RAW 264.7 co-cultured with ultra-high molecular weight polyethylene particles spontaneously differentiate into osteoclasts: an in vitro model of periprosthetic osteolysis.

Wear-particle osteolysis affects prosthesis survival leading to implant loosening up to 70% of revisions. Therapeutic strategies are increasing, however alternative testing methods to experimentally evaluate such treatments are lacking. The aim of this study was to reproduce an in vitro osteolysis model recapitulating the events that, starting from the exposure of macrophages to polyethylene, lead to the establishment of osteoclastogenesis and inflammation. Responses to polyethylene, at 3 and 7 days, in a macrophage cell line, RAW 264.7, were determined by DNA quantification, immunofluorescence, pit assay, gene expression, cytokine production and NF-kB activation. Results showed that 3 days exposure to particles could induce a significant production of Tumor Necrosis Factor alpha (p < 0.0005) and Prostaglandin E2 (p < 0.005) compared to controls. Particles also induced macrophages to spontaneously differentiate into mature and active osteoclasts, in terms of identification of multinucleated cells by Phalloidin staining and by the analysis of osteoclast-specific gene markers. In particular, at 3 days polyethylene induced a significant up-regulation of Nuclear Factor of Activated T-cells, cytoplasmic 1, Receptor Activator of Nuclear factor Kappa-B and Receptor Activator of Nuclear Factor Kappa-B Ligand genes (p < 0.0005) compared to controls. At protein level, the particles induced a significant increase of Receptor Activator of Nuclear Factor Kappa-B Ligand at day 7 over controls (p < 0.0005). Osteoclasts were capable to resorb bone even in absence of differentiating factors. The possible mechanism, beside spontaneous osteoclastogenesis mediated by wear debris, was identified in an autocrine up-regulation of Receptor activator of nuclear factor kappa-B ligand gene expression and protein synthesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 510-520, 2017.

Novel halogenated derivates of JWH-018: Behavioral and binding studies in mice.

JWH-018 is a synthetic CB1 and CB2 agonist illegally marketed as products named "Spice" or "herbal blend" for its psychoactive effects which are much higher than those produced by cannabis. In the last year, the European Monitoring Centre for Drugs and Drug Addiction reported to the Italian National Early Warning System the seizure of plant material containing new halogenated derivatives of JWH-018 (JWH-018 Cl and JWH-018 Br). The present study aimed to investigate the in vitro and in vivo activity of these two new synthetic cannabinoids in mice. In vitro competition binding experiments performed on mouse and human CB1 receptors revealed a high affinity and potency of the halogenated compounds. Synthetic cannabinoids (0.01-6 mg/kg i.p.) impaired motor activity and induced catalepsy in mice and their effects were more severe with respect to those evoked by Δ(9)-THC. Moreover, they increased the mechanical and thermal pain threshold and induced a marked hypothermia. It is interesting to note that whereas high doses of JWH-018 cause seizures, myoclonia and hyperreflexia, the halogenated compounds, in particular JWH-018Br, were less effective. Behavioral and neurological changes were prevented by the selective CB1 receptor antagonist AM 251. These data demonstrate for the first time that JWH-018 Cl and JWH-018 Br act similarly to JWH-018 while inducing less convulsive episodes and myoclonias. These data support the hypothesis that the halogenated compounds may have been introduced onto market to produce similar intoxicating effects as JWH-018 while causing less side effects.

A(2A) adenosine receptors and Parkinson's disease severity.

OBJECTIVES: In the last decade, increasing evidence suggests a key role of adenosine in Parkinson's disease (PD) and A2A adenosine receptors (A2A ARs) as an important pharmacological target in PD. An overexpression of A2A ARs has been found in putamen and in peripheral blood cells of PD patients. The primary aim of this study was to verify whether the alterations in A2A ARs in lymphocytes of PD subjects correlate with disease severity. MATERIAL AND METHODS: A consecutive sample of PD patients was enrolled. A clinical examination and a face-to-face interview were carried out. A2A ARs were investigated to verify the affinity and receptor density in lymphocyte membranes. The data were compared with those found in healthy controls. Moreover, the correlation between A2A AR density and affinity and clinical variables was evaluated in PD patients. RESULTS: In human lymphocyte membranes from PD patients, an increase in A2A AR density and a decrease in A2A AR affinity were found if compared with healthy subjects. A statistically significant correlation between the A2A AR density or affinity and specific clinical parameters as motor and cognitive impairment was detected. Patients with higher A2A AR density and lower affinity were more likely to exhibit motor complications. CONCLUSIONS: Parkinson's disease patients show an A2A AR upregulation in lymphocyte membranes if compared with healthy subjects. The correlation found between A2A AR density or affinity and clinical parameters highlights the central role of A2A AR modulation in the pharmacological treatment for PD and could suggest the putative role of A2A AR as a candidate biomarker of PD severity.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1ß (IL-1ß), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes.

BACKGROUND AND PURPOSE: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. EXPERIMENTAL APPROACH: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. KEY RESULTS: mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. CONCLUSIONS AND IMPLICATIONS: These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Adenosine and adenosine receptors: their contribution to airway inflammation and therapeutic potential in asthma.

Growing evidence emphasizes that the purine nucleoside adenosine plays an active role as local regulator in airway inflammation and pulmonary diseases. The notion that increased adenosine concentrations are associated with lung inflammation indicates the importance of this signaling pathway, which involves the activation of a family of cell surface G-protein coupled receptor subtypes named as A(1), A(2A), A(2B) and A(3). Recently, important progress has been made to better clarify the role of these receptors in a variety of inflammatory airway disorders including asthma. As a consequence, new molecules with high affinity and high selectivity for the human adenosine receptors subtypes designed to control the airway inflammatory component of asthma have been launched and are currently tested in clinical trials as anti-asthma treatments. With the availability of these molecules for testing in humans, the role of adenosine receptors in asthma can now be validated.

Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts.

OBJECTIVE: To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E(2) (PGE(2)) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). METHODS: SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N6-cyclohexyladenosine (CHA, A(1)), 2-[p-(2-carboxyethyl)-phenetyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, A(2A)), 5'-N-ethylcarboxamidoadenosine (NECA, non-selective), N6-(3-iodobenzyl)2-chloroadenosine-5'-N-methyluronamide (Cl-IB-MECA, A(3)). SFs were treated with TNF-alpha (10 ng/ml) and lipopolysaccharide (LPS) (1 microg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-alpha- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75 Hz, 1.5 mT) during the period in culture (24h). PGE(2) release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. RESULTS: TNF-alpha and LPS stimulated PGE(2) release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE(2) production. EMFs inhibited PGE(2) production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE(2) release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE(2) levels were associated to modification of COX-2 expression. CONCLUSIONS: This study supports anti-inflammatory activities of A(1) and A(2A) adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A(2A) receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes.

OBJECTIVE: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release. METHODS: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated. RESULTS: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. CONCLUSIONS: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields.

OBJECTIVE: The present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied. METHODS: Saturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug-receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated. RESULTS: Bovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types. CONCLUSIONS: PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug-receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases.

Biological abnormalities of peripheral A(2A) receptors in a large representation of polyglutamine disorders and Huntington's disease stages.

Huntington's disease is one of a group of hereditary neurodegenerative diseases characterized by a glutamine expansion (polyQ) in proteins which are expressed in various cell populations. In agreement with this widespread distribution, we have previously shown that A(2A) receptor signaling is affected in mouse brain as well as in peripheral blood cells from a small cohort of HD patients. Here we analyzed a total of 252 subjects, including 126 HD gene-positive individuals, from different clinical sites. Consistent with our previous data we show that A(2A) receptor B(max) values are robustly increased at all HD stages as well as in 32 pre-symptomatic subjects. We report that the same abnormality is present also in other polyQ but not in non-polyQ inherited neurological disorders. Finally, we demonstrate that the same peripheral cells exhibit an altered membrane fluidity, a finding that may explain the observed change in receptor density. We argue that the observed alteration in lymphocytes reflects the presence of the mutant protein, and we suggest that the measure of the A(2A) receptor binding activity might be of potential interest for a peripheral assessment of chemicals capable of interfering with the immediate toxic effects of the mutation.

Caffeine intake induces an alteration in human neutrophil A2A adenosine receptors.

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (K(D)) and density (B(max)) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5'-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors.

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.

1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

Structure-affinity relationships of a unique nicotinic ligand: N(1)-dimethyl-N(4)-phenylpiperazinium iodide (DMPP).

DMPP is a well-known nicotinic agonist that does not fit any proposed pharmacophore for nicotinic binding and represents a unique ligand among the hundreds of nicotinic agonists studied in the past decades. A systematic modulation of the chemical structure of DMPP, aimed to establish its structure-affinity relationships, is reported. The research has allowed to identify molecules such as 11c, 13c, 14c, and 28c, with affinities for alpha(4)beta(2) receptors in the low nanomolar range, some 2 orders of magnitude lower than the lead compound. The agonistic properties of the most interesting compounds have been assessed by measuring their analgesic activity on mice (hot-plate test). Another result of the research was the identification of DMPP analogues, such as 3a (K(i) = 90 nM) and 14b (K(i) = 180 nM), that maintain affinity for the central nicotinic receptor when the ammonium function is changed into an aminic one and are therefore possible leads for drug development in neurodegenerative diseases.

Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells.

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

Comparison of prazosin, terazosin and tamsulosin: functional and binding studies in isolated prostatic and vascular human tissues.

BACKGROUND: Terazosin and tamsulosin are drugs currently used in the treatment of benign prostatic hypertrophy (BPH). The potency of these two alpha(1) receptor antagonists and that of prazosin to inhibit contractions induced by noradrenaline and the binding of [(3)H]-prazosin in human prostate and four different human arterial and venous vessels (saphenous and umbilical veins, renal and mesenteric arteries) was studied. METHODS: By bioassay and binding studies, we examined the receptor affinities of different alpha(1) receptor antagonists in different human tissues. RESULTS: pKb of terazosin, tamsulosin, and prazosin obtained in the prostatic tissues (8.15, 9.64, and 8.59, respectively) were not different from those obtained in the umbilical veins (8.07, 9.56, and 8.30, respectively), in the mesenteric artery (8.27, 10.29, and 9.01, respectively), renal artery (8.35, 10.13, and 8.76, respectively) and saphenous vein (7.8, 10.3, and 9.32, respectively). IC(50) (nM) of prazosin, terazosin, and tamsulosin obtained from binding studies in membrane preparations from prostate tissue were similar to those from umbilical veins, saphenous vein, and renal artery. CONCLUSIONS: All of the evaluated drugs showed similar selectivity for prostatic vs. vascular tissues. Thus, different clinical profiles of the present drugs should not result from their differential affinity for prostatic versus vascular alpha(1)-adrenoceptors.

Effects of two-carbon bridge region methoxylation of benztropine: discovery of novel chiral ligands for the dopamine transporter.

6-Methoxylated and 8-oxygenated benztropines were prepared and evaluated for their DAT and SERT activity (binding and uptake inhibition). Methoxylation at the two-carbon bridge of benztropine produced a novel class of potent and selective DAT ligands. An interesting enantioselectivity was also observed for this new class of chiral benztropines. The inactivity of the 8-oxygenated analogues seems to point out that, unlike cocaine and its analogues, interactions of benztropine ligands with DAT may be strongly governed by the nitrogen atom.

Met-Ile-Phe-Leu derivatives: full and partial agonists of human neutrophil formylpeptide receptors.

The biological action of a series of Met-Ile-Phe-Leu analogues was analyzed on human neutrophils, to evaluate their ability to interact with formylpeptide receptors and to induce the related neutrophil responses. Three in vitro assays were carried out: receptor binding, chemotaxis and superoxide anion release. Our results demonstrate that formyl-Met-Ile-Phe-Leu derivatives act as more potent full agonists than formyl-Met-Leu-Phe, the tripeptide normally used as a model chemoattractant for the study of cell functions. On the other hand, the presence of N-ureidoisopropyl substituent in tetrapeptides imparts weak partial agonist properties. It has furthermore been demonstrated that the C-terminal methyl esterification or amination weakly influences the properties of tetrapeptide homologues. Finally, t-Boc-Met-Ile-Phe-Leu derivatives do not appear able to interact with formylpeptide receptors.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.