Bernard Cleynhens

Synthesis and biological evaluation of a 99mTc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia

INTRODUCTION Carbonic anhydrase (CA) IX is a transmembrane protein overexpressed in many frequently occurring tumors associated with tumor hypoxia. Sulfonamides and their bioisosteres are known to inhibit CA IX activity. In this study, 4-(2-aminoethyl)benzenesulfonamide was conjugated to a tridentate ligand, N-2-picolyl-N-acetic acid and labeled with a (99m)Tc(I)-tricarbonyl moiety resulting in [(99m)Tc(CO)(3) (L)] (L=N-(pyridin-2-yl-methyl)-N[2-(4-sulfamoylphenyl)-ethyl]aminoethyl acetate) complex, [(99m)Tc]-5. Similarly the corresponding rhenium congener (Re-4) was synthesized. The in vitro CA IX affinity and inhibitory activity of Re-4 were determined and [(99m)Tc]-5 was evaluated as a tracer for in vivo visualisation of CA IX expression. METHODS Evaluation of the in vitro affinity (inhibition constant, K(i)) of Re-4 for CA isozymes I, II, IX and XII was carried out by assaying the CA catalyzed CO(2) hydration activity and efficacy studies were performed in HT 29 cell lines expressing CA IX under normoxia or hypoxia. Biodistribution studies of [(99m)Tc]-5 were performed in xenograft mice bearing CA IX expressing tumors. RESULTS The in vitro affinity of Re-4 for CA IX was 58 nM and CA IX induced acidification of extracellular medium was efficiently reduced (P<.05) in the presence of 1 mM Re-4. Biodistribution studies indicated a maximal tumor uptake of [(99m)Tc]-5 of 0.1% ID/g at 30 min post injection. CONCLUSION [(99m)Tc]-5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.

Comparative evaluation of 99Tcm-Hynic-HSA and 99Tcm-MAG3-HSA as possible blood pool agents.

SummaryTwo strategies have been used to increase the 99Tcm binding strength of human serum albumin (HSA) and thus enhance its blood retention. In a first approach, HSA was derivatized with a varying number of hydrazino nicotinyl (Hynic) side-chains using N-hydroxysuccinimidyl hydrazino nicotinate. Labelling of this albumin derivative with 99Tcm resulted in labelling yields of 90–95%. On the other hand, a 99Tcm-MAG3-HSA conjugate was prepared using the preformed chelate approach. In this way, non-specific binding of 99Tcm to HSA could be excluded. The in vitro stability of both 99Tcm-HSA derivatives was evaluted by cysteine challenge experiments and revealed a much higher stability for 99Tcm-Hynic-HSA than for 99Tcm-MAG3-HSA. The biological behaviour of the preparations was evaluated in mice and a rabbit using 125I-HSA as an internal biological standard. The blood retention of 99Tcm-MAG3-HSA decreased more rapidly than that of 125I-HSA in both animal species, whereas 99Tcm-Hynic-HSA seemed to provide a quasi-perfect 99Tcm-labelled analogue for 125I-HSA and 99Tcm-red blood cells (99Tcm-RBCs). In addition, the blood retention of 99Tcm-Hynic-HSA appeared to be similar to that of 99Tcm-RBCs in a volunteer. These results clearly indicate the superiority of 99Tcm-Hynic-HSA over 99Tcm-MAG3-HSA as a possible blood pool agent.

Investigation of the labelling characteristics of 99mTc-mercaptoacetyltriglycine

S-Benzyl-, S-benzamidomethyl- and S-benzoylmercaptoacetyltriglycine were synthesized and compared in exchange labelling experiments for the preparation of 99mTc-MAG3. The rate of exchange from 99mTc-tartrate to 99mTc-MAG3 starting from the respective precursors was determined in different conditions. Labelling proceeded most rapidly starting from the S-benzoyl protected precursor but efficient labelling was also accomplished using the more stable S-benzamidomethyl- and S-benzylmercaptoacetyltriglycine. 99mTc-MAG3 was also prepared by direct labelling of unprotected mercaptoacetyltriglycine at alkaline pH. Radiochemical purity in these conditions is mainly dependent on the pH during labelling.

Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K(i) = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with (99m)Tc, to obtain anionic and neutral (99m)Tc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K(i) = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with (99m)Tc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG₂ tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged (99m)Tc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.

Importance of the two ester functions for the brain retention of 99mTc-labelled ethylene dicysteine diethyl ester (99mTc-ECD)

99mTc-ethylene dicysteine diethyl ester (99mTc-L,L-ECD) is a neutral lipophilic tracer agent that crosses the blood-brain barrier and is retained in the brain of primates following enzymatic hydrolysis of one of the ester functions to the ionized mono-ester, mono-acid metabolite. Up to now, it is not clear whether the second ethylcarboxylate group is essential for brain uptake and retention. Therefore, we have synthesized and studied two derivatives of 99mTc-L,L-ECD that contain only one ethylcarboxylate function, namely 99mTc-labelled L- and D-ethylene cysteamine cysteine ethyl ester (99mTc-ECCE). Direct labelling of L- or D-ECCE at neutral pH and room temperature resulted for each of them in the formation of two probably diastereomeric 99mTc-complexes in a 1:1 ratio. This means that four different isomers could be isolated. The 99mTc-labelled complexes formed after labelling ECCE (A and B, in order of elution during HPLC) are slightly less lipophilic than 99mTc-L,L-ECD. In mice, all four isomers show a low brain activity at 10 min post injection (p.i.), approximately 0.1% to 0.3% of the injected dose versus 0.9% for 99mTc-L,L-ECD. The clearance from the blood is comparable with (isomers LA and LB) or slower (isomers DA and DB) than that of 99mTc-L,L-ECD. Isomers LA and DA show high liver uptake and rapid excretion to the intestines. Both 99mTc-ECCE-LB and 99mTc-ECCE-DB, the most lipophilic isomers, are cleared from the blood mainly by the kidneys and excreted more efficiently to the urine. 99mTc-ECCE-LB is characterized by a surprisingly high heart uptake (about 1.5% of i.d.) at 10 min p.i. versus 1.0% for 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) and 0.2-0.3% for the other isomers, but also lung uptake is relatively high. In the baboon, brain and heart uptake of isomers LA and LB of 99mTc-ECCE were negligible. Activity concentrated mostly in the hepatobiliary system for isomer LA and in the renal system for isomer LB. The results indicate a clear difference in biological behaviour between 99mTc-L,L-ECD and the four isomers of 99mTc-ECCE. This shows that the presence of both ester functions in 99mTc-L,L-ECD is an essential structural requirement for its brain uptake and retention in primates.

Influence of the bifunctional chelate on the biological behavior of 99mtc-labeled chemotactic peptide conjugates

Conjugates of For-MLFK and For-NleLFNleYK with S-benzyl mercaptoacetyl dipeptides containing, respectively, zero, one, and two carboxyl functions in their structures were prepared and labeled with (99m)Tc. In vitro binding studies using isolated human granulocytes indicated specific receptor binding of the radiolabeled conjugates. The fraction of granulocyte-associated activity was determined after incubation with total blood. Biodistribution studies of the (99m)Tc-peptides in normal mice revealed a very fast blood clearance proceeding mainly via the hepatobiliary system. Urinary excretion was higher for conjugates containing carboxyl functions in their ligand structures.

S-Trityl protection of bis-amino bis-thiol (BAT) chelator enables flexible derivatisation and facile labelling with technetium-99m

Abstract We have coupled S , S ′-bis-trityl N -BOC protected 1,2-ethylenedicysteamine, a bis-amino bis-thiol (BAT) tetraligand, via a propylene or ethylene spacer to several biologically active molecules including 2-nitroimidazole, desethylflumazenil, a beta-CIT analogue, glucose and 2-(2′-hydroxy-4′-aminophenyl)-1,3-benzothiazole. The conjugates were efficiently labelled with technetium-99m by consecutive heating of the S , S ′-bis-trityl protected ligand in hydrochloric acid followed by neutralisation and heating in the presence of 99m Tc-tartrate. The S , S ′-bis-trityl BAT chelator is an interesting synthon that allows both flexible derivatisation with various biologically active molecules and facile labelling with technetium-99m.

RP-HPLC separation of the diastereomers of technetium-99m labelled tropanes and identity confirmation using radio-LC-MS

99mTc-TRODAT-1 (technetium(V)-oxo-2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]]amino]-ethanethiolato(3-)) and 99mTc-TRODAT-M, the 4-methylphenyl derivative of 99mTc-TRODAT-1, are at this moment being evaluated in clinical trials as imaging agents for the central nervous dopamine transporter system. Both compounds are formed as a mixture of two major diastereomers. As the tracer concentration in preparations for clinical investigations is very low (30-150 pmol/ml), identification of these 99mTc-complexes was, up to now, carried out indirectly using X-ray diffraction analysis of the corresponding rhenium complexes which can be synthesized in gram amounts. In this study, we developed a convenient and practical reversed phase HPLC-method for purification and isolation of the respective diastereomers of 99mTc-TRODAT-1 and three of its derivatives using mixtures of solvents which are compatible with biological studies, i.e. aqueous buffers and ethanol. Furthermore, direct identity confirmation of the 99mTc-complexes using radio-LC-MS was successfully elaborated.

Characteristics and biological behaviour of 99mTc-labelled hydroxyacetyltriglycine, a potential alternative to 99mTc-MAG3.

Abstract.Substitution of the oxidation-sensitive thiol function of mercaptoacetyltriglycine (MAG3) by a hydroxyl group yields a tetraligand (hydroxyacetyltriglycine or HAG3) which is almost insensitive to oxidation and has the advantage over MAG3 that it can be stored safely without protection of the alcohol function. We found that deprotected HAG3 could be directly labelled at alkaline pH (pH≥11.5) and room temperature in high yield (>95%). Results of electrophoresis experiments suggested a comparable structure for 99mTc-HAG3 and 99mTc-MAG3, namely binding of an oxotechnetium(V)core via three deprotonated amides and a deprotonated hydroxyl group. Biodistribution studies in mice at 10 min and 30 min p.i. showed a slightly higher urinary excretion, a faster renal transit and a significantly lower hepatobiliary handling for 99mTc-HAG3 than for 99mTc-MAG3. In a baboon, the 1-h plasma clearance of 99mTc-HAG3 was clearly higher than that of 99mTc-MAG3. Its plasma protein binding was in the same order as that of Hippuran and much lower than that of 99mTc-MAG3. Evaluation in a human volunteer confirmed the favourable biological characteristics of 99mTc-HAG3, namely a rapid renal excretion, a high 1-h plasma clearance and a negligible hepatobiliary handling. The results indicate that 99mTc-HAG3 may be an easy-to-prepare and practical substitute for 99mTc-MAG3 with improved renal excretion characteristics.

Preclinical evaluation of carbon-11 and fluorine-18 sulfonamide derivatives for in vivo radiolabeling of erythrocytes

BackgroundTo date, few PET tracers for in vivo labeling of red blood cells (RBCs) are available. In this study, we report the radiosynthesis and in vitro and in vivo evaluation of 11C and 18F sulfonamide derivatives targeting carbonic anhydrase II (CA II), a metallo-enzyme expressed in RBCs, as potential blood pool tracers. A proof-of-concept in vivo imaging study was performed to demonstrate the feasibility to assess cardiac function and volumes using electrocardiogram (ECG)-gated positron emission tomography (PET) acquisition in comparison with cine magnetic resonance imaging (cMRI) in rats and a pig model of myocardial infarction.MethodsThe inhibition constants (Ki) of CA II were determined in vitro for the different compounds by assaying CA-catalyzed CO2 hydration activity. Binding to human RBCs was estimated after in vitro incubation of the compounds with whole blood. Biodistribution studies were performed to evaluate tracer kinetics in NMRI mice. ECG-gated PET acquisition was performed in Wistar rats at rest and during pharmacological stress by infusing dobutamine at 10 μg/kg/min and in a pig model of myocardial infarction. Left ventricular ejection fraction (LVEF) and volumes were compared with values from cMRI.ResultsThe Ki of the investigated compounds for human CA II was found to be in the range of 8 to 422 nM. The fraction of radioactivity associated with RBCs was found to be ≥90% at 10- and 60-min incubation of tracers with heparinized human blood at room temperature for all tracers studied. Biodistribution studies in mice indicated that 30% to 67% of the injected dose was retained in the blood pool at 60 min post injection. A rapid and sustained tracer uptake in the heart region with an average standardized uptake value of 2.5 was observed from micro-PET images. The LVEF values obtained after pharmacological stress in rats closely matched between the cMRI and micro-PET values, whereas at rest, a larger variation between LVEF values obtained by both techniques was observed. In the pig model, a good agreement was observed between PET and MRI for quantification of left ventricular volumes and ejection fraction.ConclusionsThe 11C and 18F sulfonamide derivatives can be used for efficient in vivo radiolabeling of RBCs, and proof-of-concept in vivo imaging studies have shown the feasibility and potential of these novel tracers to assess cardiac function.