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Urotensin II (UII) and its paralog peptide, urotensin II-related peptide (URP), exert not only common but also divergent actions through the activation of UT, a specific membrane-bound receptor that belongs to the 1A G protein-coupled receptor subclass. In this study, we have designed and synthesized new URP analogues in which the intracyclic Trp residue was replaced with natural, unnatural, and constrained amino acids to determine important physicochemical features for receptor binding and activation. The biological data, highlighting the potent agonistic behavior of [Tiq(4)]URP and [Tpi(4)]URP, also suggest that the Trp residue, and more specifically the indole ring, is not critical for receptor interaction and could in fact be involved in the intramolecular stabilization of the bioactive conformation of URP. Finally, these analogues, which are intracyclic constrained URP-based agonists, could represent useful pharmacological tools for the study of the urotensinergic system.

A one-pot procedure for the highly diastereoselective synthesis of 1-carbamoyl-4-amino-1,2,4,5-tetrahydroindolo[2,3-c]azepin-3-one derivatives is described. Using 2-formyl-L-tryptophan as a bifunctional building block, a catalyst-free Ugi-three-component reaction (Ugi-3CR) was developed to present trisubstituted indoloazepinones in good yields and excellent diastereomeric excess.

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α-helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues in a 'helical' fragment., (Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.)

The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), but Ang IV also acts as a weak agonist for the Ang II-receptor (AT1), implying the need for stable and highly selective Ang IV-analogues. Here we present the screening of novel Ang IV-analogues, selected on basis of high affinity for IRAP, high selectivity (compared to aminopeptidase N and the AT1 receptor) and resistance against proteases. The selected compound IVDE77 possesses a number of advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (Ki 1.71 nM), (ii) it does not activate the AT1 receptor, (iii) it is easily radiolabeled with tritium and (iv) it is resistant to proteolysis, even in human plasma. In addition, pre-treatment of intact CHO-K1 cells with IVDE77 led to a virtually complete inhibition of subsequent intracellular accumulation of [(3)H]IVDE77-IRAP complexes. IVDE77 thus represents the first Ang IV-analogue able to abolish IRAP-availability completely at the cell surface in vitro. In summary, IVDE77 is a useful tool for the detection of IRAP under physiological conditions, and may contribute to elucidating the mechanism of Ang IV to ascertain which functions are IRAP-dependent., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To address the different types of pain (e.g. acute, chronic, neuropathic) different classes of medications, mainly non-steroidal anti-inflammatory drugs and narcotics (opioids), are used. More specifically, the alleviation or treatment of moderate to severe pain states commonly invokes the use of opioids. Unfortunately, their chronic administration induces various undesirable side effects, such as for example physical dependence and tolerance. One strategy to overcome these major side effects and to prolong the antinociceptive efficiency of the applied drugs involves the creation of multifunctional compounds which contain hybridized structures. Combination of opioid agonist and antagonist pharmacophores in a single chemical entity has been considered and extensively investigated, but opioids have also been combined with other bioactive neurotransmitters and peptide hormones that are involved in pain perception (e.g. substance P, neurotensin, cholecystokinin, cannabinoids, melanocortin ligands, etc.). Such novel chimeras (also called designed multiple ligands or twin/triplet drugs), may interact independently with their respective receptors and potentially result in more effective antinociceptive properties. The designed multiple ligands presented in this work include opioid-non-opioid peptide dimer analogs, mixed peptidic- non-peptidic bifunctional ligands and dual non-peptidic dimers. The main focus herein is placed on the design and biological evaluation of these multiple opioid compounds, rather than the synthetic approach and preparation.

Background: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite., Methods: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications., Results: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity., Conclusion: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Radioligand binding studies revealed that Ang IV binds to insulin-regulated aminopeptidase (IRAP)/'AT(4) receptors' with high affinity. Yet, as these experiments were routinely carried out in the presence of chelators, only the catalytic zinc-depleted apo-form of IRAP was labelled. While the chelators remove the catalytic zinc from IRAP and protect Ang IV from proteolytic degradation, the aminopeptidase N selective inhibitor '7B' only exerts the latter effect. By using 7B along with the new stable Ang IV-analog [(3) H]AL-11, we here show that the native enzyme is only a low-affinity target for Ang IV., (© 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Française de Pharmacologie et de Thérapeutique.)

Background: An important limiting factor in the development of centrally acting pharmaceuticals is the blood-brain barrier (BBB). Transport of therapeutic peptides through this highly protective physiological barrier remains a challenge for peptide drug delivery into the central nervous system (CNS). Because the most common strategy to treat moderate to severe pain consists of the activation of opioid receptors in the brain, the development of active opioid peptide analogues as potential analgesics requires compounds with a high resistance to enzymatic degradation and an ability to cross the BBB., Results: Herein we report that tetrapeptide analogues of the type H-Dmt1-Xxx2-Yyy3-Gly4-NH2 are transported into the brain after intravenous and subcutaneous administration and are able to activate the μ- and δ opioid receptors more efficiently and over longer periods of time than morphine. Using the hot water tail flick test as the animal model for antinociception, a comparison in potency is presented between a side chain conformationally constrained analogue containing the benzazepine ring (BVD03, Yyy3: Aba), and a "ring opened" analogue (BVD02, Yyy3: Phe). The results show that in addition to the increased lipophilicity through amide bond N-methylation, the conformational constraint introduced at the level of the Phe3 side chain causes a prolonged antinociception. Further replacement of NMe-D-Ala2 by D-Arg2 in the tetrapeptide sequence led to an improved potency as demonstrated by a higher and maintained antinociception for AN81 (Xxx2: D-Arg) vs. BVD03 (Xxx2: NMe-D-Ala). A daily injection of the studied opioid ligands over a time period of 5 days did however result in a substantial decrease in antinociception on the fifth day of the experiment. The compact opioid agonist-NK1 antagonist hybrid SBCHM01 could not circumvent opioid induced tolerance., Conclusions: We demonstrated that the introduction of a conformational constraint has an important impact on opioid receptor activation and subsequent antinociception in vivo. Further amino acid substitution allowed to identify AN81 as an opioid ligand able to access the CNS and induce antinociception at very low doses (0.1 mg/kg) over a time period up to 7 hours. However, tolerance became apparent after repetitive i.v. administration of the investigated tetrapeptides. This side effect was also observed with the dual opioid agonist-NK1 receptor antagonist SBCHM01.

Two synthetic routes for the synthesis of amino-triazolodiazepine (Ata) scaffolds are presented. The scope of both of these proceeding through key intra- and intermolecular Huisgen cycloaddition reactions is discussed. The replacement of the His-Pro dipeptide segment in angiotensin IV by the dipeptide mimetic Ata-Gly and subsequent biological evaluation in two inhibitory enzyme assays validated the use of the Ata moiety as a His mimic given the equipotency of both peptidic analogs.

Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH(2) (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.

Dermorphin analogues, containing a (S)- and (R)-4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold (Aba) and the α-methylated analogues as conformationally constrained phenylalanines, were prepared. Asymmetric phase-transfer catalysis was unable to provide the (S)-α-Me-o-cyanophenylalanine precursor for (S)-α-MeAba in acceptable enantiomeric purity. However, by using a Schöllkopf chiral auxiliary, this intermediate was obtained in 88 % ee. [(S)-Aba 3-Gly 4]dermorphin retained μ-opioid affinity but displayed an increased δ-affinity. The corresponding R epimer was considerably less potent. In contrast, the [(R)-α-MeAba 3-Gly 4]dermorphin isomer was more potent than its S epimer. Tar-MD simulations of both non-methylated [Aba 3-Gly 4]dermorphin analogues showed a degree of folding at the C-terminal residues toward the N terminus of the peptide, without however, adopting a stabilized β-turn conformation. The α-methylated analogues, on the other hand, exhibited a type I/I' β-turn conformation over the α-MeAba 3 and Gly 4 residues, which was stabilized by a hydrogen bond involving Tyr 5-HN and D-Ala 2-CO., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Phe(3) residue of the N-terminal tetrapeptide of dermorphin (H-Dmt-d-Ala-Phe-Gly-NH(2)) was conformationally constrained using 4- or 5-methyl-substituted 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) stereoisomeric scaffolds. Several of the synthesized peptides were determined to be high affinity agonists for the μ opioid receptor (OPRM) with selectivity over the δ opioid receptor (OPRD). Interesting effects of the Aba configuration on ligand binding affinity were observed. H-Dmt-d-Ala-erythro-(4S,5S)-5-Me-Aba-Gly-NH(2)9 and H-Dmt-threo-(4R,5S)-5-Me-Aba-Gly-NH(2)12 exhibited subnanomolar affinity for OPRM, while they possess an opposite absolute configuration at position 4 of the Aba ring. However, in the 4-methyl substituted analogues, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 was significantly more potent than the (4S)-derivative 13. These unexpected results were rationalized using the binding poses predicted by molecular docking simulations. Interestingly, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 is proposed to bind in a different mode compared with the other analogues. Moreover, in contrast to Ac-4-Me-Aba-NH-Me, which adopts a β-turn in solution and in the crystal structure, the binding mode of this analogue suggests an alternative receptor-bound conformation.

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β-Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e-β-MePhe⁶ substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP-N or the AT₁ receptor. This indicates an important role of the orientation of the Phe⁶ for inducing selectivity. Pro⁵ replacement with 2-aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT₁ affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides., (Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.)

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.

Insulin regulated aminopeptidase (IRAP) recognises "AT(4)-receptor" ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [(3)H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (Demaegdt et al., 2009). Here, we show that, contrary to [(3)H]Ang IV, [(3)H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [(3)H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [(3)H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable "AT(4)-receptor" ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The in vivo efficacy of stable and unstable "AT(4)-receptor" ligands could therefore differ., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Recently, a new derivative of angiotensin (Ang) II, called "Ang A," has been discovered to be present in plasma of healthy humans and, in increased concentrations, in end-stage renal failure patients. The objectives of the study were to investigate the blood pressure and renal hemodynamic responses to Ang A in normotensive and hypertensive rats and in genetically modified mice and the binding properties of Ang A to Ang II type 1 (AT(1)) or Ang II type 2 (AT(2)) receptors. Intravenous and intrarenal administration of Ang A induced dose-dependent pressor and renal vasoconstrictor responses in normotensive rats, which were blocked by the AT(1) receptor antagonist candesartan but were not altered by the AT(2) receptor ligands PD123319, CGP42112A, or compound 21. Similar responses were observed after intravenous administration in spontaneously hypertensive rats. Deletion of AT(1a) receptors in mice almost completely abolished the pressor and renal vasoconstrictor responses to Ang A, indicating that its effects are mediated via AT(1a) receptors. Ang A was less potent than Ang II in vivo. The in vitro study demonstrated that Ang A is a full agonist for AT(1) receptors, with similar affinity for AT(1) and AT(2) receptors as Ang II. Overall, the responses to Ang A and Ang II were similar. Ang A has no physiological role to modulate the pressor and renal hemodynamic effects of Ang II.

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

This study reports on new proteolytically stable, pharmacologically active endomorphin analogues, incorporating Dmt(1), Achc(2), pFPhe(4), or βMePhe(4) unnatural amino acids. Consistent with earlier results, it was found that the analogues carrying Dmt(1) and Achc(2) residues displayed the highest μ-opioid receptor affinities, depending upon the configuration of the incorporated Achc(2). Combination of such derivatives with pFPhe(4) or βMePhe(4) yielded further compounds with variable binding potencies. Combined application of Dmt(1), cis-(1S,2R)Achc(2), and pFPhe(4) (compound 16) resulted in the most potent analogue. Ligand stimulated [(35)S]GTPγS binding assays indicated that the analogues retained their agonist activities and opioid receptor specificities. NMR and molecular modeling studies of the analogues containing βMePhe(4) or pFPhe(4) confirmed the predominance of bent structures, however, it is apparent that bent structures are energetically more favored than random/extended structures for all studied compounds.

Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis. Several genes coding for xenobiotic biotransformation enzymes were found to be positively regulated upon exposure to 4-Me(2)N-BAVAH. For CYP1A1/2B1/3A2, these observations were confirmed by qRT-PCR and immunoblot analysis. In addition, significantly higher 7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activity levels were measured. These effects were accompanied by an increased expression of CCAAT/enhancer binding protein alpha and hepatic nuclear factor (HNF)4α, but not of HNF1α. Finally, 4-Me(2)N-BAVAH was found to induce histone H3 acetylation at the proximal promoter of the albumin, CYP1A1 and CYP2B1 genes, suggesting that chromatin remodelling is directly involved in the transcriptional regulation of these genes. In conclusion, histone deacetylase inhibitors prove to be efficient agents for better maintaining a differentiated hepatic phenotype in rat hepatocyte cultures., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: The clinical treatment of various types of pain relies upon the use of opioid analgesics. However most of them produce, in addition to the analgesic effect, several side effects such as the development of dependence and addiction as well as sedation, dysphoria, and constipation. One solution to these problems are chimeric compounds in which the opioid pharmacophore is hybridized with another type of compound to incease antinociceptive effects. Neurotensin-induced antinociception is not mediated through the opioid system. Therefore, hybridizing neurotensin with opioid elements may result in a potent synergistic antinociceptor., Results: Using the known structure-activity relationships of neurotensin we have synthesized a new chimeric opioid-neurotensin compound PK20 which is characterized by a very strong antinociceptive potency. The observation that the opioid antagonist naltrexone did not completely reverse the antinociceptive effect, indicates the partial involvement of the nonopioid component in PK20 in the produced analgesia., Conclusions: The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores overlap partially, expresses high antinociceptive tail-flick effects after central as well as peripheral applications.

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side-chain-functionalized N(α)-propargyl lysine derivatives, conjugates containing a ⁹⁹(m)Tc-based imaging probe for SPECT and two different moieties (e.g., tumor-targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the Cu(I)-catalyzed alkyne-azide cycloaddition in efficient one-pot protocols. This strategy was successfully applied to the preparation of a ⁹⁹(m)Tc-labeled conjugate comprising a tumor-targeting peptide sequence (bombesin(7-14)) and a low-molecular-weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin-releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional ⁹⁹(m)Tc-based radiopharmaceuticals with significant potential for a multitude of applications.

Overexpression of the gastrin-releasing peptide receptor (GRPR) in a variety of human carcinomas has provided a means of diagnosis and treatment. Previously we reported a metabolically stable (N(α)His)Ac-βAla-βAla-[Cha(13),Nle(14)]BBS(7-14) analogue with high affinity for the GRPR. We have also shown that the biodistribution pattern of this fairly lipophilic, radiolabeled peptide can be enhanced by glycation, which is easily carried out by Cu(I)-catalyzed cycloaddition. Herein, we further elaborate this "click approach" in the synthesis of a new series of triazole-based chelating systems as alternatives to the (N(α)His)Ac chelator for labeling with the (99m)Tc(CO)(3) core. The bombesin analogues, containing these new chelating systems, were evaluated with regard to their synthesis and in vitro and in vivo properties, and were compared with their (N(α)His)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor-to-background ratios.

We report on the characteristics of the radical-ion-driven dissociation of a diverse array of β-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., β-Phe), and to a substantially lower extent, carbonyl-containing (e.g., β-Glu and β-Gln) amino acid side chains, lead to N-Cβ bond cleavage in the corresponding β-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of β-amino acids reveals a shift in cleavage priority from the N-Cβ to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven β-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced delta/mu antagonists, as determined by the functional [S(35)]GTPgammaS binding assay, the dimerization of potent Aia-based 'parent' ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The Pictet-Spengler reaction is known as a useful tool for the synthesis of constrained analogs of tryptophan. Herein, we present the further cyclization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl esters with 1-(1'-aminoalkyl) side chain. These transformations lead to heterocyclic structures which can find useful applications in medicinal chemistry as peptide mimetics.

A thorough comparative study to demonstrate the properties of a new microwave labile backbone amide linker is presented. A cyclic pentapeptide, cyclo(Trp-Gln-Gly-beta-Ala-Phe), was used as a model and synthesized following 16 different conditions. The new backbone amide linker is stable towards acid and base at r.t., and can be cleaved at elevated temperature in trifluoroacetic acid under microwave irradiation, avoiding the use of aggressive reagents like HF. The new linker is compatible with Boc- as well as Fmoc-strategy and allows the cleavage of acid labile side chain protective groups at r.t., prior to cleavage of the cyclic pentapeptide from the resin.

'AT(4) receptors' through which Angiotensin IV (Ang IV) improves memory acquisition, were recently identified as insulin regulated aminopeptidase (IRAP). Radioligand binding studies have hitherto been performed with iodinated Ang IV in the presence of divalent cation chelators EDTA and 1,10-phenanthrolin. Hence, they referred to the apo-form of IRAP. Presently, binding of [(3)H]Ang IV and [(3)H]AL-11, a stable Ang IV analog, was compared on Chinese hamster ovary (CHO-K1) and mouse hippocampal (P40H1) cell membranes. With chelators, their high affinity sites showed the same pharmacological profile as for [(125)I]Ang IV binding. Without chelators, only high affinity binding was perceived for [(3)H]AL-11. The same pharmacological profile was recorded in both membrane preparations; it was different from the one in the presence of chelators and corresponded to catalytically active IRAP (despite the concurrent presence of aminopeptidase N (APN) in P40H1 cell membranes). This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile.

The histidine residue in angiotensin IV was replaced by various conformationally constrained amino acids. The substitution of the His(4)-Pro(5) dipeptide sequence by the constrained Trp analogue Aia-Gly, in combination with beta(2)hVal substitution at the N-terminus, provided a new stable analogue H-(R)-beta(2)hVal-Tyr-Ile-Aia-Gly-Phe-OH (AL-40) that is a potent ligand for the Ang IV receptor IRAP and selective versus AP-N and the AT1 receptor.

The increased expression of the neurotensin (NT) receptor NTS1 by different cancer cells, such as pancreatic adenocarcinoma and ductal breast cancer cells, as compared to normal epithelium, offers the opportunity to target these tumors with radiolabeled neurotensin analogues for diagnostic or therapeutic purposes. The aim of the present study was to design and synthesize new neurotensin radioligands and to select a lead molecule with high in vivo tumor selectivity for further development. Two series of neurotensin analogues bearing DTPA were tested: a series of NT(8-13) analogues, with DTPA coupled to the α-NH(2), sharing the same peptide sequence with analogues previously developed for radiolabeling with technetium or rhenium, as well as an NT(6-13) series in which DTPA was coupled to the ε-NH(2) of Lys(6). Changes were introduced to stabilize the bonds between Arg(8)-Arg(9), Pro(10)-Tyr(11), and Tyr(11)-Ile(12) to provide metabolic stability. Structure-activity studies of NT analogues have shown that the attachment of DTPA induces an important loss of affinity unless the distance between the chelator and the NT(8-13) sequence, which binds to the NTS1 receptor, is increased. The doubly stabilized DTPA-NT-20.3 exhibits a high affinity and an elevated stability to enzymatic degradation. It shows specific tumor uptake and high tumor to blood, to liver, and to intestine activity uptake ratios and affords high-contrast planar and SPECT images in an animal model. The DTPA-NT-20.3 peptide is a promising candidate for imaging neurotensin receptor-positive tumors, such as pancreatic adenocarcinoma and invasive ductal breast cancer. Analogues carrying DOTA are being developed for yttrium-90 or lutetium-177 labeling.

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug-target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

The selective and efficient synthesis of novel tridentate metal chelating systems containing two 1,4-disubstituted 1,2,3-triazole heterocycles obtained via the copper(I)-catalyzed cycloaddition of alkynes and azides (click reaction) is described. The constructs are shown to be efficient ligand systems for the chelation of fac-[M(CO)(3)(H(2)O)(3)](+) (M=(99m)Tc, Re) yielding well- defined and stable complexes. The organometallic (99m)Tc conjugates are suitable for application as diagnostic radiotracers for single photon emission computed tomography (SPECT) as demonstrated in vivo with a fragment of the tumor-targeting bombesin peptide functionalized with a di-1,2,3-triazole chelator and radiolabeled with [(99m)Tc(CO)(3)](+). Starting from readily available dialkyne precursors, the central chelating systems are formed as the conjugates are assembled by click reaction with azide-functionalized entities. Depending on the nature of the azide substrates employed (e.g. lipophilic or hydrophilic residues) pharmacologically relevant characteristics of the final metal conjugate such as hydrophilicity or overall charge can be readily modulated. The procedures described also enable the facile introduction of other probes into the metal conjugate, providing access to potential multimodal imaging agents.

Replacement of the constrained phenylalanine analogue 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the opioid Dmt-Tic-Gly-NH-Bn scaffold by the 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]azepin-3-one (Aia) and 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffolds has led to the discovery of novel potent mu-selective agonists (Structures 5 and 12) as well as potent and selective delta-opioid receptor antagonists (Structures 9 and 15). Both stereochemistry and N-terminal N,N-dimethylation proved to be crucial factors for opioid receptor selectivity and functional bioactivity in the investigated small peptidomimetic templates. In addition to the in vitro pharmacological evaluation, automated docking models of Dmt-Tic and Dmt-Aba analogues were constructed in order to rationalize the observed structure-activity data.

The synthesis, biological evaluation, and conformational analysis of 4-amino-indolo[2,3-c]azepin-3-one (Aia)-containing SRIF mimetics are reported. Different subtype selectivities are observed depending on the N- and C-terminal substituents of the D-Aia-Lys dipeptide mimetic. An sst(5)-selective analogue with subnanomolar binding affinity was obtained that is the most potent agonist reported to date. A nonselective mimetic with high potency was also identified. This study allows a better definition of the bioactive conformation of the essential D-Trp side chain in the somatostatin pharmacophore.

Purpose: Neurotensin (NT) and its high affinity receptor (NTR1) are involved in several neoplastic processes. Thus, NT-based radiopharmaceuticals are potential tracers for targeted diagnosis and therapy of NTR-positive tumours. A new analogue based on NT(8-13), NT-XIX, with the three enzymatic cleavage sites stabilised, was synthesised and tested., Methods: The synthesis was performed by Boc strategy. Labelling with (99m)Tc/(188)Re was performed using the tricarbonyl technique. Metabolic stability was tested in vitro and in vivo. NT-XIX was further characterised in vitro in HT-29 cells and in vivo in nude mice with HT-29 xenografts., Results: NT-XIX showed much longer half-lives than non-stabilised analogues. Binding to NTR1 was highly specific, although the affinity was lower than that of natural NT. Bound activity rapidly internalised into HT-29 cells and 50% remained trapped after 24 h. In the time-course biodistribution, the highest uptake was found in the tumour at all p.i. times. In vivo uptake was specific, and accumulation of activity in the kidneys was low. Radioactivity clearance from healthy organs was faster than that from the tumour, resulting in improved tumour-to-tissue ratios and good SPECT/CT imaging. Treatment with (188)Re-NT-XIX (30 MBq, in three or four fractions) decreased tumour growth by 50% after 3 weeks., Conclusion: The high in vivo stability and the favourable in vivo behaviour makes NT-XIX an excellent candidate for the imaging and therapy of NTR1-positive tumours.

A strategy was developed to directly assemble 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]-azepin-3-ones on solid-phase-supported peptide sequences. Fmoc- and Boc-based strategies were investigated. The Fmoc-strategy approach strongly depends on the peptide sequence being synthesized while the Boc-based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia-analogs of several bioactive peptides containing one or more Trp-residues which were shown to be important for biological recognition., (Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.)

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

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.

Angiotensin IV, a metabolite of angiotensin II, inhibits the enzyme insulin regulated aminopeptidase or IRAP and also, although with lower potency, aminopeptidase-N (AP-N). When both beta (2)-homo amino acid- and beta (3)-homo amino acid substitutions were used, allowed the identification of H-( R)beta (2)hVal-Tyr-Ile-His-Pro-beta (3)hPhe-OH as a potent and stable Ang IV analog with high selectivity for IRAP versus AP-N and the AT1 receptor.

The constitutional similarity with different secondary structure preference between the Aba-Gly and the spiro-Aba-Gly scaffolds were exploited to design the novel endomorphin-2 analogs Tyr-spiro-( R/ S)-Aba-Gly-Phe-NH(2) ( 1 and 2) and Tyr-( R/ S)-Aba-Gly-Phe-NH(2) ( 3 and 4). The ( R)-spiro analog 1 was found to be a potent and selective micro-opioid agonist/partial agonist ( K (imicro) = 29.3 nM, IC(50) = 50 nM, K(e) = 0.57). NMR experiments and molecular modeling indicated that its backbone adopts mainly a beta-turn in aqueous solution.

Objective: Benzoylaminoalkanohydroxamic acids, including 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), are structural analogues of Trichostatin A, a naturally occurring histone deacetylase inhibitor (HDACi). 4-Me(2)N-BAVAH has been shown to induce histone hyperacetylation and to inhibit proliferation in Friend erythroleukaemia cells in vitro. However, the molecular mechanisms have remained unidentified., Materials and Methods: In this study, we evaluated the effects of 4-Me(2)N-BAVAH on proliferation in non-malignant cells, namely epidermal growth factor-stimulated primary rat hepatocytes., Results and Conclusion: We have found that 4-Me(2)N-BAVAH inhibits HDAC activity at non-cytotoxic concentrations and prevents cells from responding to the mitogenic stimuli of epidermal growth factor. This results in an early G(1) cell cycle arrest that is independent of p21 activity, but instead can be attributed to inhibition of cyclin D1 transcription through a mechanism involving inhibition of nuclear factor-kappaB activation. In addition, 4-Me(2)N-BAVAH delays the onset of spontaneous apoptosis in primary rat hepatocyte cultures as evidenced by down-regulation of the pro-apoptotic proteins Bid and Bax, and inhibition of caspase-3 activation.

A novel opioid peptide antagonist analogue, [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe, derived from the potent, delta-receptor selective TIPP tetrapeptide (Tyr-Tic-Phe-Phe) series was synthesized and radiolabeled by catalytic tritiation of its iodinated precursor peptide. The purified radioprobe exhibited a specific activity of 2.15 TBq/mmol (58 Ci/mmol). The novelty of this compound is that it contains structurally modified tyrosine residue (2',6'-dimethyltyrosine, Dmt1) replacing tyrosine (Tyr1) at the N-terminus, and beta-methyl substituted phenylalanine (betaMePhe3) at the third position. As the configuration of betaMePhe3 side-chain might be different due to diastereomerism, and accordingly can alter the biological activity, both unlabeled threo (2S,3R and 2R,3S) diastereomeric analogues were also prepared and included in this study. The affinity and selectivity (delta-opioid versus mu-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potencies were determined in [35S]GTPgammaS binding experiments using Chinese Hamster Ovary (CHO) cells selectively expressing delta- or mu-opioid receptors. The equilibrium binding of the radiolabeled peptide derivative [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe to rat brain membranes was saturable and the Scatchard analysis indicated a single binding site with a Kd of 0.3 nM and a Bmax of 127 fmol/mg protein. A study of [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe binding displacement by various receptor-type specific opioid ligands showed the rank order of competitor's potency delta > mu > kappa, suggesting selective labeling of opioid delta-sites. In the functional tests, the (2S,3R) and (2R,3S) peptides exhibited partial agonist behaviour by weakly stimulating regulatory G-proteins in CHO cell membranes transfected with different receptors. Both isomers were quite weak partial agonists at the delta-receptor and reasonable partial agonists at the mu-receptor, with a prevalence of (2S,3R) over (2R,3S) for the mu-receptor. Consistent with these observations both stereomers competitively inhibited the stimulation of [35S]GTPgammaS binding induced by the prototype delta-agonist peptide (pClPhe4)-DPDPE in delta(m) CHO cell membranes, and still the (2S,3R) compound exerted more potent delta-antagonist effect. [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe represents a high affinity new radioligand and also constitute further example of the influence of beta-methyl substitution on the potency and selectivity of TIPP analogues, thus becoming a valuable biochemical and pharmacological tool in opioid research.

The stereoisomers of N-phthaloyl-protected amino acids and dipeptidomimetics were separated on macrocyclic glycopeptide and cellulose-based chiral stationary phases (CSPs) in the RP and polar-ionic modes. The effects of the organic modifier, the mobile phase composition, and the pH on the separations were investigated. Optimization of these separations was achieved through variation of the mobile-phase additive combinations. The elution sequence was determined for some of the samples. A practical application for the monitoring of the reaction conditions for N-phthaloylation of (S)-Phe was demonstrated.

The synthesis and biological evaluation of four peptidomimetic analogs of somatostatin based on a constrained Trp residue, 3-amino-indolo[2,3-c]azepin-2-one (Aia), are reported. It is shown that dipeptidomimetics with a D-Aia-Lys sequence, functionalized with N- and C-terminal aromatic substituents, display a good selectivity for both sst4 and sst5. This study allowed us to identify a new highly potent sst5 agonist with good selectivity over the other receptors, except versus sst4.

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.

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH(2)) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH(2)) by replacing the His-d-Phe and His-d-Nal(2') fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx=D-Phe/pCl-D-Phe/D-Nal(2')). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.

Aim: Bombesin (BBS) receptors are potential targets for diagnosis and therapy of breast and prostate tumors. To overcome the rapid degradation of natural BBS some modifications were introduced at positions 13 and 14. Additionally, a spacer was inserted between the chelator and the binding sequence in order to further improve the in vivo uptake. The analogues were labeled with the [(99m)Tc(CO)(3)]-core and tested., Methods: Stability was analyzed in vitro in human plasma. Binding affinity and internalization were determined in vitro in prostate carcinoma PC-3 cells. Biodistribution studies and single photon emission computed tomography/X-ray computed tomography (SPECT/CT) imaging were performed in nude mice with PC-3 tumor xenografts., Results: The changes introduced in the BBS(7-14) sequence substantially increased plasma stability. Affinity for gastrin releasing-peptide (GRP) receptors on PC-3 cells was comparable to that of the unmodified analogue with Kd<1 nM. The presence of a spacer in the molecule induced an increment in the in vivo uptake in pancreas and PC-3 xenografts (GRP receptor-positive tissues). The increase in pancreas and tumor uptake was higher when both spacer and stabilization are present in the same molecule. Moreover, in vivo uptake was highly specific. The tumor was clearly visualized by SPECT/CT., Conclusions: The modifications in the BBS(7-14) sequence led to a higher plasma stability while binding affinity remained unaffected. Stabilization resulted in improved biodistribution with better tumor to non-tumor ratios. However, the insertion of a spacer had a greater influence on the biodistribution. Analogues with both spacer and stabilization are the most promising radiopharmaceuticals for targeting GRP receptor-positive tumors.

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.