LASSBio‐2039 anti‐inflammatory activity in vivo and in vitro
Authors: da Costa Salomé Dayana , de Morais Cordeiro Natália , Freitas Rosana Helena Coimbra Nogueira , Manssour Carlos Alberto , Fernandes Patricia Dias 
Published: November 2023

LASSBio‐2039 is a n‐acylhydrazone derived from LASSBio‐1524 designed as IKK‐β inhibitor, an enzyme that participates of NF‐κB activation and is involved with most of the inflammatory events. Our aim was to evaluate a possible anti‐inflammatory effect of LASSBio–2039 using in vivo and in vitro models.

In vivo tests used Swiss webster mice (n= 6–8). Animals were treated with LASSBio‐2039 (1, 30 or 100 μmol/kg, p.o) 1 h before experiments. Formalin‐induced licking model consists in two phases. The 1st (nociceptive phase) and the 2nd (inflammatory phase) after formalin injection (20 μL/paw). Carrageenan‐induced cell migration into the subcutaneous air pouch (SAP) consists in the injection of a phlogistic agent into the cavity. Exudates were collected from SAP for cell counting and cytokines measurement by ELISA. In vitro assays used RAW 264.7 cell line LPS‐activated preincubated with the compound (1, 10 and 30 μM) for nitric oxide (NO) measurement and cell viability assay (reduction of bromide of 3‐(4,5‐dimethylthiazol‐2‐yl) ‐2,5‐difenltetrazolium (MTT) to formazan after 24h of incubation). Results are expressed as mean±SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p<0,05). Tests followed animal testing standards under the protocol DFBCICB015‐04/16.

In the formalin‐induced licking response the compound did not affect the first phase (1 μmol/kg = 14 ± 5 sec, 30 μmol/kg = 12 ± 4 sec and 100 μmol/kg = 10 ± 3 sec, compared to vehicle‐treated group = 25 ± 6 sec). On the other hand, second phase was significantly reduced by higher doses (1 μmol/kg = 122 ± 55 sec, 30 μmol/kg = 87* ± 29 sec and 100 μmol/kg = 81* ± 25 sec, compared to vehicle‐treated group = 160 ± 44 sec). All doses inhibited cellular migration: 1 μmol/kg inhibited 41% (57* ± 15 cells × 103/mL), 30 μmol/kg inhibited 45.2% (53* ± 19 cells × 103/mL) and 100 μmol/kg inhibited 43.1% (55 ± 10* cells × 103/mL), compared to the group that received carrageenan = 96 ± 23 cells × 103/mL (group that received PBS into cavity = 2 ± 0.9 cells × 103/mL). IL1‐β and TNF‐α production were reduced after treatment. IL1‐β: 1 μmol/kg = 2,720 ± 920*pg/mL, 30 μmol/Kg = 4,340 ± 2,169 pg/mL and 100 μmol/kg = 3,365 ± 1,822* pg/mL, compared to PBS group = 496 ± 376 pg/mL and carrageenan group = 5,699 ± 1,417 pg/mL. TNF‐α: 1 μmol/kg = 398 ± 150* pg/mL, 30 μmol/kg = 675 ± 183 pg/mL and 100 μmol/kg = 605 ± 374 pg/mL, compared to PBS group = 276 ± 127 pg/mL and carrageenan group = 849 ± 104 pg/mL. LASSBio‐2039 did not reduce cell viability. NO production was reduced at all concentrations: 1 μM = 73 ± 4.4 μM, 10 μM = 39* ± 18 μM and 30 μM = 21* ± 14 μM, compared LPS‐stimulated group = 85 ± 20 μM (unstimulated group = 4 ± 2 μM).

LASSBio‐2039 reduced NO production without interfering with cellular viability. It also reduced paw licking time in the second phase of the test, suggesting that this substance may have an anti‐inflammatory effect. In the SAP model, LASSBio‐2039 was able to reduce cellular migration and cytokines production. These results suggest that this compound can be considered as a new anti‐inflammatory prototype.

Support or Funding Information

Financial support: Capes, CNPq, FAPERJ and Instituto Vital Brazil (donation of animals).

Technical Support: Alan Minho