István Kertész

Binding site of salsolinol: its properties in different regions of the brain and the pituitary gland of the rat

It has been recently shown that salsolinol (SAL) is present in the hypothalamic neuroendocrine dopaminergic (NEDA) system and appears to be a selective and potent stimulator of prolactin (PRL) secretion in the rat. Furthermore, the lack of interference of SAL with 3H-spiperone binding in the striatum and the anterior lobe (AL) of the pituitary gland has been also demonstrated. These data clearly indicate that SAL does not act at the dopamine (DA) D(2) receptors, and suggest that SAL supposedly has a binding site through which the secretion of PRL may be affected. Therefore, binding of 3H-SAL to different regions of the central nervous system (CNS) has been investigated. Specific and saturable binding has been detected in the striatum, cortex, median eminence and in the hypothalamus as well as in the AL and the neuro-intermediate lobe (NIL) of the pituitary gland. K(D) values of the bindings were in the nanomolar range in all tissue tested. 3H-SAL displacing activity of several agonists and antagonists of known DA receptors have also been tested. It has been found that DA and in a lesser extent, apomorphine could displace 3H-SAL, but other DA receptor specific ligands have not been able to affect it. Furthermore, several pharmacologically active compounds, selected on the basis of their influence on DA synthesis, transport mechanisms and signal transduction, have also been tested. Neither mazindol (a selective DA transporter inhibitor) nor clonidine (an alpha(2)-adrenoreceptor agonist) could alter SAL binding. At the same time, L-dopa, carbidopa, benserazide and alpha-methyldopa were able to displace 3H-SAL. The possible changes in SAL binding due to physiological and pharmacological stimuli, like suckling stimulus and reserpine pretreatment (that blocks vesicular monoamine transport in DA terminals), respectively, have also been investigated. In the NIL of the pituitary gland and in the median eminence of the hypothalamus the binding decreased following 10 min of suckling stimulus compared to the binding detected in the same tissues obtained from mothers separated from their pups for 4h and not allowed to be suckled. At the same time, there were no changes in the binding at the AL and striatum. Following reserpine pretreatment that has completely prevented PRL releasing effect of SAL, the binding was significantly augmented. These results support our assumption that SAL should have specific binding sites through which it can affect PRL secretion. Furthermore, it clearly suggests that it may regulate DAergic neurotransmission of NEDA neurons by an altered intracellular or intraterminal synthesis and/or distribution of hypophysiotropic DA.

Synthesis of (E)- and (Z)-fluoro-olefin analogues of potent dipeptidyl peptidase IV inhibitors

Abstract ( E )- and ( Z )-fluoro-olefin analogues of potent dipeptidyl peptidase IV inhibitors were synthesized. A Wadsworth–Horner–Emmons reaction, followed by amide formation and reduction of the amide were used for the construction of the α-fluoro-α,β-unsaturated amine functionality.

β-Fluorinated proline derivatives: potential transition state inhibitors for proline selective serine dipeptidases

Abstract Three new types of β-fluorinated proline derivatives were synthesized as potential transition state inhibitors for proline selective serine dipeptidases. The fluorophosponate derived from protected proline was tested as a Wadsworth–Horner–Emmons reagent for the synthesis of fluoro-olefin-containing pseudodipeptides.

Efficient synthesis of an (aminooxy) acetylated-somatostatin derivative using (aminooxy)acetic acid as a 'carbonyl capture' reagent.

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated‐somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a ‘carbonyl capture’ reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4‐fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography. Copyright

In vivo imaging of Aminopeptidase N (CD13) receptors in experimental renal tumors using the novel radiotracer 68Ga-NOTA-c(NGR)

PURPOSE Aminopeptidase N (APN/CD13) plays an important role in tumor neoangiogenic process and the development of metastases. Furthermore, it may serve as a potential target for cancer diagnosis and therapy. Previous studies have already shown that asparagine-glycine-arginine (NGR) peptides specifically bind to APN/CD13. The aim of the study was to synthesize and investigate the APN/CD13 specificity of a novel (68)Ga-labeled NOTA-c(NGR) molecule in vivo using miniPET. METHODS c[KNGRE]-NH2 peptide was conjugated with p-SCN-Bn-NOTA and was labeled with Ga-68 ((68)Ga-NOTA-c(NGR)). Orthotopic and heterotopic transplanted mesoblastic nephroma (NeDe) bearing Fischer-344 rats were prepared, on which biodistribution studies and miniPET scans were performed for both (68)Ga-NOTA-c(NGR) and ανβ3 integrin selective (68)Ga-NODAGA-[c(RGD)]2 tracers. APN/CD13 receptor expression of NeDe tumors and metastases was analyzed by western blot. RESULTS (68)Ga-NOTA-c(NGR) was produced with high specific activity (5.13-5.92GBq/μmol) and with excellent radiochemical purity (95%<), at all cases. Biodistribution studies in normal rats showed that uptake of the (68)Ga-NOTA-c(NGR) was significantly (p⩽0.05) lower in abdominal organs in comparison with (68)Ga-NODAGA-[c(RGD)]2. Both radiotracers were mainly excreted from the kidney. In NeDe tumor bearing rats higher (68)Ga-NOTA-c(NGR) accumulation was found in the tumors than that of the (68)Ga-NODAGA-[c(RGD)]2. Using orthotopic transplantation, metastases were developed which showed specific (68)Ga-NOTA-c(NGR) uptake. Western blot analysis confirmed the presence of APN/CD13 expression in NeDe tumors and metastases. CONCLUSION Our novel radiotracer (68)Ga-NOTA-c(NGR) showed specific binding to the APN/CD13 expressed ortho- and heterotopic transplanted NeDe tumors. Therefore, (68)Ga-NOTA-c(NGR) is a suitable tracer for the detection of APN/CD13 positive tumors and metastases in vivo.

Synthesis of 2′,6′‐dimethyltyrosine containing tritiated delta opioid‐receptor selective antagonist dipeptide ligands with extraordinary affinity

A new class of δ opioid antagonists was recently discovered in which the sequence Tyr-Tic was used as a message domain. The substitution of Tyr1 by Dmt enhanced the δ selectivity and antagonist activity. The excellent properties of these ligands stimulated us to prepare the corresponding tritiated derivatives. Peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, with a reduction in opioid activity. To avoid this side-reaction we synthesised the N,N-dimethylated analogue (N,N(Me)2-Dmt-Tic-OH), which was found to be stable. On the basis of this result, we prepared diiodinated analogues of H-Dmt-Tic-OH and N,N(Me)2-Dmt-Tic-OH to undergo catalytic dehalotritiation. Products of high specific radioactivity were obtained: 44.67 Ci/mmol for [3H]Dmt-Tic-OH and 59.88 Ci/mmol for [3H]N,N(Me)2-Dmt-Tic-OH. Copyright

The Influence of the Combination of Carboxylate and Phosphinate Pendant Arms in 1,4,7-Triazacyclononane-Based Chelators on Their 68Ga Labelling Properties

In order to compare the coordination properties of 1,4,7-triazacyclononane (tacn) derivatives bearing varying numbers of phosphinic/carboxylic acid pendant groups towards 68Ga, 1,4,7-triazacyclononane-7-acetic-1,4-bis(methylenephosphinic) acid (NOPA) and 1,4,7-triazacyclononane-4,7-diacetic-1-[methylene(2-carboxyethyl)phosphinic] acid (NO2AP) were synthesized using Mannich reactions with trivalent or pentavalent forms of H-phosphinic acids as phosphorus components. Stepwise protonation constants logK1–3 12.06, 3.90 and 1.95, and stability constants with GaIII and CuII, logKGaL 24.01 and logKCuL 16.66, were potentiometrically determined for NOPA. Both ligands were labelled with 68Ga and compared with NOTA (tacn-N,N′,N″-triacetic acid) and NOPO, a TRAP-type [tacn-N,N′,N″-tris(methylenephosphinic acid)] chelator. At pH 3, NOPO and NOPA showed higher labelling efficiency (binding with lower ligand excess) at both room temperature and 95 °C, compared to NO2AP and NOTA. Labelling efficiency at pH = 0–3 correlated with a number of phosphinic acid pendants: NOPO >> NOPA > NO2AP >> NOTA; however, it was more apparent at 95 °C than at room temperature. By contrast, NOTA was found to be labelled more efficiently at pH > 4 compared to the ligands with phosphinic acids. Overall, replacement of a single phosphinate donor with a carboxylate does not challenge 68Ga labelling of TRAP-type chelators. However, the presence of carboxylates facilitates labelling at neutral or weakly acidic pH.

Synthesis and pharmacological characterization of a novel, highly potent, peptidomimetic δ-opioid radioantagonist, [3H]Tyr-Tic-(2S, 3R)-β-MePhe-Phe-OH

Abstract [ 3 H]Tyr-Tic-( 2S , 3R )-β-MePhe-Phe-OH (where Tic: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) with a specific radioactivity of 53.7 Ci/mmol was synthesized and characterized in receptor binding assays at 25°C in rat brain membranes. The specific binding was saturable and displayed high affinity, with a K D of 0.16±0.005nM and B max of 85.9±6.3fmol/mg protein. NaCl increased its affinity by about 4-fold in membranes of rat brain and Chinese Hamster Ovary Cells stably transfected with the human δ-opioid receptors (hDOR-CHO) showing that the new ligand is an antagonist. The prototypic δ-opioid ligands were much more potent than μ- or κ-specific ligands in competition assays. The autoradiographic distribution of the binding sites of the new ligand agreed with the known locations of the δ-opioid receptors in rat brain. The unlabeled new ligand was about 7-fold more potent than the parent peptide in competing for the binding sites of [ 3 H]Tyr-Tic-( 2S , 3R )-β-MePhe-Phe-OH in rat brain membranes. Likewise, the threo -β-methyl analog was 3.8-fold more potent than the parent compound in antagonizing the effect of DPDPE in the [ 35 S]GTPγS functional assay in hDOR-CHO membranes. The new, highly potent, conformationally constrained antagonist may be a valuable pharmacological tool in understanding the structural and topographical requirements of peptide ligand binding to the δ-opioid receptors.

Synthesis and binding characteristics of [3H] H-Tyr-Ticψ[CH2-NH] Cha-Phe-OH, a highly specific and stable δ-opioid antagonist

Abstract Substitution of the Phe 3 aromatic ring in H-Tyr-Ticψ[CH 2 -NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticψ[CH 2 -NH]Cha-Phe-OH (TICP[ψ]), showing substantially increased δ-opioid antagonist potency and high δ selectivity. TICP[ψ] was radiolabeled by catalytic tritiation of its precursor Tyr(3′,5′-I 2 ) 1 TICP[ψ]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25°C, an equilibrium dissociation constant ( K d ) of 0.35 nM and a receptor density ( B max ) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for δ-opioid receptors, whereas its binding to μ and κ receptors is weak. A study of [H 3 ]TICP[ψ] binding displacement by various receptor-selective opioids showed the following rank order of potency: δ > κ = μ. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling δ-opioid receptors, both in vitro and in vivo.

Multiparametric labeling optimization and synthesis of 68Ga-labeled compounds applying a continuous-flow microfluidic methodology

AbstractThe synthesis and functional evaluation of a wide variety of radiolabeled chelator—biomolecule conjugates with high specific activity and radiochemical purity are crucial to development of personalized nuclear medicine. An excellent platform technology for achieving this objective involves use of generator-produced positron emission tomography (PET)-radionuclide 68Ga. Currently, applied manual methodology for optimization and development for new labeling techniques offers only slow screening with relatively high precursor consumption. A capillary-based microfluidic synthesis module with online high-performance liquid chromatography (HPLC) was constructed for the optimization of reaction parameters of 68Ga-PET tracers. This approach enables performance of 68Ga-labeling reactions in 10 μL volumes, followed by sample analysis. The high-throughput capacity of the system allows very rapid optimization. The optimal pH and ligand concentration from the experiments were utilized directly to the production of 68Ga-NODAGA-(RGD)2 and 68Ga-NOPO-RGD. Applying optimal parameters to production of these aforementioned radiopharmaceuticals allowed their synthesis with high radiochemical purity (over 95%) and with surprisingly negligible retention of residual activity in the system.