G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M 2 acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M 2 Trp 422 in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists. G protein-coupled receptors (GPCRs)4 have outstanding importance as targets for drug action (1, 2). Conformational changes underlying the inactive-to-active receptor switch in GPCRs are in the focus of current research. In general, the receptor transmembrane helices (TMs) rearrange, allowing the intracellular loop region to unfold and to stimulate neighboring G proteins (3, 4). Conformational changes include extracellular receptor regions, and a critical role of the second extracellular loop (E2) for GPCR activation and ligand binding has emerged (5-9). Rational development of agonistic drugs for GPCR activation requires deeper insight into such conformational changes. Because GPCRs are hardly accessible for crystallization, indirect approaches are applied that often involve modification of the receptor protein such as receptor mutagenesis, introduction of metal ion sites or disulfide bridges, or covalent linkage of moieties for fluorescence resonance energy transfer.Allosteric small molecules allow the study of native receptors. An increasing number of GPCRs is known to contain allosteric sites (10, 11); cinacalcet is the first allosteric GPCR modulator that has recently entered the market (12). Allosteric sites are located outside the orthosteric area that is occupied...

Interstitial ciprofloxacin concentrations in soft tissues were measured by microdialysis following intravenous administration of 200 mg to each of eight healthy volunteers. Interstitial ciprofloxacin concentrations were significantly lower than corresponding total serum drug concentrations; the interstitium-to-serum concentration ratios ranged from 0.55 to 0.73. An in vitro simulation based on interstitial pharmacokinetics showed a substantially lower antimicrobial activity than did the simulation based on serum pharmacokinetics. Thus, ciprofloxacin concentrations at the site of effect may be subinhibitory although effective concentrations are attained in serum.

Aims Skin blister fluid sampling, in vivo microdialysis and saliva sampling are commonly employed as surrogates for the measurement of drug concentrations in peripheral compartments. Although expected to exhibit comparable results, data derived from these techniques have never been directly compared. Thus, the aim of the present study was to evaluate the comparability of these techniques. Methods Paracetamol, a model drug with low protein binding, was administered to seven healthy volunteers at an oral dose of 2000 mg. Subsequently, tissue kinetics were measured simultaneously in cantharides induced skin blisters, microdialysates of subcutaneous-and skeletal muscle-tissue and saliva and compared to serum concentrations. Results Mean ratio (AUC blister /AUC serum ) was 0.88 (95% CI, 0.50-1.26), mean ratio (AUC muscle /AUC serum ) was 1.08 (0.67-1.49), mean ratio (AUC subcutaneous /AUC serum ) was 0.96 (0.41-1.51) and mean ratio (AUC saliva /AUC serum ) was 1.83 (1.39-2.27). In this study the concentration profiles after single oral administration differed among the three methods. The time course of the concentration peripheral compartment /concentration serum -ratios showed that cantharides blister and microdialysate concentrations closely paralleled serum levels. An equilibration period of less than 2 h had to be taken into account for blister measurements. In contrast, saliva concentrations were significantly higher than corresponding serum concentrations. Conclusions Skin blister sampling and microdialysis closely mirrored corresponding serum concentrations and, thus, proved to be suitable techniques for the assessment of peripheral compartment pharmacokinetics. In contrast, saliva data overestimated the corresponding serum concentrations.