Nicola Antonio Colabufo

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]

Selective κ Opioid Antagonists nor-BNI, GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. Results In binding assays, the three antagonists showed no detectable affinity (K i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α2C-adrenoceptor (K i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (K B = 3.7 µM). JDTic bound to the noradrenaline transporter (K i = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. Conclusions Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α1A-adrenoceptors). This may contribute to GNTIs severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

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.

Small and Innovative Molecules as New Strategy to Revert MDR

Multidrug resistance (MDR) is a complex phenomenon principally due to the overexpression of some transmembrane proteins belonging to the ATP binding cassette (ABC) transporter family. Among these transporters, P-glycoprotein (P-gp) is mostly involved in MDR and its overexpression is the major cause of cancer therapy failure. The classical approach used to overcome MDR is the co-administration of a P-gp inhibitor and the classic antineoplastic drugs, although the results were often unsatisfactory. Different classes of P-gp ligands have been developed and, among them, Tariquidar has been extensively studied both in vitro and in vivo. Although Tariquidar has been considered for several years as the lead compound for the development of P-gp inhibitors, recent studies demonstrated it to be a substrate and inhibitor, in a dose-dependent manner. Moreover, Tariquidar structure–activity relationship studies were difficult to carry out because of the complexity of the structure that does not allow establishing the role of each moiety for P-gp activity. For this purpose, SMALL molecules bearing different scaffolds such as tetralin, biphenyl, arylthiazole, furoxane, furazan have been developed. Many of these ligands have been tested both in in vitro assays and in in vivo PET studies. These preliminary evaluations lead to obtain a library of P-gp interacting agents useful to conjugate chemotherapeutic agents displaying reduced pharmacological activity and appropriate small molecules. These molecules could get over the limits due to the antineoplastic-P-gp inhibitor co-administration since pharmacokinetic and pharmacodynamic profiles are related to a dual innovative drug.

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.

Synthesis and Preclinical Evaluation of Novel PET Probes for P-Glycoprotein Function and Expression

UNLABELLED P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. A P-gp substrate (7) and an inhibitor (6) were labeled with (11)C, resulting in potential tracers of P-gp function and expression. METHODS 6 and 7 were labeled using (11)CH(3)I. (11)C-verapamil was prepared as published previously, using (11)C-methyl triflate. MicroPET scans (with arterial sampling) and biodistribution studies were performed in rats pretreated with saline, cyclosporin A (CsA, 50 mg/kg), or cold 6 (15 mg/kg). RESULTS The radiochemical yields of (11)C-6 and (11)C-7 were approximately 30% with a total synthesis time of 45 min. Cerebral distribution volumes (DV) of (11)C-6 (2.35 +/- 0.11) and (11)C-7 (1.86 +/- 0.15) in saline-treated rats were higher than of (11)C-verapamil (0.64 +/- 0.12). DVs of (11)C-7 and (11)C-verapamil were significantly increased by CsA (to 5.26 +/- 0.14 and 5.85 +/- 0.32, respectively). The DV of (11)C-6 was reduced by cold 6 (to 1.65 +/- 0.03). Its uptake was also reduced (up to 67%) in several peripheral organs that express P-gp. CONCLUSIONS (11)C-7 is a novel tracer of P-gp function with higher baseline uptake than (11)C-verapamil. Upregulation of P-gp function in response to treatment (which is hard to detect with (11)C-verapamil) may be detectable using (11)C-7 and PET. Because (11)C-6 shows specific binding in target organs, this compound is the first PET tracer allowing measurement of P-gp expression.

A multireceptorial binding reinvestigation on an extended class of σ ligands: N-[ω-(indan-1-yl and tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine reveal high affinities towards σ1 and EBP sites

New 1-[omega-(2,3-dihydro-1H-inden-1-yl)- and (2,3-dihydro-5-methoxy-1H-inden-1-yl)alkyl]- and 1-[omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- and (6-methoxy- or 6-fluoro-1,2,3,4-tetrahydronaphthalen-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine were synthesized, as homologous compounds of an existing series of sigma ligands, in order to carry out sigma receptor subtypes structure-affinity relationships. The new compounds and some of their related analogues, already reported, were tested in new multireceptorial radioligand binding assays. As reference compounds, the known sigma(1) ligands SA 4503, BD 1008 and NE 100 were also prepared and tested. All reported compounds showed high sigma(1) affinity assayed by (+)-[(3)H]-pentazocine on guinea-pig brain (apparent K(i)=1.75-72.2 nM) and moderate or low sigma(2) affinity by [(3)H]-DTG on rat liver, in contrast with previous results. One tertiary amine function spaced by a five-membered chain from a phenyl group is the structural feature shared by the most active compounds 26 and 43 and some reference sigma(1) ligands. The reported sigma(1) ligands, including reference compounds, also demonstrated a high affinity towards EBP (Delta(8)-Delta(7) sterol isomerase) site (apparent K(i)=0.48-14.8 nM) and some of them (37 and 44) were good ligands at L-type Ca(++) channel. 1-[4-(2,3-Dihydro-1H-inden-1-yl)butyl]-3,3-dimethylpiperidine (26) was the best mixed sigma(1) and EBP ligand (apparent K(i)=1.75 and 1.54 nM, respectively) with a good selectivity versus sigma(2) receptor (138- and 157-fold, respectively).