Francesco Berardi

Serotonin 5-HT7 receptor agents: structure-activity relationships and potential therapeutic applications in central nervous system disorders

Since its discovery in the 1940s in serum, the mammalian intestinal mucosa, and in the central nervous system, serotonin (5-HT) has been shown to be involved in virtually all cognitive and behavioral human functions, and alterations in its neurochemistry have been implicated in the etiology of a plethora of neuropsychiatric disorders. The cloning of 5-HT receptor subtypes has been of importance in enabling them to be classified as specific protein molecules encoded by specific genes. The 5-HT(7) receptor is the most recently classified member of the serotonin receptor family. Since its identification, it has been the subject of intense research efforts driven by its presence in functionally relevant regions of the brain. The availability of some selective antagonists and agonists, in combination with genetically modified mice lacking the 5-HT(7) receptor, has allowed for a better understanding of the pathophysiological role of this receptor. This paper reviews data on localization and pharmacological properties of the 5-HT(7) receptor, and summarizes the results of structure-activity relationship studies aimed at the discovery of selective 5-HT(7) receptor ligands. Additionally, an overview of the potential therapeutic applications of 5-HT(7) receptor agonists and antagonists in central nervous system disorders is presented.

Antiproliferative and cytotoxic effects of some σ2 agonists and σ1 antagonists in tumour cell lines

To establish the activity of σ ligands at σ1 and σ2 receptor, we chose two tumour cell lines, the human SK-N-SH neuroblastoma and the rat C6 glioma lines, which express σ2 receptors at a high density and σ1 receptors in their high-affinity or low-affinity state. We tested the σ2 receptor agonist PB28 and the σ2 antagonist AC927, and (+)-pentazocine and NE100 as agonist and antagonist, respectively, at σ1 receptors, with regard to antiproliferative and cytotoxic effects. In addition, 1,3-di(2-tolyl)guanidine (DTG) and haloperidol were tested as reference compounds displaying nearly equipotent σ affinity (σ2>σ1 and σ1>σ2, respectively). In both SK-N-SH and C6 cells, PB28 and NE100 displayed the most potent results both in antiproliferative and cytotoxic assay while AC927 and (+)-pentazocine were inactive in both assays. The cytotoxic and antiproliferative effects of DTG and haloperidol reflected their σ1 antagonist activity and σ2 agonist activity. Moreover, our results in the tumour cell lines correlated well with those for σ2 activity found previously in a functional assay in the guinea-pig bladder. These findings establish a new model for evaluating both σ2 and σ1 receptor activity of σ ligands, which could be useful for developing new ligands having mixed σ2 agonist/σ1 antagonist activity as potential antineoplastic agents.

Perspectives of P-Glycoprotein Modulating Agents in Oncology and Neurodegenerative Diseases: Pharmaceutical, Biological, and Diagnostic Potentials

Human ATP binding cassette (ABC) transporters belong to a family of 49 genes classified into seven subfamilies: ABCA, ABC-B, ABC-C, ABC-D, ABC-E, ABC-F, ABC-G. Some of these transporters are involved in multidrug resistance (MDR), in particular ABC-B1, better known as Pglycoprotein (P-gp), ABC-G2, better known as breast cancer resistance protein (BCRP), and ABC-C1-6, also known as multidrug resistance associated proteins (MRP1-6). 3 These transporters are overexpressed in several tumor cell lines and are responsible for drug efflux out of the cells. They use the energyofATPhydrolysis to extrude compoundsbya complex translocation process. Three models for P-gp translocation have been suggested: (1) pore, (2) flippase, and (3) hydrophobic vacuum cleaner models. In the pore model, drugs binding P-gp to the cytosol are transported out through a protein channel. In the flippase model, P-gp flips drugs that are transported from the inner to the outer compartment of the plasmamembrane against a concentration gradient. In the hydrophobic vacuum cleaner model, molecules recognized by P-gp in the lipid bilayer enter the protein from the membranous site and exit through the central cavity. P-gp contains 12 transmembrane helices organized in two membrane spanning domains (MSDs), each containing six transmembrane helices, and two nucleotide binding domains (NBDs) responsible for ATP binding. BCRP is a “half transporter” because it is formed by only one MSD and one NBD although it dimerizes to be fully active. MRPs differ from P-gp because they display three MSDs, and the additional domain contains five transmembrane domains. This review will focus on the physiological and pathological role of P-gp and will highlight the involvement of this protein both in MDR of tumors and in the physiological function of several barriers. P-gp overexpression is a significant factor in chemotherapy failure due to the ability of this pump to limit the cell accumulation of antineoplastic drugs. Moreover, P-gp is expressed in barriers such as the blood-brain barrier (BBB), bloodcerebro spinal fluid (B-CSF) barrier, and blood-testis barrier (BTB). It modulates the absorption and excretion of xenobiotics across these barriers. P-gp is localized at the apical membranes of liver, kidney, placenta, and the villus tip of enterocytes in the gut. In the gut, P-gp displays a strategic activity modulating access of drugs to the CYP3A4 enzyme, thereby regulating drug metabolism and absorption. 1.1. P-gp Involvement in Cancer and CNS Diseases.MDR is a complex phenomenon that is caused by tumor microenvironment changes or cancer cell-specific factors. Cancer cell-specific factors can occur at different levels: (i) increased drug efflux or decreased drug influx; (ii) drug inactivation; (iii) drug target modification; (iv) apoptosis evasion. The first of these mechanisms can be mediated by plasma membrane transporters such as P-gp. Asmentioned above, high levels of P-gp are reported in the luminal membrane of the endothelial cells constituting the BBB, B-CSF, and BTB. This strategic localization gives P-gp a crucial physiologically role in keeping drugs in the blood. P-gp exerts a protective function in the BBB; indeed, recent studies have reported a potential correlation between P-gp activity and/or expression in CNS disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and epilepsy. AD, a neurodegenerative disorder characterized by a progressive loss of cognitive function, evolves as several forms of dementia that display insoluble β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). Aβ secretion is *To whom correspondence should be addressed. Phone: þ39-0805442727. Fax þ39-080-5442231. E-mail: colabufo@farmchim.uniba.it. Abbreviations: Aβ, β-amyloid; AB, apical-basolateral; ABC, ATP binding cassette; AD, Alzheimer’s disease; AML, acute myelogenous leukemia; ATP, adenosine 50-triphosphate; BA, basolateral-apical; Bmax, maximal bound; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; B-CSF, blood-cerebro spinal fluid; BTB, blood-testis barrier; Caco-2, human colonic carcinoma; calcein-AM, calcein acethoxymethyl ester; CD56þ, peripheral blood mononuclear cells; CHOP, cyclophosphamide, hydroxydaunorubicin (adriamycin), oncovin (vincristine), prednisone/prednisolone; CNS, central nervous system; C-PIB, C-Pittsburgh compound B; CYP3A4, cytochrome P-4503A4; DV, distribution volume; FDA, Food and Drug Administration; F-FDDNP, F(2-(1-{6-[(2-[F]fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile; FMZ, flumazenil; GI, gastrointestinal; HCT-8, human colorectale adenocarcinoma; ISF, interstitial fluid; Kd, dissociation constant; LBs, Lewy bodies; LLC-PK1, porcine kidney cell line; LRP1, low density lipoprotein receptor-related protein; MDCK, Madin-Darby canine kidney; MDE, multidrug efflux; MDR, multidrug resistance;MPPþ, 1-methyl-4-phenylpyridinium;MRP,multidrug resistance associated proteins;MSA, multisystem atrophy;MSD, membrane spanning domain; MTD, maximum tolerated dose; MTL, medial temporal lobe; NBD, nucleotide binding domain; NFTs, neurofibrillary tangles; NSCLC, non-small-cell lung cancer; PAHG, hippocampus, parahippocampal, ambient gyrus; Papp, apparent permeability; PD, Parkinson’s disease; PET, positron emission tomography; P-gp, Pglycoprotein; PSP, progressive supranuclear palsy; SCLC, small-cell lung cancer; SNP, single nucleotide polymorphism; SPECT, single photon emission computed tomography; T8, T-suppressor cells; TLE, temporal lobe epilepsy.

Cyclohexylpiperazine derivative PB28, a σ2 agonist and σ1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer

σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ2 agonist/σ1 antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ2 receptor expression, by high and low σ1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ2 receptor affinity and low σ1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC50 in nanomolar range), a consistent time exposure-independent increase of G0-G1-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ2 agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]

5-HT1A Receptor, an Old Target for New Therapeutic Agents

The serotonin receptor subtype 5 HT(1A) was one of the first serotonin receptor subtypes pharmacologically characterized. Over the last twenty years the 5 HT(1A) receptor has been the object of intense research efforts as witnessed by the 5 HT(1A) acting drugs marketed as anxiolytics. In recent years, several new chemical entities targeting the 5 HT(1A) receptor (alone or in combination with other molecular targets) have been proposed for novel therapeutic indications (neuroprotection, cognitive impairment, Parkinson Disease and related disorders, pain treatment). The present review will focus on those 5 HT(1A) receptor agents that entered preclinical trials starting from 2000.

Developments in fluorescent probes for receptor research

Early reports on the identification of fluorescent probes for receptors date back to mid-1970s. Fluorescent probes were initially used to visualize molecular targets in an analogous way to the use of fluorescent antibodies but with the same resolution as isotopically labelled ligands. In parallel to the rapid development of techniques, such as fluorescence correlation spectroscopy, multi-photon excitation fluorescence microscopy, fluorescence polarization and in vivo fluorescence imaging, fluorescent probes are becoming multifaceted tools in life science. The present review will focus on how the design of fluorescent ligands for receptors has evolved to meet the needs of most recent fluorescence applications.

Structural Modifications of N-(1,2,3,4-Tetrahydronaphthalen-1-yl)-4-Aryl-1-piperazinehexanamides: Influence on Lipophilicity and 5-HT7 Receptor Activity. Part III

Starting from the previously reported 5-HT 7 receptor agents 4-7 with N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinehexanamide structure, the 1-(2-methylthiophenyl)-, 1-(2-diphenyl)-, 1-(2-isopropylphenyl)-, and 1-(2-methoxyphenyl)piperazine derivatives 8-31 were designed with the primary aim to obtain new compounds endowed with suitable physicochemical properties for rapid and extensive penetration into the brain. The affinities for 5-HT 7, 5-HT 1A, and D 2 receptors of compounds 8-31 were assessed, and several compounds displayed 5-HT 7 receptor affinities in the nanomolar range. Among these, N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (25) showed high 5-HT 7 receptor affinity (Ki = 0.58 nM), high selectivity over 5-HT 1A and D 2 receptors (324- and 245-fold, respectively), and agonist properties (maximal effect = 82%, EC 50 = 0.60 microM). After intraperitoneal injection in mice, 25 rapidly reached the systemic circulation and entered the brain. Its brain concentration-time profile paralleled that in plasma, indicating that 25 rapidly and freely distributes across the blood-brain barrier. Compound 25 underwent N-dealkylation to the corresponding 1-arylpiperazine metabolite.

LP-211 is a brain penetrant selective agonist for the serotonin 5-HT7 receptor

We have determined the pharmacological profile of the new serotonin 5-HT(7) receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT(7) receptor (5-HT(7)(-/-)) and their 5-HT(7)(+/+) sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA-7). In vitro binding assays revealed that RA-7 possessed higher 5-HT(7) receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP-211, and to a lesser degree RA-7, induced hypothermia in 5-HT(7)(+/+) but not in 5-HT(7)(-/-) mice. Our results suggest that LP-211 can be used as a 5-HT(7) receptor agonist in vivo.

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump.

ABC Pumps and Their Role in Active Drug Transport

Pharmacokinetic limitations affect drug bioavailability determining the loss or the reduction of the pharmacological effects. The Gastro Intestinal tract (GI) and the Blood Brain Barrier (BBB) are the most important restrictive and selective physiological lines of defense of the organism. Although several parameters such as LogP, LogD and K(a) have been extensively employed for determining drug bioavailability, the active transports, present in these biological barriers, play an important role for dosing and limiting cell drugs concentration. In particular, ATP Binding Cassette (ABC) transporters are involved in the active transport both in GI and BBB. Their strategic activity and biochemical and pharmacological role are herein treated.