Epidepride nanoSPECT neuroimaging can trace the progressive alteration of D₂R expression in striatum and midbrain caused by long-lasting MK-801 treatment. Besides diagnosing illness stage of disease, [¹²³I]epidepride can be a useful tool to evaluate therapeutic effects of antipsychotic drug in chronic MK-801-induced rat schizophrenia model.     

Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

ObjectiveThe goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the α_vβ₃ integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process.MethodsA hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing an RGD motif for binding to the α_vβ₃integrin, a metal chelator (DOTA) for radiolabeling with [⁶⁴Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [¹²³I] was synthesized, labeled with [⁶⁴Cu] and [¹²³I], and evaluated in vitro as a potential imaging agent.ResultsThe peptide was synthesized and labeled with [⁶⁴Cu] and [¹²³I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of > 98%. c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for α_vβ₃ integrins (Kd = 83.4 + 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2.ConclusionsThese results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity.     

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.     

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.     

[11 C]Rhodamine-123: Synthesis and biodistribution in rodents

IntroductionRhodamine-123 is a known substrate for the efflux transporter, P-glycoprotein (P-gp). We wished to assess whether rhodamine-123 might serve as a useful substrate for developing probes for imaging efflux transporters in vivo with positron emission tomography (PET). For this purpose, we aimed to label rhodamine-123 with carbon-11 (t_1/2 = 20.4 min) and to study its biodistribution in rodents.Methods[¹¹ C]Rhodamine-123 was prepared by treating rhodamine-110 (desmethyl-rhodamine-123) with [¹¹ C]methyl iodide. The biodistribution of this radiotracer was studied with PET in wild-type mice and rats, in efflux transporter knockout mice, in wild-type rats pretreated with DCPQ (an inhibitor of P-gp) or with cimetidine (an inhibitor of organic cation transporters; OCT), and in P-gp knockout mice pretreated with cimetidine. Unchanged radiotracer in forebrain, plasma and peripheral tissues was also measured ex vivo at 30 min after radiotracer administration to wild-type and efflux transporter knockout rodents.Results[¹¹ C]Rhodamine-123 was obtained in 4.4% decay-corrected radiochemical yield from cyclotron-produced [¹¹ C]carbon dioxide. After intravenous administration of [¹¹ C]rhodamine-123 to wild-type rodents, PET and ex vivo measurements showed radioactivity uptake was very low in brain, but relatively high in some other organs such as heart, and especially liver and kidney. Inhibition of P-gp increased uptake in brain, heart, kidney and liver, but only by up to twofold. Secretion of radioactivity from kidney was markedly reduced by OCT knockout or pretreatment with cimetidine.Conclusions[¹¹ C]Rhodamine-123 was unpromising as a PET probe for P-gp function and appears to be a strong substrate of OCT in kidney. Cimetidine appears effective for blocking OCT in kidney in vivo.     

Quantification of GABAA receptors in the rat brain with [123I]Iomazenil SPECT from factor analysis-denoised images

PurposeIn vivo imaging of GABA_A receptors is essential for the comprehension of psychiatric disorders in which the GABAergic system is implicated. Small animal SPECT provides a modality for in vivo imaging of the GABAergic system in rodents using [¹²³I]Iomazenil, an antagonist of the GABA_A receptor. The goal of this work is to describe and evaluate different quantitative reference tissue methods that enable reliable binding potential (BP) estimations in the rat brain to be obtained.MethodsFive male Sprague–Dawley rats were used for [¹²³I]Iomazenil brain SPECT scans. Binding parameters were obtained with a one-tissue compartment model (1TC), a constrained two-tissue compartment model (2TC_c), the two-step Simplified Reference Tissue Model (SRTM2), Logan graphical analysis and analysis of delayed-activity images. In addition, we employed factor analysis (FA) to deal with noise in data.ResultsBP_ND obtained with SRTM2, Logan graphical analysis and delayed-activity analysis was highly correlated with BP_F values obtained with 2TC_c (r = 0.954 and 0.945 respectively, p < 0.0001). Equally significant correlations were found between values obtained with 2TC_c and SRTM2 in raw and FA-denoised images (r = 0.961 and 0.909 respectively, p < 0.0001). Scans of at least 100 min are required to obtain stable BP_ND values from raw images while scans of only 70 min are sufficient from FA-denoised images. These images are also associated with significantly lower standard errors of 2TC_c and SRTM2 BP values.ConclusionReference tissue methods such as SRTM2 and Logan graphical analysis can provide equally reliable BP_ND values from rat brain [¹²³I]Iomazenil SPECT. Acquisitions, however, can be much less time-consuming either with analysis of delayed activity obtained from a 20-minute scan 50 min after tracer injection or with FA-denoising of images.     

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: Influence of isomeric substitution on radiolabeling and target cell residualization

IntroductionN-succinimidyl 4-guanidinomethyl-3-[^⁎I]iodobenzoate ([^⁎I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [¹³¹I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[¹³¹I]iodobenzoate (iso-[¹³¹I]SGMIB) wherein this bulky group was moved from ortho to meta position.MethodsBoc₂-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc₂-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors — trastuzumab (Tras) and a nanobody 5 F7 (Nb) — were labeled using iso-[^⁎I]SGMIB and [^⁎I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.ResultsWhen the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc₂-iso-[¹³¹I]SGMIB were significantly higher than those for Boc₂-[¹³¹I]SGMIB (70.7 ± 2.0% vs 56.5 ± 5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[¹³¹I]SGMIB than with [¹³¹I]SGMIB (Nb, 33.1 ± 7.1% vs 28.9 ± 13.0%; Tras, 45.1 ± 4.5% vs 34.8 ± 10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5 F7 Nb indicated similar residualizing capacity over 6 h; however, at 24 h, radioactivity retained intracellularly for iso-[¹³¹I]SGMIB-Nb was lower than for [¹²⁵I]SGMIB-Nb (46.4 ± 1.3% vs 56.5 ± 2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[¹²⁵I]SGMIB and [¹³¹I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [¹³¹I]SGMIB-Tras.ConclusionGiven the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.     

In vivo evaluation of [123I]MNI-420: A novel single photon emission computed tomography radiotracer for imaging of adenosine 2A receptors in brain

IntroductionDysregulation of adenosine 2A (A_2A) receptor function in brain has been implicated in multiple psychiatric and neurodegenerative disorders, including schizophrenia and Parkinson's disease, making the development of an imaging agent to study A_2A receptors in both healthy brain and disease states desirable. In this study, [¹²³I]MNI-420 was evaluated as a potential single photon emission computed tomography (SPECT) radiotracer for imaging A_2A receptors in brain.MethodsTwo adult male monkeys (Macaca fascicularis) and three adult female baboons (Papio anubis) were anesthetized and imaged on Neurofocus SPECT cameras. Baboons underwent baseline and displacement studies using varying doses of caffeine (2.0–20 mg/kg). Baseline and pre-blocking experiments with multiple doses of preladenant (0.01–1.2 mg/kg), a highly selective A_2A antagonist, were performed in cynomolgus monkeys.ResultsFollowing bolus intravenous (i.v.) injection, [¹²³I]MNI-420 rapidly entered the non-human primate brain. The regional brain accumulation of [¹²³I]MNI-420 matched the known distribution of A_2A receptors in brain (highest in the striatum). Striatum to cerebellum ratios and binding potentials of around 3.0–3.5 and 2.0–2.5, respectively, were measured in monkey and baboon brain. A dose-dependent occupancy was observed following i.v. injection of caffeine at pseudo-equilibrium conditions during displacement experiments. Pre-treatment with preladenant blocked specific binding in A_2A rich regions in a dose-dependent fashion.ConclusionsThe data indicate that [¹²³I]MNI-420 holds promise as a SPECT radiotracer for imaging A_2A receptors in brain and further evaluation is warranted, in order to determine its utility as a SPECT radiotracer for imaging of A_2A in brain.     

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

IntroductionTwo 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA_A/benzodiazepine receptor by comparing them to [¹¹C]flumazenil in rhesus monkey.MethodsRadiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [¹⁸F]AH114726 and [¹⁸F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results[¹⁸F]GEH120348 and [¹⁸F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [¹⁸F]AH114726 and [¹⁸F]GEH120348 were determined in a non-human primate and directly compared with [¹¹C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [¹¹C]flumazenil and the known concentrations of GABA_A/benzodiazepine receptors in the monkey brain. [¹⁸F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [¹¹C]flumazenil. In contrast, [¹⁸F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.ConclusionThe ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [¹¹C]flumazenil, support that [¹⁸F]AH114726 is a suitable option for imaging the GABA_A receptor.     

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

IntroductionWith a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors.Methods5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N^?-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N^α-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-d-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations.ResultsThe radiochemical yields for Iodogen and [¹³¹I]IB-Mal-d-GEEEK labeling were 83.6 ± 5.0% (n = 10) and 59.6 ± 9.4% (n = 15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-d-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65 ± 0.61% ID/g vs. 2.92 ± 0.24% ID/g at 8 h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios.ConclusionsTaken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-d-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²⁹I) and PET imaging (¹²⁴I) of patients with HER2-expressing tumors.     

[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging

PurposeCXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [^99mTc]O₂-AMD3100 as a potential SPECT tracer for imaging of CXCR4.MethodAMD3100 was labelled with [^99mTc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100.ResultsAMD3100 was labelled with technetium-99 m with a radiochemical yield of > 98%. The tracer was stable in PBS and mouse plasma for at least 6 h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC₅₀ values of 240 ± 10 μM, and 92 ± 5 μM for [¹²⁵I]SDF-1α and [^99mTc]O₂-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20 mg/kg) decreased the uptake in these organs (p < 0.05). [^99mTc]O₂-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p < 0.05).Conclusion[^99mTc]O₂-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [^99mTc O₂-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the non-invasive imaging of CXCR4 receptors.     

Biodistribution and radiation dosimetry in humans of [11C]FLB 457, a positron emission tomography ligand for the extrastriatal dopamine D2 receptor

Purpose[¹¹C]FLB 457, a radioligand with very high affinity and selectivity for dopamine D_2/3 receptors, is used to measure receptor binding in extrastriatal regions showing low density of the receptors. The purpose of this study was to estimate the whole-body biodistribution of radioactivity and the radiation absorbed doses to organs after intravenous injection of [¹¹C]FLB 457 in healthy human subjects.MethodsWhole-body images were acquired for 2 h after an injection of [¹¹C]FLB 457 in six healthy humans. Radiation absorbed doses were estimated by the MIRD scheme implemented in OLINDA/EXM 1.1 software.ResultsOrgans with the longest residence time were the liver, lungs, and brain. The organs with the highest radiation doses were the kidneys, liver, and pancreas. The effective dose delivered by [¹¹C]FLB 457 is 5.9 μSv/MBq, similar to those of other ¹¹C-labeled tracers.ConclusionsThis effective dose would allow multiple scans in the same individual based on prevailing maximum recommended-dose guidelines in the USA and Europe.     

Evaluation of metabolism,plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

Introduction(−)-[¹⁸ F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α₄β₂ receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.MethodsPlasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min.ResultsA fraction of 15% ± 2% of (−)-[¹⁸ F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[¹⁸ F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[¹⁸ F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts.Discussion(−)-[¹⁸ F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.     

A heterodimeric [RGD-Glu-[64Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors

IntroductionIn the present study, we describe a ⁶⁴Cu-radiolabeled heterodimeric peptide conjugate for dual α_vβ₃/GRPr (α_vβ₃ integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for α_vβ₃ integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting).MethodsRGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization–mass spectrometry (ESI-MS). Radiolabeling of the conjugate with ⁶⁴Cu produced [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥ 95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model.ResultsA competitive displacement receptor binding assay in human prostate PC-3 cells using ¹²⁵I-[Tyr⁴]BBN as the radioligand showed high binding affinity of [RGD-Glu-[^natCu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09 ± 0.34 nM). A similar assay in human, glioblastoma U87-MG cells using ¹²⁵I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvβ₃ integrin (518 ± 37.5 nM). In vivo studies of [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86 ± 1.01 %ID/g, 1 h post-intravenous injection (p.i.)) and prolonged retention (4.26 ± 1.23 %ID/g, 24 h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4 h p.i.ConclusionsThe favorable pharmacokinetics and enhanced tumor uptake of ⁶⁴Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy.     

Synthesis,metabolite analysis,and in vivo evaluation of [11C]irinotecan as a novel positron emission tomography (PET) probe

IntroductionIrinotecan is a semisynthetic derivative of camptothecin that exerts potent antitumor activity by inhibiting topoisomerase I. Despite much research into the complex pharmacokinetic profile and pharmacodynamic effects of irinotecan, unpredictable and severe side effects are still commonly observed. In this study, we synthesized [¹¹C]irinotecan as a positron emission tomography (PET) probe, performed the metabolite analysis, and evaluated the biodistribution and kinetics of [¹¹C]irinotecan using small animal PET.Methods[¹¹C]Irinotecan was synthesized by two routes using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation. Metabolites in the plasma of mice following injection of [¹¹C] irinotecan were investigated using a combination of column-switching high-performance liquid chromatography (HPLC) and on-line solid-phase extraction (SPE). Whole-body PET studies were conducted in wild-type mice and P-glycoprotein and breast cancer resistance protein (Pgp/Bcrp) knockout mice.Results[¹¹C]Irinotecan was successfully synthesized by the two abovementioned routes. Decay-corrected radiochemical yields based on [¹¹C]carbon dioxide using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation were 8.8 ± 2.0% (n = 8) and 16.9 ± 2.9 % (n = 5), respectively. Metabolite analysis of the plasma of mice following injection of [¹¹C]irinotecan was successfully performed using the column-switching HPLC and on-line SPE combination resulting in greater than 87 % recovery of radioactivity from HPLC. In the PET study in mice, the radioactivity levels in the brain, liver, and small intestine were slightly increased by inhibition of the Pgp/Bcrp function for more than 30 min after [¹¹C]irinotecan injection. This result demonstrated that in vivo behavior of [¹¹C] irinotecan and radioactive metabolites are influenced by the Pgp/Bcrp function.ConclusionPET studies using [¹¹C]irinotecan combined with metabolite analysis may be a useful tool for evaluating irinotecan pharmacokinetics and toxicity.     

2-[18F]Fluoroethanol and 3-[18F]fluoropropanol: facile preparation,biodistribution in mice,and their application as nucleophiles in the synthesis of [18F]fluoroalkyl aryl ester and ether PET tracers

Introduction2-[¹⁸F]Fluoroethoxy and 3-[¹⁸F]fluoropropoxy groups are common moieties in the structures of radiotracers used with positron emission tomography. The objectives of this study were (1) to develop an efficient one-step method for the preparation of 2-[¹⁸F]fluoroethanol (2-[¹⁸F]FEtOH) and 3-[¹⁸F]fluoropropanol (3-[¹⁸F]FPrOH); (2) to demonstrate the feasibility of using 2-[¹⁸F]FEtOH as a nucleophile for the synthesis of 2-[¹⁸F]fluoroethyl aryl esters and ethers; and (3) to determine the biodistribution profiles of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH in mice.Methods2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were prepared by reacting n-Bu₄N[¹⁸F]F with ethylene carbonate and 1,3-dioxan-2-one, respectively, in diethylene glycol at 165 °C and purified by distillation. 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were prepared by coupling 2-[¹⁸F]FEtOH with 4-fluorobenzoyl chloride and 1-fluoro-4-nitrobenzene, respectively. Biodistribution and PET/CT imaging studies of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were performed in normal female Balb/C mice.ResultsThe preparation of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH took 60 min, and their decay-corrected yields were 88.6 ± 2.0% (n = 9) and 65.6 ± 10.2% (n = 5), respectively. The decay-corrected yields for the preparation of 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were 36.1 ± 5.4% (n = 3) and 27.7 ± 10.7% (n = 3), respectively. Imaging/biodistribution studies in mice using 2-[¹⁸F]FEtOH showed high initial radioactivity accumulation in all major organs followed by very slow clearance. On the contrary, by using 3-[¹⁸F]FPrOH, radioactivity accumulated in all major organs was cleared rapidly, but massive in vivo defluorination (31.3 ± 9.57%ID/g in bone at 1 h post-injection) was observed.ConclusionsUsing 2-[¹⁸F]FEtOH/3-[¹⁸F]FPrOH as a nucleophile is a competitive new strategy for the synthesis of 2-[¹⁸F]fluoroethyl/3-[¹⁸F]fluoropropyl aryl esters and ethers. Our biodistribution data emphasize the importance of in vivo stability of PET tracers containing a 2-[¹⁸F]fluoroethyl or 3-[¹⁸F]fluoropropyl group due to high background and high bone uptake resulting from 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH, respectively. This is especially important for their aryl ester derivatives which are prone to in vivo hydrolysis.     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.