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Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H2 O2 . Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H2 O2 . An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H2 O2 . Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo.

We report intermolecular transition metal frustrated Lewis pairs (FLPs) based on zirconocene aryloxide and phosphine moieties that exhibit a broad range of small molecule activation chemistry that has previously been the preserve of only intramolecular pairs. Reactions with D2, CO2, THF, and PhCCH are reported. By contrast with previous intramolecular examples, these systems allow facile access to a variety of steric and electronic characteristics at the Lewis acidic and Lewis basic components, with the three-step syntheses of 10 new intermolecular transition metal FLPs being reported. Systematic variation to the phosphine Lewis base is used to unravel steric considerations, with the surprising conclusion that phosphines with relatively small Tolman steric parameters not only give highly reactive FLPs but are often seen to have the highest selectivity for the desired product. DOSY NMR spectroscopic studies on these systems reveal for the first time the nature of the Lewis acid/Lewis base interactions in transition metal FLPs of this type.

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure–activity relationships of this indole series of DP receptor antagonists will also be discussed.

The synthesis and the EP(1) receptor binding affinity of 2,3-diarylthiophene derivatives are described. The evaluation of the structure-activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity for the human EP(1) receptor and a selectivity greater than 100-fold against the EP(2), EP(3), EP(4), DP, FP, and IP receptors and greater than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the way they are distributed in the brain.

Rationale Prostaglandin D 2 (PGD 2 ) is released from mast cells during the allergic response. Since PGD 2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic Prostaglandin D synthase (PGDS) and the two PGD 2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. Methods DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression was revealed by immunohistochemistry. Results In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: 1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, 2) both DP mRNA and CRTH2 were detected in eosinophils and 3) CRTH2 was detected on a subset of infiltrating T-cells. Although DP mRNA could not be detected in the T-cells invading the nasal mucosa, it was found to be expressed in the T-cells present in the lymph node and the thymus from normal individuals. Conclusions This study indicates that cells capable of producing PGD 2 are present in the nasal mucosa and that both PGD 2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.

Recent work has reported the first electroanalytical detection of pindolol using reduced graphene oxide (RGO) modified glassy carbon electrodes [S. Smarzewska and W. Ciesielski, Anal. Methods, 2014, 6, 5038] where it was reported that the use of RGO provided significant improvements in the electroanalytical signal in comparison to a bare (unmodified) glassy carbon electrode. We demonstrate, for the first time, that the electroanalytical quantification of pindolol is actually possible using bare (unmodified) screen-printed graphite electrodes (SPEs). This paper addresses the electroanalytical determination of pindolol utilising RGO modified SPEs. Surprisingly, it is found that bare (unmodified) SPEs provide superior electrochemical signatures over that of RGO modified SPEs. Consequently the electroanalytical sensing of pindolol is explored at bare unmodified SPEs where a linear range between 0.1 μM–10.0 μM is found to be possible whilst offering a limit of detection (3σ) corresponding to 0.097 μM. This provides a convenient yet analytically sensitive method for sensing pindolol. The optimised electroanalytical protocol using the unmodified SPEs, which requires no pre-treatment (electrode polishing) or electrode modification step (such as with the use of RGO), was then further applied to the determination of pindolol in urine samples. This work demonstrates that the use of RGO modified SPEs have no significant benefits when compared to the bare (unmodified) alternative and that the RGO free electrode surface can provide electro-analytically useful performances. Financial support for this research was supplied by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

In this paper in situ bismuth, antimony, tin modified electrodes and combinations thereof are explored towards the model target analytes cadmium(II) and lead(II), chosen since they are the most widely studied, to explore the role of the underlying electrode substrate with respect to boron-doped diamond, glassy carbon, and screen-printed graphite electrodes. It is found that differing electrochemical responses are observed, dependent upon the underlying electrode substrate. The electrochemical response using the available range of metallic modifications is only ever observed when the underlying electrode substrate exhibits relatively slow electron transfer properties; in the case of fast electron transfer properties, no significant advantages are evident. Furthermore these bismuth modified systems which commonly employ a pH 4 acetate buffer, reported to ensure the bismuth(III) stability upon the electrode surface can create create a problem when sensing at low concentrations of heavy metals due to its high background current. It is demonstrated that a simple change of pH can allow the detection of the target analytes (cadmium(II) and lead(II)) at levels below that set by the World Health Organisation (WHO) using bare graphite screen-printed electrodes.

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

Cobalt phthalocyanine (CoPC) compounds have been reported to provide electrocatalytic performances towards a substantial number of analytes. In these configurations, electrodes are typically constructed via drop casting the CoPC onto a supporting electrode substrate, while in other cases the CoPC complex is incorporated within the ink of a screen-printed sensor, providing a one-shot economical and disposable electrode configuration. In this paper we critically compare CoPC modified electrodes prepared by drop casting CoPC nanoparticles (nano-CoPC) onto a range of carbon based electrode substrates with that of CoPC bulk modified screen-printed electrodes in the sensing of the model analytes L-ascorbic acid, oxygen and hydrazine. It is found that no “electrocatalysis” is observed towards L-ascorbic acid using either of these CoPC modified electrode configurations and that the bare underlying carbon electrode is the origin of the obtained voltammetric signal, which gives rise to useful electroanalytical signatures, providing new insights into literature reports where “electrocatalysis” has been reported with no clear control experiments undertaken. On the other hand true electrocatalysis is observed towards hydrazine, where no such voltammetric features are witnessed on the bare underlying electrode substrate.

We introduce the concept of screen-printed back-to-back electroanalytical sensors where in this facile and generic approach, screen-printed electrodes are printed back-to-back with a common electrical connection to the two working electrodes with the counter and reference electrodes for each connected in the same manner as a normal “traditional” screen-printed sensor would be. This approach utilises the usually redundant back of the screen-printed sensor, converting this “dead-space” into a further electrochemical sensor which results in improvements in the analytical performance. In the use of the back-to-back design, the electrode area is consequently doubled with improvements in the analytical performance observed with the analytical sensitivity (gradient of a plot of peak height/analytical signal against concentration) doubling and the corresponding limit-of-detection being reduced. We also demonstrate that through intelligent electrode design, a quadruple in the observed analytical sensitivity can also be realised when double microband electrodes are used in the back-to-back configuration as long as they are placed sufficiently apart such that no diffusional interaction occurs. Such work is generic in nature and can be facilely applied to a plethora of screen-printed (and related) sensors utilising the commonly overlooked redundant back of the electrode providing facile improvements in the electroanalytical performance.

It has recently been demonstrated that 2-aminoacetophenone (2-AA) is a chemical indicator in exhaled air/breath of Pseudomonas aeruginosa infection associated with progressive life threatening decline of lung function in cystic fibrosis sufferers [Scott-Thomas et al., BMC Pulm. Med., 2010, 10, 56]. Currently the detection of 2-AA involves laboratory based instrumentation such as mass spectrometry and a hand-held point-of-care type breath device would be ideal in providing real-time results within seconds to accelerate patient care decision-making processes. To this end, we demonstrate proof-of-concept that the chemical marker 2-AA, indicative of Pseudomonas aeruginosa infection, can be measured using electrochemical based sensing strategies. A range of commercially available electrode substrates are explored demonstrating for the first time that 2-AA is electrochemically active within aqueous based solutions providing an (electro)analytical signal. Glassy carbon, boron-doped diamond and platinum electrodes have been explored towards the electrochemical oxidation of 2-AA. Electrode fouling is observed requiring pre-treatment in the form of mechanical polishing between voltammetric scans and measurements. To alleviate this, screen-printed graphite electrodes are shown to be a more viable option for implementation into breath sensing devices and overcome the fouling problem since due to their low cost and disposable nature, a new electrode can be used for each measurement. The analytical utility of the platinum, screen-printed and boron-doped diamond electrodes were found to correspond to 6.85, 7.66 and 4.86 mM respectively. The challenges associated with the electrochemical sensing of 2-AA in breath that need to be overcome are discussed. This generic approach where electrochemical based technology is used to provide measurements for chemical markers in exhaled air/breath for medical diagnostics termed electrochemical breathprints (ec-breathprints), has the potential to be developed into a hand-held point-of-care breath diagnostic tool for identifying Pseudomonas aeruginosa infection in exhaled air/breath.

The prostaglandin E 2 (PGE 2 ) EP 4 subtype is one of four prostanoid receptors that use PGE 2 as the preferred ligand. We have investigated the agonist-mediated regulation of EP 4 using a multifaceted approach. Short-term (30 min) agonist challenge of recombinant EP 4 expressed in human embryonic kidney 293 cells (EP 4 -HEK293 cells) with PGE 2 (1 μM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [ 3 H]PGE 2 specific binding sites. These events correlated with sequestration of EP 4 , as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [ 32 P]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP 4 -HEK293 cells (10 μM forskolin or 1 mM 8-bromo-cAMP) did not induce EP 4 desensitization, sequestration, or phosphorylation. In contrast, stimulation of protein kinase C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE 2 -induced adenylyl cyclase activity and increased EP 4 phosphorylation, but did not induce sequestration or a reduction in [ 3 H]PGE 2 specific binding sites. EP 4 receptors containing a third intracellular loop deletion [EP 4 (del. 215–263)] or a carboxyl-terminal tail truncation [EP 4 (del. 355)] of EP 4 were used to demonstrate that the C-terminal tail governs sequestration as well as phosphorylation of the receptor.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

The structure-activity relationship of a series of styrylpyridine analogs of MK-0476 (montelukast, Singulair) is described. This work has led to the identification of a number of potent and orally active cysLT1 receptor (LTD4 receptor) antagonists including 2ab (L-733,321) as an optimized candidate.

A novel method is reported, whereby screen-printed electrodes (SPELs) are combined with dispersive liquid–liquid microextraction. In-situ ionic liquid (IL) formation was used as an extractant phase in the microextraction technique and proved to be a simple, fast and inexpensive analytical method. This approach uses miniaturized systems both in sample preparation and in the detection stage, helping to develop environmentally friendly analytical methods and portable devices to enable rapid and onsite measurement. The microextraction method is based on a simple metathesis reaction, in which a water-immiscible IL (1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide, [Hmim][NTf2]) is formed from a water-miscible IL (1-hexyl-3-methylimidazolium chloride, [Hmim][Cl]) and an ion-exchange reagent (lithium bis[(trifluoromethyl)sulfonyl]imide, LiNTf2) in sample solutions. The explosive 2,4,6-trinitrotoluene (TNT) was used as a model analyte to develop the method. The electrochemical behavior of TNT in [Hmim][NTf2] has been studied in SPELs. The extraction method was first optimized by use of a two-step multivariate optimization strategy, using Plackett–Burman and central composite designs. The method was then evaluated under optimum conditions and a good level of linearity was obtained, with a correlation coefficient of 0.9990. Limits of detection and quantification were 7 μg L−1 and 9 μg L−1, respectively. The repeatability of the proposed method was evaluated at two different spiking levels (20 and 50 μg L−1), and coefficients of variation of 7 % and 5 % (n = 5) were obtained. Tap water and industrial wastewater were selected as real-world water samples to assess the applicability of the method. The authors would like to thank the Spanish Ministry of Science and Innovation (project n. CTQ2011-23968), Generalitat Valenciana (Spain) (projects n. ACOMP/2013/072 and PROMETEO/2012/038) and Universidad de Alicante (Spain) (project n. GRE12-45) for the financial support. E.F. also thanks Generalitat Valenciana for her fellowship.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1iloprostprostaglandin F2alphaprostaglandin D2U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, MB28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

The production and abuse of new psychoactive substances, known as "legal highs" which mimic traditional drugs of abuse is becoming a global epidemic. Traditional analytical methodologies exist which can provide confirmatory analysis but there is a requirement for an on-the-spot analytical screening tool that could be used to determine whether a substance, or sample matrix contains such legal, or formally "legal highs". In this paper the electrochemical sensing of (±)-methcathinone and related compounds at a range of commercially available electrode substrates is explored. We demonstrate for the first time that this class of "legal highs" are electrochemically active providing a novel sensing protocol based upon their electrochemical oxidation. Screen-printed graphite sensing platforms are favoured due to their proven ability to be mass-produced providing large numbers of reliable and reproducible electrode sensing platforms that preclude the requirement of surface pre-treatment such as mechanical polishing as is the case in the use of solid/re-usable electrode substrates. Additionally they hold potential to be used on-site potentially being the basis of an on-site legal high screening device. Consequently the electroanalytical sensing of (±)-methcathinone (3a), (±)-4′-methylmethcathinone [3b, 4-MMC, (±)-mephedrone] and (±)-4′-methyl-N-ethylcathinone (3c, 4-MEC) is explored using screen-printed sensing platforms with the effect of pH explored upon the analytical response with their analytical efficiency evaluated towards the target legal highs. Interesting at pH values below 6 the voltammetric response quantitatively changes from that of an electrochemically irreversible response to that of a quasi-reversible signature which can be used analytically. It is demonstrated for the first time that the electroanalytical sensing of (±)-methcathinone (3a), (±)-mephedrone (3b) and 4-MEC (3c) are possible with accessible linear ranges found to correspond to 16–200 μg mL(−1) for 3a (at pH 12) and 16–350 μg mL(−1) for both 3b and 3c in pH 2, with limits of detection (3σ) found to correspond to 44.5, 39.8 and 84.2 μg mL(−1) respectively. Additionally adulterants that are commonly incorporated into cathinone legal highs are electrochemically explored at both pH 2 and 12.

The electroanalytical sensing of Rohypnol® (flunitrazepam) is reported for the first time utilising screen-printed graphite electrodes without the requirement for any additional pre-treatment or modification. The methodology is shown to be useful for quantifying low levels (μg mL(-1)) of Rohypnol® in not only buffered solutions but also two internationally favoured drinks: Coca Cola™ and the alcopop WKD™ without any sample pre-treatment. The current analytical approaches for the sensing of Rohypnol® are also summarised within this paper. The niche of this electroanalytical protocol is the lack of the requirement of any pre-treatment of the sample/beverage or electrode modification (cleaning, pre-treatment etc.) for the determination of Rohypnol® in beverages and offers a potential rapid, cost-effective, yet suitably sensitive and accurate screening solution to the problem posed by coloured drinks to products such as the colour changing 'Smart Cup'.

Improvements in analytical methods for the determination and quantification of methylcytosine in DNA are vital since it has the potential to be used as a biomarker to detect different diseases in the first stage such as in the case of carcinomas and sterility. In this work we utilized screen printed graphite electrodes (SPGE) for studying the electrochemical response of all free DNA bases, methylcytosine and short oligonucleotides by cyclic voltammetry (CV) and square wave voltammetry (SWV). CV and SWV responses of free DNA bases and methylcytosine have been investigated by using SPGE platforms and the feasibility of detecting and quantifying cytosine and methylcytosine as free DNA moieties has been evaluated as a function of pH, concentration and the presence of the other free DNA bases in solution simultaneously. Repeatability of using SWV has been performed for the electrochemical behavior of both 250 μM cytosine and 250 μM methylcytosine in the presence of 25 μM guanine, with coefficient of variations of 6.9% and 2.6% respectively based upon peak current (N = 5). Six-mer oligonucleotides with a sequence 5′-XXXCGC-3′, where the XXX motif corresponds to TTT, TTA, TAA and AAA have been performed using SWV in 0.1 M acetate buffer pH 5.0 to explore how the DNA base position effects the electrooxidation of guanine and cytosine into the oligonucleotide. Furthermore SWV comparisons of the electrooxidation of the oligonucleotides 5′-CGCGCG-3′ and its methylated 5′-mCGmCGmCG-3′ have been performed with concentrations in acetate buffer solutions, and the interaction of both oligonucleotides with the graphitic surface of the SPGE has been demonstrated by fitting well-known adsorption models such as Freundlich and Langmuir kinetics according to the SWV current response of guanine, cytosine and methylcytosine into the oligonucleotide.

Screen printed carbon nanotube electrodes (SPEs) are explored as electroanalytical sensing platforms for the detection of capsaicin in both synthetic capsaicin solutions and capsaicin extracted from chillies and chilli sauces utilising both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). It is found that the technique which is most applicable to the electroanalytical detection of capsaicin depends upon the analyte concentration: for the case of low capsaicin concentrations, CV is a more appropriate method as capsaicin exhibits characteristic voltammetric waves of peak heights relevant to the capsaicin concentration; but for the case of high capsaicin concentrations where the voltammetric waves merge and migrate out of the potential window, EIS is shown to be a more appropriate technique, owing to the observed linear increases in R(ct) with increasing concentration. Furthermore, we explore different types of screen printed carbon nanotube electrodes, namely single- and multi- walled carbon nanotubes, finding that they are technique-specific: for the case of low capsaicin concentrations, single-walled carbon nanotube SPEs are preferable (SW-SPE); yet for the case of EIS at high capsaicin concentrations, multi-walled carbon nanotube SPEs (MW-SPE) are preferred, based upon analytical responses. The analytical performance of CV and EIS is applied to the sensing of capsaicin in grown chillies and chilli sauces and is critically compared to 'gold standard' HPLC analysis.

We demonstrate the first example of the fabrication of co-planar 50 μm (width) screen printed graphite microbands (length: 20 mm), fabricated entirely via screen printing which are characterised both microscopically and electrochemically via cyclic voltammetry and evaluated towards the sensing of NADH offering a competitive limit of detection (3σ) of 0.24 μM. The fabricated electrodes are also shown to be extended to gold screen printed microbands which are evaluated towards the sensing of chromium(VI) offering a limit of detection (3σ) of 2.65 μM. These microbands are seen to be the first produced entirely through screen printed technology potentially allowing disposable, mass produced microbands to be realised.

A new class of potent ligand for the human peripheral cannabinoid (hCB 2 ) receptor is described. Two indole analogs 13 and 17 exhibited nanomolar potencies (K i ) with good selectivity for the hCB 2 receptor over the human central cannabinoid (hCB 1 ) receptor.

Structure-activity studies leading to the discovery of a new series of non-quinoline cysLT 1 receptor (LTD 4 receptor) antagonists are described. These studies demonstrated that the quinoline ring system of montelukast ( 5 ) may be replaced by an appropriately substituted thienopyridine system, yielding potent compounds. Two other molecular features of montelukast, the terminal phenyl ring substitution and the vinyl link, were also reevaluated. These studies led to the identification of 1 (L-740,515) , a compound with optimized in vitro and in vivo biological profiles.

Replacement of the quinoline pharmacophore of verlukast by alkylthiazoles and benzothiazoles has lead to the discovery of a new series of potent and orally active LTD4 receptor antagonists. The synthesis and structure activity relationships of this series of compounds are described.

A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3′-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristic of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 PGE2 > PGF2ɑ = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector into HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissues showed that the hDP receptor has a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.

Structure-activity studies leading to the discovery of 1 (MK-0476) are described. The initial compound of this series, 2, was a potent leukotriene D4 (LTD4) antagonist, but was also a peroxisomal enzyme inducer in the mouse. Structure-activity relationships around the thioether chain were explored to remove this undesirable feature. It was found that alkyl substituents in the s position relative to the carboxylic acid reduce the potency as a peroxisomal enzyme inducer while preserving the LTD4 antagonistic properties. Dialkyl substitution essentially eliminates the enzyme induction. The optimal styryl quinoline 1 exhibited high in vitro potency and in vivo activity on oral dosing without significant liver enzyme induction in the mouse.

We report the fabrication of gold screen printed macro electrodes which are electrochemically characterised and contrasted to polycrystalline gold macroelectrodes with their potential analytical application towards the sensing of chromium(III) and (VI) critically explored. It is found that while these gold screen printed macro electrodes have electrode kinetics typically one order of magnitude lower than polycrystalline gold macroelectrodes as is measured via a standard redox probe, in terms of analytical sensing, these gold screen printed macro electrodes mimic polycrystalline gold in terms of their analytical performance towards the sensing of chromium(III) and (VI), whilst boasting additional advantages over the macro electrode due to their disposable one-shot nature and the ease of mass production. An additional advantage of these gold screen printed macro electrodes compared to polycrystalline gold is the alleviation of the requirement to potential cycle the latter to form the required gold oxide which aids in the simplification of the analytical protocol. We demonstrate that gold screen printed macro electrodes allow the low micro-molar sensing of chromium(VI) in aqueous solutions over the range 10 to 1600 μM with a limit of detection (3σ) of 4.4 μM. The feasibility of the analytical protocol is also tested through chromium(VI) detection in environmental samples.

Dimethyl sulfoxide-differentiated U937 (dU937) cells express high affinity G-protein-coupled receptors for leukotriene (LT)D4 and LTB4 and, as described here, specific binding sites for LTC4. The specific binding of [3H]LTC4 was of low affinity (KD = 26 nM) and high abundance (Bmax = 33 pmol/mg of protein), as compared to LTD4 and LTB4 receptors. In addition, although [3H]LTC4 specific binding was enhanced by divalent cations, it was not inhibited by nonhydrolyzable GTP analogs. [3H]LTC4 specific binding to dU937 cell membranes does not have, therefore, the characteristics of binding to a G-protein-coupled receptor. Competition for [3H]LTC4 specific binding to dU937 cell membranes by leukotrienes and related analogs, including N-methylated LTC4, as well as glutathione, suggested a dependence on the presence of an arachidonic acid backbone, although varying degrees of saturation were well tolerated, and that the glutathione moiety of LTC4 in particular was important in determining affinity. The possibility that [3H]LTC4 specific binding was to a member of the glutathione S-transferase (GST) family of enzymes, such as LTC4 synthase, cytosolic GST, or microsomal GST, was therefore investigated. [3H]LTC4 specific binding sites could be separated from LTC4 synthase and cytosolic GSTs by differential detergent solubilization, but cofractionated with microsomal GST during solubilization and subsequent anion exchange chromatography. In membranes that were depleted of LTC4 synthase and cytosolic GSTs, 125I-azido-LTC4 (a photoaffinity probe based on LTC4) specifically photolabeled in a cation-dependent manner a 17-kDa polypeptide that was comparable in mass to the microsomal GST polypeptide. Furthermore, [3H]LTC4 bound specifically to purified human microsomal GST with the same characteristics as to the endogenous dU937-cell membrane specific binding sites. The principal [3H]LTC4 specific binding site present in dU937 cells, therefore, is not a G-protein-coupled receptor, LTC4 synthase, or cytosolic GSTs, but is microsomal GST. Finally, the 1:3 stoichiometry of [3H]LTC4 specific binding to purified microsomal GST is consistent with the enzyme functioning as a homotrimer.

The styryl quinoline thioether 5 (L-699,392) is a potent and orally active leukotriene D4 antagonist. The structure-activity studies leading to its discovery are described.

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3-I, hEP3-II and hEP3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 >> PGF2 alpha = iloprost > PGD2 >> U46619. In addition, the EP3-selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1-selective antagonist AH6909 gave IC50 values of 2-7 microM and the EP2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP3 receptor having comparable ligand binding properties.

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.

Screen printing is widely used to fabricate disposable and economical electrochemical sensors and has helped us to establish the route from ‘lab-to-market’ for a plethora of sensors. We overview recent developments in the field where screen printed electrochemical sensors are utilised. Starting with their fundamental understanding, through to highlighting new developments in bulk metal and mediator modified electrodes, as well as novel advantageous electrode designs, we demonstrate the wide and diverse range of applications that sensors based on this fabrication approach have achieved.

A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.

The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D 4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [ 3 H]Leukotriene D 4 -specific binding to THP-1 cell membranes was of high affinity ( K D = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [ 3 H]leukotriene D 4 -specific binding with the following rank order of potency: leukotriene D 4 ⪢ leukotriene E 4 〉 leukotriene C 4 In addition, leukotriene D 4 -receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [ 3 H]leukotriene D 4 competition assays. These data show that [ 3 H]leukotriene D 4 -specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D 4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D 4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N -glycosylation, resulted in a 6-fold decrease in the number of detectable [ 3 H]leukotriene D 4 -specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [ 3 H]leukotriene D 4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D 4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.

A structurally new series of potent leukotriene D4 antagonists has evolved from modification of L-695,499.

The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.

The leukotriene D4 receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D4 receptor found in human lung and guinea-pig lung preparations. [3H]Leukotriene D4 specific binding in differentiated U937 cell membranes is of high affinity (KD = 0.35 nM), saturable (Bmax = 287 fmol/mg protein), with differentiation resulting in a 3–5-fold increase in the number of detectable binding sites. [3H]Leukotriene D4-specific binding in differentiated U937 cell membranes displays several features of G-protein-cupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [3H]leukotriene D4-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D4 receptor types were observed. [3H]Leukotriene D4 equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E4 and leukotriene C4, although leukotriene D4 and the leukotriene D4-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [3H]leukotriene D4-specific binding in all three membranes preparations. In conclusion, the leukotriene D4 receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.

The leukotriene (LT) B4 receptor has been characterized in the human monocyte leukemia THP-1 cell line. Scatchard analysis of [3H]LTB4 specific binding to THP-1 cell membranes revealed a single population of high affinity (KD = 56 pM) and saturable (2000 receptors/cell) binding sites. [3H]LTB4 specific binding was enhanced by divalent cations, but inhibited by both monovalent cations and a non-hydrolysable GTP analogue. Treatment with GTP analogue resulted in a concentration-dependent reduction in the number of high affinity binding sites, accompanied by the appearance of an equal number of binding sites of lower affinity (KD = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB4 receptors (KD = 48 pM and 270 pM). Treatment with GTP analogue, however, converted all these detectable binding sites to the lower affinity state. These data suggest that the LTB4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB4 receptors present in these two cell types was also substantiated by target-size analysis by radiation inactivation, which estimated a comparable molecular mass of 56.5 kDa and 52.8 kDa for the THP-1 cell and PMN LTB4 receptors, respectively. Finally, the presence of a single LTB4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [3H]LTB4 equilibrium binding assay incubations resulted in complete photolysis of [3H]LTB4. Subsequent resolution of the tritiated PMN proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed one major radioactive peak migrating with an apparent molecular weight of 61,000. This peak was identified as the LTB4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB4 or the LTB4-receptor antagonist, L-662,328. Photolabelling was also partially inhibited by pretreatment with GTP analogue, consistent with G-protein uncoupling reagents reducing receptor affinity without complete inhibition. In summary, the LTB4 receptor identified in human myeloid cells is a G-protein coupled receptor with interconvertible high and low affinity states, having a molecular mass of 53-61 kDa.

Potent and selective EP3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24-95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.

A new, potent, orally active leukotriene D 4 receptor antagonist has been discovered. The structure -activity relationship leading to L-695,499 is described.

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.

Multiple-protein delivery has been proven to be a critical consideration for promoting tissue regeneration. Many polymeric composite biomaterials have been designed and used for modulating dual-protein delivery to enhance tissue regeneration in vitro or in vivo. However, the fabrication conditions and low water contents within the portions of these composite matrices that determine protein release rates are not optimal for maintaining the stability of encapsulated macromolecular therapeutics. In this proof-of-concept work, we aim to resolve this deficiency by single-step fabrication of affinity hydrogels capable of independently delivering two or more proteins. Selective protein-binding sites were incorporated into poly(ethylene glycol) hydrogels via copolymerization with glycidyl methacrylate-iminodiacetic acid (GMIDA) ligands to modulate release of two model proteins, lysozyme and hexahistidine tagged green fluorescent protein (hisGFP), via two distinct matrix-binding mechanisms, namely electrostatic interaction and metal-ion chelation. Differing from composite matrices for dual-protein delivery, the results reported herein indicate that injectable monolithic affinity hydrogels are capable of rapidly encapsulating multiple therapeutic agents under mild physiological conditions and independently controlling their localized delivery. Most importantly, these affinity hydrogels retain high water permeabilities throughout the entire device, characteristics that are necessary for maintaining the stability and viability of encapsulated proteins and cells.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Two different series of very potent and selective EP(3) antagonists have been reported: a novel series of ortho-substituted cinnamic acids [Belley, M., Gallant, M., Roy, B., Houde, K., Lachance, N., Labelle, M., Trimble, L., Chauret, N., Li, C., Sawyer, N., Tremblay, N., Lamontagne, S., Carriere, M.-C., Denis, D., Greig, G. M., Slipetz, D., Metters, K. M., Gordon, R., Chan, C. C., Zamboni, R. J. Bioorg. Med. Chem. Lett.2005, 15, 527] and the acylsulfonamides of ortho-(arylmethyl)cinnamates. [(a) Juteau, H., Gareau, Y., Labelle, M., Sturino, C. F., Sawyer, N., Tremblay, N., Lamontagne, S., Carriere, M.-C., Denis, D., Metters, K. M. Bioorg. Med. Chem. 2001, 9, 1977; (b) Juteau, H., Gareau, Y., Labelle, M., Lamontagne, S., Tremblay, N., Carriere, M.-C., Denis, D., Sawyer, N., Metters, K. M. Bioorg. Med. Chem. Lett.2001, 11, 747] The structural differences between the two series, along with their biological activity in vivo, in vitro, and metabolism, are analyzed. Some of those compounds, including hybrids containing the best structural features of both series, possess K(i) as low as 0.6 nM on the EP(3) receptor.

A series of novel ortho -substituted cinnamic acids have been synthesized, and their binding activity and selectivity on the four prostaglandin E 2 receptors evaluated. Many of them are very potent and selective EP 3 antagonists ( K i 3–10 nM), while compound 9 is a very good and selective EP 2 agonist ( K i 8 nM). The biological profile of the EP 2 agonist 9 in vivo and the metabolic profile of selected EP 3 antagonists are also reported.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Triaryl compounds bearing an ortho-substituted propionic acid moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinities of key compound on all eight human prostanoid receptors is reported.

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.