Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

What happens under the flag of direct observation, and how that matters: A qualitative study in general practice residency.

INTRODUCTION: In competency-based medical education, direct observation (DO) of residents' skills is scarce, notwithstanding its undisputed importance for credible feedback and assessment. A growing body of research is investigating this discrepancy. Strikingly, in this research, DO as a concrete educational activity tends to remain vague. In this study, we concretised DO of technical skills in postgraduate longitudinal training relationships. METHODS: Informed by constructivist grounded theory, we performed a focus group study among general practice residents. We asked residents about their experiences with different manifestations of DO of technical skills. A framework describing different DO patterns with their varied impact on learning and the training relationship was constructed and refined until theoretical sufficiency was reached. RESULTS: The dominant DO pattern was ad hoc, one-way DO. Importantly, in this pattern, various unpredictable, and sometimes unwanted, scenarios could occur. Residents hesitated to discuss unwanted scenarios with their supervisors, sometimes instead refraining from future requests for DO or even for help. Planned bi-directional DO sessions, though seldom practiced, contributed much to collaborative learning in a psychologically safe training relationship. DISCUSSION AND CONCLUSION: Patterns matter in DO. Residents and supervisors should be made aware of this and educated in maintaining an open dialogue on how to use DO for the benefit of learning and the training relationship.

Patterns of direct observation and their impact during residency: general practice supervisors' views.

CONTEXT: Direct observation (DO) of residents' performance, despite the importance that is ascribed to it, does not readily fit in with the practice of postgraduate medical education (PGME); it is infrequent and the quality of observation may be poor in spite of ongoing efforts towards improvement. In recent literature, DO is mostly portrayed as a means to gather information on the performance of residents for purposes of feedback and assessment. The role of DO in PGME is likely to be more complex and poorly understood in the era of outcome-based education. By exploring the possible complexity of DO in workplace learning, our research aims to contribute to a better use of DO in the practice of PGME. METHODS: Constructivist grounded theory informed our data collection and analysis. Data collection involved focus group sessions with supervisors in Dutch general practice who were invited to discuss the manifestations, meanings and effects of DO of technical skills. Theoretical sufficiency was achieved after four focus groups, with a total of 28 participants being included. RESULTS: We found four patterns of DO of technical skills: initial planned DO sessions; resident-initiated ad hoc DO; supervisor-initiated ad hoc DO, and continued planned DO sessions. Different patterns of DO related to varying meanings, such as checking or trusting, and effects, such as learning a new skill or experiencing emotional discomfort, all of them concerning the training relationship, patient safety or residents' learning. CONCLUSIONS: Direct observation, to supervisors, means much more than gathering information for purposes of feedback and assessment. Planned DO sessions are an important routine during the initiation phase of a training relationship. Continued planned bidirectional DO sessions, although infrequently practised, potentially combine most benefits with least side-effects of DO. Ad hoc DO, although much relied upon, is often hampered by internal tensions in supervisors, residents or both.

Raising enthusiasm for the medical care of elderly patients: a concept mapping study to find elements for an elderly friendly medical curriculum.

BACKGROUND: To deliver high quality of care for the growing population of older patients more geriatricians are needed. However, the interest of medical students for a career in geriatrics is lagging behind due to a lack of exposure, the nature of the work, and the low status and financial rewards. So far, only isolated interventions aimed at enhancing interest and/or attitudes with regard to geriatrics have been studied, pointing to the need for a broader-based strategy. The goal of this research is to find elements for a curriculum framework that can raise medical students' enthusiasm for the medical care of elderly patients. METHODS: We used the concept mapping method developed by Trochim. This computer-assisted procedure consists of five steps: brainstorming, prioritizing and clustering with several experts, followed by processing by the computer and analysis. RESULTS: The views that were generated were grouped into the following clusters: a patient-centered medical curriculum, a curriculum representative of patient population, geriatrics presented as intellectually challenging and emotionally appealing, senior-friendly role models, a clear professional perspective. The results are presented in the form of a graphic chart. CONCLUSIONS: An agenda to discuss the necessary actions for drastic curricular reforms in medical schools is set. This may give some guidance to this urgent, but highly complicated issue how to make medical student enthusiastic for the medical care for elderly patients.

[A career in elderly care medicine; an option for today's medical student? : Medical students' interest in elderly care medicine]. / Een carrière als specialist ouderengeneeskunde; iets voor de huidige geneeskundestudent? : Belangstelling geneeskundestudenten voor ouderengeneeskunde.

BACKGROUND: Due to the rise of older patients with multi-morbidity, we need more elderly care physicians. However, not all available training slots for the elderly care medicine specialty have been fully utilized in recent years. To assess medical student interest in this specialty as well as potential causes for this interest we explored the interest of medical students in the profession of elderly care physician, as well their perception of this profession, both in the 'old curriculum' and in a 'new curriculum', where the new curriculum had a mandatory elderly care medicine clerkship and more competency-related learning. METHOD: At VUmc 120 final year medical students were asked to complete a questionnaire in 2014 about professional preferences and professional characteristics. The same questionnaire had been presented five years earlier, in 2009, to 150 medical students at the end of their final year. RESULTS: The response rates were 100% and 85% respectively. Of the students in the new curriculum 16,7% considered a career in elderly care medicine. This percentage was 9,4% for students in the old curriculum (p = 0,087). The characteristics of the profession that appealed most to the students, but were not considered applicable to elderly care medicine were: diagnostics skills, acute complaints, visible results. The professional characteristics that students found to be very much applicable to this specialty, but less attractive for their future profession were: psychosocial, chronic and terminal conditions. DISCUSSION: We observe a trend that students in the new curriculum are more interested in the profession of elderly care physician, even though this interest remains limited. We recommend that the basic medical training, both in the bachelor phase and in a mandatory elderly care medicine clerkship, focus more on demonstrating that the characteristics students find appealing in the medical profession are indeed present in this speciality. Also, the basic training should concentrate more on guidance and treatment of patients with chronic and terminal conditions.

Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.

Why medical students do not choose a career in geriatrics: a systematic review.

BACKGROUND: While the demand for doctors specialised in the medical care of elderly patients is increasing, the interest among medical students for a career in geriatrics is lagging behind. METHODS: To get an overview of the different factors reported in the literature that affect the (low) interest among medical students for a career in geriatrics, a systematic literature search was conducted using PubMed, Embase, PsycINFO, and ERIC. Quality assessment criteria were applied. RESULTS: Twenty studies met the criteria and were included in the review. In relation to the nature of the work, the preference of medical students is young patients, and acute somatic diseases that can be cured. The complexity of the geriatric patient deters students from choosing this specialty. Exposure by means of pre-clinical and particularly clinical education increases interest. The lack of status and the financial aspects have a negative influence on interest. CONCLUSION: Exposure to geriatrics by means of education is necessary. The challenge in geriatric education is to show the rewarding aspects of the specialty.

Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2.

The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

Multiple binding sites for small-molecule antagonists at the CC chemokine receptor 2.

The chemokine receptor CCR2 is a G protein-coupled receptor that is activated primarily by the endogenous CC chemokine ligand 2 (CCL2). Many different small-molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all these antagonists lack clinical efficacy, and therefore a better understanding of their mechanism of action is warranted. In this study, we examined the pharmacological properties of small-molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist (125)I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 [N-(2-(((3S,4S)-1-((1r,4S)-4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide] and CCR2-RA, were radiolabeled to study the binding site in greater detail. We discovered that [(3)H]INCB3344 and [(3)H]CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we examined CCR2 inhibition in multiple functional assays, including a novel label-free whole-cell assay. INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA showed a noncompetitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified into two groups with different binding sites and thereby different modes of inhibition. We have provided further insights in CCR2 antagonism, and these insights are important for the development of novel CCR2 inhibitors.

Adenosine A2B receptor agonism inhibits neointimal lesion development after arterial injury in apolipoprotein E-deficient mice.

OBJECTIVE: The A(2B) adenosine receptor (A(2B)R) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A(2B)R deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A(2B)R agonism protects against injury-induced intimal hyperplasia. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A(2B) receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A(2B) receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583-treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583. CONCLUSIONS: Our data show that activation of the adenosine A(2B) receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A(2B) receptor agonism as a new therapeutic approach in the prevention of restenosis.

Kinetic profiling and functional characterization of 8-phenylxanthine derivatives as A2B adenosine receptor antagonists.

A2B adenosine receptor (A2BAR) antagonists have therapeutic potential in inflammation-related diseases such as asthma, chronic obstructive pulmonary disease and cancer. However, no drug is currently clinically approved, creating a demand for research on novel antagonists. Over the last decade, the study of target binding kinetics, along with affinity and potency, has been proven valuable in early drug discovery stages, as it is associated with improved in vivo drug efficacy and safety. In this study, we report the synthesis and biological evaluation of a series of xanthine derivatives as A2BAR antagonists, including an isothiocyanate derivative designed to bind covalently to the receptor. All 28 final compounds were assessed in radioligand binding experiments, to evaluate their affinity and for those qualifying, kinetic binding parameters. Both structure-affinity and structure-kinetic relationships were derived, providing a clear relationship between affinity and dissociation rate constants. Two structurally similar compounds, 17 and 18, were further evaluated in a label-free assay due to their divergent kinetic profiles. An extended cellular response was associated with long A2BAR residence times. This link between a ligand's A2BAR residence time and its functional effect highlights the importance of binding kinetics as a selection parameter in the early stages of drug discovery.

Putative role of the adenosine A(3) receptor in the antiproliferative action of N (6)-(2-isopentenyl)adenosine.

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N (6)-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A(3) receptor (hA(3)R) ligands with affinities in the high nanomolar range (K (i) values of 159 and 649 nM, respectively). These values were comparable to the observed K (i) value of adenosine on hA(3)R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A(3)R. In a functional assay in Chinese hamster ovary cells transfected with hA(3)R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A(3)R antagonist VUF5574. Both IPA and reference A(3)R agonist 2-chloro-N (6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A(3)R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A(3)R-independent mechanism, as was previously reported for other A(3)R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A(3)R.

Diagnoses during follow-up of patients presenting with fatigue in primary care.

BACKGROUND: Little is known about the distribution of diagnoses that account for fatigue in patients in primary care. We evaluated the diagnoses established within 1 year after presentation with fatigue in primary care that were possibly associated with the fatigue. METHODS: We conducted a prospective observational cohort study with 1-year follow-up. We included adult patients who presented with a new episode of fatigue between June 2004 and January 2006. We extracted data on diagnoses during the follow-up period from the patients' medical records as well as data on pre-existing chronic diseases. RESULTS: Of the 571 patients for whom diagnostic data were available, 268 (46.9%) had received one or more diagnoses that could be associated with fatigue. The diagnoses were diverse and mostly included symptom diagnoses, with main categories being musculoskeletal (19.4%) and psychological problems (16.5%). Clear somatic pathology was diagnosed in 47 (8.2%) of the patients. Most diagnoses were not made during the consultation when fatigue was presented. INTERPRETATION: Only a minority of patients were diagnosed with serious pathology. Half of the patients did not receive any diagnosis that could explain their fatigue. Nevertheless, because of the wide range of conditions and symptoms that may explain or co-occur with the fatigue, fatigue is a complex problem that deserves attention not only as a symptom of underlying specific disease.

The impact of scan length on the exposure levels in 16- and 64-row multidetector computed tomography: a phantom study.

RATIONALE AND OBJECTIVES: Higher patient exposure levels have been reported for 64-row multidetector computed tomography (MDCT) compared to 16-row MDCT. The objective of this study was to make a thorough comparison by evaluating the impact of scan length on the exposure levels at 16-row MDCT and 64-row MDCT. MATERIALS AND METHODS: Dose-length product (DLP) values were determined to compare exposure levels in 16- and 64-row MDCT. This phantom study does not deal with a possible reduction in image quality induced by an increase in scattered radiation in 64-row MDCT compared to 16-row MDCT. RESULTS: The exposure levels of 64-row MDCT (scan slice thickness, 0.5 mm) are up to 18% lower than those of 16-row MDCT at slice thickness 0.5 mm when scanning an object larger than 12.3 cm. At this value, the plots of the 16- and 64-row DLP values versus scan length cross. The DLP curves of 1- and 2-mm slice thickness 16-row MDCT are in closer resemblance to those of 0.5-mm 64-row MDCT. The respective exposure levels of 1- and 2-mm slice thickness 16-row MDCT exceed those of 0.5-mm 64-row MDCT by up to 4% and 3%, with intersections of 30 and 25 cm, respectively. CONCLUSION: Lower effective doses are obtained in 64-row MDCT compared to 16-row MDCT (0.5-mm slice thickness) provided that scan length exceeds 12.3, 30, and 25 cm, for 16-row MDCT slice thickness of 0.5, 1, and 2 mm, respectively. Reduced effective dosage in 64-row MDCT compared to 16-row MDCT has not been demonstrated before. Differences in object size may thus explain discrepancies between previous studies with regard to the exposure levels at 64-slice CT compared to 16-slice CT.

Opportunities for Students to Prescribe: An Evaluation of 185 Consultations in the Student-run Cardiovascular Risk Management Programme.

Involving students in cardiovascular risk management (CVRM) could offer patients a structured CVRM programme and students a valuable learning opportunity. We describe and evaluate a student-run CVRM programme that was set up to offer primary prevention to patients with known risk factors in a general practitioner's practice. During a consultation, two undergraduate medical students assessed the patients' actual risk and formulated a CVRM plan, which they discussed with the patient after approval by a GP. After the consultations, patients were asked to complete evaluation/feedback questionnaires. From December 2014 to December 2015, 185 consultations were carried out by 46 students. Feedback questionnaires of 153 consultations were returned, in which patient satisfaction was 8.43 (1-10, min-max). The cardiovascular risk of 95 patients was determined, and in >50% patients, it was 'high'. Participating students and GPs were enthusiastic about the (pharmacotherapy) learning opportunities and improved CVRM care while contributing to real patient care in this CVRM programme.

The daily practice of (suspected) coeliac disease management by general practitioners: A qualitative approach.

BACKGROUND: General practitioners (GPs) play a crucial role in diagnosing coeliac disease (CD). However, data on GP management of (suspected) CD patients is sparse. OBJECTIVES: To provide insights into the daily practice of diagnosis, treatment, and follow-up of CD by GPs. METHODS: A qualitative study using topic list-based semi-structured in-depth interviews with Dutch GPs with more than five years' experience carried out between January and March 2017. GPs were purposively sampled. The number of GPs interviewed depended on when data saturation was reached. We applied content analysis to the semi-structured interviews. RESULTS: Seven GPs were interviewed, five of whom were female. Analysis of the interviews resulted in three main themes: 'awareness,' 'diagnostics' and 'management.' Vague gastrointestinal symptoms and diarrhoea were often mentioned as a possible presentation of CD. Antibodies were used in CD diagnosis, although some GPs would start a gluten-free diet as a first diagnostic tool. Some GPs diagnosed CD only based on positive antibodies without referring to secondary care or duodenal biopsy analysis. GPs mentioned no role for primary care physicians in the follow-up of CD and noted the important role of dieticians in CD management. CONCLUSION: The different views of GPs on how to diagnose and monitor CD could be a basis for further research to improve CD detection rate and CD care.

A two-state model for the kinetics of competitive radioligand binding.

BACKGROUND AND PURPOSE: Ligand-receptor binding kinetics is receiving increasing attention in the drug research community. The Motulsky and Mahan model, a one-state model, offers a method for measuring the binding kinetics of an unlabelled ligand, with the assumption that the labelled ligand has no preference while binding to distinct states or conformations of a drug target. As such, the one-state model is not applicable if the radioligand displays biphasic binding kinetics to the receptor. EXPERIMENTAL APPROACH: We extended the Motulsky and Mahan model to a two-state model, in which the kinetics of the unlabelled competitor binding to different receptor states (R1 and R2 ) can be measured. With this extended model, we determined the binding kinetics of unlabelled N-5'-ethylcarboxamidoadenosine (NECA), a representative agonist for the adenosine A1 receptor. Subsequently, an application of the model was exemplified by measuring the binding kinetics of other A1 receptor ligands. In addition, limitations of the model were investigated as well. KEY RESULTS: The kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [3 H]-NECA was used. The model was further validated by good correlation between simulated results and the experimental data. CONCLUSION: The two-state model is sufficient to analyse the binding kinetics of an unlabelled ligand, when a radioligand shows biphasic association characteristics. We expect this two-state model to have general applicability for other targets as well.

Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligand.

Recently, the development of the fluorinated PET tracer [18F]1a for imaging of CB2 receptors in the central nervous system was reported. [18F]1a showed high CB2 affinity and selectivity over the CB1 subtype, but rapid biotransformation in mice. In addition to the amide hydrolysis, oxidative N-dealkylation and carbazole oxidation were postulated as main metabolic pathways. Based on these results, novel carbazole derivatives with additional 6-substituents (23a, 24a), modified hydrogenation state (26a) and enlarged fluoroalkyl substituent (13a, 13b) were synthesized and pharmacologically evaluated. The key step in the synthesis of substituted carbazoles 23a, 24a and 26a was a Fischer indole synthesis. Nucleophilic substitution of tosylated lactate 5 by carbazole anion provided the fluoroisopropyl derivatives 13a and 13b. Partial hydrogenation of the aromatic carbazole system (26a) was not tolerated by the CB2 receptor. A methylsulfonyl moiety in 6-position (24a) led to considerably reduced CB2 affinity, whereas a 6-methoxy moiety (23a) was well tolerated. An additional methyl moiety in the fluoroethyl side chain of 1a resulted in fluoroisopropyl derivatives 13 with unchanged high CB2 affinity and CB2: CB1 selectivity. Compared with the fluoroethyl derivative 1a, the carbazole N-atom of the fluoroisopropyl derivative 13a (Ki(CB2) = 2.9 nM) is better shielded against the attack of CYP enzymes as formation of N-oxides was not observed and N-dealkylation took place to a less amount.

Optimization of the metabolic stability of a fluorinated cannabinoid receptor subtype 2 (CB2) ligand designed for PET studies.

The central CB2 receptor represents a promising target for the treatment of neuroinflammatory diseases as CB2 activation mediates anti-inflammatory effects. Recently, the F-18 labeled PET radiotracer [18F]7a was reported, which shows high CB2 affinity and high selectivity over the CB1 subtype but low metabolic stability due to hydrolysis of the amide group. Based on these findings twelve bioisosteres of 7a were synthesized containing a non-hydrolysable functional group instead of the amide group. The secondary amine 23a (Ki = 7.9 nM) and the ketone 26a (Ki = 8.6 nM) displayed high CB2 affinity and CB2:CB1 selectivity in in vitro radioligand binding studies. Incubation of 7a, 23a and 26a with mouse liver microsomes and LC-quadrupole-MS analysis revealed a slightly higher metabolic stability of secondary amine 23a, but a remarkably higher stability of ketone 26a in comparison to amide 7a. Furthermore, a logD7.4 value of 5.56 ±â€¯0.08 was determined for ketone 26a by micro shake-flask method and LC-MS quantification.

Kinetics of human cannabinoid 1 (CB1) receptor antagonists: Structure-kinetics relationships (SKR) and implications for insurmountable antagonism.

While equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In this study, we therefore conducted kinetic assays for nine 1-(4,5-diarylthiophene-2-carbonyl)-4-phenylpiperidine-4-carboxamide derivatives and included the CB1 antagonist rimonabant as a comparison. For this we newly developed a dual-point competition association assay with [3H]CP55940 as the radioligand. This assay yielded Kinetic Rate Index (KRI) values from which structure-kinetics relationships (SKR) of hCB1 receptor antagonists could be established. The fast dissociating antagonist 6 had a similar receptor residence time (RT) as rimonabant, i.e. 19 and 14 min, respectively, while the slowest dissociating antagonist (9) had a very long RT of 2222 min, i.e. pseudo-irreversible dissociation kinetics. In functional assays, 9 displayed insurmountable antagonism, while the effects of the shortest RT antagonist 6 and rimonabant were surmountable. Taken together, this study shows that hCB1 receptor antagonists can have very divergent RTs, which are not correlated to their equilibrium affinities. Furthermore, their RTs appear to define their mode of functional antagonism, i.e. surmountable vs. insurmountable. Finally, based on the recently resolved hCB1 receptor crystal structure, we propose that the differences in RT can be explained by a different binding mode of antagonist 9 from short RT antagonists that is able to displace unfavorable water molecules. Taken together, these findings are of importance for future design and evaluation of potent and safe hCB1 receptor antagonists.