Toxicological in vitro and subchronic evaluation of LASSBio-596.

LASSBio-596, 2-[4-(1,4-tiazinan-4-ylsulfonyl) phenylcarbamoyl] benzoic acid, is an achiral compound containing a subunit carboxylic amide, was capable of preventing induced mechanical and morphological changes in the lungs that commonly caused the onset of asthma. Previous studies to determine the acute toxicity of oral LASSBio-596 at dose of 2000mg/kg caused no deaths in any of the tested animals. To further evaluate the safety of LASSBio-596, in vitro and in vivo tests were carried out. Regarding to in vitro test were used renal, hepatic, pulmonary, cardiac, neurologic and intestinal cell lines. They were evaluated using neutral red (NR) and [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assays. Micronuclei also was performed. Concerning to in vivo was performed subchronic on Wistar rats at doses of 10, 50, and 250mg/kg and zebrafish test. The in vitro tests results showed the safety of LASSBio-596. However, subchronic toxicity study results revealed changes in the blood parameters of amylase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose and creatine kinase (CK) which is used for cardiotoxicity evaluation, although, did not identify any histopathological alterations. However, zebrafish test demonstrated cardiac damage. It was impossible to estimate the no-observed-adverse-effect-levels and lowest observed-adverse-effect level due to the presence of cardiotoxicity in all tested doses.

Stimulation of the proximal tubule Na+-ATPase activity by adenosine A(2A) receptor.

The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.