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A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo , which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC 50 's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Research on snapshot multispectral imaging has been popular in the remote sensing community due to the high demands of video-rate remote sensing system for various applications. Existing snapshot multispectral imaging techniques are mainly of a fixed wavelength type, which limits their practical usefulness. This paper describes a tunable multispectral snapshot system by using a dual prism assembly as the dispersion element of the coded aperture snapshot spectral imagers (CASSI). Spectral tuning is achieved by adjusting the air gap displacement of the dual prism assembly. Typical spectral shifts of about 1 nm at 400 nm and 12 nm at 700 nm wavelength have been achieved in the present design when the air-gap of the dual prism is changed from 4.24 mm to 5.04 mm. The paper outlines the optical designs, the performance, and the pros and cons of the dual-prism CASSI (DP-CASSI) system. The performance of the system is illustrated by TracePro TM ray tracing, to allow researchers in the field to repeat or to validate the results presented in this paper.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.

Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a high-throughput screen (HTS). An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2 inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structure-activity relationships. The optimized compound 21 is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3-dioxygenase 1 (IDO1) and with improved human whole blood stability., Competing Interests: The authors declare no competing financial interest.

The N -methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca 2+ and Na + . NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimer's disease. Recently, we have disclosed GNE-0723 ( 1 ), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure-activity relationships, despite the structural similarity to GNE-0723. GNE-5729 ( 13 ), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.

NAMPT inhibitors may show potential as therapeutics for oncology. Throughout our NAMPT inhibitor program, we found that exposed pyridines or related heterocyclic systems in the left-hand portion of the inhibitors are necessary pharmacophores for potent cellular NAMPT inhibition. However, when combined with a benzyl group in the center of the inhibitors, such pyridine-like moieties also led to consistent and potent inhibition of CYP2C9. In an attempt to reduce CYP2C9 inhibition, a parallel synthesis approach was used to identify central benzyl group replacements with increased Fsp3. A spirocyclic central motif was thus discovered that was combined with left-hand pyridines (or pyridine-like systems) to provide cellularly potent NAMPT inhibitors with minimal CYP2C9 inhibition. Further optimization of potency and ADME properties led to the discovery of compound 68, a highly potent NAMPT inhibitor with outstanding efficacy in a mouse tumor xenograft model and lacking measurable CYP2C9 inhibition at the concentrations tested.

The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

Herein we report the optimization efforts to ameliorate the potent CYP3A4 time-dependent inhibition (TDI) and low aqueous solubility exhibited by a previously identified lead compound from our NAMPT inhibitor program (1, GNE-617). Metabolite identification studies pinpointed the imidazopyridine moiety present in 1 as the likely source of the TDI signal, and replacement with other bicyclic systems was found to reduce or eliminate the TDI finding. A strategy of reducing the number of aromatic rings and/or lowering cLogD7.4 was then employed to significantly improve aqueous solubility. These efforts culminated in the discovery of 42, a compound with no evidence of TDI, improved aqueous solubility, and robust efficacy in tumor xenograft studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Intranasal delivery of DNA vaccines has become a popular research area recently. It offers some distinguished advantages over parenteral and other routes of vaccine administration. Nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (NALT). Different kinds of DNA vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (RSV) etc. DNA vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. The biggest obstacle to DNA vaccines is low immunogenicity. One of the approaches to enhance the efficacy of DNA vaccine is to improve DNA delivery efficiency. This review provides insight on the development of intranasal DNA vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of DNA vaccines using non-viral delivery agents.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

Prolonged inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is a strategy for targeting cancer metabolism. Many NAMPT inhibitors undergo NAMPT-catalyzed phosphoribosylation (pRib), a property often correlated with their cellular potency. To understand this phenomenon and facilitate drug design, we analyzed a potent cellularly active NAMPT inhibitor (GNE-617). A crystal structure of pRib-GNE-617 in complex with NAMPT protein revealed a relaxed binding mode. Consistently, the adduct formation resulted in tight binding and strong product inhibition. In contrast, a biochemically equipotent isomer of GNE-617 (GNE-643) also formed pRib adducts but displayed significantly weaker cytotoxicity. Structural analysis revealed an altered ligand conformation of GNE-643, thus suggesting weak association of the adducts with NAMPT. Our data support a model for cellularly active NAMPT inhibitors that undergo NAMPT-catalyzed phosphoribosylation to produce pRib adducts that retain efficient binding to the enzyme., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 μM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 μM, A2780 cell culture IC50 = 0.000 49 μM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.

The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50=19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC50=121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC50 = 11 nM; PC-3 antiproliferative IC50 = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC50 = 10nM; A2780 antiproliferative IC50 = 7 nM) in complex with the NAMPT protein was also determined., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt-7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model.

Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 μM; A2780 IC50 = 0.032 μM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).

Despite their discovery in the early 20th century and intensive study over the last 20 years, nicotinic acetylcholine receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low nanomolar binding affinities and excellent subtype selectivity while acting as partial agonists at α4β2-nAChRs. Their favorable antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad screening toward other common neurotransmitter receptors were also carried out to further evaluate their safety profile and eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the clinic as new drug candidates for treating depression.

Traumatic brain injury (TBI) leads to a rapid and excessive increase in glutamate concentration in the extracellular milieu, which is strongly associated with excitotoxicity and neuronal degeneration. N-acetylaspartylglutamate (NAAG), a prevalent peptide neurotransmitter in the vertebrate nervous system, is released along with glutamate and suppresses glutamate release by actions at pre-synaptic metabotropic glutamate autoreceptors. Extracellular NAAG is hydrolyzed to N-acetylaspartate and glutamate by peptidase activity. In the present study PGI-02776, a newly designed di-ester prodrug of the urea-based NAAG peptidase inhibitor ZJ-43, was tested for neuroprotective potential when administered intraperitoneally 30 min after lateral fluid percussion TBI in the rat. Stereological quantification of hippocampal CA2-3 degenerating neurons at 24 h post injury revealed that 10 mg/kg PGI-02776 significantly decreased the number of degenerating neurons (p<0.05). Both average latency analysis of Morris water maze performance and assessment of 24-hour memory retention revealed significant differences between sham-TBI and TBI-saline. In contrast, no significant difference was found between sham-TBI and PGI-02776 treated groups in either analysis indicating an improvement in cognitive performance with PGI-02776 treatment. Histological analysis on day 16 post-injury revealed significant cell death in injured animals regardless of treatment. In vitro NAAG peptidase inhibition studies demonstrated that the parent compound (ZJ-43) exhibited potent inhibitory activity while the mono-ester (PGI-02749) and di-ester (PGI-02776) prodrug compounds exhibited moderate and weak levels of inhibitory activity, respectively. Pharmacokinetic assays in uninjured animals found that the di-ester (PGI-02776) crossed the blood-brain barrier. PGI-02776 was also readily hydrolyzed to both the mono-ester (PGI-02749) and the parent compound (ZJ-43) in both blood and brain. Overall, these findings suggest that post-injury treatment with the ZJ-43 prodrug PGI-02776 reduces both acute neuronal pathology and longer term cognitive deficits associated with TBI., (Copyright © 2011 Elsevier B.V. All rights reserved.)

AMOP-H-OH (sazetidine-A; 6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) and some sulfur-bearing analogues were tested for their activities in vitro against human alpha4beta2-, alpha4beta4-, alpha3beta4*- and alpha1*-nicotinic acetylcholine receptors (nAChRs). AMOP-H-OH was also assessed in an antidepressant efficacy model. AMOP-H-OH and some of its analogues have high potency and selectivity for alpha4beta2-nAChRs over other nAChR subtypes. Effects are manifested as partial agonism, perhaps reflecting selectivity for high sensitivity (alpha4)(3)(beta2)(2)-nAChRs. More prolonged exposure to AMOP-H-OH and its analogues produces inhibition of subsequent responses to acute challenges with full nicotinic agonists, again selectively for alpha4beta2-nAChRs over other nAChR subtypes. The inhibition is mediated either via antagonism or desensitization of nAChR function, but the degree of inhibition of alpha4beta2-nAChRs is limited by the partial agonist activity of the drugs. Certain aspects of the in vitro pharmacology suggest that AMOP-H-OH and some of its analogues have a set of binding sites on alpha4beta2-nAChRs that are distinct from those for full agonists. The in vitro pharmacological profile suggests that peripheral side effects of AMOP-H-OH or its analogues would be minimal and that their behavioral effects would be dominated by central nAChR actions. AMOP-H-OH also has profound and high potency antidepressant-like effects in the forced swim test. The net action of prolonged exposure to AMOP-H-OH or its analogues, as for nicotine, seems to be a selective decrease in alpha4beta2-nAChR function. Inactivation of nAChRs may be a common neurochemical endpoint for nicotine dependence, its treatment, and some of its manifestations, including relief from depression.

A series of heteroaromatic analogs of pregabalin has been identified that possess anticonvulsant activity in the DBA/2 mouse model. The methods of synthesis and preliminary pharmacology are discussed herein.

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells., ((c) 2005 Wiley-Liss, Inc.)

Cytogenetic and molecular cytogenetic analyses were performed on four sublines derived from a newly established, SV40T-immortalized nasopharyngeal (NP) cell line, NP69, with two of the sublines expressing LMP1, an Epstein-Barr virus-encoded gene. A total of seven cytogenetically related subclones were identified, all having highly complex karyotypes with massive numerical and structural rearrangements. Centromeric rearrangements in the form of isochromosomes and whole-arm translocations were prevalent. A cytogenetic sign of gene amplification [i.e., homogeneously staining region (HSR)] was detected at 1q25 in all metaphase cells analyzed. Multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) was used to confirm the karyotypic interpretations. Furthermore, multicolor COBRA-FISH also showed that part of the HSR contained chromosome 20 material. Extensive clonal evolution could be observed by the assessment of karyotypic variation among different subclones and individual metaphase cells. The evaluation of clonal evolution enabled the identification of the temporal order of chromosome aberrations during cell immortalization and malignant transformation. A striking karyotypic similarity was found between sublines expressing LMP1 and an NP carcinoma cell line, with loss of genetic material from chromosome arm 3p being an important recurrent observation. More interestingly, the karyotypic features of NP69 were also similar to those of many epithelial malignancies. Our observations suggest that serial transformation of NP cell lines might provide a useful in vitro model for the study of the multistep neoplastic transformation of NP cells.

Objectives: This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis., Methods: The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis., Results: In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization., Conclusion: The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

Undifferentiated nasopharyngeal carcinoma (NPC) is closely associated with EBV infection, and the EBV-encoded latent membrane protein 1 (LMP1) is frequently detected in NPC. However, little is known about the pathologic roles of LMP1 in this disease. Recently, we reported the morphologic transformation and increased expression of the LAMC2 and ITGalpha6 genes in LMP1-expressing NPC cell lines. In this study, we further examine the effects of LMP1 in an immortalized nasopharyngeal epithelial cell line called NP69. This cell line was established from primary nonmalignant nasopharyngeal epithelial cells and may represent a model of premalignant nasopharyngeal epithelial cells. LMP1 induced many phenotypic changes in NP69 cells. These include morphologic transformation, increased cell proliferation, anchorage-independent growth, resistance to serum free-induced cell death, and enhanced cell migration and invasion. In addition, expression array analysis identified 28 genes that demonstrated a more than 2-fold difference in expression of NP69 cells expressing LMP1 when compared with a vector control. Two of the up-regulated genes (VEGF and vimentin) identified have been previously reported as LMP1 targets. The majority of the identified genes are associated with cell growth, differentiation, cell shape, and invasion. The present findings support the proposed roles of LMP1 in promoting cell transformation, migration, and invasion in premalignant nasopharyngeal epithelial cells. The present study also indicates the activation of the Ras/MAPK pathway in LMP1-expressing cells, which may be involved in mediating some of the transforming effects of LMP1 observed in nasopharyngeal epithelial cells.

Many distinct regions of 3p show frequent allelic losses in a wide range of tumour types. Previously, the BLU candidate tumour suppressor gene (TSG) encoded by a gene-rich critical deleted region in 3p21.3 was found to be inactivated rarely in lung cancer, although expression was downregulated in a subset of lung tumour cell lines. To elucidate the role of BLU in tumorigenesis, we analysed BLU promoter methylation status in tumour cell lines and detected promoter region hypermethylation in 39% lung, 42% breast, 50% kidney, 86% neuroblastoma and 80% nasopharyngeal (NPC) tumour cell lines. Methylation of the BLU promoter region correlated with the downregulation of BLU transcript expression in tumour cell lines. Expression was recovered in tumour cell lines treated with 5-aza 2-deoxycytidine. Exogenous expression of BLU in neuroblastoma (SK-N-SH) and NSCLC (NCI-H1299) resulted in reduced colony formation efficiency, in vitro. Furthermore, methylation of the BLU promoter region was detected in primary sporadic SCLC (14%), NSCLC (19%) and neuroblastoma (41%). As frequent methylation of the RASSF1A 3p21.3 TSG has also been reported in these tumour types, we investigated whether BLU and RASSF1A methylation were independent or related events. No correlation was found between hypermethylation of RASSF1A and BLU promoter region CpG islands in SCLC or neuroblastoma. However, there was association between RASSF1A and BLU methylation in NSCLC (P=0.0031). Our data suggest that in SCLC and neuroblastoma, RASSF1A and BLU methylations are unrelated events and not a manifestation of a regional alteration in epigenetic status, while in NSCLC there may be a regional methylation effect. Together, these data suggest a significant role for epigenetic inactivation of BLU in the pathogenesis of common human cancers and that methylation inactivation of BLU occurs independent of RASSF1A in SCLC and neuroblastoma tumours.

Loss of E-cadherin (E-cad) has been associated with progression and poor survival in nasopharyngeal carcinoma (NPC). In this study, we investigated the role of methylation on E-cad inactivation in NPC cell lines, as well as in NPC tissue samples. Using 6 NPC cell lines, we found that methylation of the E-cad 5' CpG island promoter region was correlated with the loss of both mRNA and E-cad protein expression in these cell lines. In addition, using 29 NPC and 10 non-malignant nasopharyngeal samples, we also observed 5' CpG methylation of the E-cad gene in 52% (15 out of 29) NPC samples, but in only 10% (1 out of 10) of the non-malignant nasopharyngeal tissues. Our findings indicate that 5' CpG island methylation of the E-cad gene may play an important part in the inactivation of E-cad in NPC. Our results also suggest that reducing the methylation of the E-cad gene may be a potential therapeutic strategy for NPC.

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein-Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFbeta inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.

Aims: To evaluate the clinicopathological significance of p16 expression in the surgical management of squamous cell carcinomas of the oral cavity, oropharynx, hypopharynx, and larynx., Method: p16 expression in 225 head and neck squamous cell carcinomas (HNSCCs) was studied using an immunohistochemical method and paraffin wax embedded tumour tissues. Associations between p16 expression and clinicopathological features were investigated., Results: Decreased p16 expression was found in 48% of the tumours. There was a higher frequency of decreased p16 expression in tumours of the larynx compared with those from the pharynx and oral cavity. There was a significant correlation between decreased p16 expression and more advanced T stage. There was no significant correlation between p16 expression and sex, age, tumour grade, nodal metastasis, recurrence, or survival., Conclusion: There was a high frequency of downregulation of p16 expression in HNSCC. Tumours of the larynx had a significantly higher frequency of weak p16 expression compared with tumours of the oral cavity and pharynx. Downregulation of p16 contributed to cellular proliferation, resulting locally in a more advanced tumour. It had no prognostic significance for nodal metastasis and survival.

A principal mechanism of calcium-mediated neuronal injury is the activation of neutral proteases known as calpains. Proteolytic substrates for calpain include receptor and cytoskeletal proteins, signal transduction enzymes and transcription factors. Recently, calpain inhibitors have been shown to provide benefit in rat models of focal head injury and focal cerebral ischemia. The present study sought to investigate, in experiment 1, the time course of calpain-mediated cytoskeletal injury in a mouse model of diffuse head injury by measuring the 150- and 145-kDa alpha-spectrin breakdown products (SBDP). Secondly, in experiment 2, we examined the effect of early (20 min postinjury) administration of the novel calpain inhibitor SJA6017 on functional outcome measured 24 h following injury and its effect on posttraumatic alpha-spectrin degradation. Lastly, in experiment 3, we examined the effect of delayed (4 or 6 h postinjury) administration of SJA6017 on 24-h postinjury functional outcome. In experiment 1, isoflurane-anesthetized male CF-1 mice (18-22 g) were subjected to a 750 g-cm weight drop-induced injury and were sacrificed for SBDP analysis at postinjury times of 30 min, and 1, 2, 6, 24 and 48 h (plus sham). In experiments 2 and 3, mice were injured as described, and delivered a single tail vein injection of either SJA6017 (0.3, 1, or 3 mg/kg) or vehicle (administered immediately, 4 or 6 h postinjury [3 mg/kg]). Functional outcome was evaluated in both studies, and, in experiment 2, 24-h postinjury assessment of SBDPs was determined. Following injury, the level of SBDP 145 was significantly different from sham at 24 and 48 h in cortical and at 24 h in the hippocampal tissues and at 48 h in the striatum. Immediate postinjury administration of SJA6017 resulted in a dose-related improvement in 24-h functional outcome (p < 0.05 at 3 mg/kg). Significance was maintained after a 4-h delay of the 3 mg/kg, but was lost after a 6-h delay. Despite improvement in functional outcome at 24 h, SJA6017 did not reduce spectrin breakdown in cortical or hippocampal tissues. These results support a role for calpain-mediated neuronal injury and the potential for a practical therapeutic window for calpain inhibition following traumatic brain injury. However, measurements of regional spectrin degradation may not be the most sensitive marker for determining the effects of calpain inhibition.

The aim of the present study was to evaluate the clinicopathologic significance of p53 and p21 expression in lingual squamous cell carcinomas. Immunohistochemical staining was performed with p53 and p21 monoclonal antibodies on surgical specimens from 87 patients who underwent primary surgical treatment for lingual carcinoma between 1976 and 1996. We found positive expression of p53 in 45 (52%) of 87 cases and of p21 in 49 (56%) of 87 cases. There was no correlation of p53 and p21 expression with cancer stage, T stage, nodal metastasis, and tumor grade. Univariate analysis revealed that p21 expression, tumor stage, T stage, and nodal stage were significant prognostic factors for survival. However, only p21 expression and tumor stage were significant independent prognostic factors for survival in a multivariate Cox regression analysis. Overexpression of p21 but not p53 has prognostic value for survival in the surgical treatment of lingual carcinomas. The combination of stage with p21 expression is recommended for evaluation of prognosis and for management planning.

Background: Chinese nasopharyngeal carcinoma (NPC) specimens were analyzed by microsatellite allelotyping to evaluate their usefulness as biomarkers for cancer detection., Materials and Methods: A panel of eight microsatellite markers localized to chromosomes 3p, 6p, 9p, I1q and 14q were used to study 47 specimens., Results: A moderately low loss of heterozygosity (LOH) frequency (8% to 35%) was observed; 23 (49%) specimens showed LOH with at least one marker while microsatellite instability (MSI) was detected in 10 specimens (21%)., Conclusion: Poor survival was significantly associated with LOH detected by a chromosome 3 marker. Interestingly, tumors with multiple genetic alterations were significanty associated with earlier staging

To define the patterns of genetic imbalances in nasopharyngeal carcinoma (NPC), we studied 30 primary NPC tumors with comparative genomic hybridization (CGH). The common sites of chromosomal gains were found in descending order of frequency in 12p11.2-p12 (36%), 12q14-q21 (33%), 2q24-q31 (23%), 1q31-qter (20%), 3q13 (20%), 1q13.3 (20%), 5q21 (17%), 6q14-q22 (13%), 7q21 (13%), 8q11.2-q23 (13%) and 18q12-qter (13%). The common sites of chromosomal loss were at 3p14-p21 (20%), 11q23-qter (20%), 16q21-qter (17%) and 14q24-qter (13%). Correlation with clinicopathologic features showed that 3p loss was associated with a significantly higher risk of death related to recurrence as compared with patients without 3p loss (50% vs. 9%, P=.029). The presence of 16q loss was associated with more advanced stage tumors (stages I & II: 6% vs. stages III & IV: 33%, P=.046). We conclude that consistent patterns of genetic imbalances can be observed in NPC. Deletion of 3p and 16q were associated with higher risk of tumor recurrence and advanced stage cancer.

Hemidesmosome (HD) is a transmembrane complex that mediates attachment of epithelial cells to the basement membrane. Abnormal expression of HD components has been reported in several types of human cancers and is believed to play a role in tumor invasion and metastasis. Using differential gene display, we have identified downregulation of BPAG1 expression in nasopharyngeal carcinoma cells. BPAG1 is a major component of hemidesmosome. In the present study, we have extended our work to investigate the expression pattern of other components in the HD complex, namely, BPAG2, ITGalpha6 and ITGbeta4 in three distinct biological groups of nasopharyngeal epithelial cells: (a) non-malignant nasopharyngeal epithelial cells established from primary culture of nasopharyngeal explants, (b) non-malignant nasopharyngeal epithelial cells immortalized by viral oncogenes, SV40 or HPV16E6E7, and (c) nasopharyngeal carcinoma (NPC) cells. Both non-malignant primary cultured nasopharyngeal epithelial cells and immortalized nasopharyngeal epithelial cell lines expressed all the HD components examined, although the immortalized cells expressed a lower level of HD components compared with the non-malignant nasopharyngeal cells established from primary culture. In contrast, downregulation of HD components is commonly observed in nasopharyngeal carcinoma cells. Loss of HD expression in NPC may be associated with the undifferentiated properties of NPC cells and may have prognostic significance.

Objectives: To study the relationship between the size of neopharynx after total laryngectomy and long-term swallowing function by means of scintigraphy., Study Design: The width of pharyngeal remnant was measured during surgery in 11 patients. Their swallowing function was assessed by scintigraphy 8 to 10 years after surgery., Methods: The width of pharyngeal remnant at its narrowest point in both relaxed and stretched state was measured during surgery. Postoperative scintigraphy data on swallowing were obtained and computed along 3 lines, (1) transit time through neopharynx, (2) percentage of bolus transferred, and (3) swallowing efficiency. The relationship between the 2 sets of data was analyzed by linear regression analysis., Results: (1) No statistically significant relationship was found between the size of neopharynx and swallowing function. (2) All patients are clinically asymptomatic., Conclusions: The swallowing function is not affected by the size of the neopharynx in 11 patients with pharyngeal remnant width ranging from 3 to 8 cm (stretched).

Background: The endoscopic and histologic findings before and after radiotherapy (RT) for nasopharyngeal carcinoma (NPC) were correlated to study the sensitivity and specificity of endoscopic findings in predicting histologic results. The efficiacy of endoscopic examination and post-RT multiple site biopsies in detecting persistent disease was also evaluated., Methods: Seven hundred forty-six patients were evaluated. Pre-RT, biopsies were taken from both sides of the nasopharynx to assess the extent of tumor. Four to 16 weeks after RT, routine six-site biopsy specimens were taken from both roofs, lateral, and posterior walls of the nasopharynx and repeated 2 weeks later. Endoscopic findings of exophytic growth, nodule, ulcer, and submucosal bulge were considered "residual tumor," others were considered "no residual tumor." Persistent disease was defined as positive histologic findings 12 weeks after RT., Results: Before RT, sensitivity of endoscopic findings and biopsy specimens in detecting malignancy were 99.7% and 94.2%, respectively. After RT, sensitivity and specificity of endoscopic findings in predicting positive histologic findings were 29% and 85.8%, respectively, with a positive predictive value of 34.9% and a negative predictive value of 82.2%. Of positive histologic findings, 27.7% were missed in the first session of biopsies; 33.5% of those with positive histologic findings turned out to have persistent disease. For prediction of persistent disease, the sensitivity and specificity of endoscopic findings were 40.4% and 84.4%, with a positive predictive value of 16.3% and a negative predictive value of 95%, and that of histologic findings in the first session of biopsies were 59.6% and 88.3%, respectively, with a positive predictive value of 27.7% and a negative predictive value of 96.7%., Conclusions: Endoscopic findings alone have low sensitivity in predicting persistent disease, multiple sites biopsy specimens are indicated. Because only 1.9% of patients with endoscopic findings of "no residual tumor" and negative histologic findings in first session of biopsies had persistent disease, this group can be spared second biopsies. Repeat biopsies are indicated for those with endoscopic findings of "residual tumor" or positive histologic findings in first session of biopsies to improve detection of persistent disease.

Background: This study aimed at resolving the clinicopathological significance of p53 and p21 expression in patients undergoing surgical treatment for laryngeal carcinoma., Materials and Methods: p53 and p21 expression were studied by immunohistochemical method on surgical specimens of 193 patients. The expressions of p53 and p21 were correlated with the clinicopathological data., Results: Of the 193 tumors, 60% had positive p53 expression and 46% had positive p21 expression. Nodal metastasis was significantly correlated with p53 overexpression, supraglottic involvement and moderate- to poor-differentiation of the tumor. There was significant cumulative effect with increasing risk of nodal metastasis with increasing number of risk factors: 0% without any risk factor, 13% with 1 risk factor, 37% with 2 risk factors and 55% with all 3 risk factors., Conclusion: Positive expressions p53 and p21 were common in laryngeal carcinoma. Overexpression of p53 but not p21 was associated with nodal metastasis. The use of p53 expression has added value in providing more accurate prognostic analysis of risk of nodal metastasis in conjunction with site of tumor involvement and tumor grade.

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been recognized as important events in the carcinogenesis of many cancers, including squamous cell carcinoma of head and neck (SCCHN). However, microsatellite alterations have not been documented in SCCHN from Chinese patients. We investigated the frequency and clinical significance of LOH and MSI in 30 SCCHN from Hong Kong Chinese using polymerase chain reaction on 17 microsatellite markers on chromosomes 3p, 4q, 7q, 9p, 17p and 18q. LOH was present in nine tumours (30%) and MSI in four (13%). The incidence of LOH (7/13; 53.8%) in hypopharyngeal-laryngeal cancers was significantly higher than that (2/17; 11.8%) in the oral cancers (P=0.020). LOH was more often detected at the loci on chromosomes 7 and 9. Patients with tumours having LOH had slightly poorer outcome compared with those without, although the differences did not reach statistical significance. Our data show that the incidence of microsatellite alterations in SCCHN from Hong Kong Chinese is low. However, LOH may be one of the genetic mechanisms in the carcinogenesis of a subset of SCCHN (hypopharyngeal-laryngeal cancers).

Nasopharyngeal carcinoma (NPC) is a common cancer among southern Chinese. The profile of gene expression in NPC cells is largely unknown. In this study, we have examined differential gene expression in non-malignant and malignant nasopharyngeal epithelial (NPE) cells using a cDNA array hybridization method. A total of 42 genes were identified to be expressed in either non-malignant and malignant NPE cells or both. Thirteen of these genes were overexpressed in malignant NPE cells. These includes nuclear factor (NF90), FOS-related antigen 1 (FRA- 1), cytoplasmic dynein light chain (HDLC1), replication factor C (RFC1), nucleoside diphosphate kinase B, UV excision repair protein (RAD23A), insulin-like growth factor receptor II, transcription initiation factor TFIID subunit (TAFII31), growth factor receptor-bound protein 2 (GRB2), UV excision repair protein (RAD23B), glutathione peroxidase, Y box binding protein 1 and heat shock protein 86. In contrast, expression of nine genes was suppressed in malignant NPE cells. These includes calgranulin A, calgranulin B, neutrophil activating protein (ENA-78), heat shock protein 27, integrin beta-1, integrin beta-4, cyclin-dependent kinase inhibitor 1A (p21), interleukin-8 and tyrosine protein kinase receptor (RET). Differential expression of calgranulin A, calgraunlin B, ENA-78, FRA-1 and NF90 in non-malignant and malignant nasopharyngeal epithelial cells was confirmed by RT-PCR analysis.