The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one} has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). When given systemically, the compound has an efficacy and potency comparable to those of a benzodiazepine anxiolytic such as alprazolam or diazepam. However, this compound is differentiated from a classical benzodiazepine anxiolytic by a lack of efficient anti-panic-like activity, absence of anticonvulsant properties, and lack of effects on motor performance and cognitive function at anxiolytic doses (0.3 to 3 mg͞kg i.p.). No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (>10 mg͞kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ͞nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ͞nociceptin in adaptive behavioral fear responses to stress.T he ORL1 orphan receptor was identified from a human cDNA library on the basis of close homology (Ϸ65% in the transmembrane domains) with the -, ␦-, and -opioid receptors (1, 2). Classical opioid ligands do not bind to ORL1, but orphanin FQ͞nociceptin (OFQ͞N), a 17-amino acid neuropeptide purified from brain extracts, was found to be the natural ligand of the G protein-coupled receptor ORL1 (3, 4). OFQ͞N, its precursor peptide, and its receptor ORL1 are located in corticolimbic regions involved in the integration of the emotional components of fear and stress as well as in the spinal cord, with a pattern distinct from that of opioid peptides and receptors in rodents (5-9). The expression of OFQ͞N or its receptor in the amygdaloid complex, septohippocampal region, periaqueductal gray matter, locus coeruleus, and dorsal raphe nucleus suggests that major brain neuronal systems may be sensitive to the action of OFQ͞N. Such sensitivity has widespread implications for many aspects of behavior including arousal, attention, neuroendocrine control, fear, and anxiety (10). In brain slices, OFQ͞N has potent inhibitory actions on neurons in the dorsal raphe nucleus, the locus coeruleus, the periaqueductal gray matter, and the amygdala (11)(12)(13)(14). In general, OFQ͞N plays an inhibitory role on synaptic transmission in the central nervous system and thereby may contribute to a reduction in responsiveness to stress. When given intracerebroventricularly to rodents, OFQ͞N reduces elementary stress-induced physiological respon...

The ATP-dependent drug efflux pump P-glycoprotein (P-gp) affects the absorption and disposition of many compounds. P-gp may also play role in clinically significant drug-drug interactions. Therefore, it is important to find potential substrates or inhibitors of P-gp early in the drug discovery process. To identify compounds that interact with this transporter, several P-gp assays were validated and compared by testing a set of 28 reference compounds, including inhibitors of cytochrome P450 3A4 (CYP3A4). The assays included in silico predictions, inhibition assays (based on cellular uptake of rhodamine-123 or calcein AM), and functional assays (ATPase activity assay and transcellular transport assay, the latter for a subset of compounds). In addition, species differences were studied in an indirect fluorescence indicator screening assay and test systems expressing porcine, mouse, or human P-gp. Our results suggest that several P-gp assays should be used in combination to classify compounds as substrates or inhibitors of P-gp. Recommendations are given on screening strategies which can be applied to different phases of the drug discovery and development process.

The inhibition of human cytochrome P450s (CYPs) is one of the most common mechanisms which can lead to drug-drug interactions. The inhibition of CYPs can be reversible (competitive or non-competitive) or irreversible. Irreversible inhibition usually derives from activation of a drug by CYPs into a reactive metabolite, which tightly binds to the enzyme active site, leading to a long lasting inactivation. This process is called "mechanism based inhibition" or "suicide inhibition". The irreversible inactivation usually implies the formation of a covalent bond between the metabolite and the enzyme, which can lead to hapten formation and can in some cases trigger an autoimmune-response. For these reasons it is of utmost importance to study the mechanism of the CYP inhibition of new potential drugs as early as possible during the drug discovery process. The literature on CYPs is vast and covers numerous aspects of their biology and biochemistry, however to our knowledge there is no general and systematic review focusing on mechanism-based inhibitors; we have reviewed the literature and compiled all the available data on chemical entities, which are known to be CYP suicide inhibitors. Each compound is reported together with its chemical structure, the CYP isoform and the parameters describing the inactivation. Literature references are reported together with their PMID (PubMed ID number) to allow a fast retrieval of the papers. This review offers a quick reference to help predict liabilities of new chemical entities without carrying out extensive in vitro work, and will hopefully help in designing safer drugs.

The effects of sulfaphenazole, 1, on typical activities catalyzed by human cytochromes P450 of the 1A, 3A, and 2C subfamilies expressed in yeast were studied. 1 acts as a strong, competitive inhibitor of CYP 2C9 (K(i) = 0.3 +/- 0.1 microM); it is much less potent toward CYP 2C8 and 2C18 (K(i) = 63 and 29 microM, respectively) and fails to inhibit CYP 1A1, 1A2, 3A4, and 2C19. From difference visible spectroscopy experiments using microsomes of yeast expressing various human P450s, 1 selectively interacts only with CYP 2C9 with the appearance of a peak at 429 nm as expected for the formation of a P450 Fe(III)-nitrogenous ligand complex (Ks = 0.4 +/- 0.1 microM). Comparative studies of the spectral interaction and inhibitory effects of twelve compounds related to 1 with CYP 2C9 showed that the aniline function of 1 is responsible for the formation of the iron-nitrogen bond of the 429 nm-absorbing complex and is necessary for the inhibitory effects of 1. The study of two new compounds synthesized during this work, in which the N-phenyl group of 1 was replaced with either an ethyl group or a 3,4-dichlorophenyl group, showed that the presence of an hydrophobic substituent at position 1 of the pyrazole function of 1 is required for a strong interaction with CYP 2C9. A model for the binding of 1 in the CYP 2C9 active site is proposed; that takes into account three major interactions that should be at the origin of the high-affinity and specific inhibitory effects of 1 toward CYP 2C9: (i) the binding of its nitrogen atom to CYP 2C9 iron, (ii) an ionic interaction of its SO2N- anionic site with a cationic residue of CYP 2C9, and (iii) an interaction of its N-phenyl group with an hydrophobic part of the protein active site.