Virtual screening and biological evaluation of novel antipyretic compounds.

Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally (i.v.) or centrally (i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors.

Central mediators involved in the febrile response: effects of antipyretic drugs.

Fever is a complex signal of inflammatory and infectious diseases. It is generally initiated when peripherally produced endogenous pyrogens reach areas that surround the hypothalamus. These peripheral endogenous pyrogens are cytokines that are produced by leukocytes and other cells, the most known of which are interleukin-1ß, tumor necrosis factor-α, and interleukin-6. Because of the capacity of these molecules to induce their own synthesis and the synthesis of other cytokines, they can also be synthesized in the central nervous system. However, these pyrogens are not the final mediators of the febrile response. These cytokines can induce the synthesis of cyclooxygenase-2, which produces prostaglandins. These prostanoids alter hypothalamic temperature control, leading to an increase in heat production, the conservation of heat, and ultimately fever. The effect of antipyretics is based on blocking prostaglandin synthesis. In this review, we discuss recent data on the importance of prostaglandins in the febrile response, and we show that some endogenous mediators can still induce the febrile response even when known antipyretics reduce the levels of prostaglandins in the central nervous system. These studies suggest that centrally produced mediators other than prostaglandins participate in the genesis of fever. Among the most studied central mediators of fever are corticotropin-releasing factor, endothelins, chemokines, endogenous opioids, and substance P, which are discussed herein. Additionally, recent evidence suggests that these different pathways of fever induction may be activated during different pathological conditions.

Involvement of PGE2 and RANTES in Staphylococcus aureus-induced fever in rats.

This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 × 10(10) colony-forming units (CFU)/cavity, and body temperature (T(b)) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T(b), whereas the two higher doses promoted similar long-lasting and sustained T(b) increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T(b) increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 µg/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

A crucial role for IL-6 in the CNS of rats during fever induced by the injection of live E. coli.

Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 have been established as important mediators of fever induced by lipopolysaccharide (LPS) from Gram-negative bacteria. Whether these pro-inflammatory cytokines are also important in mediating fever induced by live bacteria remains less certain. We therefore investigated the following: (1) the synthesis of TNF-α, IL-1ß, and IL-6 during E. coli-induced fever and (2) the effect of blocking the action of cytokines within the brain on E. coli-induced fever. Body or tail skin temperature (bT or Tsk, respectively) was measured by biotelemetry or telethermometry, every 30 min, during 6 or 24 h. Depending on the number of colony-forming units (CFU) injected i.p., administration of E. coli induced a long-lasting increase in bT of male Wistar rats. The duration of fever did not correlate with the number of CFU found in peritoneal cavity or blood. Because 2.5 × 10(8) CFU induced a sustained fever without inducing a state of sepsis/severe infection, this dose was used in subsequent experiments. The E. coli-induced increase in bT was preceded by a decrease in Tsk, reflecting a thermoregulatory response. TNF-α, IL-1ß, and IL-6 were detected at 3 h in serum of animals injected i.p. with E. coli. In the peritoneal exudates, TNF-α, IL-1ß, and IL-6 were detected at 0.5 and 3 h after E. coli administration. Moreover, both IL-1ß and IL-6, but not TNF-α, were found in the cerebrospinal fluid (CSF) and hypothalamus of animals injected with E. coli. Although pre-treatment (i.c.v., 2 µl, 15 min before) with anti-IL-6 antibody (anti-IL-6, 5 µg) reduced E. coli-induced fever, pre-treatment with either IL-1 receptor antagonist (IL-1ra, 200 µg) or soluble TNF receptor I (sTNFRI, 500 ng) had no effect on the fever response. In conclusion, replicating E. coli promotes an integrated thermoregulatory response in which the central action of IL-6, but not IL-1 and TNF, appears to be important.

Cyclooxygenase-independent mechanism of ibuprofen-induced antipyresis: the role of central vasopressin V1 receptors.

This study compared the antipyretic effects of ibuprofen and indomethacin regarding the efficacy in blocking fevers induced by lipopolysaccharide (LPS from E. coli) or pyrogenic mediators that act on prostaglandin (PG)-dependent and PG-independent pathways. The content of PGE2 in the cerebrospinal fluid (CSF) and the dependence on central arginine vasopressin (AVP) release by both antipyretics were also compared during the reduction of LPS-induced fever. Finally, we investigated the effect of ibuprofen on hypothalamic cytokine content during LPS-induced fever. Ibuprofen (intraperitoneally, i.p.) dose-dependently inhibited the fever induced by LPS (intravenously, i.v.). Indomethacin (2 mg/kg) and ibuprofen (10 mg/kg) reduced the fever induced by i.c.v. injection of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, or arachidonic acid (AA). Ibuprofen, but not indomethacin, inhibited i.c.v. endothelin-1- and pre-formed pyrogenic factor (PFPF)-induced fever. Neither ibuprofen nor indomethacin affected fever by PGE2 , PGF(2α) , or corticotrophin-releasing factor (CRF); however, both reduced the CSF PGE2 content after LPS. Bilateral injection of the AVP V(1) receptor antagonist d(CH2)5 Tyr(Me)AVP into the ventral septal area blocked both ibuprofen- and indomethacin-induced antipyresis. Ibuprofen did not modify the hypothalamic increase in either IL-1ß or IL-6 induced by LPS. In conclusion, although the antipyretic effect of ibuprofen involves the blockage of central production of PGE2 and the endogenous release of AVP, differently from low dose of indomethacin, ibuprofen not only reduced the fever induced by PGE2 -dependent, but also, that induced by PGE2 -independent endogenous pyrogens. Moreover, ibuprofen does not affect the hypothalamic synthesis/release of IL-1ß and IL-6.

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus.

BACKGROUND AND PURPOSE: Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE(2) content in plasma, CSF and hypothalamus induced by either LPS or ET-1. EXPERIMENTAL APPROACH: Rats were given (i.p.) dipyrone (120 mg·kg(-1)) or indomethacin (2 mg·kg(-1)) 30 min before injection of LPS (5 µg·kg(-1), i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE(2) levels were determined in the plasma, CSF and hypothalamus by elisa. KEY RESULTS: LPS or ET-1 induced fever and increased CSF and hypothalamic PGE(2) levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE(2) but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE(2) levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE(2) levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE(2) levels. Dipyrone also reduced the increase in the venous plasma PGE(2) concentration induced by LPS. CONCLUSIONS AND IMPLICATIONS: These findings confirm that PGE(2) does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE(2) synthesis.

Inflammatory mediators involved in the nociceptive and oedematogenic responses induced by Tityus serrulatus scorpion venom injected into rat paws.

The present study investigated the role of kinins, prostaglandins (PGs) and nitric oxide (NO) in mechanical hypernociception, spontaneous nociception and paw oedema after intraplantar (ipl) injection of Tityus serrulatus venom (Tsv) in male Wistar rats. Tsv was ipl-injected in doses of 0.01-10microg/paw. Pre-treatment (30min prior) with DALBK (100nmol/paw) and icatibant (10nmol/paw), B1 and B2 selective kinin receptor antagonists, L-NAME (50mg/kg, i.p., a non-selective nitric oxide synthase inhibitor) or celecoxib, selective COX-2 inhibitor, was given 1h prior per os (5mg/kg, p.o.), significantly reduced the hypernociceptive response (Von Frey method), the spontaneous nociception (determined by counting the number of flinches) and paw oedema (plethysmometer method) induced by Tsv at doses of 1.0 and 10microg/paw for both nociceptive and oedematogenic responses, respectively. Nevertheless, indomethacin (5mg/kg, i.p., 30min prior) was ineffective in altering all of these events. The results of the present study show that Tsv, injected ipl into the rat paw, causes a dose-dependent paw oedema, mechanical hypernociception and flinches (a characteristic biphasic response) in which kinins and NO are substantially involved. Although celecoxib was effective against the oedema and pain caused by Tsv, COX-2 does not seem to be involved in the inflammatory response caused by Tsv.

CCR1 and CCR5 chemokine receptors are involved in fever induced by LPS (E. coli) and RANTES in rats.

This study, besides examining the involvement of CCR1 and CCR5 receptors in the LPS-induced fever (lipopolysaccharide, Escherichia coli) in male Wistar rats, evaluated if RANTES (regulated on activation, normal T cells expressed and secreted) injected into the preoptic area of the anterior hypothalamus (AH/POA) would promote an integrated febrile response via these receptors. Moreover, the effects of selective and non-selective cyclooxygenase blockers on both fever and the level of prostaglandin (PG)E(2) in the cerebrospinal fluid (CSF) after injection of RANTES into the AH/POA were also investigated. Met-RANTES, CCR1 and CCR5 receptor antagonist, reduced LPS-evoked fever dose dependently. RANTES microinjected into the AH/POA increased the rectal temperature of rats dose dependently and caused a significant decrease in the tail skin temperature and an increase (at 2.5 and 5 h) of the levels of PGE(2) in the CSF. Met-RANTES prevented the fever induced by RANTES. Ibuprofen abolished the fever caused by RANTES between 60 min and 2.5 h, and it reduced the temperature until the end of observation period. Celecoxib blocked the RANTES-induced fever, while indomethacin reduced it in the last 60 min of the experimental period. At 2.5 and 5 h all antipyretics brought the CSF PGE(2) level near to the control. These results indicate that CCR1 and CCR5 receptors are involved in the fever induced by systemic LPS and intrahypothalamic RANTES. RANTES promotes an integrated febrile response accompanied by an increase of CSF PGE(2). The inhibitory effects of celecoxib and ibuprofen suggest that PGE(2) was generated via COX-2. As indomethacin dissociates fever and the decrease of PGE(2) level during the RANTES-induced fever, an alternative COX-2-independent pathway or other mechanisms of action of celecoxib and ibuprofen might be considered.