Impurity profiling of N,N'-ethylenebis-l-cysteine diethyl ester (Bicisate).

A HPLC-UV-CAD method with a HILIC column for impurity profiling of the 99mTc chelating agent bicisate has been developed and evaluated. Bicisate and its impurities were separated by means of isocratic elution on a zwitterionic stationary phase using a mixture of 7.5mmol/L trifluoroacetic acid and acetonitrile (47.5:52.5 V/V) as the mobile phase. Five different bicisate batches of a manufacturer were tested using the method. In addition LC-MS experiments were conducted in order to identify the impurities. The predominant impurities found were the oxidation product (disulfide), the monoester of ethylene dicysteine and an unknown compound with an m/z of 293 in ESI positive mode. A new degradation product of bicisate, bicisate lactam, was identified during sample solution stability assessment.

Carbon-11 and Fluorine-18 Radiolabeled Pyridopyrazinone Derivatives for Positron Emission Tomography (PET) Imaging of Phosphodiesterase-5 (PDE5).

The cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type 5 (PDE5) plays an important role in various pathologies including pulmonary arterial hypertension and cardiomyopathy. PDE5 represents an important therapeutic and/or prognostic target, but noninvasive assessment of PDE5 expression is lacking. The purpose of this study was to develop and evaluate pyridopyrazinone derivatives labeled with carbon-11 or fluorine-18 as PDE5-specific PET tracers. In biodistribution studies, highest PDE5-specific retention was observed for [11C]-12 and [18F]-17 in the lungs of wild-type mice and in the myocardium of transgenic mice with cardiomyocyte-specific PDE5 overexpression at 30 min postinjection. In vivo dynamic microPET images in rats revealed that both tracers crossed the blood-brain barrier but brain retention was not PDE5-specific. Both [11C]-12 and [18F]-17 showed specific binding to PDE5 in myocardium of transgenic mice; however [18F]-17 showed significantly higher PDE5-specific inhibitable binding than [11C]-12.

Evaluation of PET radioligands for in vivo visualization of phosphodiesterase 5 (PDE5).

INTRODUCTION: The cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type 5 (PDE5) is considered to play an important role in various etiologies such as pulmonary arterial hypertension (PAH) and chronic heart failure. This PDE5 modulation represents an important prognostic and/or therapeutic target; however, there is currently no method to non-invasively evaluate the PDE5 expression levels in vivo. METHODS: Radiolabeled tracers were prepared by N-alkylation of the corresponding precursors with [(11)C]methyl trifluoromethanesulfonate ([(11)C]CH3OTf) or 2-[(18)F]fluoroethyl trifluoromethanesulfonate ([(18)F]FEtOTf). Biodistribution of radiolabeled tracers was studied in NMRI mice and their specific binding to PDE5 was investigated by comparing their lung retention as the enzyme is abundantly expressed in this organ. RESULTS: The overall radiochemical yields ranged between 24% and 60% for labeled radiotracers with radiochemical purity of>99%. The highest retention in the lungs at 30min post injection was observed for vardenafil derivatives [(11)C]-7 and [(18)F]-11 and the retention of the ethoxyethyl pyrazolopyrimidine derivative [(11)C]-37 was moderate. The other investigated compounds [(11)C]-8, [(11)C]-14, [(11)C]-21 and [(11)C]-33 showed lower retention in lungs in agreement with their lower in-vitro affinity for PDE5. CONCLUSION: Among the different radiolabeled PDE5 inhibitors evaluated in this study, the vardenafil derivatives [(11)C]-7 and [(18)F]-11 are found to be promising tracers for in vivo visualization of PDE5.

Synthesis and biological evaluation of 68Ga labeled bis-DOTA-3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) as a PET tracer for in vivo visualization of necrosis.

The aim of the present study was to develop a (68)Ga labeled bis-DOTA derivative of benzylidene-bis-indole and compare the in vivo stability and biodistribution with that of the previously reported bis-DTPA derivate for in vivo imaging of necrosis using PET. Uptake of the tracer was evaluated in a mouse model of Fas-mediated hepatic apoptosis in correlation with histochemical stainings. The novel (68)Ga labeled tracer showed an improved in vivo stability and could therefore be used for selective non-invasive imaging of necrotic cell death using PET.

New transient receptor potential vanilloid subfamily member 1 positron emission tomography radioligands: synthesis, radiolabeling, and preclinical evaluation.

The transient receptor potential vanilloid subfamily member 1 (TRPV1) cation channel is known to be involved in pain nociception and neurogenic inflammation, and accumulating evidence suggests that it plays an important role in several central nervous system (CNS)-related disorders. TRPV1-specific positron emission tomography (PET) radioligands can serve as powerful tools in TRPV1-related (pre)clinical research and drug design. We have synthesized several potent TRPV1 antagonists and accompanying precursors for radiolabeling with carbon-11 or fluorine-18. The cinnamic acid derivative [(11)C]DVV24 and the aminoquinazoline [(18)F]DVV54 were successfully synthesized, and their biological behavior was studied. In addition, the in vivo behavior of a (123)I-labeled analogue of iodo-resiniferatoxin (I-RTX), a well-known TRPV1 antagonist, was evaluated. The binding affinities of DVV24 and DVV54 for human TRPV1 were 163 ± 28 and 171 ± 48 nM, respectively. [(11)C]DVV24, but not [(18)F]DVV54 or (123)I-RTX, showed retention in the trigeminal nerve, known to abundantly express TRPV1. Nevertheless, it appears that ligands with higher binding affinities will be required to allow in vivo imaging of TRPV1 via PET.

Synthesis and biological evaluation of [¹¹C]SB366791: a new PET-radioligand for in vivo imaging of the TRPV1 receptor.

INTRODUCTION: The transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor, a non-selective cation channel, is known for its key role in pain nociception and neurogenic inflammation. TRPV1 expression has been demonstrated in diverse tissues and an essential role for TRPV1 in various disorders has been suggested. A TRPV1-specific PET-radioligand can serve as a useful tool for further in vivo research in animals and directly in humans. In this study, we report the synthesis and biological evaluation of a carbon-11 labelled analogue of N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) which was reported as a specific high-affinity antagonist for TRPV1. METHODS: The new tracer was evaluated with respect to log D and biodistribution in control, pretreated and TRPV1⁻/⁻ mice. The percentage of radiometabolites of [¹¹C]SB366791 was determined in mouse plasma and brain. RESULTS: [¹¹C] SB366791 was obtained in good yield (69%±11%; isolated amounts 3034-5032MBq) and high specific activity (390±215 GBq/µmol). The tracer was efficiently cleared from blood and all major organs via hepatobiliary and renal pathways. Initial brain uptake was high (1.6% ID) and wash-out from brain was rapid. The retention of [¹¹C] SB366791 in the trigeminal nerve of control mice was prominent. The in vitro binding affinity of SB366791 was determined to be 280±56 nM and 780±140 nM for human and rat TRPV1, respectively. CONCLUSIONS: [¹¹C] SB366791 has favourable biodistribution characteristics in mice. However the obtained low binding affinity for TRPV1 may not be sufficient to use the current compound as PET tracer.

Radioiodinated phenylalkyl malonic acid derivatives as pH-sensitive SPECT tracers.

INTRODUCTION: In vivo pH imaging has been a field of interest for molecular imaging for many years. This is especially important for determining tumor acidity, an important driving force of tumor invasion and metastasis formation, but also in the process of apoptosis. METHODS: 2-(4-[(123)I]iodophenethyl)-2-methylmalonic acid (IPMM), 2-(4-[(123)I]iodophenethyl)-malonic acid (IPM), 2-(4-[(123)I]iodobenzyl)-malonic acid (IBMM) and 4-[(123)I]iodophthalic acid (IP) were radiolabeled via the Cu(+) isotopic nucleophilic exchange method. All tracers were tested in vitro in buffer systems to assess pH driven cell uptake. In vivo biodistribution of [(123)I]IPMM and [(123)I]IPM was determined in healthy mice and the pH targeting efficacy in vivo of [(123)I]IPM was evaluated in an anti-Fas monoclonal antibody (mAb) apoptosis model. In addition a mouse RIF-1 tumor model was explored in which tumor pH was decreased from 7.0 to 6.5 by means of induction of hyperglycemia in combination with administration of meta-iodobenzylguanidine. RESULTS: Radiosynthesis resulted in 15-20% for iodo-bromo exchange and 50-60% yield for iodo-iodo exchange while in vitro experiments showed a pH-sensitive uptake for all tracers. Shelf-life stability and in vivo stability was excellent for all tracers. [(123)I]IPMM and [(123)I]IPM showed a moderately fast predominantly biliary clearance while a high retention was observed in blood. The biodistribution profile of [(123)I]IPM was found to be most favorable in view of pH-specific imaging. [(123)I]IPM showed a clear pH-related uptake pattern in the RIF-1 tumor model. CONCLUSION: Iodine-123 labeled malonic acid derivates such as [(123)I]IPM show a clearly pH dependent uptake in tumor cells both in vitro and in vivo which allows to visualize regional acidosis. However, these compounds are not suitable for detection of apoptosis due to a poor acidosis effect.

Radiolabeling and preliminary biological evaluation of a (99m)Tc(CO)(3) labeled 3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) derivative as a potential SPECT tracer for in vivo visualization of necrosis.

N,N'-bis(diethylenetriamine pentaacetic acid)-3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) (bis-DTPA-BI) was radiolabeled with (99m)Tc(CO)(3). The resulting (99m)Tc(CO)(3)-bis-DTPA-BI was characterized (LC-MS) and evaluated as a potential SPECT tracer for imaging of necrosis in Wistar rats with a reperfused partial liver infarction and Wistar rats with ethanol induced muscular necrosis. To study the specificity, uptake of (99m)Tc(CO)(3)-bis-DTPA-BI was also studied in a mouse model of Fas-mediated hepatic apoptosis. The obtained results indicate that (99m)Tc(CO)(3)-bis-DTPA-BI displays selective uptake in necrotic tissue and can be used for in vivo visualization of necrosis by SPECT.

99mTc-tricarbonyl labeled agents for cell labeling: development, biodistribution in normal mice and preliminary in vitro evaluation.

We have developed four (99m)Tc(CO)(3)-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. (99m)Tc(CO)(3)-hexadecylamino-N,N'-diacetic acid (negatively charged), (99m)Tc(CO)(3)-hexadecylamino-N-alpha-picolyl-N'-acetic acid (uncharged), (99m)Tc(CO)(3)-N,N'-dipicolylhexadecylamine (positively charged), (99m)Tc(CO)(3)-N-hexadecylaminoethyl-N'-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with (99m)Tc-d,l-HMPAO and [(18)F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for (99m)Tc(CO)(3)-N-hexadecylaminoethyl-N'-aminoethylamine after 60min incubation (compared to 55% and 23% for (99m)Tc-d,l-HMPAO and [(18)F]FDG, respectively) while in plasma-rich medium, (99m)Tc(CO)(3)-N,N'-dipicolylhexadecylamine was best bound (54%, similar to the binding of (99m)Tc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent ((99m)Tc(CO)(3)-N,N'-dipicolylhexadecylamine, logP=1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent (99m)Tc-d,l-HMPAO, (99m)Tc(CO)(3)-N,N'-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.

Synthesis and biological evaluation of (11)C-labeled beta-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging.

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled beta-galactosyl triazoles 1-(beta-d-galactopyranosyl)-4-(p-[(11)C]methoxyphenyl)-1,2,3-triazole ([(11)C]-6) and 1-(beta-d-galactopyranosyl)-4-(6-[(11)C]methoxynaphthyl)-1,2,3-triazole ([(11)C]-13). The precursors for the radiolabeling and the non-radioactive analogues (6 and 13) were synthesized using straightforward 'click' chemistry. In vitro incubation experiments of 6 with beta-galactosidase in the presence of o-nitrophenyl beta-d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of beta-galactosidase activity. Radiolabeling of both precursors was performed using [(11)C]methyl iodide as alkylating agent at 70 degrees C in DMF in the presence of a small amount of base. The logP values were -0.1 and 1.4, respectively, for [(11)C]-6 and [(11)C]-13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [(11)C]-6 was mainly cleared via the renal pathway, while the more lipophilic [(11)C]-13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [(11)C]-13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [(11)C]-13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.