Madeleine Antonsson

Activation of the GABAB receptor inhibits transient lower esophageal sphincter relaxations in dogs

BACKGROUND & AIMS Transient lower esophageal sphincter relaxation (TLESR) appears to be the most frequent motor event responsible for gastroesophageal reflux. Because TLESRs are considered to be triggered by activation of gastric mechanoreceptors, and because the gamma-aminobutyric acid type B (GABA(B))-receptor agonist baclofen is known to inhibit transmitter release from mechanosensitive afferents, the effects of baclofen on TLESRs in the dog were assessed. METHODS A total of 183 recordings of the pharyngeal, esophageal, lower esophageal sphincter, and gastric pressures as well as measurement of esophageal pH were performed in 15 awake dogs. Racemic baclofen, its enantiomers, and the GABA(B)-receptor antagonist CGP36742 were administered before stimulation of TLESRs by a liquid meal and air insufflation. The pharmacodynamics of baclofen were compared with its pharmacokinetics. RESULTS Baclofen dose-dependently inhibited TLESRs, with a 50% effective dose (ED(50)) of 1.0 micromol/kg after intravenous administration. The maximal inhibition amounted to approximately 80%. Intragastric baclofen was almost equally effective (ED(50), 1.8 micromol/kg), compatible with the complete oral availability of the drug (100%). The inhibitory effect of baclofen resided in the pharmacologically active R enantiomer, and CGP36742 reduced some of the effects of baclofen. CONCLUSIONS Baclofen is a potent and efficacious inhibitor of TLESRs and reflux in the dog. Activation of the GABA(B) receptor may be a new approach to the treatment of reflux disease.

In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids

Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unheplful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (Vu,brain). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with Vu,brain microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the Vu,brain of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.

Structure−Brain Exposure Relationships in Rat and Human Using a Novel Data Set of Unbound Drug Concentrations in Brain Interstitial and Cerebrospinal Fluids

New experimental methodologies were applied to measure the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) and the unbound CSF-to-plasma concentration ratio (K(p,uu,CSF)) in rats for 43 structurally diverse drugs. The relationship between chemical structure and K(p,uu,brain) was dominated by hydrogen bonding. Contrary to popular understanding based on the total brain-to-plasma concentration ratio (logBB), lipophilicity was not a determinant of unbound brain exposure. Although changing the number of hydrogen bond acceptors is a useful design strategy for optimizing K(p,uu,brain), future improvement of in silico prediction models is dependent on the accommodation of active drug transport. The structure-brain exposure relationships found in the rat also hold for humans, since the rank order of the drugs was similar for human and rat K(p,uu,CSF). This cross-species comparison was supported by K(p,uu,CSF) being within 3-fold of K(p,uu,brain) in the rat for 33 of 39 drugs. It was, however, also observed that K(p,uu,CSF) overpredicts K(p,uu,brain) for highly effluxed drugs, indicating lower efflux capacity of the blood-cerebrospinal fluid barrier compared to the blood-brain barrier.

Development of a high-throughput brain slice method for studying drug distribution in the central nervous system.

New, more efficient methods of estimating unbound drug concentrations in the central nervous system (CNS) combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (Vu,brain). Although the brain slice method is the most reliable in vitro method for measuring Vu,brain, it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4 to 5 h of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programs. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit of buffer volume. The use of casette experiments for investigating Vu,brain in a linear drug concentration range increased the throughput of the method. The Vu,brain for the model compounds ranged from 4 to 3000 ml · g brain–1, and the sources of variability are discussed. The optimized setup of the brain slice method allows precise, robust estimation of Vu,brain for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.

In silico prediction of unbound brain-to-plasma concentration ratio using machine learning algorithms.

Distribution over the blood-brain barrier (BBB) is an important parameter to consider for compounds that will be synthesized in a drug discovery project. Drugs that aim at targets in the central nervous system (CNS) must pass the BBB. In contrast, drugs that act peripherally are often optimised to minimize the risk of CNS side effects by restricting their potential to reach the brain. Historically, most prediction methods have focused on the total compound distribution between the blood plasma and the brain. However, recently it has been proposed that the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) is more relevant. In the current study, quantitative K(p,uu,brain) prediction models have been built on a set of 173 in-house compounds by using various machine learning algorithms. The best model was shown to be reasonably predictive for the test set of 73 compounds (R(2)=0.58). When used for qualitative prediction the model shows an accuracy of 0.85 (Kappa=0.68). An additional external test set containing 111 marketed CNS active drugs was also classified with the model and 89% of these drugs were correctly predicted as having high brain exposure.

Discovery of (3-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone (AZD1979), a Melanin Concentrating Hormone Receptor 1 (MCHr1) Antagonist with Favorable Physicochemical Properties

A novel series of melanin concentrating hormone receptor 1 (MCHr1) antagonists were the starting point for a drug discovery program that culminated in the discovery of 103 (AZD1979). The lead optimization program was conducted with a focus on reducing lipophilicity and understanding the physicochemical properties governing CNS exposure and undesired off-target pharmacology such as hERG interactions. An integrated approach was taken where the key assay was ex vivo receptor occupancy in mice. The candidate compound 103 displayed appropriate lipophilicity for a CNS indication and showed excellent permeability with no efflux. Preclinical GLP toxicology and safety pharmacology studies were without major findings and 103 was taken into clinical trials.

Discovery of Agonists of Cannabinoid Receptor 1 with Restricted Central Nervous System Penetration Aimed for Treatment of Gastroesophageal Reflux Disease

Agonists of the cannabinoid receptor 1 (CB1) have been suggested as possible treatments for a range of medical disorders including gastroesophageal reflux disease (GERD). While centrally acting cannabinoid agonists are known to produce psychotropic effects, it has been suggested that the CB1 receptors in the periphery could play a significant role in reducing reflux. A moderately potent and highly lipophilic series of 2-aminobenzamides was identified through focused screening of GPCR libraries. Development of this series focused on improving potency and efficacy at the CB1 receptor, reducing lipophilicity and limiting the central nervous system (CNS) exposure while maintaining good oral absorption. Improvement of the series led to compounds having excellent potency at the CB1 receptor and high levels of agonism, good physical and pharmacokinetic properties, and low penetration into the CNS. A range of compounds demonstrated a dose-dependent inhibition of transient lower esophageal sphincter relaxations in a dog model.

Identification of positron emission tomography (PET) tracer candidates by prediction of the target-bound fraction in the brain

BackgroundDevelopment of tracers for imaging with positron emission tomography (PET) is often a time-consuming process associated with considerable attrition. In an effort to simplify this process, we herein propose a mechanistically integrated approach for the selection of tracer candidates based on in vitro measurements of ligand affinity (Kd), non-specific binding in brain tissue (Vu,brain), and target protein expression (Bmax).MethodsA dataset of 35 functional and 12 non-functional central nervous system (CNS) PET tracers was compiled. Data was identified in literature for Kd and Bmax, whereas a brain slice methodology was used to determine values for Vu,brain. A mathematical prediction model for the target-bound fraction of tracer in the brain (ftb) was derived and evaluated with respect to how well it predicts tracer functionality compared to traditional PET tracer candidate selection criteria.ResultsThe methodology correctly classified 31/35 functioning and 12/12 non-functioning tracers. This predictivity was superior to traditional classification criteria or combinations thereof.ConclusionsThe presented CNS PET tracer identification approach is rapid and accurate and is expected to facilitate the development of novel PET tracers for the molecular imaging community.

Effects of a novel potent melanin-concentrating hormone receptor 1 antagonist, AZD1979, on body weight homeostasis in mice and dogs.

Melanin‐concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non‐human primates and dogs express two MCH receptors (MCH1 and MCH2). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood.

Different in vitro and in vivo profiles of substituted 3‐aminopropylphosphinate and 3‐aminopropyl(methyl)phosphinate GABAB receptor agonists as inhibitors of transient lower oesophageal sphincter relaxation

BACKGROUND AND PURPOSE Gastro‐oesophageal reflux is predominantly caused by transient lower oesophageal sphincter relaxation (TLOSR) and GABAB receptor stimulation inhibits TLOSR. Lesogaberan produces fewer CNS side effects than baclofen, which has been attributed to its affinity for the GABA transporter (GAT), the action of which limits stimulation of central GABAB receptors. To understand the structure–activity relationship for analogues of lesogaberan (3‐aminopropylphosphinic acids), and corresponding 3‐aminopropyl(methyl)phosphinic acids, we have compared representatives of these classes in different in vitro and in vivo models.