Synthesis and biological evaluation of a nonsteroidal bromine-76-labeled androgen receptor ligand 3-[76Br]bromo-hydroxyflutamide

INTRODUCTION: Androgen receptors (ARs) are overexpressed in normal tissues and in most primary and metastatic prostate cancers. In our efforts to develop a nonsteroidal AR-specific imaging agent, we synthesized (+/-)-3-[(76)Br]bromo-hydroxyflutamide ((76)Br-), an analog of hydroxyflutamide, the active metabolite of the AR antagonist ligand flutamide. MATERIALS AND METHODS: (76)Br- was synthesized in three steps, starting with commercially available compounds. Labeling of (76)Br- was achieved through the nucleophilic opening of an epoxide intermediate, and a labeled compound was obtained in high specific activity and good radiochemical yield. RESULTS AND DISCUSSION: (+/-)-3-Bromo-hydroxyflutamide has a significantly higher affinity for ARs compared to hydroxyflutamide, its parent compound. The androgen target-tissue uptake of (76)Br- in diethylstilbestrol-treated male rats was examined; however, AR-mediated uptake was minimal due most likely to the rapid metabolic debromination of the radiolabeled ligand. CONCLUSIONS: This study is part of our first look at a novel class of nonsteroidal AR antagonists as positron emission tomography (PET) imaging agents, which are alternatives to steroidal AR agonist-based imaging agents. Although (76)Br- has a significant affinity for ARs, it showed limited promise as a PET imaging agent because of its poor target-tissue distribution properties.     

Characterization of bromine-76-labelled 5-bromo-6-nitroquipazine for PET studies of the serotonin transporter.

The development of suitable radioligands for brain imaging of the serotonin transporter is of great importance for the study of depression and other affective disorders. The potent and selective serotonin transporter ligand, 5-iodo-6-nitro-2-piperazinylquinoline, has been labelled with iodine-123 and used as a radioligand for single photon emission computerized tomography. To evaluate the potential of the bromine-76-labelled analogue, 5-bromo-6-nitroquipazine, as a radioligand for positron emission tomography (PET), its brain distribution and binding characteristics were examined in rats. In vivo brain distribution and ex vivo autoradiography demonstrated that [76Br]5-bromo-6-nitroquipazine enters the brain rapidly. The regional brain distribution of [76Br]5-bromo-6-nitroquipazine was consistent with the known distribution of serotonin transporters in the midbrain, pons, thalamus, striatum, and neocortex. Specific binding was inhibited by the selective serotonin reuptake inhibitor citalopram. The peripheral metabolism in plasma was rapid, but more than 90% of the radioactivity in brain represented unchanged radioligand 2 h postinjection (p.i.). A preliminary PET study was also performed in a baboon. Following the intravenous injection of [76Br]5-bromo-6-nitroquipazine in a baboon, there was a conspicuous accumulation of radioactivity in thalamus, striatum, and pons. The radioactivity in these brain regions was 1.5 times higher than in the cerebellum at 3 h and 2.5-4 times higher at 24 h. A rapid metabolism of the radioligand in plasma was observed (38% unchanged after 5 min). The results indicate that [76Br]5-bromo-6-nitroquipazine has potential for PET imaging of the serotonin transporter.     

A simple synthesis of bromine-77-labeled alkyl bromides

Bromine-77 labeled primary, secondary and benzylic alkyl bromides have been prepared in no-carrier-added form by the reaction of the corresponding alcohols with Na⁷⁷Br and chlorotrimethylsilane in acetonitrile solution. This represents a new and very simple method for radiobromination, and has been used to prepare ⁷⁷Br-12-bromododecanoic acid useful for studying myocardial metabolism.     

Evaluation of a bromine-76-labeled progestin 16alpha,17alpha-dioxolane for breast tumor imaging and radiotherapy: in vivo biodistribution and metabolic stability studies

INTRODUCTION: Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. They can be used as targets for diagnostic imaging and radiotherapy. METHOD: 16alpha,17alpha-[(R)-1'-alpha-(5-[(76)Br]Bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione ([(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (3)), a PR ligand with relative binding affinity (RBA)=65 and log P(o/w)=5.09+/-0.84, was synthesized via a two-step reaction, and its tissue biodistribution and metabolic stability were evaluated in estrogen-primed immature female Sprague-Dawley rats. RESULTS: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was synthesized in 5% overall yield with specific activity being 200-1250 Ci/mmol. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 demonstrated high PR-mediated uptake in the target tissue uterus (8.72+/-1.84 %ID/g at 1 h) that was reduced by a blocking dose of unlabeled progestin R5020, but the nonspecific uptake in blood and muscle (2.11+/-0.14 and 0.89+/-0.16 %ID/g at 1 h, respectively) was relatively high. [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 was stable in whole rat blood in vitro, but it was not stable in vivo due to the fast metabolism that occurred in the liver, resulting in the formation of a more polar radioactive metabolite and free [(76)Br]bromide. The level of free [(76)Br]bromide in blood remained high during the experiment (2.11+/-0.14 %ID/g at 1 h and 1.52+/-0.24 %ID/g at 24 h). The tissue distribution of [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 at 1 and 3 h was compared with that of the (18)F analogs, [(18)F]FFNP fluoro furanyl norprogesterone (FFNP) 1 and ketal 2. CONCLUSION: [(76)Br]16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione 3 may have potential for imaging PR-positive breast tumors at early time points, but it is not suitable for imaging at later times or for radiotherapy.     

Comparison of fluorine-18 and bromine-76 imaging in positron emission tomography

State of the art positron emission tomography (PET) systems allow for scatter and attenuation correction. However, the size of the structure being studied and the region of interest (ROI) chosen also influence the accuracy of measurements of radioactive concentration. Furthermore, the limited spatial resolution of PET tomographs, which depends, among other factors, on the range of positrons in matter, can also contribute to a loss in quantitation accuracy. In this paper we address the influence of positron range, structure size and ROI size on the quantitation of radioactive concentration using PET. ECAT EXACT HR+ (HR+) and ECAT 953B/31 (ECAT 953B) PET systems were used in phantom acquisitions performed with two radioisotopes with different positron ranges. The 3D Hoffman phantom was scanned on both scanners with both radioisotopes, to visually analyse the image quality. A resolution phantom having six spheres of different diameters in a Plexiglas cylinder was used to calculate the values of the contrast recovery coefficient or hot spot recovery coefficient and of the spill-over or cold spot recovery coefficient under different imaging conditions used in clinical routine at our institution. Activity ratios were varied between 2 and 30 or between 0.4 and 200 by filling the spheres with fluorine-18 or bromine-76 respectively and the cylinder with ¹¹C. Dynamic scans were performed on each scanner. Data were reconstructed using the same parameters as are used in clinical protocols. The variations in sphere and cylinder activities with time were fitted using the function M(t)=k ₁·A(t)+k ₂.B(t), where M(t) is the radioactivity concentration measured in an ROI placed on each sphere and A(t) and B(t) represent the true radioactivity concentrations present at time t in the spheres and in the cylinder respectively. k ₁ and k ₂ are factors representing the contrast recovery coefficient and the spill-over from surrounding activity on measurements respectively. The visual analysis of images obtained using a 3D Hoffman phantom showed that image resolution and image contrast between different regions are radioisotope dependent and clearly better when using ¹⁸F. Linear profiles taken on these images confirmed the visual assessment. For a given scanner, the k ₁ values obtained with ¹⁸F were systematically higher than those measured using ⁷⁶Br in the same machine (especially for the smaller spheres) when using the same ROI. For a sphere of a particular diameter, the use of a wider ROI resulted in lower quantitative accuracy when using the same isotope and the same camera. Lower quantitative accuracy was found for smaller spheres for all ROI sizes used in image analysis. For the same scanner and for a similar imaging situation (same sphere and same ROI), it was found that k ₁ and k ₂ values depend on the radioisotope used. For the same isotope and tomograph, the k ₁ values obtained decreased with the size of the structures imaged, as well as with the increase in ROI size. The use of a tomograph with better spatial resolution (HR+, rather than ECAT 953B) greatly increased the k ₁ values for ¹⁸F while only a mild improvement in these values was observed for ⁷⁶Br. The use of ⁷⁶Br led to k ₂ values that were slightly higher than those measured using ¹⁸F. These differences may have been due to the difference in the range of the positrons emitted by the radioisotopes used in this study. The measurements performed in this study show that the comparison of studies obtained on the same camera depends on the radioisotope used and may require the adaptation of ROI size between examinations. Marked differences are visible if the positron ranges of such radioisotopes are very different. Therefore, when employing commercially available tomographs and imaging protocols used in clinical routine, the effects of differences in positron range on image quality and quantitation are noticeable and correction for these effects may be of importance. With the arrival of PET imaging systems with better spatial resolution (close to 2 mm full-width at half-maximum for animal PET systems), positron range will have an increasing influence on the image quality and on the choice of radioisotope for a given application. Received 30 January and in revised form 18 March 1999     

Comparison of fluorine-18 and bromine-76 imaging in positron emission tomography.

State of the art positron emission tomography (PET) systems allow for scatter and attenuation correction. However, the size of the structure being studied and the region of interest (ROI) chosen also influence the accuracy of measurements of radioactive concentration. Furthermore, the limited spatial resolution of PET tomographs, which depends, among other factors, on the range of positrons in matter, can also contribute to a loss in quantitation accuracy. In this paper we address the influence of positron range, structure size and ROI size on the quantitation of radioactive concentration using PET. ECAT EXACT HR+ (HR+) and ECAT 953B/31 (ECAT 953B) PET systems were used in phantom acquisitions performed with two radioisotopes with different positron ranges. The 3D Hoffman phantom was scanned on both scanners with both radioisotopes, to visually analyse the image quality. A resolution phantom having six spheres of different diameters in a Plexiglas cylinder was used to calculate the values of the contrast recovery coefficient or hot spot recovery coefficient and of the spill-over or cold spot recovery coefficient under different imaging conditions used in clinical routine at our institution. Activity ratios were varied between 2 and 30 or between 0.4 and 200 by filling the spheres with fluorine-18 or bromine-76 respectively and the cylinder with 11C. Dynamic scans were performed on each scanner. Data were reconstructed using the same parameters as are used in clinical protocols. The variations in sphere and cylinder activities with time were fitted using the function M(t)=k1. A(t)+k2.B(t), where M(t) is the radioactivity concentration measured in an ROI placed on each sphere and A(t) and B(t) represent the true radioactivity concentrations present at time t in the spheres and in the cylinder respectively. k1 and k2 are factors representing the contrast recovery coefficient and the spill-over from surrounding activity on measurements respectively. The visual analysis of images obtained using a 3D Hoffman phantom showed that image resolution and image contrast between different regions are radioisotope dependent and clearly better when using 18F. Linear profiles taken on these images confirmed the visual assessment. For a given scanner, the k1 values obtained with 18F were systematically higher than those measured using 76Br in the same machine (especially for the smaller spheres) when using the same ROI. For a sphere of a particular diameter, the use of a wider ROI resulted in lower quantitative accuracy when using the same isotope and the same camera. Lower quantitative accuracy was found for smaller spheres for all ROI sizes used in image analysis. For the same scanner and for a similar imaging situation (same sphere and same ROI), it was found that k1 and k2 values depend on the radioisotope used. For the same isotope and tomograph, the k1 values obtained decreased with the size of the structures imaged, as well as with the increase in ROI size. The use of a tomograph with better spatial resolution (HR+, rather than ECAT 953B) greatly increased the k1 values for 18F while only a mild improvement in these values was observed for 76Br. The use of 76Br led to k2 values that were slightly higher than those measured using 18F. These differences may have been due to the difference in the range of the positrons emitted by the radioisotopes used in this study. The measurements performed in this study show that the comparison of studies obtained on the same camera depends on the radioisotope used and may require the adaptation of ROI size between examinations. Marked differences are visible if the positron ranges of such radioisotopes are very different. Therefore, when employing commercially available tomographs and imaging protocols used in clinical routine, the effects of differences in positron range on image quality and quantitation are noticeable and correction for these effects may be of importance. (ABSTRACT TRUNCATED)     

Synthesis and in vivo evaluation of 2 high-affinity 76Br-labeled sigma2-receptor ligands.

The sigma(2)-receptor has been shown to be upregulated in proliferating tumors cells. The purpose of this study was to compare 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) and 2 new (76)Br-radiolabeled compounds that have a high affinity and selectivity for the sigma(2)-receptor. These are 5-bromo-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-2,3-dimethoxybenzamide (compound (1)) and 5-bromo-N-(2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)ethyl)-2-methoxybenzamide (compound (2)). METHODS: Two sigma(2)-receptor-binding ligands were prepared, from the corresponding tributylstannyl precursors using standard electrophilic chemistry, (76)Br-compound (1) ((76)Br-1) and (76)Br-compound (2) ((76)Br-2). (18)F-FLT, (76)Br-1, and (76)Br-2 were compared using allograft tumors of the EMT-6 cell line (mouse mammary adenocarcinoma) in biodistribution studies at 5 min, 0.5, 1, and 2 h. Imaging of (76)Br-1 and (18)F-FLT was also performed at 2 and 1 h, respectively. RESULTS: (76)Br-1 and (76)Br-2 were synthesized with yields between 50% and 70% with high specific activity. Both compounds showed uptake into the tumor with tumor-to-normal tissue ratios of (76)Br-1 being greater than both (76)Br-2 and (18)F-FLT. Except for the liver and kidney, all ratios were greater than 1 and uptake into the tumor was shown with microPET imaging for (76)Br-1. CONCLUSION: We were able to synthesize two (76)Br-radiolabeled compounds with a high yield and specific activity that target the sigma(2) receptor with high affinity and selectivity. The studies presented show that both of the flexible benzamide compounds can identify EMT-6 breast tumors in vivo. (76)Br-1 also has higher tumor-to-normal tissue ratios when compared with (76)Br-2 and (18)F-FLT. The high affinity and low nonspecific binding of (76)Br-1 indicates that it can be a potential PET radiotracer for imaging solid tumors.     

Preparation of a bromine-76 labelled analogue of epibatidine: a potent ligand for nicotinic acetylcholine receptor studies.

Epibatidine analogues have been labelled with I-123 for single photon emission computed tomography and with short half-life positron emitters (C-11 and F-18) for PET. For easier radiopharmacological studies the bromo analogue of epibatidine (norchlorobromoepibatidine or exo-7-azabicyclo-2-(2-bromo-5-pyridyl)-[2.2.1]heptane) was labelled with Br-76, a longer half-life positron emitter, (T1/2 = 16.2h). [76Br]-norchlorobromoepibatidine was prepared by using a Cu+ assisted bromodeiodination exchange from the iodo analogue in reducing conditions at 190 degrees C. The tracer purified by RP-HPLC was obtained in 70% radiochemical yield with a specific radioactivity of 20 GBq/micromol. Radiochemical and chemical purities measured by radio-TLC and HPLC were >98%.     

Synthesis and biological evaluation of a fluorine-18-labeled nonsteroidal androgen receptor antagonist, N-(3-[18F]fluoro-4-nitronaphthyl)-cis-5-norbornene-endo-2,3-dicarboxylic imide

INTRODUCTION: Androgen receptor (AR), which is overexpressed in most prostate cancers, is the target of androgen ablation and antiandrogen therapies: it is also the target for the receptor-mediated imaging of AR-positive prostate cancer using radiolabeled ligands. Previous AR imaging agents were based on a steroidal core labeled with fluorine. To develop a novel class of nonsteroidal imaging agents, with binding and pharmacological characteristics that are more similar to those of clinically used AR antagonists, we synthesized N-(3-fluoro-4-nitronaphthyl)-cis-5-norbornene-endo-2,3-dicarboxylic imide (3-F-NNDI), an analog of recently reported AR antagonist ligands. METHODS: 3-F-NNDI was synthesized in six steps starting with 1-nitronaphthalene, with fluorine incorporation as the final step. The labeling of 3-F-NNDI with fluorine-18 was achieved through a novel, extremely mild, S(N)Ar displacement reaction of an o-nitro-activated arene trimethylammonium salt, and 3-[(18)F]F-NNDI was prepared in high specific activity. RESULTS AND DISCUSSION: 3-F-NNDI was found to have an AR-binding affinity similar to that of its parent compound. In vitro assays demonstrated high stability of the labeled compound under physiological conditions in buffer and in the blood. Androgen target tissue uptake in diethylstilbestrol-pretreated male rats, however, was minimal, probably because of extensive metabolic defluorination the radiolabeled ligand. CONCLUSIONS: This study is part of our first look at a novel class of nonsteroidal AR antagonists as positron emission tomography (PET) imaging agents that are alternatives to steroidal AR agonist-based imaging agents. Although 3-[(18)F]F-NNDI has significant affinity for AR, it showed limited promise as a PET imaging agent because of its poor target tissue distribution properties.     

Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography

IntroductionBromine-76-radiolabeled analogues of previously reported high-affinity A₃ adenosine receptor (A₃AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography.MethodsThe radiosyntheses were accomplished by oxidative radiobromination on the N⁶-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats.ResultsWe prepared an agonist ligand {[⁷⁶Br](1′S,2′R,3′S,4′R,5′S)-4′-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1′-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2′,3′-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/μmol and an antagonist ligand {[⁷⁶Br](1′R,2′R,3′S,4′R,5′S)-4′-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2′,3′-diol (MRS5147)} in 65% radiochemical yield with a specific activity of 22 GBq/μmol. The resultant products exhibited the expected high affinity (K_i～0.6 nM) and specific binding at the human A₃AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A₃AR, over a 2-h time course, which suggests the presence of a specific A₃AR retention mechanism.ConclusionWe were able to compare uptake of the [⁷⁶Br]-labeled antagonist MRS5147 to [⁷⁶Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A₃AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.     

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor

IntroductionThe cannabinoid receptor type 2 (CB₂) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB₂ receptor ligands. Some of the compounds demonstrated to be good CB₂-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their ¹⁸F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB₂ receptor expression. Here, we report the radiosynthesis of two ¹⁸F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB₂-specific radioligand.Methods4-[¹⁸F]fluorobenzyl amine [¹⁸F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH₄ and then coupled with acid chlorides 11 and 12 to afford [¹⁸F]-13 and [¹⁸F]-14. In vitro CB₂ receptor binding assay was performed using U87 cells transduced with CB₂ and CB₁ receptor. Ex vivo autoradiography was performed with [¹⁸F]-14 on spleen and on CB₂- and CB₁-expressing and wild-type U87 subcutaneous tumors grown in mice.ResultsThe radiochemical yields of [¹⁸F]-13 and [¹⁸F]-14 were 10%–15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/μmol. The dissociation constant Kd for [¹⁸F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [¹⁸F]-14 in the CB₂-expressing tumor.ConclusionTwo new [¹⁸F]-labeled CB₂ ligands have been synthesized. Compound [¹⁸F]-14 appears to be a potential PET imaging agent for the assessment of CB₂ receptor expression; however, poor solubility restrain its use in vivo.     

Synthesis and preliminary evaluation of a carbon-11-labeled adenosine transporter blocker [(11)C]KF21562

We prepared an (11)C-labeled adenosine transporter blocker, [1-methyl-(11)C]-3-[1-(6,7-dimethoxyquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H, 3H)-quinazolinedione ([(11)C]KF21652) and examined its potential as a positron emission tomography (PET) ligand for mapping adenosine transporters in the brain and peripheral organs. The log P(7.4) value of KF21652 was 3.14, and the K(i) value was 13 nM for adenosine transporters using [(3)H]nitrobenzylthioinosine as a radioligand. In mice, the highest initial uptake was found in the liver, followed by the kidney and small intestine. The brain uptake was very low. The radioactivity level slightly increased with time in the liver and small intestine, but decreased in the other organs. Coinjection of carrier KF21652 slightly decreased the uptake of [(11)C]KF21562 only in the liver, but not in any other organs. Ex vivo autoradiography of the rat brain showed that [(11)C]KF21652 was scarcely incorporated into the brain. On the other hand, in vitro autoradiography showed the binding of [(11)C]KF21562 to adenosine transporters with high nonspecific binding. These results show that the compound is not a suitable PET ligand for mapping adenosine transporters.     

Synthesis and biological evaluation of carbon-11- and fluorine-18-labeled 2-oxoquinoline derivatives for type 2 cannabinoid receptor positron emission tomography imaging

IntroductionThe type 2 cannabinoid (CB₂) receptor is part of the endocannabinoid system and has been suggested as a mediator of several central and peripheral inflammatory processes. Imaging of the CB₂ receptor has been unsuccessful so far. We synthesized and evaluated a carbon-11- and a fluorine-18-labeled 2-oxoquinoline derivative as new PET tracers with high specificity and affinity for the CB₂ receptor.MethodsTwo 2-oxoquinoline derivatives were synthesized and radiolabeled with either carbon-11 or fluorine-18. Their affinity and selectivity for the human CB₂ receptor were determined. Biological evaluation was done by biodistribution, radiometabolite and autoradiography studies in mice.ResultsIn vitro studies showed that both compounds are high affinity CB₂-specific inverse agonists. Biodistribution study of the tracers in mice showed a high in vivo initial brain uptake and fast brain washout, in accordance with the low CB₂ receptor expression levels in normal brain. A persistently high in vivo binding to the spleen was observed, which was inhibited by pretreatment with two structurally unrelated CB₂ selective inverse agonists. In vitro autoradiography studies with the radioligands confirmed CB₂-specific binding to the mouse spleen.ConclusionWe synthesized two novel CB₂ receptor PET tracers that show high affinity/selectivity for CB₂ receptors. Both tracers show favourable characteristics as radioligands for central and peripheral in vivo visualization of the CB₂ receptor and are promising candidates for primate and human CB₂ PET imaging.     

Synthesis and evaluation of fluorine-18-labeled SA4503 as a selective sigma1 receptor ligand for positron emission tomography

The [(18)F]fluoromethyl analog of the sigma(1) selective ligand 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA4503) ([(18)F]FM-SA4503) was prepared and its potential evaluated for the in vivo measurement of sigma(1) receptors with positron emission tomography (PET). FM-SA4503 had selective affinity for the sigma(1) receptor (K(i) for sigma(1) receptor, 6.4 nM; K(i) for sigma(2) receptor, 250 nM) that was compatible with the affinity of SA4503 (K(i) for sigma(1) receptor, 4.4 nM; K(i) for sigma(2) receptor, 242 nM). [(18)F]FM-SA4503 was synthesized by (18)F-fluoromethylation of O-demethyl SA4503 in the radiochemical yield of 2.9-16.6% at the end of bombardment with a specific activity of 37.8-283 TBq/mmol at the end of synthesis. In mice, the uptake of [(18)F]FM-SA4503 in the brain was gradually increased for 30 min after injection, and then decreased. In the blocking study, brain uptake was significantly decreased by co-injection of haloperidol to 32% of control, and FM-SA4503 to 52% of control. In PET study of the monkey brain, high uptake was found in the cerebral cortex, thalamus and striatum. The radioactivity level of [(18)F]FM-SA4503 in the brain regions gradually increased over a period of 120 min after injection, followed by a stable plateau phase until 180 min after injection. In pretreatment with haloperidol measurement of the monkey brain, the radioactivity level was 22-32% and 11-25% of the baseline at 60 and 180 min, respectively, after injection, suggesting high receptor-specific binding. [(18)F]FM-SA4503 showed specific binding to sigma(1) receptors in mice and monkeys; therefore, [(18)F]FM-SA4503 has the potential for mapping sigma(1) receptors in the brain.     

Effects of 5-bromo-2-deoxyuridine and 2-deoxy-D-glucose on radiation-induced micronuclei in mouse bone marrow

Effects of 2-deoxy-D-glucose (2-DG) and 5-bromo-2-deoxyuridine (BrdU) on 60Co gamma-radiation-induced damage in normal mouse bone marrow have been investigated. Cytogenetic damage as measured by the frequency of cells with micronuclei was studied at various time intervals after irradiation. Intravenous administration of 2-DG (0.1 ml, 1 g/kg body weight) just before or after whole-body irradiation (absorbed dose 2.5 Gy) significantly reduced the micronuclei fraction as compared with irradiated controls. Incorporation of BrdU given intraperitoneally in 13 injections (0.3 ml) (total dose 2 g/kg body weight) at intervals of 1 h increased the micronuclei frequency, but administration of 2-DG significantly decreased cytogenetic damage. The results have interesting implications for improving tumour radiotherapy.     

Synthesis and autoradiographic evaluation of a novel high-affinity Tc-99m ligand for the 5-HT2A receptor

The synthesis and in vitro autoradiography of a novel Tc-99m ligand with subnanomolar affinity to the 5-HT_2A receptor is reported. The complex combines the 4-(4-fluoro)-benzoyl piperidine portion derived from the 5-HT_2A receptor antagonist ketanserin with a neutral oxotechnetium(V) chelate in form of a mixed ligand “3+1” unit containing the SNS/S donor set. The analogous rhenium compound has been synthesized as a surrogate for the Tc-99m complex for use in receptor binding assays and for complete structural characterization. In competition experiments the Tc-99 complex as well as its Re analogue display subnanomolar affinity toward the 5-HT_2A receptor (K_i 0.44 nM for Tc, 0.25 nM for Re). The subnanomolar 5-HT_2A receptor binding of the Re complex was confirmed by functional in vitro antagonism of contractile effects evoked by 5-HT in rat arterial tissue. Re 1 inhibited 5-HT-induced, 5-HT_2A receptor-mediated contractions of isolated rat tail artery in a competitive fashion and possessed nanomolar affinity ( pA₂=9.08, pA₂ representing the negative decadic logarithm of the Re 1/5-HT_2A-receptor dissociation constant [mol/L]). Like ketanserin, Re 1 displayed moderate affinity for adrenergic _1D ( pA₂=8.23) and histamine H₁ receptors ( pA₂=8.00), and was >600-fold up to 10,700-fold less active at several neurotransmitter receptor subtypes. In vitro autoradiographic studies clearly indicate the accumulation of the Tc-99m compound in 5-HT_2A-receptor-rich areas of the brain. This enrichment can be blocked by 5-HT_2A receptor antagonists such as mianserin and ketanserin and is therefore specific.     

In vivo evaluation of copper-64-labeled monooxo-tetraazamacrocyclic ligands

Copper-64 (T_1/2=12.7 h; β⁺: 0.653 MeV, 17.4%; β⁻: 0.578 MeV, 39%) has applications in positron emission tomography (PET) imaging and radiotherapy, and is conveniently produced on a biomedical cyclotron. Tetraazamacrocyclic ligands are the most widely used bifunctional chelators (BFCs) for attaching copper radionuclides to antibodies and peptides due to their relatively high kinetic stability. In this paper, we evaluated three monooxo-tetraazamacrocyclic ligands with different ring sizes and oxo group positions. H1 [1,4,7,10-tetraazacyclotridecan–11-one], H2 [1,4,8,11-tetraazacyclotetradecan-5-one] and H3 [1,4,7,10-tetraazacyclotridecan-2-one] were radiolabeled with ⁶⁴Cu in high radiochemical yields under mild conditions. The three ⁶⁴Cu-labeled complexes are all +1 charged, as determined by their electrophoretic mobility. While they demonstrated >95% stability in rat serum out to 24 h, both biodistribution and microPET imaging studies revealed high uptake and long retention of the compounds in major clearance organs (e.g., blood, liver and kidney), which suggests that ⁶⁴Cu dissociated from the complexes in vivo. Of the three complexes, ⁶⁴Cu-2⁺, which has a cyclam backbone (1,4,8,11-tetraazacyclotetradecane), exhibited the lowest nontarget organ accumulation. The data from these studies may invalidate the candidacy of the monooxo-tetraazamacrocyclics as BFCs for copper radiopharmaceuticals. However, the data presented here suggest that neutral or negatively charged Cu(II) complexes of tetraazamacrocyclic ligands with a cyclam backbone (tetradecane) are optimal for copper radiopharmaceutical applications.     

Synthesis of 2'-deoxy-2'-[18F]fluoro-5-bromo-1-beta-D-arabinofuranosyluracil ([18F]-FBAU) and 2'-deoxy-2'-[18F]fluoro-5-chloro-1-beta-D-arabinofuranosyl-uracil ([18F]-FCAU), and their biological evaluation as markers for gene expression

[(18)F]-FBAU and [(18)F]-FCAU have been synthesized and evaluated in vivo as markers for HSV1-tk gene expression. At 2 hours, uptake of [(18)F]-FBAU and [(18)F]-FCAU in HSV1-tk-positive tumors was 7.9-fold and 6.0-fold higher than the control tumors, respectively. Micro-PET images also showed very high uptake in HSV-tk tumors. Compared to [(14)C]-FMAU, total uptake of [(18)F]-FBAU and [(18)F]-FCAU was similar in tk-positive cells, but the uptake ratio (tk+/wild) was higher. [(18)F]-FBAU and [(18)F]-FCAU appear to be potential PET imaging agents for gene expression.     

Synthesis and autoradiographic evaluation of a novel high-affinity Tc-99m ligand for the 5-HT2A receptor

The synthesis and in vitro autoradiography of a novel Tc-99m ligand with subnanomolar affinity to the 5-HT_2A receptor is reported. The complex combines the 4-(4-fluoro)-benzoyl piperidine portion derived from the 5-HT_2A receptor antagonist ketanserin with a neutral oxotechnetium(V) chelate in form of a mixed ligand “3+1” unit containing the SNS/S donor set. The analogous rhenium compound has been synthesized as a surrogate for the Tc-99m complex for use in receptor binding assays and for complete structural characterization. In competition experiments the Tc-99 complex as well as its Re analogue display subnanomolar affinity toward the 5-HT_2A receptor (K_i 0.44 nM for Tc, 0.25 nM for Re). The subnanomolar 5-HT_2A receptor binding of the Re complex was confirmed by functional in vitro antagonism of contractile effects evoked by 5-HT in rat arterial tissue. Re 1 inhibited 5-HT-induced, 5-HT_2A receptor-mediated contractions of isolated rat tail artery in a competitive fashion and possessed nanomolar affinity ( pA₂=9.08, pA₂ representing the negative decadic logarithm of the Re 1/5-HT_2A-receptor dissociation constant [mol/L]). Like ketanserin, Re 1 displayed moderate affinity for adrenergic α_1D ( pA₂=8.23) and histamine H₁ receptors ( pA₂=8.00), and was >600-fold up to 10,700-fold less active at several neurotransmitter receptor subtypes. In vitro autoradiographic studies clearly indicate the accumulation of the Tc-99m compound in 5-HT_2A-receptor-rich areas of the brain. This enrichment can be blocked by 5-HT_2A receptor antagonists such as mianserin and ketanserin and is therefore specific.     

Synthesis and regional rat brain distribution of [11C]MDL 72222: a 5HT3 receptor antagonist

MDL 72222, an antagonist of 5HT₃ receptors, was labeled with a specific radioactivity of 340–400 mCi/μmol by alkylation of the nor-precursor with [¹¹C]CH₃I. The yield of the synthesis, starting from [¹¹C]methyliodide to the purified product and corrected for decay, was good ∼70–75%. After i.v. injection, [¹¹C]MDL 72222 diffuses readily in the central nervous system but is not detected as metabolites in brain and blood, during 1 h study carried out in rats. The time course and distribution of [¹¹C]MDL 72222 was assessed in various organs (liver, lung, kidney, heart, whole brain) and in blood; the organ uptake was rapid and large; the highest accumulation was found in the lung. The regional brain distribution shows initial uptake and subsequent retention of tracer in favor of the cerebral cortex. The level of brain radioactivity was not reduced by pretreatment with a 1000-fold excess of unlabeled MDL 72222. These results suggest that [¹¹C]MDL 72222 is of limited interest for 5HT₃ receptor binding studies in brain in vivo, presumably mainly because of large non-specific binding.