Peter Aranyi

A novel orally active inhibitor of HLE.

Human leukocyte elastase (HLE) is a serine proteinase, capable of degrading a variety of structural matrix proteins. SSR69071 2-[(4-isopropyl-6-methoxy-1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methoxy]-9-(2-piperidin-1-ylethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one was selected as a novel orally active HLE inhibitor for treatment of chronic obstructive pulmonary diseases, asthma, emphysema, cystic fibrosis and several inflammatory diseases (WO 01/44245 A1) (J. Pharm. Exp. Ther., submitted for publication).

A novel specific binding site for homophthalazines in the rat brain

The specific binding sites of a homophthalazine, girisopam, in rat brain have been localized by qualitative and quantitative autoradiography. This substance exerts strong anxiolytic and antipsychotic effects both in rodents and in humans. High labeling was present in all major components of the extrapyramidal system, such as the caudate-putamen, globus pallidus, subthalamic nucleus, substantia nigra, and the extrapyramidal portion of the accumbens nucleus and the olfactory tubercle, while specific labeling was not seen in any other brain areas including the cerebral cortex, thalamus, cerebellum or brainstem areas. This novel distribution of girisopam is consistent with its antipsychotic effect and anxiolytic properties and may provide a morphological basis for further studies to elucidate the mechanisms of action of homophthalazines in the central nervous system.

Autoradiographic localization and quantitative determination of specific binding sites of anxiolytic homophthalazines (formerly called 2,3-benzodiazepines) in the striato-pallido-nigral system of rats

Homophthalazines (2,3-benzodiazepin-derivates, such as tofisopam, nerisopam, girisopam) constitute a drug family with strong anxiolytic and antipsychotic potencies. By autoradiography, all of these drugs showed a specific distribution pattern of binding sites exclusively in brain areas which relate to the striato-pallido-nigral system, while no specific label was found in any other brain areas in the rat. Quantitative analyses of the autoradiograms by computerized densitometry, as well as by a receptor binding assay on 32 microdissected brain areas showed very high concentrations of tritiated homophthalazines in the glubus pallidus, caudate nucleus, putamen and the substantia nigra. Relatively high density of binding sites was measured in the nucleus accumbens, the olfactory tubercle, the entopeduncular nucleus and the subthalamic nucleus. Concentrations measured in the cerebral cortical areas, cerebellum or brainstem nuclei did not differ from the background. No significant differences were found between the homophthalazines investigated in terms of the distribution patterns or density of binding sites.

A novel specific binding site for anxiolytic homophthalazines in the rat brain

Radioligand binding studies were performed in order to elucidate the mechanism of action of anxiolytic‐neuroleptic homophthalazines. Rat striatal membrane preparations were found to bind 3H‐EGIS 6775 [3H‐GYKI‐52 322, 3H‐(I‐(4‐aminophenyl)‐4‐methyl‐7,8‐dimethoxy‐5H‐homophthalazine)] in a specific and displaceable manner. Several other brain regions tested were devoid of similar binding activity. Saturation analysis revealed that binding affinity was in the 10−5‐10−7 M range. Binding was enhanced by Mg2+ ions and, to a smaller extent by Ca2+ ions. The binding principle was sensitive to heat or trypsin treatment. This specific binding site appears, according to competition studies, different from the receptors whose presence in the rat striatum has been reported earlier.

Prolyl Oligopeptidase Inhibition by N-Acyl-pro-pyrrolidine-type Molecules †

Three novel, N-acyl-pro-pyrrolidine-type, inhibitors of prolyl oligopeptidase (POP) with nanomolar activities were synthesized and their binding analyzed to the host enzyme in the light of X-ray diffraction and molecular modeling studies. We were interested in the alteration in the binding affinity at the S3 site as a function of the properties of the N-terminal group of the inhibitors. Our studies revealed that, for inhibitors with flat aromatic terminal groups, the optimal length of the linker chain is three C-C bonds, but this increases to four C-C bonds if there is a bulky group in the terminal position. Molecular dynamics calculations indicate that this is due to the better fit into the binding pocket. A 4-fold enhancement of the inhibitor activity upon replacement of the 4-CH2 group of the proline ring by CF2 is a consequence of a weak hydrogen bond formed between the fluorine atom and the hydroxy group of Tyr473 of the host enzyme. There is notably good agreement between the calculated and experimental free energies of binding; the average error in the IC50 values is around 1 order of magnitude.

Drotaverine interacts with the L-type Ca2+ channel in pregnant rat uterine membranes

The effect of the isoquinoline derivative, drotaverine on the specific binding of [(3)H]nitrendipine and [(3)H]diltiazem to pregnant rat uterine membranes was examined. Drotaverine inhibited the specific [(3)H]nitrendipine and [(3)H]diltiazem bindings with IC(50) values of 5.6 and 2.6 microM, respectively. Saturation studies showed that diltiazem caused a significant increase in the maximum binding density without changing the K(D) of [(3)H]nitrendipine while drotaverine increased both the K(D) and the B(max) of [3H]nitrendipine. The dissociation kinetics of both [3H]nitrendipine and [(3)H]diltiazem were accelerated by drotaverine. These results suggest that drotaverine has a negative allosteric interaction with the binding sites for 1,4-dihydropyridines and 1,5-benzothiazepines on the L-type Ca(2+) channel in pregnant rat uterine membranes, which may have implications as to the potential usefulness of this drug in aiding child delivery.

Effect of novel adenosine A3 receptor antagonist SSR161421 in allergic sheep models

Sheep is a species that shows 85% overall identity of the adenosine A3 receptor with its human ortholog. In this study we demonstrate a role for the adenosine A3 receptor in ovine allergic airway responses. In isolated tracheal preparations taken from sheep naturally sensitive to Ascaris suum, antigen challenge evoked airway smooth muscle contraction. The smooth muscle contractile response was dependent on the applied Ascaris suum antigen dose. The response to a threshold dose of antigen was enhanced in the presence of an adenosine A3 agonist AB-MECA (10-6M), suggesting that adenosine A3 receptor function contributed to the enhanced allergen response. This was confirmed by showing that a selective adenosine A3 receptor antagonist SSR16142, inhibited the increased antigen response with EC50=2•10M. We then tested the effects of SSR161421, given intravenously, on antigen-induced responses in allergic sheep following inhalation of Ascaris suum, in-vivo. SSR161421 (0.3-3 mg/kg) provided dose-dependent inhibition of the antigen-induced late bronchoconstrictor responses and the post antigen-induced airway hyper responsiveness. This is the first demonstration that a selective adenosine A3 receptor antagonist inhibits antigen-induced responses in a species with an A3 receptor that bears significant similarity to its human counterpart. Correspondence to: Endre G Mikus, LabMagister Training and Science Ltd., H-1045 Budapest To street 1-5, Hungary; E-mail: endre.mikus@labmagister.com