CAP-232

Since the conventional and adjuvant analgesics have limited effectiveness frequently accompanied by serious side effects, development of novel, potent pain killers for chronic neuropathic and inflammatory pain conditions is a big challenge. Somatostatin (SS) regulates endocrine, vascular, immune and neuronal functions, cell proliferation through 5 G_i protein-coupled receptors (SST₁-SST₅). SS released from the capsaicin-sensitive peptidergic sensory nerves mediates anti-inflammatory and antinociceptive effects without endocrine actions via SST₄. The therapeutic use of the native SS is limited by its diverse biological actions and short plasma elimination half-life. Therefore, SST₄ selective SS analogues could be promising analgesic and anti-inflammatory drug candidates with new mode of action. TT-232 is a cyclic heptapeptide showing great affinity to SST₄ and SST₁. Here, we report the in silico SST₄ receptor binding mechanism, in vitro binding (competition assay) and cAMP- decreasing effect of TT-232 in SST₄-expressing CHO cells, as well as its analgesic and anti-inflammatory actions in chronic neuropathic pain and arthritis models using wildtype and SST₄-deficient mice. TT-232 binds to SST₄ with similar interaction energy (-11.03 kcal/mol) to the superagonist J-2156, displaces somatostatin from SST₄ binding (10 nM to 30 µM) and inhibits forskolin-stimulated cAMP accumulation (EC₅₀: 371.6 ± 58.03 nmol; E_max: 78.63 ± 2.636 %). Its i.p. injection (100, 200 µg/kg) results in significant, 35.7 % and 50.4 %, analgesic effects upon single administration in chronic neuropathic pain and repeated injection in arthritis models in wildtype, but not in SST₄-deficient mice. These results provide evidence that the analgesic effect of TT-232 is mediated by SST₄ activation, which might open novel drug developmental potentials. Chemical compounds Chemical compounds studied in this article TT-232 (PubChem CID: 74053735).

Somatostatin and its analogues, known as somatostatin receptor ligands (SRLs), have been reported to attenuate weight gain in some clinical settings. However, their direct effects on preadipocytes are barely investigated. Therefore, this study aimed to evaluate the influence of SRLs on preadipocytes and to further explore the potential mechanisms. Cell Counting Kit‐8 assay, Oil Red O staining, triglyceride contents measurements, quantitative polymerase chain reaction (qPCR) and western blot were used to investigate the effects of SRLs on preadipocytes. We found that three SRLs (octreotide, TT232 and pasireotide) inhibited cell viability after 8–48 h but not 4 h. Further western blot results showed that they significantly suppressed activation of PI3K/Akt pathway. Besides, lipid accumulation was also significantly inhibited by these SRLs. Moreover, mRNA levels of some critical adipogenic markers, including Pparg, Cebpa, Fasn, Fabp4, Acaca and Lpl, were downregulated by the treatments of all these SRLs. Consistently, the protein expression of peroxisome proliferator‐activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα) and fatty acid synthase (FAS) was also suppressed by SRLs. SRLs inhibit the proliferation and lipogenesis in preadipocytes. Their inhibitory effects on cell proliferation may be mediated by the downregulated PI3K/Akt pathway, and the suppressive actions on lipogenesis may be related to the decreased PPARγ and C/EBPα expression.