Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr(3)-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [(125)I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [(125)I]TOCA, as a reference, [(125)I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [(125)I]Gluc-S- and [(125)I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst(1)-sst(5), all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst(2). In contrast, internalization into sst(2)-expressing cells (in % of [(125)I]Tyr(3)-octreotide ([(125)I]TOC)) as well as the EC(50,R) of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [(123)I]Gal-S-TOCA and [(123)I]Gluc-S-TOCA (internalization, 190% +/- 12% and 265% +/- 20%, respectively, vs 168% +/- 6% of [(125)I]TOC for [(123)I]Mtr-TOCA; EC(50,R), 2.62 +/- 0.07 and 2.96 +/- 0.14, respectively, vs 1.81 +/- 0.07 for [(123)I]Mtr-TOCA). The tumor accumulation of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 +/- 2.2 and 26.2 +/- 5.6%ID/g) and comparable to that of [(125)I]Mtr-TOCA (25.1 +/- 4.4%ID/g). [(125)I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [(125)I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA was also slightly higher than that of [(125)I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 +/- 0.3 and 2.2 +/- 0.4, respectively, vs 4.0 +/- 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([(125)I]Glucuron-TOCA vs [(125)I]Gal-S- and [(125)I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [(123)I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.     

Synthesis and evaluation of N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide: a deuterium-substituted radioligand for peripheral benzodiazepine receptor

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]2) is a potent ligand (IC(50): 1.71 nM) for peripheral benzodiazepine receptor (PBR). However, in vivo evaluation on rodents and primates showed that this ligand was unstable and rapidly metabolized to [(18)F]F(-) by defluorination of the [(18)F]fluoromethyl moiety. In this study, we designed a deuterium-substituted analogue, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]5) as a radioligand for PBR to reduce the in vivo metabolic rate of the non-deuterated [(18)F]2. The design principle was based on the hypothesis that the deuterium substitution may reduce the rate of defluorination initiated by cleavage of the C-H bond without altering the binding affinity for PBR. The non-radioactive 5 was prepared by reacting diiodomethane-d(2) (CD(2)I(2), 6) with a phenol precursor 7, followed by treatment with tetrabutylammonium fluoride. The ligand [(18)F]5 was synthesized by the alkylation of 7 with [(18)F]fluoromethyl iodide-d(2) ([(18)F]FCD(2)I, [(18)F]9). Compound 5 displayed a similar in vitro affinity to PBR (IC(50): 1.90 nM) with 2. In vivo evaluation demonstrated that [(18)F]5 was metabolized by defluorination to [(18)F]F(-) as a main radioactive component, but its metabolic rate was slower than that of [(18)F]2 in the brain of mice. The deuterium substitution decreased the radioactivity level of [(18)F]5 in the bone of mouse, augmented by the percentage of specific binding to PBR in the rat brain determined by ex vivo autoradiography. However, the PET image of [(18)F]5 for monkey brain showed high radioactivity in the brain and skull, suggesting a possible species difference between rodents and primates.     

Synthesis and evaluation of novel F-18 labeled 4-aminoquinazoline derivatives: potential PET imaging agents for tumor detection

Three novel (18)F-labeled 4-aminoquinazoline derivatives, N-(3-chloro-4-fluorophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]1), N-(3-ethynylphenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]2), and N-(3-bromophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]3) were synthesized and radiolabeled by two-step reaction with overall radiochemical yield of 21-24% (without decay corrected). Then we carried out their biodistribution experiments in S180 tumor-bearing mice. Results showed that they had certain concentration accumulation in tumor and fast clearance from muscle and blood. It was encouraging that [(18)F]3 was competitive among three (18)F-labeled 4-aminoquinazoline derivatives in some aspects such as tumor/muscle uptake ratio reaching 7.70 at 60 min post-injection, tumor/blood uptake ratio reaching 6.61 at 120 min post-injection. So we compared radioactivity characteristics of [(18)F]3 with those of [(18)F]-FDG and L-[(18)F]-FET in the same animal model. The absolute radioactivity uptake of [(18)F]3 in tumor reached 3.31 at 60 min p.i., which was slightly higher than [(18)F]-FDG (2.16) and L-[(18)F]-FET (2.75) at the same time phase. For [(18)F]3, tumor/muscle uptake ratio peaked 7.70 at 60 min, which was obviously superior to those of [(18)F]-FDG and L-[(18)F]-FET at all time points. The tumor/brain uptake ratios of [(18)F]3 were 10.36, 17.42, 41.11 at 30 min, 60 min and 120 min post-injection, respectively, and are much higher than those of L-[(18)F] FET (2.54, 2.92 and 2.95) and [(18)F]-FDG (0.61, 1.02 and 1.33) at the same time points. All these results indicate that [(18)F]3 is promising to become a potential PET tumor imaging agent.     

Synthesis of a [6-pyridinyl-18F]-labelled fluoro derivative of WAY-100635 as a candidate radioligand for brain 5-HT1A receptor imaging with PET

In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.     

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90?min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15?min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.     

Synthesis and radiosynthesis of [(18)F]FPhEP, a novel alpha(4)beta(2)-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors

FPhEP (1, (+/-)-2-exo-(2'-fluoro-3'-phenyl-pyridin-5'-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3'-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP (1, K(i) of 0.24 nM against [(3)H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives (3a,b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields=17% for 1, 9% for 3a and 8% for 3b). FPhEP (1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives (3 a,b-1mg) and the activated K[(18)F]F-Kryptofix(222) complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [(18)F]FPhEP ([(18)F]-1, 2.22-3.33 GBq, 66-137 GBq/micromol) was obtained after semi-preparative HPLC (Symmetry C18, eluent aq 0.05 M NaH(2)PO(4)/CH(3)CN, 80:20 (v:v)) in 75-80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [(18)F]fluoride production batch (10-20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity (K(D) 660 pM) of [(18)F]FPhEP ([(18)F]-1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [(18)F]FPhEP ([(18)F]-1) and [(18)F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [(18)F]FPhEP ([(18)F]-1) has potential for in vivo imaging nAChRs in the brain with PET.     

Meta-iodobenzylguanidine derivatives containing a second guanidine moiety

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. To investigate whether an additional guanidine function in the structure of MIBG will yield analogues that may potentially enhance tumor-to-target ratios, two derivatives-one with a guanidine moiety and another with a guanidinomethyl group at the 4-position of MIBG-were prepared. In the absence of any uptake-1 inhibiting conditions, the uptake of 4-guanidinomethyl-3-[(131)I]iodobenzylguanidine ([(131)I]GMIBG) by SK-N-SH cells in vitro was 1.7+/-0.1% of input counts, compared to a value of 40.3+/-1.4% for [(125)I[MIBG suggesting that guanidinomethyl group at the 4-position negated the biological properties of MIBG. On the other hand, 4-guanidino-3-[(131)I]iodobenzylguanidine ([(131)I]GIBG) had an uptake (5.6+/-0.3%) that was 12-13% that of [(125)I]MIBG (46.1+/-2.7%), and the ratio of uptake by control over DMI-treated (nonspecific) cultures was higher for [(131)I]GIBG (20.9+/-0.3) than [(125)I]MIBG itself (15.0+/-2.7). The exocytosis of [(131)I]GIBG and [(125)I]MIBG from SK-N-SH cells was similar. The uptake of [(131)I]GIBG in the mouse target tissues, heart and adrenals, as well as in a number of other tissues was about half that of [(125)I]MIBG. These results suggest that substitution of guanidine functions, especially a guanidinomethyl group, in MIBG structure may not be advantageous.     

Aromatic radiofluorination and biological evaluation of 2-aryl-6-[18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

To develop agents for radionuclide imaging Aβ plaques in vivo, we prepared three fluorine-substituted analogs of arylbenzothiazole class; compound 2 has a high affinity for Aβ (K(i)=5.5nM) and the specific binding to Aβ in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogs in radiolabeled form as an Aβ plaques-specific positron emission tomography (PET) imaging probe, we investigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18 (t(1/2)=110min) and diaryliodonium tosylate precursors (12, 13a-e and 14). 2-Aryl-6-[(18)F]fluorobenzothiazoles ([(18)F]1-3) were synthesized in efficiently short reaction times (40-60min) with high radiochemical yields (19-40%), purities (>95%) and specific activities (85-118GBq/μmol). Tissue distribution studies showed that high radioactivity of [(18)F]2 accumulated in the brain with rapid clearance in healthy mice. Radioactive metabolites were analyzed in brain samples of mice and corresponded to 81% of parent remained by 30min after a tail-vein injection. These results suggest that [(18)F]2 is a promising probe for evaluation of Aβ plaques imaging in brain using PET.     

Syntheses of F-18 labeled fluoroalkyltyrosine derivatives and their biological evaluation in rat bearing 9L tumor

We hereby report the synthesis of four fluorine-18 labeled tyrosine derivatives, 3-(2-[(18)F]fluoroethyl)tyrosine ([(18)F]1, [(18)F]ortho-FET), 3-(3-[(18)F]fluoropropyl)tyrosine ([(18)F]2, [(18)F]ortho-FPT) O-methyl-[3-(2-[(18)F]fluoroethyl)]tyrosine ([(18)F]3, [(18)F]MFET), and O-methyl-[3-(3-[(18)F]fluoropropyl)]tyrosine ([(18)F]4, [(18)F]MFPT). The fluorine-18 labeled tyrosine derivatives were prepared by the displacement reaction of the ethyl and propyl tosylates with K[(18)F]/K2.2.2 in acetonitrile under no-carrier-added (NCA) conditions, followed by hydrolysis with 4N HCl. The biological properties of labeled compounds were evaluated in rats bearing 9L tumor after an intravenous injection and PET image was obtained. The tumor/blood and tumor/brain ratios were 2.06, 2.92 for [(18)F]1, 2.25, 4.05 for [(18)F]2, 2.88, 1.90 for [(18)F]3, and 2.00, 2.60 for [(18)F]4 at 60 min post injection, respectively. The PET image showed localized accumulation of PET tracers in 9L glioma of the rat.     

Synthesis and in vivo evaluation of [18F]N-(2-benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine, a novel σ1 receptor PET imaging agent

N-(2-Benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine (6, σ(1)K(i)=2.6 nM) was radiolabeled with fluorine-18 to provide a potential σ(1) receptor radioligand for use in positron emission tomography (PET). Radiofluorination of the appropriate tosylate precursor furnished [(18)F]6 with a specific activity of 45 GBq/μmol, in an average radiochemical yield of 18% and greater than 98% radiochemical purity. MicroPET imaging in Papio hamadryas baboon brain revealed [(18)F]6 uptake consistent with σ receptor distribution, and specificity for σ receptors was demonstrated in a haloperidol pre-treated animal. [(18)F]6 possesses suitable properties for PET imaging of σ(1) receptors, and further investigation of this σ(1) receptor tracer is warranted.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

Improvement of pharmacokinetics of radioiodinated Tyr(3)-octreotide by conjugation with carbohydrates

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr(3)-octreotide (TOC). Glucose ([(125)I]Gluc-TOC), maltose ([(125)I]Malt-TOC), and maltotriose ([(125)I]Mtr-TOC) derivatives of [(125)I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [(125)I]Gluc-TOC and [(125)I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [(125)I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [(125)I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5-3.5. While tumor uptake of [(125)I]TOC (6.7 +/- 2.6%ID/g), [(125)I]Malt-TOC (5.3 +/- 1.9%ID/g), and [(125)I]Mtr-TOC (4.9 +/- 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [(125)I]Gluc-TOC was significantly increased (10.1 +/- 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and gamma-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.     

A dual fluorinated and iodinated radiotracer for PET and SPECT imaging of β-amyloid plaques in the brain

We report a fluorinated and iodinated radiotracer as a probe for PET/SPECT to detect of β-amyloid (Aβ) plaques in the brain of patients with Alzheimer's disease (AD). We successfully designed and synthesized the fluorinated and iodinated aurone derivative (3) and its radiolabels ([(125)I]3 and [(18)F]3). In binding experiments in vitro, 3 showed high affinity for Aβ aggregates (K(i)=6.81nM). In brain sections of AD model mice, 3 intensely stained Aβ plaques. Furthermore, a specific plaque labeling signal was observed on the autoradiography of postmortem AD brain sections using [(125)I]3. In biodistribution experiments using normal mice, [(125)I]3 and [(18)F]3 displayed good uptake into and a rapid washout from the brain, properties highly desirable for Aβ imaging agents. These results suggest that 3 may function as a PET/SPECT dual imaging agent for detecting Aβ plaques in AD brains.     

Synthesis and evaluation of 18F- and 11C-labeled phenyl-galactopyranosides as potential probes for in vivo visualization of LacZ gene expression using positron emission tomography

3-Hydroxy-2-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, a derivative of the chromogenic beta-galactosidase (beta-gal) substrate o-nitrophenyl beta-D-galactopyranoside (ONPG) was synthesized using a Koenigs-Knorr glycosylation reaction. It was alkylated with 2-[(18)F]fluoroethyl triflate or [(11)C]methyl triflate, followed by deacetylation of the sugar hydroxyl groups to obtain radiolabeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenyl beta-D-galactopyranoside ([(18)F]-2c) and 3-[(11)C]methoxy-2-nitrophenyl beta- d-galactopyranoside ([(11)C]-3c), which were evaluated as potential reporter probes for in vivo visualization of LacZ gene expression with positron emission tomography (PET). In vitro, [(18)F]- 2c and [(11)C]-3c were good substrates of beta-gal and showed, respectively, a 7.5- and 2.5-fold higher uptake into beta-gal expressing cells (LacZ cells) compared to control cells. However, reversed-phase HPLC analysis of the LacZ cell lysate and supernatant showed that labeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenol, the hydrolysis product formed by beta-gal-mediated cleavage of [(18)F]-2c, substantially leaked out of the cells, which would lead to loss of PET signal. In a microPET study of [(18)F]-2c in a mouse with a beta-gal expressing tumor, high retention was observed in liver and kidneys, but only negligible accumulation was seen in the tumor. As a general conclusion, it can be stated that the synthesized PET tracers [ (18)F]-2c and [(11)C]-3c are not suitable for use as LacZ reporter probes. Further structural modifications to improve the diffusion over the tumor cell membrane and to increase retention in beta-gal expressing cells may lead to more favorable in vivo imaging probes.     

Site-specific labeling of annexin V with F-18 for apoptosis imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.     

Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques

Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.     

N-[18F]fluoroethyl-4-piperidyl acetate ([18F]FEtP4A): A PET tracer for imaging brain acetylcholinesterase in vivo

N-[(18)F]Fluoroethyl-4-piperidyl acetate ([(18)F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [(18)F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[(18)F]fluoroethyl bromide ([(18)F]FEtBr) at 130 degrees C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [(18)F]FEtP4A was synthesized by reacting P4A with 2-[(18)F]fluoroethyl iodide ([(18)F]FEtI) or 2-[(18)F]fluoroethyl triflate ([(18)F]FEtOTf in improved radiochemical yields, compared with [(18)F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [(18)F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of (18)F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood-brain barrier and to enter the brain. In the rat brain, only [(18)F]fluoroethyl-4-piperidinol ([(18)F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [(18)F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [(18)F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [(11)C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.     

Synthesis of [18F]-labeled (6-fluorohexyl)triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography

Lipophilic cations such as phosphonium salts penetrate the hydrophobic barriers of the plasma and mitochondrial membranes and accumulate in mitochondria in response to the negative inner-transmembrane potentials. Thus, as newly developed noninvasive imaging agents, [(18)F]-labeled phosphonium salts may serve as molecular "voltage sensor" probes to investigate the role of mitochondria, particularly in myocardial disease. The present study reports the radiosynthesis of (6-fluorohexyl)triphenylphosphonium salt (3) as a potential agent for myocardial imaging by using positron emission tomography (PET). The reference compound of (6-[(18)F]fluorohexyl)triphenylphosphonium salt ([(18)F]3) was synthesized with 74% yield via three-step nucleophilic substitution reactions. The reference compound was radiolabeled via two-step nucleophilic substitution reactions of no-carrier-added [(18)F]fluoride with the precursor hexane-1,6-diyl bis(4-methylbenzenesulfonate) in the presence of Kryptofix 2.2.2 and K(2)CO(3). The radiolabeled compound was synthesized with 15-20% yield. The radiochemical purity was >98% by analytical HPLC, and the specific activity was >6.10-6.47 TBq/μmol. The cellular uptake assay showed preferential uptake of [(18)F]3 in cardiomyocytes. The results of biodistribution and micro-PET imaging studies of [(18)F]3 in mice and rats showed preferential accumulation in the myocardium. The results suggest that this compound would be a promising candidate for myocardial imaging.     

18F]FMDAA1106 and [18F]FEDAA1106: two positron-emitter labeled ligands for peripheral benzodiazepine receptor (PBR)

We synthesized and evaluated N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethyl-5-methoxybenzyl)acetamide ([(18)F]-FMDAA1106) and N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoroethyl-5-methoxybenzyl)acetamide ([(18)F]FEDAA1106) as two potent radioligands for peripheral benzodiazepine receptors (PBR). [(18)F]FMDAA1106 and [(18)F]FEDAA1106 were respectively synthesized by fluoroalkylation of the desmethyl precursor DAA1123 with [(18)F]FCH(2)I and [(18)F]FCH(2)CH(2)Br. Ex vivo autoradiograms of [(18)F]FMDAA1106 and [(18)F]FEDAA1106 binding sites in the rat brains revealed that a high radioactivity was present in the olfactory bulb, the highest PBR density region in the brain.     

Evaluation of 5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine and its analogues as PET radioligands for imaging nicotinic acetylcholine receptors

A novel series of compounds derived from the high-affinity nicotinic acetylcholine receptor (nAChR) ligand, 5-(2-(4-pyridinyl)vinyl)-6-chloro-3-((1-methyl-2-(S)-pyrrolidinyl)methoxy)pyridine (Me-p-PVC), originally developed by Abbott Laboratories, was characterized in vitro in nAChR binding assays at 37 degrees C to show K(i) values in the range of 9-611 pm. Several compounds of this series were radiolabeled with (11)C and evaluated in vivo in mice and monkeys as potential candidates for PET imaging of nAChRs. [(11)C]Me-p-PVC (K(i) =56 pm at 37 degrees C; logD = 1.6) was identified as a radioligand suitable for the in vivo imaging of the alpha 4 beta 2* nAChR subtype. Compared with 2-[(18)F]FA, a PET radioligand that has been successfully used in humans and is characterized by a slow kinetic of brain distribution, [(11)C]Me-p-PVC is more lipophilic. As a result, [(11)C]Me-p-PVC accumulated in the brain more rapidly than 2-[(18)F]FA. Pharmacological evaluation of Me-p-PVC in mice demonstrated that the toxicity of this compound was comparable with or lower than that of 2-FA. Taken together, these results suggest that [(11)C]Me-p-PVC is a promising PET radioligand for studying nAChR occupancy by endogenous and exogenous ligands in the brain in vivo.