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The creation of a candidate drug molecule is very complex and involves multiple cycles of different assays and tests across diverse scientific disciplines. Attrition is generally high, and currently only 10–20% of all drug discovery projects result in a novel drug. A leading cause of attrition is poor efficacy, which is often only discovered late when entering human trials. Therefore early, translatable assessment of whether the candidate molecule will modulate the target of interest in a safe and effective manner is critical. A key aspect of this assessment (which is sometimes not given the focus that is warranted) is the generation of detailed knowledge of the interaction between drug molecules and targets, and how this translates to disease modulation. In this chapter we will look at how quantitative in vitro pharmacology and mechanistic enzymology methods can be applied to characterize the action of novel compounds in terms of potency, specificity and deeper understanding of mechanisms of action, with reference to specific examples. A detailed knowledge of the drug–target interactions from the molecular to the cellular level enables better prediction of efficacy and safety and contributes to mechanistic PKPD modelling to aid dose setting and translation through to patients.

Purpose: The strategy of combining endocrine therapy with PI3K-mTOR inhibition has shown promise in estrogen receptor (ER)–positive breast cancer, but new agents and combinations with a better therapeutic index are urgently needed. Taselisib is a potent, selective, beta-isoform–sparing PI3 kinase inhibitor. Patients and Methods: 30 patients with ER-positive, metastatic breast cancer who had failed prior endocrine therapy were treated with escalating doses of taselisib (2 or 4 mg in an intermittent or continuous schedule) combined with tamoxifen 20 mg once daily in this phase 1b study using a “rolling six” design. Results: Taselisib combined with tamoxifen was generally well tolerated, with treatment-emergent adverse events as expected for this class of drugs, including diarrhea (13 patients, 43%), mucositis (10 patients, 33%), and hyperglycemia (8 patients, 27%). No dose-limiting toxicities were observed. Objective responses were seen in 6 of 25 patients with RECIST-measurable disease (ORR 24%). Median time to disease progression was 3.7 months. Twelve of 30 patients (40%) had disease control for 6 months or more. Circulating tumor (ct)DNA studies using next-generation tagged amplicon sequencing identified early indications of treatment response and mechanistically relevant correlates of clinical drug resistance (e.g., mutations in KRAS, ERBB2) in some patients. Conclusions: Taselisib can be safely combined with tamoxifen at the recommended phase 2 dose of 4 mg given once daily on a continuous schedule. Preliminary evidence of antitumor activity was seen in both PIK3CA mutant and wild-type cancers. The randomized phase 2 part of POSEIDON (testing tamoxifen plus taselisib or placebo) is currently recruiting.

IntroductionSelf-monitoring of blood pressure (BP) in pregnancy could improve the detection and management of pregnancy hypertension, while also empowering and engaging women in their own care. Two linked trials aim to evaluate whether BP self-monitoring in pregnancy improves the detection of raised BP during higher risk pregnancies (BUMP 1) and whether self-monitoring reduces systolic BP during hypertensive pregnancy (BUMP 2).Methods and analysesBoth are multicentre, non-masked, parallel group, randomised controlled trials. Participants will be randomised to self-monitoring with telemonitoring or usual care. BUMP 1 will recruit a minimum of 2262 pregnant women at higher risk of pregnancy hypertension and BUMP 2 will recruit a minimum of 512 pregnant women with either gestational or chronic hypertension. The BUMP 1 primary outcome is the time to the first recording of raised BP by a healthcare professional. The BUMP 2 primary outcome is mean systolic BP between baseline and delivery recorded by healthcare professionals. Other outcomes will include maternal and perinatal outcomes, quality of life and adverse events. An economic evaluation of BP self-monitoring in addition to usual care compared with usual care alone will be assessed across both study populations within trial and with modelling to estimate long-term cost-effectiveness. A linked process evaluation will combine quantitative and qualitative data to examine how BP self-monitoring in pregnancy is implemented and accepted in both daily life and routine clinical practice.Ethics and disseminationThe trials have been approved by a Research Ethics Committee (17/WM/0241) and relevant research authorities. They will be published in peer-reviewed journals and presented at national and international conferences. If shown to be effective, BP self-monitoring would be applicable to a large population of pregnant women.Trial registration numberNCT03334149

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Background: The European Academy of Paediatric Dentistry (EAPD) proposes this best-practice guidance to help practitioners to decide when and how to use local analgesia to control pain in children, adolescents, and medically compromised individuals during the delivery of oral health care. Methods: A search of different databases was conducted using all terms relevant to the subject. Relevant papers were identified after a review of their titles, abstracts or full papers. Three workshops were held during the corresponding EAPD interim seminar in Torino (Italy) in 2017. Several statements were agreed upon and, furthermore, knowledge gaps were identified. Results: An important outcome was that when local analgesia administered appropriately—correct choice of agent(s) and dosage, proper route of administration—it is, firstly, clinically effective for pain-control in treating children and, secondly, it carries a very low risk of morbidity including adverse or side-effects. Furthermore, several gaps in knowledge were identified during the workshop which indicates future research needs. Most importantly it remains unsatisfactory that in several European countries the most frequently used injectable local analgesic agent, articaine, is not approved for usage in children below the age of 4 years. Conclusion: When considering the dental demand to treat vulnerable (medically compromised) children and adolescents in a safe, painless, less-invasive and effective way, there seems to be an urgent need to close these gaps in knowledge. © 2017, European Academy of Paediatric Dentistry.

Background: Published preclinical findings provided new insights into the functional 'cross-talk' between the oestrogen receptor (ER) and the progesterone receptor (PR) in breast cancer (BC) (Mohammed et al., Nature, 2015). Addition of a PR agonist to anti-oestrogens directly modifies ERa chromatin binding and the transcriptional response in breast cancer cells, and is anti-proliferative in vitro and in vivo. Megestrol Acetate (MA), an off-patent semi-synthetic derivative of progesterone, has been licensed for many years as treatment for ER+ metastatic BC. There is also good evidence for the effectiveness of MA as a supportive therapy to ameliorate endocrine therapy-related hot flushes. Trial design: PIONEER is a three-arm, open label, multi-centre randomized phase II pre-surgical window trial evaluating effects of 15 days of preoperative therapy with Letrozole (LET), or LET plus MA 40mg, or LET plus MA 160mg in postmenopausal women with ER+ HER2- invasive primary BC. Eligibility criteria: Inclusion CriteriaPostmenopausal women with histologically confirmed invasive BC, ≥T1c, clinical NX or N0-N3, ER+ (Allred≥3), and HER2-2 groups of patients are potentially eligible: Cohort A: Patients whose cancers have been deemed to be operable by the Multi-Disciplinary Team (MDT) with surgery planned for the next 2-6 weeks Cohort B: Patients with early or locoregionally advanced BC planned for primary endocrine therapy either in lieu of surgery or as neoadjuvant therapy before surgery ECOG performance status of 0-2Adequate Liver, Renal, Bone marrow function Exclusion CriteriaHormone replacement therapy in the last 6 monthsTreatment with tamoxifen or an aromatase inhibitor (AI) in the last 6 monthsA progestogen-containing intrauterine system in situ, unless removed prior to randomisationMetastatic disease on presentationRecurrent BC (patients with a new primary invasive BC are eligible) Specific aims: The primary endpoint is % change in proliferation between baseline and day 15 tumor biopsies, measured by Ki67 IHC assessment. Secondary endpoints include: expression of Aurora Kinase A, Caspase 3 and AR/PR/EMT markers by IHC; and safety endpoints. Exploratory endpoints: transcription factor mapping (ChIP-seq) and identification of differential ERa-associated proteins (RIME) on paired fresh-frozen tumor samples. PIONEER will help determine whether there is value in conducting a Phase III adjuvant study to investigate the longer term benefit of combining an AI with MA, and if so, at what dose (40mg vs. 160mg). Statistical methods: Patients are randomized in a 1:1.5:1.5 ratio into arms A:B:C. Based on results from previous clinical trials, a mean 66% reduction in Ki67 is anticipated for LET alone (arm A), and a 77.5% reduction for the combination arms B and C, based on preclinical data. Present and target accrual: Patients are being recruited in Cambridge, Cornwall, Belfast & London with 6 other UK sites due to open q3/4 2018. At the time of submission, 29 patients had been recruited. A recruitment total of 189 patients is required. Citation Format: Kumar SS, Benson J, McIntosh S, King P, Dougall G, Kateb A, Vallier A-L, Jones L, Qian W, Provenzano E, Caldas C, Pantziarka P, Carroll J, Baird RD. PIONEER- Pre-operative wIndOw study of letrozole plus PR agonist megestrol acetate versus letrozole aloNE in post-menopausal patients with ER-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-01-07.

Background and Purpose The discovery of DP2 as a second receptor for PGD2 has prompted the search for antagonists as potential novel therapies based on the associations between PGD2 and disease. Here we describe the biochemical and pharmacological properties of 4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-acetic acid (AZD1981), a novel DP2 receptor antagonist. Experimental Approach Binding to DP2, functional receptor pharmacology and selectivity were studied in both human and animal systems. Key Results AZD1981 displaced radio-labelled PGD2 from human recombinant DP2 with high potency (pIC50 = 8.4). Binding was reversible, non-competitive and highly selective against a panel of more than 340 other enzymes and receptors, including DP1 (>1000-fold selective). AZD1981 inhibited DP2-mediated shape change and CD11b up-regulation in human eosinophils, shape change in basophils and chemotaxis of human eosinophils and Th2 cells with similar potency. AZD1981 exhibited good cross-species binding activity against mouse, rat, guinea pig, rabbit and dog DP2. Evaluation in mouse, rat or rabbit cell systems was not possible as they did not respond to DP2 agonists. Agonist responses were seen in guinea pig and dog, and AZD1981 blocked DP2-mediated eosinophil shape change. Such responses were more robust in the guinea pig, where AZD1981 also blocked DP2-dependent eosinophil emigration from bone marrow. Conclusions and Implications AZD1981 is a DP2 antagonist that blocks functional responses in eosinophils, Th2 cells and basophils. It exhibited similar potency irrespective of the cell type, DP2 agonist or species used. This selective orally active agent is currently under clinical evaluation as a potential therapeutic agent in respiratory diseases including asthma.

Novel indole-3-thio-, 3-sulfonyl- and 3-oxy-aryl-1-acetic acids are reported which are potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). Optimization required maintenance of high CRTh2 potency whilst achieving a concomitant reduction in rates of metabolism, removal of cyp p450 inhibition and minimization of aldose reductase and aldehyde reductase activity. High quality compounds suitable for in vivo studies are highlighted, culminating in the discovery of AZD1981 (22).

A novel series of biaryl phenoxyacetic acids was discovered as potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A hit compound 4 was discovered from high throughput screening. Modulation of multiple aryl substituents afforded both agonists and antagonists, with small changes often reversing the mode of action. Understanding the complex SAR allowed design of potent antagonists such as potential candidate 34.

Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). In this setting they are routinely exposed to cigarette smoke and a range of pathogens including bacteria and viruses. The gene expression changes that result from these challenges may contribute to the initiation and progression of the disease. Understanding such changes is therefore of great interest and could aid the discovery of novel therapeutics. To study this, we stimulated monocyte-derived macrophages (MDM) from smokers and non-smokers with either cigarette smoke extract (CSE) or bacterially derived lipopolysaccharide (LPS) and profiled global transcriptional changes using Affymetrix arrays. LPS and CSE stimulation elicited markedly different transcriptome profiles with the former agent producing a larger number of significant changes. The CSE evoked changes showed some overlap with those observed when comparing habitual smokers with non-smokers, although the latter changes were generally of a more subtle nature. Detailed pathway analyses indicated that a number of genes involved in host defence were regulated following CSE stimulation and in MDM from smokers. In particular the interferon gamma (IFNgamma)-signalling pathway was significantly down-regulated following CSE stimulation, a finding that was confirmed by RT-PCR analysis. Furthermore, these changes were associated with suppressed release of the IFNgamma-induced chemokines, CXCL10 and CXCL9 from CSE treated MDM. In summary, our data provides evidence that smoking alters key mechanisms of host defence in macrophages. Such changes may explain the increased susceptibility of COPD patients to the lung infections that are associated with exacerbations of this disease.

The first step in the biological assessment of compounds synthesized by medicinal chemists usually involves testing in a range of in vitro biochemical/pharmacological assays. The primary purpose of these experiments is to generate robust estimates of drug properties including potency, affinity, intrinsic activity, or efficacy, as well as providing an understanding of the mechanism of action of the compounds. This information is used by medicinal chemists to generate structure activity relationships that drive the optimization of compounds appropriate for testing in more complex in vitro, ex vivo, and ultimately in vivo assays. This chapter outlines the biological systems typically used in modern drug discovery programs, describing the measurements made and highlighting the properties of various different classes of drugs and the principles underlying their analysis. The subsequent testing and analysis of compounds in animal models is also described, and the utility of such models for predicting efficacy, “dose-to-man,” and safety issues is discussed.

The first step in the biological assessment of compounds synthesised by medicinal chemists usually involves testing in a range of in vitro biochemical/pharmacological assays. The primary purpose of these experiments is to generate robust estimates of drug parameters including potency, affinity, intrinsic activity or efficacy as well as providing an understanding of the mechanism of action of the compounds. This information is used by medicinal chemists to generate structure activity relationships and ultimately aids the design of new therapeutic agents. This article outlines the principles underlying the pharmacological analyses typically performed, describing the measurements made and highlighting the properties of various different classes of agonists and antagonists. Finally, the application of these principles and the associated analyses to the drug discovery process are exemplified.

Starting with the weak agonist indomethacin, a series of potent, selective CRTh2 (DP(2)) antagonists have been discovered as potential treatments for asthma, allergic rhinitis and other inflammatory diseases.

The pharmacological classification of P2 receptors owes its origin to the pioneering efforts of Geoff Burnstock and those who followed him, research that was conducted primarily in physiological experimental systems. Over recent years, the techniques of molecular biology have been increasingly applied in the study of P2 receptors while, at the same time, advances in their pharmacological analysis have been limited by a lack of potent and selective agonist or antagonist ligands. This has resulted in a classification scheme which is largely structural in nature, with relatively little contribution from pharmacology. Our endeavours in this area have been directed towards the discovery of ligands with which the pharmacological analysis and definition of P2 receptors could be advanced, the ultimate goal being the design of therapeutic agents. This article will describe some of our experiences in this challenging but rewarding area.

By using a pharmacophore model, a geometrical representation of the features necessary for molecules to show a particular biological activity, it is possible to search databases containing the 3D structures of molecules and identify novel compounds which may possess this activity. We describe our experiences of establishing a working 3D database system and its use in rational drug design. By using muscarinic M(3) receptor antagonists as an example, we show that it is possible to identify potent novel lead compounds using this approach. Pharmacophore generation based on the structures of known M(3) receptor antagonists, 3D database searching, and medium-throughput screening were used to identify candidate compounds. Three compounds were chosen to define the pharmacophore: a lung-selective M(3) antagonist patented by Pfizer and two Astra compounds which show affinity at the M(3) receptor. From these, a pharmacophore model was generated, using the program DISCO, and this was used subsequently to search a UNITY 3D database of proprietary compounds; 172 compounds were found to fit the pharmacophore. These compounds were then screened, and 1-[2-(2-(diethylamino)ethoxy)phenyl]-2-phenylethanone (pA(2) 6.67) was identified as the best hit, with N-[2-(piperidin-1-ylmethyl)cycohexyl]-2-propoxybenz amide (pA(2) 4. 83) and phenylcarbamic acid 2-(morpholin-4-ylmethyl)cyclohexyl ester (pA(2) 5.54) demonstrating lower activity. As well as its potency, 1-[2-(2-(diethylamino)ethoxy)phenyl]-2-phenylethanone is a simple structure with limited similarity to existing M(3) receptor antagonists.

1. In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor. 2. The P2Y1-receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pK(B) of 5.8 and 6.0, respectively). 3. The selective P2Y(ADP) antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pK(A)) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 microM) approximately 100 fold greater than its known P2Y(ADP) receptor affinity, had no effect on ADP-induced increases in [Ca2+]i. 4. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPgammaS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. 5. The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgammaS and ATP were lower in platelets. 6. The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system. 7. The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors. 8. In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2Y(ADP)-receptors.

The present study attempted to classify pharmacologically the α1-adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta. In the ear artery, the agonist effects of phenylephrine were antagonized by 5-methyl urapidil (pA2=7.90; Schild slope=0.85) and BMY 7378 (pA2=6.11; Schild slope=0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5-methyl urapidil data set could be fitted to a two-receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03. The effects of clonidine in the ear artery were competitively antagonised by 5-methyl urapidil (pKB=7.91) and BMY 7378 (pKB=5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype. In the aorta, the effects of phenylephrine were antagonized by 5-methyl urapidil (pA2=7.95; Schild slope=1.11) and BMY 7378 (pA2=9.08; Schild slope=0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two-receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00. The alkylating agent, chloroethylclonidine (CEC) elicited concentration-dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 μm, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 μm, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration-effect curves. The affinity of clonidine in the rabbit ear artery (pKA=6.17) was found to be significantly different to its affinity in the rat thoracic aorta (pKA=7.12) suggesting that this agonist activates different α1-adrenoceptor subtypes in the two tissues. These results suggest that heterogeneous populations of α1-adrenoceptors are present in both tissues. In the ear artery, the profile of antagonist and agonist activity is most consistent with α1A-adrenoceptors being the predominant receptor subtype. The second receptor population does not appear to correspond to any of the recognized α1-adrenoceptor subtypes. In the aorta α1D-adrenoceptors appear to predominate, with α1A-adrenoceptors being the most likely candidate for the second receptor population. British Journal of Pharmacology (1997) 120, 247–258; doi:10.1038/sj.bjp.0700917

Background During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis. Methods Satellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56+ cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised. Results Large-scale isolation of CD56+ cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers. Conclusions We have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function. Electronic supplementary material The online version of this article (doi:10.1007/s13539-012-0097-z) contains supplementary material, which is available to authorized users.

Background Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema. Methods A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry. Results Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin. Conclusions Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.

The discovery of DP2 as a second receptor for PGD2 has prompted the search for antagonists as potential novel therapies based on the associations between PGD2 and disease. Here we describe the biochemical and pharmacological properties of 4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-acetic acid (AZD1981), a novel DP2 receptor antagonist.Binding to DP2 , functional receptor pharmacology and selectivity were studied in both human and animal systems.AZD1981 displaced radio-labelled PGD2 from human recombinant DP2 with high potency (pIC50 = 8.4). Binding was reversible, non-competitive and highly selective against a panel of more than 340 other enzymes and receptors, including DP1 (1000-fold selective). AZD1981 inhibited DP2 -mediated shape change and CD11b up-regulation in human eosinophils, shape change in basophils and chemotaxis of human eosinophils and Th2 cells with similar potency. AZD1981 exhibited good cross-species binding activity against mouse, rat, guinea pig, rabbit and dog DP2 . Evaluation in mouse, rat or rabbit cell systems was not possible as they did not respond to DP2 agonists. Agonist responses were seen in guinea pig and dog, and AZD1981 blocked DP2 -mediated eosinophil shape change. Such responses were more robust in the guinea pig, where AZD1981 also blocked DP2 -dependent eosinophil emigration from bone marrow.AZD1981 is a DP2 antagonist that blocks functional responses in eosinophils, Th2 cells and basophils. It exhibited similar potency irrespective of the cell type, DP2 agonist or species used. This selective orally active agent is currently under clinical evaluation as a potential therapeutic agent in respiratory diseases including asthma.

Chronic Obstructive Pulmonary Disease (COPD) is a disease characterized by a largely irreversible airflow obstruction and a persistent, excessive inflammatory response. Alveolar macrophages (AMs) are increased in the lungs of COPD patients, and act as orchestrators of the inflammatory response, releasing a range of mediators to coordinate recruitment and activation of leukocytes. Attempts to treat the inflammatory component of COPD with anti-inflammatory drugs such as steroids has met with limited success. In this study, we compared the ability of the phosphodiesterase IV (PDEIV) inhibitor Cilomilast, the steroid Budesonide, and the p38 mitogen activated protein kinase inhibitor BIRB-796 to inhibit tumour necrosis factor alpha (TNFα) and interleukin 6 (IL-6) releases from AMs isolated from COPD lung transplant tissue. All studies were carried out with appropriate ethical approval and written, informed consent was obtained from each subject. Cilomilast had little effect on cytokine release from AMs. There was considerable variability in the responsiveness of AMs to Budesonide, with a subset of AMs responding poorly to Budesonide. BIRB-796 inhibited TNFα release from all AM donors, including those that responded poorly to steroids. Treatment with BIRB-796 and Budesonide together gave an additive decrease in TNFa release. These results suggest that a p38 inhibitor may provide advantages over existing anti-inflammatory treatments for COPD, either as an add-on to existing therapy, or to treat patients who respond poorly to steroids.

1. The operational model of agonism (Black & Leff, 1983) has been extended to describe the interaction between a partial agonist and a full agonist at the same receptor. The derived equation explicitly describes the interaction and allows the affinity (and efficacy) of the partial agonist to be estimated by direct fitting of raw experimental agonist concentration-effect (E/[A]) curve data. 2. The model was used to analyse experimental E/[A] curve data generated for the interaction between pilocarpine (partial agonist) and carbachol (full agonist) at the M3-muscarinic receptor mediating contraction of the guinea-pig isolated trachea. Pilocarpine affinity estimates obtained by operational model-fitting were compared with those obtained by use of the null method (Stephenson, 1956). These analyses demonstrated that the two methods gave comparable results (mean pKB estimates were 5.79 and 5.86 for the operational model and null method respectively). 3. When multiple concentrations of partial agonist are used, simultaneous operational model-fitting of all the E/[A] curve data allows the competitive nature of the interaction to be studied. 4. We conclude that operational model-fitting is a valid and analytically simple alternative to the conventional null method of analysing full/partial agonist interactions.

Two plausible theories of agonist action — the isomerization and ternary complex mechanisms — predict that agonist affinity measured using the inactivation method may be subject to overestimation. Moreover, the greater the intrinsic efficacy, the greater the predicted error. But are these predictions correct, and are they borne out by experimental data? Paul Leff and colleagues argue that accurate agonist affinity constants can be measured despite these predictions, but that effort must be made to detect any potential errors that might be anticipated from theory.

Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs. MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients. In MDMs, PGE(2) potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-alpha release (p[A](50) 8.51+/-0.11, maximum inhibition 95.9+/-4.8%). In human AMs, PGE(2) also inhibited TNF-alpha release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE(2) curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP(4)-receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE(2) in both AMs and MDMs were mediated by activation of EP(4) and EP(2) receptors. These data indicate that both E-prostanoid(4) and E-prostanoid(2) selective agonists may have anti-inflammatory properties in lung diseases where macrophages play a role.

This paper describes the development of a QSAR model for the rational control of functional duration of topical long-acting dual D(2)-receptor/beta(2)-adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease. A QSAR model highlighted the importance of lipophilicity and ionization in controlling beta(2) duration. It was found that design rules logD(7.4) > 2, secondary amine pK(a) > 8.0, yielded ultra-long duration compounds. This model was used successfully to guide the design of long- and ultra-long-acting compounds. The QSAR model is discussed in terms of the exosite model, and the plasmalemma diffusion microkinetic hypothesis, for the control of beta(2) duration. Data presented strongly suggests that beta(2) duration is primarily controlled by the membrane affinity of these compounds.

This paper describes the rationale for the development of dual dopamine D2-receptor and beta2-adrenoceptor agonists as potential treatments for the symptoms of chronic obstructive pulmonary disease (COPD). The putative involvement of pulmonary sensory afferent nerves in mediating the key COPD symptoms of breathlessness, cough and excess sputum production is outlined and the hypothesis that activation of D2-receptors on such nerves would modulate their activity is developed. This premise was tested, in a range of animal models, using the first of a novel class of dual dopamine D2-receptor and beta2-adrenoceptor agonists, sibenadet HCl (Viozan, AR-C68397AA). In the course of these studies it was demonstrated that sibenadet, through activation of D2-receptors, inhibited discharge of rapidly adapting receptors and was effective in reducing reflex-induced tachypnoea, mucus production and cough in the dog. Sibenadet, through its activation of beta2-adrenoceptors, was also shown to be an effective bronchodilator with a prolonged duration of action following topical administration to the lungs. These studies also indicated that sibenadet had a wide therapeutic ratio with respect to expected undesirable side-effects such as emesis and cardiovascular disturbances. These results provided a compelling rationale for the initiation of a clinical development programme with sibenadet for the treatment of COPD.

In his article in last month's issue of TiPS (14. 89–91), Douglas Craig discussed the application of the Cheng-Prusoff relationship to the analysis of antagonists in functional experiments. Craig described how the translation of this method from biochemistry to pharmacology can be accompanied by errors due to theoretical misunderstanding. He also emphasized the lower level of rigour associated with this means of estimating antagonist affinity constants compared with Schild analysis. In this article, Paul Leff and Iain Dougall itextend Craig's arguments by describing some additional ways in which the application of the Cheng-Prusoff method to pharmacological experiments are likely to result in misinterpretation and erroneous estimates of antagonist affinity.

Experiments conducted on human tissue samples are a key component of modern drug discovery programs and complement the use of animal tissue based assays in this process. Such studies can (i) enhance our understanding of disease pathophysiology, (ii) increase (or decrease) confidence that modulating the function of particular molecular targets will have therapeutic benefit (iii) allow comparison of the activities of different agents on particular mechanisms/processes and (iv) provide information on the potential safety risks associated with targets. All of this information is critical in identifying the targets that are most likely to deliver efficacious and safe medicines to address unmet clinical needs. With the introduction of new technologies, human tissue samples are also increasingly being incorporated into drug project screening cascades, including their use in high throughput assays. Improved access to human tissue would undoubtedly further extend the utility of this valuable resource in the drug discovery process.

1. The present study attempted to classify pharmacologically the alpha 1-adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta. 2. In the ear artery, the agonist effects of phenylephrine were antagonized by 5-methyl urapidil (pA2 = 7.90; Schild slope = 0.85) and BMY 7378 (pA2 = 6.11; Schild slope = 0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5-methyl urapidil data set could be fitted to a two-receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03. 3. The effects of clonidine in the ear artery were competitively antagonised by 5-methyl urapidil (pKB = 7.91) and BMY 7378 (pKB = 5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype. 4. In the aorta, the effects of phenylephrine were antagonized by 5-methyl urapidil (pA2 = 7.95; Schild slope = 1.11) and BMY 7378 (pA2 = 9.08; Schild slope = 0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two-receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00. 5. The alkylating agent, chloroethylclonidine (CEC) elicited concentration-dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 microM, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 microM, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration-effect curves. 6. The affinity of clonidine in the rabbit ear artery (pKA = 6.17) was found to be significantly different to its affinity in the rat thoracic aorta (pKA = 7.12) suggesting that this agonist activates different alpha 1-adrenoceptor subtypes in the two tissues. 7. These results suggest that heterogeneous populations of alpha 1-adrenoceptors are present in both tissues. In the ear artery, the profile of antagonist and agonist activity is most consistent with alpha 1A-adrenoceptors being the predominant receptor subtype. The second receptor population does not appear to correspond to any of the recognized alpha 1-adrenoceptor subtypes. In the aorta alpha 1D-adrenoceptors appear to predominate, with alpha 1A-adrenoceptors being the most likely candidate for the second receptor population.

1. In isolated ring preparations of the rabbit saphenous vein, prostaglandin E2 (PGE2) caused well-defined, stable and concentration-dependent relaxations of KCl contracted tissues with a mean potency (p[A50]) of 9.39. 2. The prostanoid EP-receptor agonists, PGE1, 11-deoxy PGE1, 16,16-dimethyl PGE2 and misoprostol were all full agonists in this preparation. The EP2-receptor selective agonists, butaprost and AH13205, and the EP1/EP3-receptor selective agonist, sulprostone, also relaxed this tissue but were at least 300 times less potent than PGE2. 3. Prostaglandin D2 (PGD2), the DP-receptor agonist, BW245C, and the IP-receptor agonist, cicaprost, caused concentration-dependent relaxations of the rabbit saphenous vein but were at least 60 times less potent than PGE2. 4. The selective EP4-receptor antagonist, AH23848B (30 microM), antagonized the PGE2 concentration-effect (E/[A]) curves yielding a pA2 estimate of 4.96. The EP1/DP-receptor antagonist, AH6809 (10 microM), had no effect on the location of PGE2 E/[A] curves. 5. The stable thromboxane A2-mimetic, U46619, elicited concentration-dependent contractions of the rabbit saphenous vein (p[A50] = 8.01) however, PGE2 and prostaglandin F2 alpha (PGF2 alpha) were unable to produce a contractile response. The response to U46619 was competitively antagonized by the TP-receptor antagonist, GR32191B, yielding a pKB estimate of 7.08. 6. In the rabbit isolated ear artery, PGE2, misoprostol and AH13205 relaxed tissues pre-contracted with phenylephrine. PGE2 (p[A50] = 7.04) and misoprostol were equipotent, whereas AH13205 was some 40 fold less potent. AH23848B (30 microM) and AH6809 (1 and 10 microM) caused no significant shift in the location of PGE2 E/[A] curves. 7. These data suggest that the rabbit isolated saphenous vein contains prostanoid, EP-, DP-, IP- and TP-receptors. Based on antagonist affinity information and agonist potency orders, the rabbit saphenous vein contains an inhibitory prostanoid EP-receptor different from that in the rabbit ear artery, but comparable to the recently described EP4-receptor.

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

1. We have attempted to estimate the affinity and efficacy of the new beta 2-adrenoceptor agonist salmeterol in the guinea-pig isolated trachea and to compare these estimates with those obtained for salbutamol. 2. The kinetics of the relaxation to salmeterol were considerably slower than those to salbutamol and the responses were poorly defined. Nevertheless, in experiments employing a cumulative curve design both salmeterol and salbutamol exhibited partial agonist behaviour. Operational model-fitting of these data indicated that the intrinsic efficacies of the two agonists were indistinguishable but that salmeterol had an affinity (pKA = 7.4) some 30 fold greater than salbutamol (pKA = 5.9). 3. Single dose experiments, using maximally effective concentrations of salmeterol or salbutamol were carried out in an attempt to quantify more reliably the relative efficacies of the two agonists. In these experiments salbutamol was found to have an efficacy (2.5) approximately 3 fold greater than that of salmeterol (0.8). 4. Full/partial agonist interaction studies were carried out to provide another estimate of the affinity of salmeterol. Isoprenaline and adrenaline were employed as full agonists in these experiments and the affinity (pKB) estimates obtained for salmeterol against these agonists were 7.3 and 7.4 respectively. These affinity estimates were similar to the pKA value (7.4) obtained by operational model-fitting. 5. Salmeterol has slightly lower efficacy (3 fold) but markedly greater affinity (30 fold) than salbutamol at beta 2-receptors. The slightly lower efficacy of salmeterol is unlikely to have clinical implications for this drug as a bronchodilator beta 2-adrenoceptor agonist in asthma.

1 Recent theoretical studies have questioned the pharmacological estimation of agonist affinity. They showed that when receptor isomerisation or ternary complex mechanisms operate, the receptor inactivation method can substantially overestimate affinity, whereas methods for partial agonist analysis are more accurate. We previously suggested that the operation of such mechanisms and therefore the presence of errors could be detected by analysing the same partial agonist by the receptor inactivation and comparative methods. This paper describes the practical application of this test. 2 The ternary complex mechanism was simulated for a partial agonist under various conditions relating receptor (R) and transducer (T) concentrations, one of which also corresponds to the receptor isomerisation mechanism. The theoretical data so generated were then analysed by the inactivation and comparative methods to quantify the magnitude of error of affinity estimation that could occur. 3 This analysis showed that for a partial agonist with approximately 85% of the activity of a full agonist, the inactivation method could produce an affinity (pKA) estimate up to 0.7 log10 units higher than that produced by the comparative method. This difference would occur when the total receptor concentration ([R0]) is less than or equal to the total transducer concentration ([T0]). It also showed that the over-estimation of affinity by the inactivation method was accompanied by drastic overestimation of Em, the maximal effect parameter. 4 The test was then exemplified using the muscarinic receptor system in the guinea-pig isolated left atrial preparation, where there is evidence that a ternary complex mechanism operates. The test agonist was pilocarpine, which produced on average 83% of the activity of the full agonist, carbachol. Pilocarpine was analysed in comparison with carbachol and by receptor inactivation in the same tissue resulting in small and statistically insignificant differences in Em (96.7% and 97.3% respectively) and pKA (5.03 and 4.95 respectively). 5 In conclusion, in this experimental system, there was no evidence for the errors in agonist affinity estimation predicted by theory. Although this conclusion only applies to this system and application of the test to others is necessary to establish the generality of the present results, further examination of the theoretical basis for the predicted errors is required.