Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist

Abstract

Pancreatic pain resulting from chronic inflammation of the pancreas is often intractable and clinically difficult to manage with available analgesics reflecting the need for more effective therapies. The mechanisms underlying pancreatitis pain are not well understood. Here, the possibility that interleukin-6 (IL-6) may promote pancreatitis pain was investigated with TB-2-081 (3-O-formyl-20R,21-epoxyresibufogenin, EBRF), a small molecule IL-6 receptor antagonist that was semi-synthetically derived from natural sources. The potential activity and mechanism of TB-2-081 were investigated following the induction of persistent pancreatitis using dibutyltin dichloride (DBTC) in rats. TB-2-081 displaces the binding of IL-6 to the human recombinant soluble IL-6 receptor with apparent high affinity and inhibits IL-6 mediated cell growth. Systemic or oral, but not intrathecal, administration of TB-2-081 reversed DBTC-induced abdominal hypersensitivity in a dose- and time-dependent manner. IL-6 levels were significantly up-regulated in the dorsal root ganglia (DRG) of rats with pancreatitis on day 6 after DBTC injection. IL-6-enhanced capsaicin-evoked release of calcitonin gene-related peptide from cultured DRG neurons was blocked by TB-2-081. Our data demonstrate that TB-2-081 acts as a systemically available and orally active small molecule IL-6 receptor antagonist. TB-2-081 effectively reduces pancreatitis-induced pain through peripheral mechanisms that are likely due to (a) increased expression of IL-6 in the DRG and (b) IL-6-mediated sensitization of nociceptive neurons. The activity of TB-2-081 implicates an important role for IL-6 in sustaining pancreatitis pain. Strategies targeting IL-6 actions through small molecule antagonists may offer novel approaches to improve the therapy of chronic pancreatitis and other chronic pain states.

Figure 1

A. Structure of TB-2-081. B Saturation of IL-6 at human recombinant sIL-6R. Half saturation concentration is 960 pM. C. Competitive binding of TB-2-081 to IL-6R (IC₅₀ = 29.4 pM). D. TB-2-081 inhibits the growth of IL-6 dependent cell line TF-1(IC₅₀ = 250 pM).

Figure 2

Effects of systemic TB-2-081 in the DBTC pancreatitis model. A. Time course of the effects of TB-2-081 in rats with pancreatitis (DBTC) or without pancreatitis (ethanol vehicle). The doses of TB-2-081 are represented as mg/kg. PBS/ethanol indicates the vehicle for TB-2-081. N=6-10 animals in all groups. (*P < 0.05). B. Dose-response curve for TB-2-081 induced reversal of abdominal hypersensitivity 15 min after subcutaneous (s.c.) administration in animals with experimental pancreatitis. C. Area under the time-effect curve for subcutaneously administered TB-2-081 against referred abdominal hypersensitivity in rats injected with DBTC. N=6-10 animals in all groups. *indicates significant difference from the control group (ethanol vehicle) (*P < 0.01).

Figure 3

Effects of orally administered TB-2-081 in the DBTC pancreatitis model. A. Time course of the effects of TB-2-081 in rats with pancreatitis (DBTC) or without pancreatitis (ethanol vehicle). The doses of TB-2-081 are represented as mg/kg. PBS/ethanol indicates the vehicle for TB-2-081. N=5-9 animals per group. B. Area under the time-effect curve for orally administered TB-2-081 against referred abdominal hypersensitivity in rats injected with DBTC. *indicates significant difference from the control group (ethanol vehicle). (*P < 0.01).

Figure 4

Pancreatitis-induced changes in IL-6 expression in the pancreas and DRG. A. Time-course of changes in levels of IL-6 in pancreata of naïve, ethanol vehicle or DBTC-treated animals (*indicates significant difference from naïve group, P<0.01). B. The levels of IL-6 in the T₈-T₁₂ DRG obtained from naïve animals or animals treated with ethanol vehicle or with DBTC-induced visceral inflammation at 6 day after injection are shown (*P < 0.01, N= 6 rats per group in all panels).

Figure 5

Effects of IL-6 on capsaicin-evoked CGRP release from adult DRG neurons in culture. A. Adult cultured DRGs were treated with capsaicin (30 nM) alone (i.e.; Control), TB-2-081 (5 μM) followed by capsaicin (TB-2-081), IL-6 (20 ng/ml) followed by capsaicin (IL-6), or IL-6 with TB-2-081 followed by capsaicin (IL-6+TB-2-081). Treatment with IL-6 increased the levels of capsaicin-evoked CGRP release by 30 % (*P = 0.01). B. Application of IL-6, soluble IL-6R (20 ng/ml) or a combination of both on the basal release of CGRP (*P = 0.02, n=6 per group in all panels).