Endre Mikus

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).

Potential L-type voltage-operated calcium channel blocking effect of drotaverine on functional models

Drotaverine is considered an inhibitor of cyclic-3′,5′-nucleotide-phophodiesterase (PDE) enzymes; however, published receptor binding data also support the potential L-type voltage– operated calcium channel (L-VOCC) blocking effect of drotaverine. Hence, in this work, we focus on the potential L-VOCC blocking effect of drotaverine by using L-VOCC–associated functional in vitro models. Accordingly, drotaverine and reference agents were tested on KCl-induced guinea pig tracheal contraction. Drotaverine, like the L-VOCC blockers nifedipine or diltiazem, inhibited the KCl-induced inward Ca2+- induced contraction in a concentration- dependent fashion. The PDE inhibitor theophylline had no effect on the KCl-evoked contractions, indicating its lack of inhibition on inward Ca2+ flow. Drotaverine was also tested on the L-VOCC–mediated resting Ca2+ refill model. In this model, the extracellular Ca2+ enters the cells to replenish the emptied intracellular Ca2+ stores. Drotaverine and L-VOCC blocker reference molecules inhibited Ca2+ replenishment of Ca2+-depleted preparations detected by agonist-induced contractions in post–Ca2+ replenishment Ca2+-free medium. Theophylline did not modify the Ca2+ store replenishment after contraction. It seems that drotaverine, but not theophylline, inhibits inward Ca2+ flux. The addition of CaCl2 to Ca2+-free medium containing the agonist induced inward Ca2+ flow and subsequent contraction of Ca2+-depleted tracheal preparations. Drotaverine, similar to the L-VOCC blockers, inhibited inward Ca2+ flow and blunted the slope of CaCl2-induced contraction in agonist containing Ca2+-free medium with Ca2+-depleted tracheal preparations. These results show that drotaverine behaves like L-VOCC blockers but, unlike PDE inhibitors using L-VOCC associated in vitro experimental models.

Assessment of the Airway Smooth Muscle Relaxant Effect of Drotaverine

Background: Drotaverine, a type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, blocks the degradation of 3’,5’-cyclic adenosine monophosphate. However, published receptor binding data showed that drotaverin also binds to the L-type voltage-operated calcium channel (L-VOCC). Based on these molecular mechanisms of action, a direct and indirect (by blocking the constrictor response) relaxant effect on airway smooth muscle can be predicted, which has not yet been assessed. Summary: Accordingly, drotaverine and reference agents were tested both on the histamine-, methacholine-, or KCl-induced contraction response and on precontracted guinea pig tracheal preparations. It was found that drotaverine not only relaxed the precontracted tracheal preparations but also decreased mediator-induced contraction. These effects of drotaverine were concentration dependent, with a significantly higher potency on the KCl-induced response, than on either the histamine or methacholine induced one. A similar result was noted for nifedipine. The PDE inhibitor, theophylline, also relaxed the precontracted preparations but was ineffective on the mediator-induced contraction in a physiologically relevant concentration range. Moreover, theophylline did not show selectivity and was the least potent relaxant among the 3 tested molecules. Key Message: These results show that drotaverine is a more potent airway smooth muscle relaxant molecule than theophylline. This enhanced potency on relaxation and inhibition of the constrictor response, at least partly, may be explained by the combined L-VOCC blocking and PDE inhibitory potential of drotaverine.

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