Anti-pneumoscystis carinni activitiy of primaquine imidazolidin-4-ones

Pneumocystis pneumonia (PCP) is one of the most frequent causes of mortality among HIV-infected patients. Primaquine (PQ) is an antimalarial 8-aminoquinoline effective against PCP when given in combination with clindamycin. This has drawn the attention of Medicinal Chemists towards the anti-PCP activity of 8-aminoquinolines, not only confined to those exhibiting antimalarial activity [1]. It is thought that anti-PCP 8-aminoquinolines exert their anti-PCP activity by acting on the electronic transport and redox system of the P. carinii pathogen [1]. Recently, our research group has been developing imidazolidin-4-one derivatives of PQ (Scheme 1), targeting novel compounds with improved therapeutic action, namely, higher resistance to metabolic inactivation, lower toxicity and equal or higher antimalarial activity than that of the parent drug [2,3]. These imidazolidin-4-ones were seen to block the transmission of rodent malaria, caused by Plasmodium berghei on BalbC mice, to the mosquito vector Anopheles stephensi [3]. The anti-PCP activity of our PQ derivatives is now under study and preliminary in vitro assays [4] show that some of the compounds exhibit slight to moderate activity after a 72 h incubation period against P. carinii. In one case, the IC50 was comparable to that of parent PQ. Both these studies and forthcoming results from ongoing biological assays will be presented and discussed.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.