The WMI Rat of Premature Cognitive Aging Presents Intrinsic Vulnerability to Oxidative Stress in Primary Neurons and Astrocytes Compared to Its Nearly Isogenic WLI Control.

The primary neuronal and astrocyte culture described here is from the stress-hyperreactive Wistar Kyoto (WKY) More Immobile (WMI) rat with premature aging-related memory deficit, and its nearly isogenic control, the Less Immobile (WLI) strain. Primary WMI hippocampal neurons and cortical astrocytes are significantly more sensitive to oxidative stress (OS) generated by administration of H2O2 compared to WLI cells as measured by the trypan blue cell viability assay. Intrinsic genetic vulnerability is also suggested by the decreased gene expression in WMI neurons of catalase (Cat), and in WMI cortical astrocytes of insulin-like growth factor 2 (Igf2), synuclein gamma (Sncg) and glutathione peroxidase 2 (Gpx2) compared to WLI. The expressions of several mitochondrial genes are dramatically increased in response to H2O2 treatment in WLI, but not in WMI cortical astrocytes. We propose that the vulnerability of WMI neurons to OS is due to the genetic differences between the WLI and WMI. Furthermore, the upregulation of mitochondrial genes may be a compensatory response to the generation of free radicals by OS in the WLIs, and this mechanism is disturbed in the WMIs. Thus, this pilot study suggests intrinsic vulnerabilities in the WMI hippocampal neurons and cortical astrocytes, and affirm the efficacy of this bimodal in vitro screening system for finding novel drug targets to prevent oxidative damage in illnesses.

I. Discovery of a novel series of CXCR3 antagonists. Multiparametric optimization of N,N-disubstituted benzylamines.

N,N-Disubstituted benzylamine derivatives have been identified as CXCR3 antagonists. Compounds were optimized to improve affinity and selectivity, to increase metabolic stability in human and mouse liver microsomes, to increase Caco-2 permeability. Optimization was supported by monitoring physico-chemical properties using both experimental and computational means. Several compounds with double-digit nanomolar CXCR3 affinity, favorable selectivity, microsomal stability, Caco-2 permeability and human hepatocyte clearance have been identified.

Interaction of SSR161421, a novel specific adenosine A(3) receptor antagonist with adenosine A(3) receptor agonists both in vitro and in vivo.

A novel adenosine A(3) receptor antagonist (SSR161421) was characterized by both receptor binding assays and pharmacological tests. Binding studies on cloned human adenosine receptors showed that SSR161421 has high affinity for adenosine hA(3) receptors (K(i)=0.37 nM) with at least 1000-fold selectivity compared to hA(1), hA(2A) and hA(2B) receptors. The receptor antagonist nature of SSR161421 was determined in a functional study on Chinese hamster ovarian cells (CHO) cells expressing human adenosine A(3) receptors. SSR161421 competitively antagonized the effect of 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) on cAMP production with a pA2 value in a luciferase reporter gene construct. In mice, intravenously administered SSR161421 inhibited the N6-(4-aminobenzyl)-adenosine-5'-N-methyl-uronamide dihydrochloride (AB-MECA) induced increase in plasma histamine levels (ED(50)=2.0mg/kg) and the Cl-IB-MECA evoked plasma extravasation (ID(50)=2.9 mg/kg) and oedema formation (ID(50)=4.6 mg/kg) in mouse ear.

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.

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).

Biochemical and pharmacological characterization of 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071), a novel, orally active elastase inhibitor.

Human leukocyte elastase (HLE) is a proteinase capable of degrading a variety of proteins. Under normal circumstances, the proteolytic activity of HLE is effectively controlled by its natural inhibitors. However, an imbalance between elastase and its endogenous inhibitors may result in several pathophysiological states such as chronic obstructive pulmonary disease, asthma, emphysema, cystic fibrosis, and chronic inflammatory diseases. It is anticipated that an orally active HLE inhibitor could be useful for the treatment of these diseases. 2-(9-(2-Piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071) is a potent inhibitor of HLE, with the inhibition constant (K(i)) and the constant for inactivation process (k(on)) being 0.0168 +/- 0.0014 nM and 0.183 +/- 0.013 10(6)/mol sr, respectively. The dissociation rate constant, k(off), was 3.11 + 0.37 10(-6)/s. SSR69071 displays a higher affinity for human elastase than for rat (K(i) = 3 nM), mouse (K(i) = 1.8 nM), and rabbit (K(i) = 58 nM) elastases. Bronchoalveolar lavage fluid from mice orally treated with SSR69071 inhibits HLE (ex vivo), and in this model, SSR69071 has a dose-dependent efficacy with an ED(50) = 10.5 mg/kg p.o. SSR69071 decreases significantly the acute lung hemorrhage induced by HLE (ED(50) = 2.8 mg/kg p.o.) in mice. Furthermore, SSR69071 prevents carrageenan- (ED(30) = 2.2 mg/kg) and HLE-induced (ED(30) = 2.7 mg/kg) paw edema in rats after p.o. administration. In conclusion, SSR69071 is a selective, orally active, and potent inhibitor of HLE with good penetration in respiratory tissues.