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Imaging the living brain and the distribution of the ligand gated channels that participate in the neurotransmission is one of the challenges that is hoped to bring new insights for the treatment of neurological diseases. Herein, we probed a new nicotinic derivative, A-186253 as a potential molecule to discriminate with high resolution the different neuronal nicotinic receptor subtypes that are expressed in distinct brain areas. Binding with a high affinity of 440 pM at the major brain alpha4beta2 receptor subtype and presenting an excellent safety margin, properties of the A-186253 were thoroughly evaluated. While autoradiography confirmed its specificity for the alpha4beta2 subtype, functional investigations revealed for short exposures a broader spectrum of action at receptors including the ganglionic alpha3beta4 and the homomeric alpha7 subtypes. Specificity was, however, observed at alpha4beta2 when receptors were exposed for several minutes with low concentration of the A-186253. In view of these promising results, the A-186253 was radiolabeled and tested in positron emission tomography on rats and pigs. Despite the high selectivity observed in vitro, the A-186253 displayed a complex binding profile and little displacement by the agonist cytisine. While the A-186253 can be valuable to discriminate receptor subtypes, improvements of this molecule must be brought for in vivo measurements.

Apomorphine has been used as a pharmacological probe of dopaminergic receptors in a variety of central nervous system disorders. The utility of apomorphine as an agent for the treatment of erectile dysfunction has also been demonstrated clinically. Apomorphine is a nonselective dopaminergic receptor agonist with potent binding affinity (Ki) of 101, 32, 26, 2.6, and 10 nM for D1, D2, D3, D4, and D5, respectively. When administered either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.), apomorphine fully evoked penile erections in conscious rats with maximum effect at 0.1 micromol/kg s.c. and 3 nmol/rat i.c.v., respectively. Apomorphine was less efficacious when injected intrathecally (i.t.) to L4-L6 spinal levels (50% at 30-100 nmol/rat i.t.). Penile erection facilitated by apomorphine was blocked by haloperidol and clozapine (i.p. and i.c.v.) but not by domperidone (a peripherally acting dopaminergic receptor antagonist). In this model using conscious rats, penile erection was significantly induced by quinpirole (D2-D3-D4 receptor agonist), but not by R(+)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol (SKF38393) and R(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzapine (SKF81297) (D1 receptor agonists), or a D2 receptor agonist R-5,6-dihydro-N,N-dimethyl-4H-imidazo[4,5,1-ij]quinolin-5-amine (PNU-95666E). The role of D4 receptors in penile erection was demonstrated using selective D4 receptor agonists [(4-phenylpiperazinyl)-methyl]benzamide (PD168077) and 5-fluoro-2-[[4-(2-pyridinyl)-1-piperazinyl]methyl]-1H-indole (CP226269), whether administered systemically (s.c.) or locally in the brain (i.c.v.). The ability of apomorphine to activate D3 receptors in relation to its proerectile activity remains to be elucidated by use of selective subtype agonists. These results suggest that the proerectile action of apomorphine in rats is mediated at supraspinal levels and that this effect is not mimicked by a D2 receptor agonist but associated with activation of D4 receptors.

Purpose: To perform a phase I trial to determine the maximum tolerated dose and the dose-limiting toxicities of paclitaxel in children with refractory or recurrent solid tumors. Paclitaxel was administered twice weekly, increasing from four to six doses every 21 to 28 days. Methods: Paclitaxel was administered as a 3-hour intravenous infusion twice weekly. The initial dose was fixed at 50 mg/m 2 /dose twice weekly for 2 weeks (four doses), every 21 days. The number of twice-weekly doses per course was increased to six in the next cohort. In subsequent cohorts, the number of twice-weekly doses per course was fixed at six, every 28 days, and dosage was increased in 25% increments. Results: Sixteen assessable patients were enrolled at three levels. Neutropenia was the dose-limiting toxicity at 65 mg/m 2 /dose, twice weekly x 6 doses, every 28 days. Nonhematologic toxicities were minor. No antitumor responses were observed. Conclusions: Protracted twice-weekly dosing of paclitaxel is limited by neutropenia. The maximum tolerated dose of paclitaxel administered twice weekly x 6 doses, every 28 days, was 50 mg/m 2 /dose.

We have discovered a novel series of N -[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl] amides that are potent openers of K ATP channels and investigated structure–activity relationships (SAR) around the 1,2-disubstituted naphthyl core. A-151892, a prototype compound of this series, was found to be a potent and efficacious potassium channel opener in vitro in transfected Kir6.2/SUR2B cells and pig bladder strips. Additionally, A-151892 was found to selectively inhibit unstable bladder contractions in vivo in an obstructed rat model of myogenic bladder function

N-[3-(1H-Imidazol-4-ylmethyl)phenyl]ethanesulfonamide (ABT-866, 1) is a novel alpha(1) agent having the unique profile of alpha(1A) (rabbit urethra, EC(50) = 0.60 microM) agonism with alpha(1B) (rat spleen, pA(2) = 5.4) and alpha(1D) (rat aorta, pA(2) = 6.2) antagonism. An in vivo dog model showed 1 to be more selective for the urethra over the vasculature than A-61603 (2), ST-1059 (3, the active metabolite of midodrine), and phenylpropanolamine (4).

Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ATP-sensitive K(+) (K(ATP)) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel 1,4-dihydropyridine K(ATP) channel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated K(ATP) channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC(50) = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC(50) = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article [Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002) J Pharmacol Exp Ther 303:387-394], A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 [(R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile] and ZD6169 [(S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide]. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective K(ATP) channel openers for the treatment of overactive bladder via myogenic etiology.

ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to inhibit spontaneous myogenic contractile activity of the urinary bladder, a mechanism hypothesized to underlie detrusor instability and symptoms of overactive bladder. However, the therapeutic utility of KCOs has been limited by a lack of differentiation of bladder versus vascular effects. In this study, we evaluated the in vivo potency and bladder selectivity of (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel dihydropyridine KCO, in a pig model of detrusor instability secondary to partial bladder outlet obstruction. For comparison, we profiled two KCOs, ((R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile (WAY-133537) and (S)-N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methyl-propionamide (ZD6169), reported previously to have improved bladder selectivity in vivo and a calcium channel blocker, nifedipine. Effective doses of A-278637, WAY-133537, ZD6169, and nifedipine to inhibit unstable contraction area under the curve by 35% and to decrease mean arterial pressure by 10% were 4.2 and 12, 109 and 51, 661 and 371, and 136 and 30 nmol/kg i.v., yielding corresponding bladder selectivity ratios of 3, 0.5, 0.6, and 0.2. Therefore, A-278637 was approximately 5- to 6-fold more bladder-selective than the other KCOs and 15-fold more selective than nifedipine, the latter approximately 4.5-fold vascular-selective. The potency of KCOs to inhibit unstable contraction in vivo was accurately predicted by their potency to inhibit spontaneous contractile activity of pig detrusor strips in vitro. These results indicate that A-278637, with enhanced potency and bladder selectivity compared with the other compounds evaluated, could serve as a useful tool in the investigation of smooth muscle K(ATP) channel openers as novel therapeutic agents for the treatment of overactive bladder.

N-[3-(1H-Imidazol-4-ylmethyl)phenyl]ethanesulfonamide, maleate (ABT-866) is a novel alpha(1)-adrenoceptor agent with mixed pharmacological properties in vitro. Compared to phenylephrine, ABT-866 demonstrates intrinsic activity at the alpha(1A)-adrenoceptor subtype present in the rabbit urethra (pD(2) = 6.22, with 80% of the phenylephrine response), reduced intrinsic activity at the alpha(1B)-adrenoceptor subtype in the rat spleen (pD(2)= 6.16, with 11% of the phenylephrine response), and no intrinsic activity at the rat aorta alpha(1D)-adrenoceptor subtype. ABT-866 also demonstrated antagonism at the rat spleen alpha(1B)-adrenoceptor (pA(2) = 5.39 +/- 0.08, slope = 1.20 +/- 0.12), and the rat aorta alpha(1D)-adrenoceptor (pA(2)= 6.18 +/- 0.09, slope = 0.96 +/- 0.13). This is in contrast to the weak non-selective activity seen with the alpha(1)-adrenoceptor agonist, phenylpropanolamine (2-amino-1-phenyl-1-propanol hydrochloride), and the alpha(1A/D)-adrenoceptor selective agonist 1-(2',5'-dimethoxyphenyl)-2-aminoethanol hydrochloride (ST-1059), the active metabolite of midodrine, that has been used clinically for the treatment of stress urinary incontinence. This study identifies a unique agent that may prove to be a valuable in vivo tool in testing the hypothesis that the alpha(1A)-adrenoceptor can be stimulated to contract the smooth muscle present in the urethra without evoking blood pressure elevations presumably caused by alpha(1B)- and alpha(1D)-adrenoceptor subtype involvements in the vasculature.

A-204176 (N-[5-(1H-imidazol-4-y1)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide) is a potent and selective alpha1A adrenoceptor agonist that binds with 17-fold and 9-fold greater affinity to the alpha1A (Ki=176 nM) than the alpha1b and alpha1d subtypes, respectively. In functional studies A-204176 is potent (pD2=6.4) and efficacious (83% of maximum control phenylephrine response) at rabbit urethra alpha1A receptors, with weaker potency and greatly reduced efficacy at rat spleen alpha1B (pD2=5.3, 11%) and rat aorta alpha1D (pD2=4.4, 10%) subtypes. In anesthetized female dogs, A-204176 is more potent than the non-selective alpha1 adrenoceptor agonist phenylpropanolamine (PPA) to increase measures of urethral tone and is more efficacious to increase pressure in the proximal region of the urethra. Significant increases on parameters of the urethral pressure profilometry were induced at 100 and 300 nmol/kg, i.v., by A-204176 and PPA, respectively. A-204176 was more potent than PPA to increase the abdominal pressure required to produce leakage. In the simultaneous measurement of intraurethral pressure and mean arterial blood pressure, A-204176 displays enhanced urethral selectivity relative to PPA. However, despite its selectivity for alpha1A versus alpha1B and alpha1D adrenoceptors in vitro, A-204176 did not display the degree of urethral selectivity in vivo that would have been expected. The observed effect of A-204176 on blood pressure may be due to the presence of extra-synaptic alpha1A adrenoceptors in the vasculature or to activation of spinal and supraspinal alpha1A adrenoceptors. These data indicate that A-204176 may represent a useful pharmacological tool to investigate the functional role of the alpha1A adrenoceptor in the urethra and to elucidate the lack of uroselectivity observed in vivo.

In functional assays, A-315456, N-[3-(cyclohexylidene-(1H-imidazol-4-ylmethyl))phenyl]ethanesulfonamide, behaved as an alpha(1D)-adrenoceptor subtype selective antagonist (pA(2)=8.34) in the rat aorta. It was 83-fold less potent at the alpha(1B)-adrenoceptor subtype expressed in the rat spleen, and was inactive at the alpha(1A)-adrenoceptor subtype expressed in the rat vas deferens. Radioligand binding assays also revealed high affinity (pK(i)=8.71) for the alpha(1D)-adrenoceptor subtype and weaker affinities at the alpha(1A)-adrenoceptor (pK(i)=6.23) and alpha(1B)-adrenoceptor (pK(i)=7.86). In comparison to its potent affinity at the alpha(1D)-adrenoceptor subtype, A-315456 was 3020-, 794- and 38-fold weaker at the dopamine D(2)-, 5-HT(1A)-, and alpha(2a)-adrenoceptors, respectively. These studies indicate that A-315456 is a potent and selective alpha(1D)-antagonist that may serve as a useful pharmacological ligand to probe the physiological role of the alpha(1D)-adrenoceptor subtype in normal and disease states.

Glioblastoma (GBM), the most common and aggressive form of glioma, is often heterogeneous, diffusely invasive and hypervascularized. Despite being aggressive, extracranial metastases are rare, suggesting constraints on hematologic dissemination or distant survival of primary GBM cells. We recently developed a novel microfluidic device, termed the CTC-iChip, for efficient, antigen agnostic capture of rare circulating tumor cells (CTCs). Using the CTC-iChip, we set out to determine whether GBM cells can be detected in the blood of mice using two orthotopic xenograft models representing diffuse (GBM8) and focal (GBM24) GBM. CTCs were identified in 5/11 and 2/5 mice bearing GBM8 and GBM24 xenografts, respectively. CTC numbers did not correlate with tumor size or latency. We then expanded our analysis to patients and identified CTCs in 12/38 GBM patients with a median 5.3 cells per ml (range: 0 to 48.2 cells per ml). To understand the molecular characteristics of GBM CTCs, we analyzed the expression of 49 GBM-specific and stem cell transcripts in single GBM CTCs from both mouse xenografts models as well as GBM patients. The expression of mesenchymal gene transcripts including VIM, TGFBR2, TGFB, and SERPINE1 was elevated in CTCs compared with cultured cells and matched primary tumor cells. Moreover, CTCs exhibited lower proliferative indices and decreased neural lineage transcripts. To determine the pathophysiologic significance of this expression profile and the prospective origin of GBM CTCs, RNA-ISH was performed on xenografts and patient tumor samples. In the highly migratory GBM8 xenograft model, broad tumor cell expression of CTC/mesenchymal transcripts was observed. However, in the GBM24 focal tumor growth model, CTC/mesenchymal gene transcripts were predominantly expressed in regions of tumor invasion, migration along fiber tracts, and around regions of necrosis. Similarly, RNA-ISH analysis of 6 GBM patient tumors revealed prominent expression of CTC/mesenchymal genes in peri-necrotic and migratory pseudopalisading cells. Finally, RNA-ISH analysis of a rare case of metastatic GBM revealed a significant enrichment of GBM/mesenchymal gene expressing tumor cells within lung and lymph node metastases. Taken together, these data provide the first evidence for the hematologic circulation of GBM cells and reveal the involvement of mesenchymal features in the pathophysiology of malignant brain tumors. Citation Format: James P. Sullivan, Brian V. Nahed, Andrew S. Chi, Marissa N. Madden, Samantha M. Oliveira, Simeon Springer, Hiroaki Wakimoto, Deepak Bhere, Khalid Shah, Phil Spuhler, Ajay M. Shah, David N. Louis, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Molecular characterization of circulating glioblastoma cells identifies a mesenchymal-like tumor cell subpopulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2014-4004

PURPOSE: To determine the response rate of the combination of cyclophosphamide and topotecan in pediatric patients with recurrent or refractory malignant solid tumors. PATIENTS AND METHODS: A total of 91 pediatric patients, 83 of whom were fully assessable for response and toxicity, received cyclophosphamide (250 mg/m2/dose) followed by topotecan (0.75 mg/m2/dose), each given as a 30-minute infusion daily for 5 days. All patients received filgrastim (5 mcg/kg) daily until the absolute neutrophil count (ANC) was ≥ 1,500 μL after the time of the expected ANC nadir. RESULTS: A total of 307 treatment courses were given to the 83 fully assessable patients. Responses (complete response plus partial response) were seen in rhabdomyosarcoma (10 of 15 patients), Ewing’s sarcoma (six of 17 patients), and neuroblastoma (six of 13 patients). Partial responses were seen in two of 18 patients with osteosarcoma and in one patient with a Sertoli-Leydig cell tumor. Twenty-three patients had either minor responses (n = 6) or stable disease (n = 17); the median number of courses administered to patients with partial or complete response was six (range, two to 13 courses), and the median administered to those with stable disease was three (range, one to 11 courses). The toxicity of the combination was limited principally to the hematopoietic system. Of 307 courses, 163 (53%) were associated with grade 3 or 4 neutropenia, 84 (27%) with grade 3 or 4 anemia, and 136 (44%) with grade 3 or 4 thrombocytopenia. Despite the severe myelosuppression, only 34 (11%) of 307 courses were associated with grade 3 or 4 infection. Nonhematopoietic toxicity of grades ≥ 3 was rare and consisted of nausea and vomiting (two courses), perirectal mucositis (one course), transaminase elevation (one course), and hematuria (two courses). CONCLUSION: The combination of cyclophosphamide and topotecan is active in rhabdomyosarcoma, neuroblastoma, and Ewing’s sarcoma. Stabilization of disease was seen in osteosarcoma, although objective responses were rare in this disease. The therapy can be given with acceptable hematopoietic toxicity with the use of filgrastim support.

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17- splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K(ATP) channels in HEK 293 cells as assessed either by changes in DiBAC(4)(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K(ATP) channels.

A critical component of the drug discovery and development process is the identification of novel pharmacophores. Such discovery efforts are, in general, facilitated by rapid and high-throughput cell-based assays of receptor/ion channel-mediated signaling processes to screen diverse chemical libraries of compounds possessing activator or inhibitor activities at the desired target. The availability of the Fluorometric Image Plate Reader (FLIPR) has made rapid assays of cellular signaling processes feasible by simultaneous kinetics measurement of cell-based fluorescence changes in a 96- or 384-well format. This unit describes the application of the FLIPR in cell-based kinetic assays for measuring membrane potential changes and intracellular calcium dynamics.

Research on the nature, structure and biological activity of the toxins present in the skin of poison-dart frogs of South America began in the Laboratory of Chemistry at the National Institutes of Health in the mid-1960s. The presence of toxins in the skin of such frogs had been discovered long ago by Indians of Western Colombia, who to this day use skin secretions from three Colombian species of dendrobatid frogs (genus Phyllobates) to poison the grooved tips of blow darts used in hunting small game and birds. Initial field work on a poison-dart frog of the Rio San Juan drainage, and preparation of extracts was first conducted by F. Marki in 1962 and then by Daly in 1964 and 1966. The toxic principles were isolated and proved on structural analysis to be steroidal alkaloids, which were named batrachotoxins.1 These were then shown to be specific and potent activators of sodium channels.2 Both the natural alkaloids and a radioactive analog have proven to be invaluable research tools for the study of sodium channels and their interaction with local anesthetics, anticonvulsants, antiarrythmics and other drugs.3 The structure of batrachotoxin and other alkaloids, subsequently isolated from frog skin, are shown in Fig. 1. These initial studies on the batrachotoxin alkaloids from the poison-dart frogs of Western Colombia might never have been extended to some sixty species of poison-frogs of the neotropical family Dendrobatidae, had not Charles W. Myers, a herpetologist working on the reptiles and amphibians of Panama, contacted Daly and proposed a collaboration on the toxicity of an extremely variable dendrobatid frog (genus Dendrobates) of the Bocas Archipelago of Panama. The initial hypothesis, namely that the more brightly colored populations would contain higher levels of toxic alkaloids, proved incorrect. However, the analyses revealed not the steroidal batrachotoxins, but instead a variety of simpler bicyclic alkaloids, including the relatively toxic pumiliotoxins and relatively nontoxic decahydroquinolines.4 The pumiliotoxins and related alkaloids later were shown to be potent myotonic/cardiotonic agents5 with modulatory effects on sodium channels.6 The initial field work by Myers and Daly led to a thirty year friendship and collaboration with the aim of analyzing the distribution, nature, structure and biological activity of alkaloids in frog skin. A major field trip by Myers and Daly in the early 1970s led to the isolation and structural determination of relatively nontoxic bicyclic histrionicotoxins,7 later established as highaffinity noncompetitive blockers of nicotinic acetylcholine receptor-channels (nAChRs).3 Over the next three decades more than 500 alkaloids of at least two dozen structural classes were discovered, most of which have, as yet, not been found elsewhere in nature.8,9 This is remarkable, since the dendrobatid frogs apparently do not synthesize any of their skin alkaloids, but instead sequester them unchanged into skin glands from dietary sources10 to be used as secreted chemical deterrents to predators. The search over the past five years for the dietary sources of the batrachotoxins, pumiliotoxins and histrionicotoxins has been frustrating, but some six classes of relatively simple decahydroquinolines, piperidines, pyrrolidines and “izidines” of dendrobatid frog skin have been found in ants, while certain of the tricyclic and spiropyrrolizidine alkaloids occur in beetles and millipedes, respectively.10,11 Fig. 1 Structures of epibatidine and other alkaloids discovered in skin extracts from poison frogs (family Dendrobatidae). Batrachotoxin from Colombian Phyllobates aurotaenia,1 pumiliotoxin B from Panamanian Dendrobates pumilio,4 histrionicotoxin from Colombian Dendrobates histrionicus,7 and epibatidine and alkaloids 251D, 251H and 341A from Ecuadorian Epipedobates tricolor.12–14,16 EMINENT SCIENTIST REVIEW

Analogs of compound 1 with a variety of azacycles and heteroaryl groups were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 10,000 nM when tested in vitro for cholinergic channel receptor binding activity (displacement of [3H](-) cytisine from whole rat brain synaptic membranes).

Analogs of A-98593 (1) and its enantiomer ABT-594 (2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays.

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily of receptors. The potential of nAChRs as targets of therapeutic intervention has been revealed, in part, by recent studies showing that a diversity of nAChR subunits exist in brain and other tissues, and that discrete subunit combinations may be involved in mediating specific neurophysiological functions and behaviors. To date, eleven gene products representing nAChRs have been identified. This unit examines the binding properties of the a4 b2 receptor in rat brain or in transformed cell lines expressing the recombinant receptor.

This study examined the binding and functional properties of human alpha7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (-)-nicotine (100 microM) for 120 h increased the Bmax values of [125I]alpha-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC50) = 970 microM) and a 6-fold upregulation was observed with the maximal concentration of (-)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (+/-)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [125I]alpha-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the alpha7 nicotinic receptor antagonist, methyllycaconitine (EC50 = 92 microM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (-)-nicotine after chronic treatment of cells with concentrations of (-)-nicotine (1000 microM) or methyllycaconitine (10 microM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505 +/- 21 pA) and 6-fold (1820 +/- 137 pA) respectively in whole cell current amplitude compared to untreated cells (267 +/- 24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of alpha7 nicotinic receptors and can differentially enhance nicotinic receptor function.

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

Recent evidence suggests the existence of a diversity of neuronal nicotinic acetylcholine receptor (nAChRs) subtypes with a wide distribution in brain, that each subtype may be involved in mediating specific functions/behaviors, and that these subtypes have a defined pharmacology that may be selectively targeted (Arneric et al., 1995a). The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favorable side-effect profiles than existing agents which generally exhibit low selectivity. This broader class of agents, collectively called cholinergic channel modulators (ChCMs) selectively activate some subtypes of nAChRs (i.e. Cholinergic Channel Activators, ChCAs) or inhibit the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs).

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, α4 and β2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 μmand a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human α4β2 receptors is (−)-nicotine > ACh > (−)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 ≫ (−)-nicotine > ACh ≫ (−)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 μmACh with known nAChR inhibitors such as dihydro-β-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50values of 80 nmand 1.5 μm. The current–voltage relationship of human α4β2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human α4β2 nAChRs are permeable to sodium and potassium ions. In the “outside-out” configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mm. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mm.

The norditerpenoid alkaloid lycaconitine (2) was synthesized from lycoctonine (3) and its affinity determined for two neuronal nicotinic acetylcholine receptor subtypes. The structure of 2 was confirmed by a combination of spectroscopic methods.

The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2(S)-azetidinylmethoxy)pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human alpha 4 beta 2 nAChR subtype (Ki = 0.05 + 0.01 nM) relative to the human alpha 7 (Ki = 148 +/- 13 nM) and the muscle alpha 1 beta 1 dg subtype expressed in Torpedo electroplax (Ki = 314 +/- 12 nM). The R-enantiomer of A-85380, A-159470, displays little enantioselectivity towards the alpha 4 beta 2 and alpha 1 beta 1 delta gamma subtypes but does not display 12-fold enantioselectivity towards the alpha 7 subtype (Ki = 1275 +/- 199 nM). (+)- and(-)-Epibatidine display similar potencies at the human human alpha 4 beta 2 (Ki = 0.04 +/- 0.02 nM and 0.07 +/- 0.02 nM, respectively), human alpha 7 (Ki = 16 +/- 2 nM and 22 +/- 3 nM, respectively) and muscle alpha 1 beta 1 delta gamma g (Ki = 2.5 +/- 0.9 nM and 5.7 +/- 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human alpha 4 beta 2 (EC50 = 0.7 +/- 0.1 microM) and ganglionic (EC50 = 0.8 +/- 0.09 microM) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 microM). Further, A-85380 can activate (EC50 = 8.9 +/- 1.9 microM) currents through channels formed by injection of the human alpha 7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the alpha 7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (-)-nicotine but less potent than (+/-)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 +/- 0.001 microM which is equipotent to (+/-)-epibatidine, and 20-fold more potent than (-)-nicotine (EC50 = 0.04 +/- 0.009 microM). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [125I] alpha-BgT sites than [3H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the alpha 4 beta 2 subtype relative to the alpha 7 and alpha 1 beta 1 delta g nAChRs than (+/-)-epibatidine.

Recent literature has shown that compounds interacting with neuronal nicotinic acetylcholine receptors (nAChRs) have the potential to be neuroprotective both in vitro and in vivo. ABT-418 is a novel ChCA that selectively stimulates discrete subtypes of the nAChRs and exhibits cognitive enhancing activity. In the present study, the neuroprotective effects of ABT-418 and (-)-nicotine, as measured by the release of lactate dehydrogenase (LDH) into the media, were investigated in a glutamate (Glu)-induced cytotoxicity assay using either primary rat cortical neurons or a human differentiated cell line, IMR 32. The neuroprotection elicited by ABT-418 and (-)-nicotine is both time and concentration dependent with an optimal concentration of 10 microM and an optimal pretreatment time of 2 h. ABT-418 remained neuroprotective and not cytotoxic to rat cortical cells following subacute exposure for 7 days. Protection appears to be mediated via an interaction with nAChRs, possibly the alpha 7 subtype, since the neuroprotection was prevented by alpha-bungaratoxin (alpha-Bgt) and methyllycaconitine (MLA), both selective alpha 7 antagonists. Removal of extracellular Ca2+ prevented the neuroprotective effects of ABT-418 and (-)-nicotine, consistent with the known ability of alpha 7 nAChRs to modulate calcium dynamics. These data support the idea that ABT-418 not only enhances cognition, but may possibly slow the progression of the neurodegenerative process.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved.

Publisher Summary Advances in the understanding of the molecular biology and pharmacology of nicotinic receptors (nAChRs) may provide targets for the development of novel and selective modulators of nAChRs in the brain. This contention is supported by the dissimilar behavioral effects observed following systemic administration of currently available nicotinic ligands. This chapter attempts to describe the biochemical and behavioral effects of (−)-nicotine, the classic Iigands [(−)-lobeline, (−)-cytisine, etc.], and the novel cholinergic channel modulators (ChCMs) [ABT-418, (±epibatidine, and GTS-21] in view of the latest molecular biological studies demonstrating the presence of multiple subunits comprising the nAChRs in the brain, ganglia, and neuromuscular junction. Some of these compounds selectively activate or block a particular nAChR subtype, and they will eventually help to define the potential physiological role of the nAChRs in the peripheral and central nervous systems. Increasing evidence suggests that the positive and negative effects of (−)-nicotine are mediated through its binding to the different subtypes in the brain and in the periphery. Based on this premise, ChCMs could selectively enhance central nAChR-mediated transmission without having the side-effect liabilities normally associated with (−)-nicotine (cardiovascular, gastrointestinal, abuse potential, and others). Identification of these ChCMs may represent a potential therapeutic strategy to ameliorate many of the CNS deficits accompanying Alzheimer's disease (AD) or other related disorders, as discussed in the chapter.

Among the losses of brain functions in Alzheimers disease (AD) are reductions in cerebral blood flow (CBF), decrements in cerebral glucose utilization and abnormal electrocorticograms (Dastur, 1985; Petit et al., 1983). Topographically, these impaired brain functions correspond to the loss of the cholinergic innervation to the cortex and hippocampus arising out of the basal forebrain (BF) (Bartus et al., 1982).

A considerable amount of evidence reveals the important roles of cancer stem cells (CSCs, also called “cancer initiating cells”) in tumor growth, metastasis, relapse and drug resistance. Our group has identified lung CSCs in both lung cancer cell lines and patient tumor samples by their elevated Aldehyde Dehydrogenase (ALDH) activity and demonstrated that NSCLC patients whose tumors are enriched in ALDH+ cells have inferior survival, and that the ALDH+ subpopulations (ranging from 0.5 to 30% of tumor cells) are self renewing, and significnatly more clonogenic and tumorigencic (in xenograft models) than the ALDH− population. To further define the molecular profile of lung CSCs and to identify key pathways involved in lung CSC function we performed genome wide microarray expression profiling of isolated ALDH+ and ALDH− cell populations from 8 non-small cell lung cancer (NSCLC) lines representing a variety of oncogenotypes to determine genes that are differentially expressed in these two populations. Strikingly we found that different lung cancers exhibited different ALDH+ cell population expression profiles but that the ALDH1A3 isozyme was one of the few commonly genes whose expression was elevated. (Previously our group and other investigators in lung and other cancers had found the ALDH1A1 and not ALDH1A3 isozyme to be a marker of the ALDH+ population). ALDH1A3 differential expression was confirmed by q RT PCR and elevated ALDH1A3 protein levels which in turn was correlated with the % of ALDH+ tumor cells in each tumor. ALDH1A3 mRNA levels in (N = 182) resected lung adenocarcinoma samples showe that the highest levels of ALDH1A3 were associated with inferior survival. Of great importance, shRNA mediated knockdown of ALDH1A3 dramatically reduced both NSCLC cell colony formation in vitro and tumor growth in vivo demonstrating the functional importance of ALDH1A3 expression in NSCLCs. However, ectopic expression of ALDH1A3 in NSCLC cells did not enhance lung cancer cell growth and tumor formation, suggesting that ALDH1A3 alone is not sufficient to promote lung cancer progression. We conclude that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity in NSCLCs of many oncogenotypes, is of potential prognostic importance, and a new therapeutic target for eliminating NSCLC stem cells.

Recent advances in detailed molecular understanding of lung cancer including complete genome sequences affords the possibility of developing molecularly targeted therapy for potentially all lung cancers. Currently the Lung Cancer Mutation Consortium (LCMC) has developed an integrated approach and network for identifying lung cancers with specific mutations and funneling these patients into clinical trials targeting the specific mutations identified. However, another approach is to use genome wide and chemical library screens to identify genetic and epigenetic changes that have been created in lung cancer cells during tumor pathogenesis that are absolutely required for the activated oncogenic pathways to function. These are often referred to as “synthetic lethal” changes and represent adaptations the cancer cell has to make to allow the “oncogene addictions” to drive tumor growth and survival. They are present in tumor but not normal cells and thus represent “acquired vulnerabilities” that can be therapeutically targeted. The NCI's “Cancer Target Discovery and Development Network” (CTD2N) exemplifies this combined genetic and pharmacologic approach. A most important subgroup of these vulnerabilities are changes that are required for the continued function and survival of a subpopulation of tumor cells that have acquired tumor initiating and often metastatic and drug resistant characteristics including many of the properties of stem cells that are referred to as cancer stem cells (CSCs) or “cancer initiating cells.” To achieve these goals we have developed a large integrated research platform. We have also: developed assays for identifying lung CSCs, “molecular portraits” (clades) that group lung cancers into subsets of clinical and molecular relevance; genome wide siRNA and chemical library functional screens to test for portrait/clade specificity. From these efforts we have learned that: There are a subset of cells within lung cancers (ranging from 0.1–30% of non small cell lung cancers, NSCLCs) and 50% + of small cell lung cancers, SCLCs) identified by elevated aldehyde dehydrogenase (ALDH) activity that have dramatically enhanced clonogenic, tumorigenic, and self renewal capacity; Patients whose tumors are enriched is such ALDH+ tumor cells have significantly impaired prognosis; The notch pathway (particularly Notch3) and ALDH1A3 are major vulnerabilities in lung CSCs; NSCLCs can be subdivided by mRNA expression profiles into “molecular portraits” (or “clades”) that have relevant clinical, oncogenotype, and drug response phenotypes; Genome wide siRNA and large chemical library screens identify targets that are specific for lung cancer over normal lung epithelial cells; The identified targets show dramatic clade and oncogenotype specificity as well as specificity for lung cancers with different responses to available chemotherapy and targeted therapy for lung cancer; The newly identified vulnerabilities provide coverage of essentially all lung cancers and as such, provide a new functional “vulnerability classification” of lung cancer. All of these findings set the stage for the development of a rational approach to developing therapy targeted at lung cancer acquired vulnerabilities including those in lung CSCs that will include personalizing the therapy for each patient. (Supported by Lung Cancer SPORE P50CA70907, DOD Prospect, CPRIT, and NCI CTD2N CA148225.)

With the plethora of receptor targets evolving as the result of nicotinic acetylcholine receptor (nAChR) cloning it is possible that new molecular entities selective for subtypes of nAChRs can be developed which are potentially free of the side effect liabilities associated with (-)-nicotine. Recent data also suggests that neuronal nAChR function can be enhanced at sites distinct from where (-)-nicotine interacts on the a subunit. nAChR agonists whose actions may occur via the selective interaction with central nAChRs subtypes, and allosteric modulators that indirectly affect ligand-gated nAChR function encompass a broader class of compounds which can be termed cholinergic channel activators (ChCAs). Functionally, ChCAs may enhance central neuronal nAChR mediated transmission while substantially reducing the side-effect liabilities normally associated with (-)-nicotine. ChCAs lacking cardiovascular or other CNS side effects associated with (-)-nicotine may thus represent a potential novel therapeutic approach to ameliorate many of the CNS deficits accompanying AD or other related disorders. ABT 418 [(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole hydrochloride] is a selective prototypic ChCA currently in development for the treatment of AD (see accompanying abstracts). ABT-418 may be a safe and effective ChCA for the potential treatment of the cognitive and emotional impairments associated with AD. The therapeutic potential of ChCAs for neuroprotection, smoking cessation, anxiety disorders, schizophrenia, attentional deficit disorder, Tourette s syndrome, analgesia and depression will be reviewed.

It is speculated that nicotine receptor ligands might be of therapeutic benefit for the treatment of obesity, anxiety, and memory loss. With the exception of nicotine, few selective high-affinity agents exist. (-)Epibatidine (1), isolated from Ecuadoran frogs, shows structural resemblence to (-)nicotine (2). Molecular modeling studies reveal that the N to N distance in 1 (5.5 A) is greater than that found in 2 (4.9 A). This distance exceeds that previously considered optimal for the nicotine receptor pharmacophore (4.8. ± 0.3 A) by about half a bond length. Nevertheless 1 binds (Ki = 0.055 nM) at [3H]nicotine-labeled receptors with higher affinity than 2 (Ki = 1.5 nM). Although our studies are still in progress, molecular superimposition and preliminary results with various structurally modified analogs suggest that it is the conformationally constrained nature of 1 that accounts for its high affinity. For example, 6-chloronicotine binds only with slightly higher affinity (Ki = 0.6 nM) than nicotine suggesting, at least for nicotine, that the chloro substituent is tolerated but does not contribute significantly to binding. Excision of the ethano bridge of deschloroepibatidine, to yield isonornicotine (Ki = 12.5 nM), results in reduced affinity. Isonicotine binds with similar (Ki = 7.3 nM) affinity. The bridged azabicycloheptane system likely fixes the structure of epibatidine in a highly receptor-preferred conformation. The pharmacophore for nicotine receptor binding will require reconsideration and should take into account the longer N-N distance of epibatidine. The N-N distance in epibatidine may be optimal, whereas that in isonornicotine is longer, and that for nicotine shorter. (Supported in part by funding from TDC/CIT.)

Background: Epithelial-to-mesenchymal transition (EMT) is an important biologic process in cancer resulting in enhanced migration, invasiveness, and therapeutic resistance. The transcription factor ZEB1 promotes EMT and tumor metastasis in several human cancers yet its role in the pathogenesis of lung cancer is less well defined. In addition to a large panel of lung cancer lines, our laboratory has developed an isogenic series of human bronchial epithelial cells (HBECs) – immortalized without viral oncoproteins – genetically manipulated with single and multiple oncogenic changes common in lung cancer (sh-p53, KRASV12, mutant EGFR, and c-MYC), providing an invaluable resource to study the progression of bronchial epithelial cells towards malignancy. Methods and Results: ZEB1 expression was analyzed by qRT-PCR across a panel of 77 non small cell lung cancer (NSCLC) lines to test for a direct correlation with ‘M’ status and indirect correlation with CDH1 expression and ‘E’ status. Genome-wide mRNA expression profiling of the NSCLC cell line cohort using Affymetrix U133 Plus 2.0 arrays was used to identify novel ZEB1-associated genes. Expression of the ephrin receptor EPHA1 significantly inversely correlated with ZEB1 expression. This was confirmed in independent siRNA experiments where knockdown of ZEB1 resulted in an increase in EPHA1 in both NSCLC cell lines and oncogenically transformed HBECs. To characterize the role of ZEB1 in the oncogenic progression of bronchial epithelial cells, ZEB1 was analyzed across our series of genetically manipulated HBECs. Expression of ZEB1 was generally absent, but was highly up-regulated with the combination of p53-knockdown, KRASV12, and c-MYC over-expression. This onco-genetic combination led to the most tumorigenic phenotype in vivo accompanied with a switch from ‘E’ to ‘M’ status. The functional importance of ZEB1 was seen with siRNA knockdown of ZEB1 resulting in a significant decrease in soft agar colonies, transwell invasiveness, and motility suggesting ZEB1 is a crucial driver of tumorigenicity in sh-p53 + KRASV12 + c-MYC transformed HBECs. To confirm the oncogenic role of ZEB1 in lung cancer, shRNA-mediated knockdown of ZEB1 was performed in several NSCLC cell lines, leading to a significant decrease in soft agar colonies, transwell invasiveness, motility, and 3D growth. Subcutaneous injection into NOD/SCID mice found inhibition of tumor growth in shZEB1 knockdown lines. Finally, loss of ZEB1 expression in NSCLCs and transformed HBECs resulted in a decrease in a “cancer stem cell” like population detected by ALDH activity. Conclusion: These findings involving mechanistic studies in NSCLC lines and oncogenically manipulated HBECs demonstrate the prominent role of ZEB1 function in EPHA1 expression, EMT, tumorigenicity, and the invasive phenotype of lung cancer, supporting the important role of ZEB1 in the pathogenesis of lung cancer making it an important new therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2011-3419

Background: Epithelial-to-mesenchymal transition (EMT) is an important biologic process in both early development and cancer whereby an epithelial cell transforms to a mesenchymal phenotype resulting in enhanced migration, invasiveness, and resistance to apoptosis. The transcription factor ZEB1 promotes EMT by repressing E-cadherin, and has been shown to promote tumor metastasis and increase resistance to standard chemotherapies in several human cancers. Its role in the tumorigenic progression of lung cancer however is less understood. Methods and Results: We analyzed genome-wide mRNA expression profiles across a cohort of 77 non small cell lung cancer (NSCLC) lines representing all common NSCLC histologies and oncogenotypes using Affymetrix GeneChip U133 Plus 2.0 arrays. Stratification of NSCLC lines by high/low ZEB1 expression identified 640 genes with significantly (p Conclusion: These findings indicate that in NSCLC, ZEB1 expression is highly variable, is associated with an mRNA gene expression signature and is inversely correlated with E-cadherin expression, a known negative target of ZEB1. analyzing an isogenic series of progressively transformed HBECs we found ZEB1 increased with tumorigenic progression and an invasive phenotype. This phenotype could in turn be reversed by knockdown of ZEB1 suggesting ZEB1 plays an important role in tumorigenic transformation of human bronchial epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2362.

Normal human lung epithelial cells that can be genetically manipulated to study the various steps in lung cancer pathogenesis and stem cell biology would be of great utility and also provide isogenic strains to develop and test new diagnostic (biomarkers), preventative and therapeutic strategies for lung cancer. Previously we immortalized normal proximal human lung bronchial epithelial cells (HBECs) for studying lung tumors arising in the central compartment of the lung and now have over 40 of these strains (growing in defined KSFM media) including many isogenic derivatives with various oncogenic changes (Can Res 2004, Can Res 2006). We have extended this by immortalizing normal distal (small airway) epithelial cells that serve as models for studying tumors arising in the peripheral compartment of the lung. Using Cdk4 and hTERT exogenous expression vectors (as used for HBECs) and SAGM defined media routinely immortalized (N= 15) human small airway epithelial cells (HSAECs) from peripheral lung. These HSAEC strains are highly enriched (>50%) for cells with stem like properties (e.g. Aldehyde dehydrogenase positivity), express basal cell markers but not Clara cell, type 1 or type 2 differentiation markers., However, HSAECs can be induced to express Clara cell, type 1 or type 2 cell markers when they are grown in matrigel (where they form cyst like structures which express such differentiation markers) and under other conditions favoring differentiation (such as insulin depletion, Ca2+ addition). HSAECs show anchorage dependent growth, do not form colonies in soft agar, and are non-tumorigenic in NOD/SCID mice. However, HSAECs can be genetically manipulated by introducing oncogenes (such as KRASV12, missense p53 and other oncogene combinations) which now allow HSAECs to progress towards a transformed phenotype (e.g. formation of soft agar colonies), and selection of a subset of HSAEC(KRAS, p53) large soft agar colonies identifies sublines that can now form poorly differentiated carcinomas. Thus, we have developed conditions to routinely immortalize lung epithelial cells derived from distal small airways (HSAECs) which grow in defined media, exhibit stem cell like characteristics, and can be genetically manipulated to give rise to poorly differentiated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3230.

The kinetics of the interactions of MPTP and its N-des-methyl-derivative (PTP) have been studied. Both were mechanism-based inhibitors as well as substrates for the enzyme. Analysis of the reaction progress-curves for the formation of the corresponding dihydropyridine derivatives allowed the kinetic parameters for the process and the partition ratio, which corresponds to the number of mol. of product formed per mol. of enzyme inactivated, to be determined for both compounds. The conversion of MPTP to its corresponding pyridinium-ion derivative through the action of MAO-B is known to be essential for its neurotoxicity. PTP has been reported not to be neurotoxic, although it appears to be a relatively good substrate for MAO-B as well as acting as a mechanism-based inhibitor. Studies of the changes in absorbance spectra during the MAO-B catalysed oxidation were consistent with the formation of the corresponding pyridinium-ion derivative (MPP+), which is known to be the effective neurotoxin, as the end-product when MPTP was oxidized. In contrast the oxidation of PTP appeared to stop at the dihydropyridine stage with no significant further oxidation to the corresponding pyridine-derivative.

Five decapeptides were prepared, each having the generic primary sequence D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-X7 -Y8-Arg9. A C-terminal beta-turn was anticipated when X was an alkyl ether of D-4-hydroxyproline in either the cis or trans geometric state and Y was either a Tic or Oic residue. Whereas cis ethers have only very weak receptor affinities, the trans ethers are significantly more potent in binding to guinea pig smooth muscle having Ki values as low as 0.16 nM. Notably, these peptides do not contain a D-aromatic amino acid at position 7 of the primary sequence.

We recently demonstrated that 2,6,diamino-N-([I-(oxotridecyl)-2-piperidinyl]methyl)-hexanamide (NPC 15437) is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme. To further investigate the interaction of NPC 15437 with PKC we expressed a series of cDNAs encoding mutant PKC molecules in COS7 cells. NPC 15437 had no effect on the protein kinase activity of mutants lacking the N-terminal region of the C1 domain. Further, NPC 15437 was a competitive inhibitor of the activation of PKCα by phorbol ester and attenuated the binding of phorbol ester to the enzyme in intact cells. The present study demonstrates that mutant enzyme constructs can be used to localize the site of interaction of NPC 15437 with PKC to residues 12–42, which encodes the pseudosubstrate binding domain and part of the first cysteine-rich repeat sequence.