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Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Histone deacetylase (HDAC)-inhibitors are well known to induce proliferative blocks and concomitant differentiation boosts in a plethora of tumor cells. Despite their promising potential as clinical therapeutics, however, the biological outcome of HDAC-inhibitors in non-tumorous cells has been poorly documented. We previously reported that the HDAC-inhibitor trichostatin A (TSA) and its metabolically more stable structural analogue 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me2N-BAVAH) cause cell cycle arrests in primary cultures of mitogen-stimulated hepatocytes. The present study was set up to explore whether this proliferative block in non-tumorous cells is also associated with inducing effects on the differentiated hepatocellular phenotype, a scenario that is usually observed in tumorous cells. In particular, the molecular actions of TSA and 4-Me2N-BAVAH on hepatic functionality and gap junctions, gatekeepers of liver homeostasis, in primary cultures of mitogen-stimulated hepatocytes are investigated. Both HDAC-inhibitors were found to promote albumin secretion and CYP1A1 gene transcription and functionality, whereas CYP2B1 gene transcription and activity were only slightly enhanced. The protein production of the gap junction component Cx26 was downregulated, whereas Cx32 expression was upregulated in response to HDAC-inhibition. Furthermore, TSA increased protein levels of the non-specific hepatocellular Cx43, whereas 4-Me2N-BAVAH rather diminished its expression. These data provide new insight into the biological impact of HDAC-inhibitors on the homeostatic balance in hepatocytes, being major executors of xenobiotic biotransformation and primary targets of drug-induced toxicity.

Histone deacetylase (HDAC) inhibitors show great pharmaceutical potential, particularly in relation to cancer. However, very little is known about their biological outcome on hepatocytes, the major executors of xenobiotic biotransformation in the organism. The current study was set up to investigate the effects of the newly synthesized HDAC inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamate (4-Me(2)N-BAVAH) on hepatocyte gap junctions and adherens junctions, being main guardians of liver homeostasis. For that purpose, freshly isolated rat hepatocytes were cultivated for 7 days either in the absence or presence of 50 microM 4-Me(2)N-BAVAH. Gap junction activity became promoted upon exposure to 4-Me(2)N-BAVAH, which was associated with elevated Cx32 protein levels. By contrast, both Cx26 and Cx43 protein levels were negatively affected. The modifications in connexin protein content were not reflected at the transcriptional level. Finally, neither the expressions nor the cellular localizations of the adherens junction building stones E-cadherin, beta-catenin and gamma-catenin were altered by 4-Me(2)N-BAVAH, a finding that is in contrast to what is commonly observed in tumor cells following exposure to HDAC inhibitors.

Introduction: Overexpression of neurotensin (NT) receptors in exocrine pancreatic cancer and other neuroendocrine cancers make them interesting targets for tumor imaging and therapy. Modifications at the cleavage bonds 8-9 and 11-12 led to the synthesis of NT-XII, NT-XIII and NT-XVIII, three new stabilized analogues. (NalphaHis)Ac was coupled to the N-terminus for labeling with [(99m)Tc]-tricarbonyl., Methods: Stability was tested in vitro in human plasma and HT-29 cells. Binding to NT1 receptors and internalization/efflux were analyzed in intact HT-29 cells. Biodistribution studies were performed in nude mice bearing HT-29 xenografts., Results: All analogues were very stable in human plasma, with half-lives of 20-21 days. Degradation in HT-29 cells was more rapid (t(1/2) of 6.5, 5 and 2.5 h for NT-XII, NT-XIII and NT-XVIII, respectively). They also showed high affinity and specificity for NT1 receptors. Bound activity was rapidly internalized at 37 degrees C. The pattern of externalization was different. NT-XII was released more slowly than NT-XIII and NT-XVIII (half of the activity still inside the cells after 24 h). Bigger differences were found in the biodistribution studies. NT-XII showed the highest tumor uptake as well as the best tumor to nontumor ratios., Conclusion: The modifications introduced in NT(8-13) increased plasma stability, maintaining unaffected the in vitro binding properties. The best biodistribution corresponded to NT-XII, which shows to be a good candidate for NT1 receptors overexpressing tumors. First clinical trials are ongoing.

Direct and indirect reversed-phase (RP) high-performance liquid chromatographic methods were developed for the separation of enantiomers of 18 unnatural beta-amino acids, including several beta-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases (CSPs) containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with a new chiral derivatizing agent (CDA), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)-NIFE), and subsequent separation of diastereomers on Discovery C18 and Hyperpep 300 C18 columns. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.

Neurotensin (NT) receptors in pancreatic and other neuroendocrine tumors are promising targets for imaging and therapeutic purposes. Here, we report on the effect of distinct changes in the peptide chain on catabolism in vitro for five radiolabeled [99mTc] neurotensin analogues having high affinity for neurotensin receptors. Substitution of NT(1-7) by (NalphaHis)Ac--the Tc-binding moiety--combined with a reduced bond 8-9 (CH2NH), N-methylation of peptide bonds or replacement of Ile(12) by tertiary leucin (Tle) led to peptide stabilization of various degrees. Biodistribution studies in nude mice bearing HT29 xenografts showed higher tumor uptake with more stable peptides, yielding high tumor to blood ratios of up to 70.

The indirect high-performance liquid chromatographic enantioresolution of beta-alkyl-substituted analogues of tyrosine, phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and tryptophan is reported. (S)-N-(4-Nitrophenoxycarbonyl)phenylalanine methoxyethyl ester, a recently developed chiral derivatizing agent, was used for pre-column derivatization of the investigated analytes. The diastereoisomers formed were analysed under reversed-phase conditions. The effects of parameters such as the amount and type of the organic modifier and the type of the stationary phase on the resolution and retention of the derivatives were investigated. Chromatographic conditions were found for the separation of all four stereoisomers of each analyte.

A new chiral derivatizing agent, (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester, (S)-NIFE, was applied for the high-performance liquid chromatographic separation of enantiomers of 19 unnatural secondary amino acids: proline, pipecolic acid analogues, piperazine-2-carboxylic acid, morpholine-3-carboxylic acid, thiomorpholine-3-carboxylic acid and analogues containing the 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydronorharmane, 1,2,3,4-tetrahydro-2-carboline and 2-benzazepine skeletons. Excellent resolutions were achieved for most of the investigated compounds by using a reversed-phase mobile phase system. The conditions of separation were optimized by variation of the mobile phase composition.

4-([(18)F]fluoro)benzoyl-neurotensin(8-13) ((18)FB-Arg(8)-Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 1) and two analogs stabilized in one and two positions ((18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 2, (18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)-Tle(12)-Leu(13)-OH, 3) were synthesized in a radiochemical yield of 25-36% and a specific activity of 5-15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumors overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). All analogs exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumors, measured by quantitative receptor autoradiography, HT-29 and WiDr cells, and to sections of tumors derived from these cell lines in mice. The radiotracers were internalized in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca(2+) of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumors, only a moderate uptake of the radioligands into the studied tumors was observed, presumed to be due to degradation in vivo and fast elimination by the kidneys. In comparison with the other analogs, the specific tumor uptake expressed as tumor-to-muscle relation was highest for the radioligand 3. The blood clearance of 3 was reduced by co-injection of peptidase inhibitors. The catabolic pathways of the radiofluorinated peptides were elucidated. The results suggest that the high binding affinity to NTR1 and the stabilization against proteolytic degradation are not yet sufficient for tumor imaging by PET.

The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

A macrocyclic glycopeptide, ristocetin A, was used as chiral stationary phase for the high-performance liquid chromatographic separation of enantiomers of 28 unnatural amino acids, such as analogues of phenylalanine, tyrosine and tryptophan, and analogues containing 1,2,3,4-tetrahydroisoquinoline, tetraline or 1,2,3,4-tetrahydro-2-carboline skeletons. Excellent resolutions were achieved for most of the investigated compounds by using reversed-phase or a new polar-organic mobile phase system. The conditions of separation were optimized by variation of the mobile phase composition, temperature and flow-rate.

The direct and indirect stereochemical resolution of the enantiomers of ring- and alpha-methyl-substituted phenylalanines and phenylalanine amides was attempted by high-performance liquid chromatographic methods. The direct separation was carried out on two chiral stationary phases, the crown-ether-based Crownpak CR(+), and the teicoplanin-based Chirobiotic T, while the indirect resolution was performed by applying pre-column derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC) and Nalpha-(2,4-dinitro-5-fluorophenyl)-L-alanine amide (Marfey's reagent, FDAA). The Chirobiotic T column was efficient in the separation of ring- and alpha-methyl-substituted phenylalanine analogues, but was ineffective for the amides of these analogues. The Crownpak CR(+) column separated the ring-substituted phenylalanines and amides, whereas the alpha-methylated analogues were coeluted. Of the two indirect methods, GITC derivatization seemed more effective than FDAA derivatization.

The isocratic retention of enantiomers of beta-methyl amino acids (beta-methyltyrosine, beta-methylphenylalanine, beta-methyl-tryptophan and beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was studied on a teicoplanin-containing chiral stationary phase at different temperatures and at different mobile phase compositions, using the reversed-phase mode. With variation of both mobile phase composition and temperature, almost baseline separations could be achieved for all four enantiomers of sterically hindered amino acids. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van 't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (delta H degree), the apparent change in entropy (delta S degree) and the apparent change in Gibbs free energy (delta G degree) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic constants (delta H degree, delta S degree and delta G degree) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.

Several agonists of luteinizing hormone-releasing hormone (LHRH) are currently used for different therapeutic purposes, but relatively little is known about their metabolic fate after administration. This paper describes the application of high-performance liquid chromatography combined with off-line fast atom bombardment mass spectrometry to identify the degradation products resulting from the incubation of LHRH analogues with proteolytic enzymes. Three analogues, containing a psi(E,CH = CH) pseudo-peptide bond were synthesized and afforded to the assay to determine the resistance against alpha-chymotrypsin and subtilisin: [Tyr5 psi(E,CH = CH)Gly6]LHRH, [Gly6 psi(E,CH = CH)D,L-Leu7]LHRH and [Pro9 psi(E,CH = CH)Gly10 ]LHRH. The pattern of peptide metabolites identified by this method indicates that alpha-chymotrypsin degrades LHRH analogues at the Trp3-Ser4 and Tyr5-Gly6 bond, while subtilisin hydrolyzes only the Tyr5-Gly6 linkage. The results also indicate a possible stabilization of native amide bonds against enzymatic degradation by neighbouring psi(E, CH = CH) modifications.

Erythro-D,L- and threo-D,L-beta-methylphenylalanine, -beta-methyltyrosine and -beta-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid were synthesized. High-performance liquid chromatographic methods were developed for the separation and identification of the enantiomers of the beta-methyl amino acids, with the application of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate as derivatizing reagents. These amino acids were incorporated into the mu-agonist/delta-antagonist opioid peptides H-beta-MeTyr-Tic-Phe-Phe-NH2, H-Tyr-Tic-beta-MePhe-Phe-NH2 and H-Tyr-Tic-Phe-beta-MePhe-NH2, and the delta-antagonist H-Tyr-beta-MeTic-Phe-Phe-OH, by solid-phase peptide synthesis. Each peptide has four stereoisomers. The peptide stereoisomers were separated on different columns and in different eluent systems and the elution order of the peptide epimers was determined.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed to obtain pure erythro[2S3S, 2R3R]- and threo[2S3R, 2R3S]-beta-methylphenylalanine. These amino acids were incorporated into an enkephalin, H-Tyr-D-Ala-Gly-beta-MePhe-Val-Val-Gly-NH2, and into a deltorphin C, H-Tyr-D-Ala-beta-MePhe-Asp-Val-Val-Gly-NH2, analogue, which yielded four diastereoisomers of the peptides. The diastereoisomers were separated on different columns and with different eluent systems. The sequence of elution of the peptide diastereoisomers was determined after hydrolysis of the peptides. For identification of the beta-methylphenylalanine enantiomers, enzymatic degradation and an RP-HPLC method were used, with application of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide as derivatizing reagent.

The stereochemical purity of psi (CH2-NH) dipeptides has been determined using gas chromatography-mass spectrometry. Different structures were found due to the derivatization procedures. A selective preparation of the linear bistrifluoroacetylated derivative and the monotrifluoroacetylated lactam makes it possible to monitor the chiral purity of the pseudodipeptides synthesized. Racemization occurring during peptide hydrolysis can be differentiated from racemization during the synthesis by using deuterium labelling. The method allows the optimization of the synthesis protocols and will be useful for further monitoring of the chiral purity of the pseudopeptides synthesized.

N-methyl- alpha -benzyl-o-aminomethylphenylacetic acid was incorporated into a cyclic somatostatin analogue in order to mimic a cis-peptide bond configuration. The high biological potency of one of the isomers of the cyclic peptide strongly argues in favour of the proposed cis-configuration of the peptide bond at that position in the parent peptide. This represents the first cis-peptide bond mimic which has high biological activity.