In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties.

BACKGROUND AND PURPOSE: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). EXPERIMENTAL APPROACH: We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. KEY RESULTS: A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.

Can 'differential display' methodologies make an impact on biological psychiatry?

The past decade has been marked by a dramatic increase in the availability of techniques to identify and clone genes that are differentially expressed in disease states and by drug treatments. The applications of such techniques to problems in biological psychiatry are manifold and the implications of discovering novel and/or known genes that are perturbed in neuropsychiatric disorders profound. While there are success stories, it is becoming ever more apparent that each of these techniques has its limitations, particularly when applied to the central nervous system. Given that these methods (e.g. differential display, RNA fingerprinting, suppression-subtractive hybridization, microarrays) are labour-intensive and potentially time-consuming, it is important to understand these limitations. For example, differential display is capable of detecting very small changes in the expression of mRNA species. Methods like suppression-subtractive hybridization are better suited to examine potential differences in rare transcripts, but only when their expression is changed substantially (currently ? 5-fold). Moreover, both the functional and morphological organization of the central nervous system present challenges that may not be encountered in other systems. In this overview, we will discuss the advantages and disadvantages of some of these approaches and their application to research in biological psychiatry.

A single amino acid residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors.

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA(A) receptors containing an alpha(5) subunit. A single amino acid residue (alpha(5)Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha(5)Ile215 with the cognate amino acid contained in the alpha(1) subunit (Val211) reduced the selectivity of RY-80 for alpha(5)beta(3)gamma(2) receptors from approximately 135- to approximately 8-fold compared with alpha(1)beta(3)gamma(2) receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for alpha(5)beta(3)gamma(2) receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the alpha(1) subunit with the cognate amino acid contained in the alpha(5) subunit (i.e., alpha(1)V211I) increased the affinities of alpha(5)-selective ligands by a approximately 20-fold and reduced by 3-fold the affinity of an alpha(1)-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of alpha(5)215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for alpha(5)-containing GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the alpha(5)I215V mutation. These data indicate that residue alpha(5)215 may not participate in formation of the lipophilic L(2) pocket that has been proposed to contribute to the unique pharmacological properties of alpha(5)-containing GABA(A) receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type alpha(5)beta(3)gamma(2) and mutant alpha(5)I215Kbeta(3)gamma(2) receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha(5)I215Vbeta(3)gamma(2) and alpha(5)I215Tbeta(3)gamma(2) receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue alpha(5)215 is a determinant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha(5) subunit.

[Expression of the human alpha-1-antitrypsin gene in Escherichia coli]. / Ekspressiia gena alpha-1-antitripsina cheloveka v kletkakh Escherichia coli.

The cDNA, containing the complete human alpha-1-antitrypsin (AT) sequence starting from codon 2, was used to construct bacterial strains producing AT. The fusion protein was obtained by junction of the AT cDNA to the fragment of an Escherichia coli ompF gene. We have also modified the AT cDNA's 5'-terminal part to construct DNAs containing ATG-codon and cDNA sequences starting from codons 1 or 2. These DNAs were inserted into E. coli expression vectors pBR322/trpII-8 and pKK223-3 that allow transcription from efficient trp- and tac-promoters. This construction resulted in the induction of a 46 kDa protein. The polypeptide produced was recognized by an antiserum raised against human alpha-1-antitrypsin protein. Truncation of the gene at its 5'-end or synthesis as a fusion OmpF-AT protein increases expression up to 10-fold, to a level of approximately 1%. On the contrary, no dependence on the promoter type has been observed. Physical properties of the recombinant proteins are discussed.