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PLoS One
2014 Jan 01;96:e99510. doi: 10.1371/journal.pone.0099510.
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LASSBio-1135: a dual TRPV1 antagonist and anti-TNF-alpha compound orally effective in models of inflammatory and neuropathic pain.
Lima CK
,
Silva RM
,
Lacerda RB
,
Santos BL
,
Silva RV
,
Amaral LS
,
Quintas LE
,
Fraga CA
,
Barreiro EJ
,
Guimaraes MZ
,
Miranda AL
.
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LASSBio-1135 is an imidazo[1,2-a]pyridine derivative with high efficacy in screening models of nociception and inflammation, presumed as a weak COX-2 inhibitor. In order to tease out its mechanism of action, we investigated others possible target for LASSBio-1135, such as TNF-α and TRPV1, to better characterize it as a multitarget compound useful in the treatment of chronic pain. TRPV1 modulation was assessed in TRPV1-expressing Xenopus oocytes against capsaicin and low pH-induced current. Modulation of TNF-α production was evaluated in culture of macrophages stimulated with LPS. In vivo efficacy of LASSBio-1135 was investigated in carrageenan and partial sciatic ligation-induced thermal hyperalgesia and mechanical allodynia. Corroborating its previous demonstration of efficacy in a model of capsaicin-induced hyperalgesia, LASSBio-1135 blocks capsaicin-elicited currents in a non-competitive way with an IC50 of 580 nM as well as low pH-induced current at 50 µM. As an additional action, LASSBio-1135 inhibited TNF-α release in these cells stimulated by LPS with an IC50 of 546 nM by reducing p38MAPK phosphorilation. Oral administration of 100 µmol x Kg(-1) LASSBio-1135 markedly reduced thermal hyperalgesia induced by carrageenan, however at 10 µmol x Kg(-1) only a partial reduction was observed at the 4th h. Neutrophil recruitment and TNF-α production after carrageenan stimulus was also inhibited by the treatment with LASSBio-1135. Modulating TRPV1 and TNF-α production, two key therapeutic targets of neuropathic pain, 100 µmol x Kg(-1) LASSBio-1135 was orally efficacious in reversing thermal hyperalgesia and mechanical allodynia produced by partial sciatic ligation 7-11 days after surgery without provoking hyperthermia, a common side effect of TRPV1 antagonists. In conclusion LASSBio-1135, besides being a weak COX-2 inhibitor, is a non-competitive TRPV1 antagonist and a TNF-α inhibitor. As a multitarget compound, LASSBio-1135 is orally efficacious in a model of neuropathic pain without presenting hyperthermia.
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24941071
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Figure 2. LASSBio-1135 inhibits TNF-α production in murine macrophages stimulated with LPS by blockade of p38 MAPK signaling pathway.A) Representative graph showing that LASSBio-1135 inhibits TNF-α release by macrophages stimulated with LPS (100 ng.ml−1) at three different concentrations. Results were analyzed using One-way ANOVA followed by Dunnett post-test (n = 3–4 experiments per group, *p<0.05 compared to DMSO group). B) Concentration-response curve of LASSBio-1135. The IC50 estimated was 642 nM (n = 3–4 experiments per concentration). D) LASSBio-1135 interferes with cell viability only at 100 µM. Results were analyzed using One-way ANOVA followed by Dunnett post-test (n = 3–4 experiments per group, *p<0.05 compared to DMSO group) C) LASSBio-1135 reduces p38 MAPK activation induced by LPS in a concentration-dependent fashion. Results were analyzed using One-way ANOVA followed by Dunnett post-test (n = 3 experiments per group, *p<0.05 compared to DMSO group).
Figure 3. LASSBio-1135 inhibits carrageenan-induced thermal hyperalgesia by reducing TNF-α production and neutrophils infiltration in the injured paw.A) Oral pre-treatment with LASSBio-1135 at 10 and 100 µmol.Kg−1 1 hour before carrageenan injection into the paw, reduces thermal hyperalgesia at different time points.Results were analyzed using Two-way ANOVA followed by Bonferroni post-test. (n = 6–8 animals per group, *p<0.05 compared to vehicle group). B) 4 hours after stimulus with carrageenan paws were removed and neutrophils migration was quantified by MPO activity. LASSBio-1135 treatment also reduces neutrophils migration Results were analyzed using One-way ANOVA followed by Dunnett post-test (n = 4 animals per group, *p<0.05 compared to vehicle group). C) The same tissue samples were used to quantify TNF-α production. LASSBio-1135 oral treatment with 10 µmol.Kg−1 and 100 µmol.Kg−1 also reduced TNF-α production in the injured paw Results were analyzed using One-way ANOVA followed by Dunnett post-test (n = 3–4 animals per group, *p<0.05 compared to vehicle group).
Figure 4. LASSBio-1135 is orally effective in reducing thermal and mechanical hypersensitivity induced by partial sciatic ligation.A) LASSBio-1135 (100 µmol.Kg−1; p.o.) inhibits thermal hyperalgesia induced by a radiant heat light source. Delta (Δ) of latency was obtained by comparing times of withdrawal before and after surgery. B) LASSBio-1135 (100 µmol.Kg−1; p.o.) also inhibits mechanical allodynia induced by Von Frey filaments. The compound was daily administered from day 5 until day 13 after surgery, 1 hour before evaluation of thermal and mechanical hypersensitivity. The withdrawal responses were determined beginning 5 days after the surgery. C) LASSBio-1135 (100 µmol.Kg−1; p.o.) did not affect baseline thermal sensibility induced by a radiant heat light source. Compound or vehicle were administered and evaluation of thermal sensitivity was performed at subsequent 1, 2, 3, 4 and 6 h. Results are expressed as mean ± SEM and analysed using Two-way ANOVA followed by Bonferroni post-test (n = 6–8 animals; *p<0.05 compared to vehicle group).
Figure 5. LASSBio-1135 chronic treatment did not promote hyperthermia.Animals submitted to partial sciatic ligation and daily treated with vehicle or LASSBio-1135 (100 µmol.Kg−1, p.o.) had their rectal temperature measured 1 hour after compound administration. Delta (Δ) of temperature was obtained comparing the measures of temperature obtained before and after surgery. Results are expressed as mean ± SEM (n = 6–8 animals).
Figure 1. LASSBio-1135 is a non-competitive TRPV1 antagonist.A) Representative recordings of inhibition produced by LASSBio-1135 at 5 µM of 1 µM capsaicin currents in TRPV1-expressing oocyte. B) Concentration-response curve of LASSBio-1135 against 1 µM capsaicin, normalized by 10 µM capsaicin applied at the end of recording. Estimated IC50 was 588 nM (n = 5–8 oocytes per concentration). C) Quantification of peak currents in capsaicin and pH 5.5 in the presence or absence of LASSBio-1135 at 50 µM, normalized by the response to capsaicin or pH 5.5 applied at the end of recording (n = 6 per group). Results were analyzed using Student t test (n = 6 oocytes per group, *p<0.05 compared to control group). D) Concentration-response curves of capsaicin and capsaicin plus 5 µM of LASSBio-1135 in TRPV1 currents. Estimated EC50 for capsaicin were 374 nM and 470 nM, alone and in the presence of LASSBio-1135, respectively (n = 5–6 oocytes in each curve). IC50 e EC50 values were determined by non-linear regression using GraphPad Prism software.
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