NOSH-sulindac (AVT-18A) is a novel nitric oxide- and hydrogen sulfide-releasing hybrid that is gastrointestinal safe and has potent anti-inflammatory, analgesic, antipyretic, anti-platelet, and anti-cancer properties

Abstract

Sulindac is chemopreventive and has utility in patients with familial adenomatous polyposis; however, side effects preclude its long-term use. NOSH-sulindac (AVT-18A) releases nitric oxide and hydrogen sulfide, was designed to be a safer alternative. Here we compare the gastrointestinal safety, anti-inflammatory, analgesic, anti-pyretic, anti-platelet, and anti-cancer properties of sulindac and NOSH-sulindac administered orally to rats at equimolar doses. Gastrointestinal safety: 6h post-administration, number/size of hemorrhagic lesions in stomachs were counted. Tissue samples were frozen for PGE2, SOD, and MDA determination. Anti-inflammatory: 1h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 5h. Anti-pyretic: fever was induced by LPS (ip) an hour before administration of the test drugs, core body temperature was measured hourly for 5h. Analgesic: time-dependent analgesic effects were evaluated by carrageenan-induced hyperalgesia. Antiplatelet: anti-aggregatory effects were studied on collagen-induced platelet aggregation of human platelet-rich plasma. Anti-cancer: We examined the effects of NOSH-sulindac on the growth properties of 12 human cancer cell lines of six different tissue origins. Both agents reduced PGE2 levels in stomach tissue; however, NOSH-sulindac did not cause any stomach ulcers, whereas sulindac caused significant bleeding. Lipid peroxidation induced by sulindac was higher than that from NOSH-sulindac. SOD activity was significantly lowered by sulindac but increased by NOSH-sulindac. Both agents showed similar anti-inflammatory, analgesic, anti-pyretic, and anti-platelet activities. Sulindac increased plasma TNFα whereas this rise was lower in the NOSH-sulindac-treated animals. NOSH-sulindac inhibited the growth of all cancer cell lines studied, with potencies of 1000- to 9000-fold greater than that of sulindac. NOSH-sulindac inhibited cell proliferation, induced apoptosis, and caused G2/M cell cycle block. These results demonstrate that NOSH-sulindac is gastrointestinal safe, and maintains the anti-inflammatory, analgesic, antipyretic, and antiplatelet properties of its parent compound sulinsac, with anti-growth activity against a wide variety of human cancer cells.

Keywords: Anti-cancer; Cyclooxygenase; Gastrointestinal; Hydrogen sulfide; Inflammation; NSAIDs; Nitric oxide; Pain; Platelet; Sulindac; Ulcer.

Graphical abstract

Fig. 1

Structural components of NOSH-sulindac. The parent compound sulindac is shown in the shaded box. The parts of the molecule that releases NO and H₂S are shown in the dotted ellipses.

Fig. 2

NOSH-sulindac is gastrointestinal safe. SUL and NOSH-SUL were administered orally at equimolar doses (0.56 mmol/kg; 200 mg/kg and 467 mg/kg for SUL and NOSH-SUL, respectively) and effects on the stomach were evaluated as indicated in Section 2.3. Panel A, shows the stomach of a vehicle-treated rat; Panel B, stomach of a SUL-treated rat showing ulceration and bleeding; Panel C, stomach of a NOSH-SUL-treated rat which is essentially devoid of ulcers. Panel D, gastric damage due to SUL, UI=135±15 mm (^†P<0.01 compared to vehicle), NOSH-SUL was gastric damage-sparing, UI=10±1 mm (^*P<0.01 compared to SUL). Photographs in Panels A–C are representative from 5 rats in each group. Results in Panel D are mean±SEM of 5 rats in each group.

Fig. 3

Effects of sulindac and NOSH-sulindac on gastric PGE₂ levels, lipid peroxidation (MDA) and superoxide dismutase (SOD). At the end of the gastrointestinal safety evaluations as described in Section 2.3, tissues from the excised stomachs of each rat was snap frozen in liquid nitrogen and processed as described in Sections 2.4–2.6. SUL and NOSH-SUL caused a significant reduction in gastric mucosal PGE₂ levels (panel A). Results are mean±SEM of 5 rats in each group, ^*P<0.05 vs vehicle group, ^†P<0.05 vs NOSH-SUL group. SUL caused an almost 9-fold increase in MDA levels, for NOSH-SUL-treated rats, MDA levels were about 2-fold higher (panel B). Results are mean±SEM for 5 rats in each group, ^†P<0.01 vs vehicle group, ^§P<0.05 vs SUL group. SUL caused a significant reduction in SOD activity, whereas NOSH-SUL did not have an effect (panel C). Results are mean±SEM of 5 rats in each group, ^†P<0.05 vs vehicle group, ^†P<0.01 vs SUL group.

Fig. 4

Anti-inflammatory properties of sulindac and NOSH-sulindac. Rat paw edema was induced by carrageenan injection as described in Section 2.9. SUL and NOSH-SUL were both equally effective in reducing paw volume at all time points (panel A). Results are mean±SEM of 5 rats in each group, ^†P<0.05 vs vehicle treated rats at all time points. SUL and NOSH-SUL also caused a significant reduction in PGE₂ levels in the paw exudate (panel B). Results are mean±SEM for 5 rats in each group, ^†P<0.01 vs vehicle, *P <0.05 vs NOSH-SUL.

Fig. 5

Effect of sulindac and NOSH-sulindac on plasma TNF-α. At the end of the gastrointestinal safety evaluations as described in Section 2.3, blood was drawn and processed as described in Section 2.7 for determination of plasma TNF-α. SUL caused a significant rise in plasma TNF-α, however, this rise was significantly less in the NOSH-SUL-treated rats. Results are mean±SEM for 5 rats in each group, *P<0.001 vs vehicle, ^§P<0.01 vs SUL.

Fig. 6

Sulindac and NOSH-sulindac reduce LPS-induced fever, raise the threshold for hyperalgesia, and show anti-platelet activity. Panel A: LPS (50 μg/kg, ip) was administered to the rats one hour before administration of SUL or NOSH-SUL at equimolar doses (0.56 mmol/kg; 200 mg/kg and 467 mg/kg for SUL and NOSH-SUL, respectively). Core body temperature was recorded at 30 min and thereafter hourly for 5 h. Results are mean±SEM for 5 rats in each group, *P<0.01 vs vehicle for both SUL and NOSH-SUL from 1–5 h. Panel B: Mechanical pain threshold was increased in a time-dependent manner by SUL and NOSH-SUL. Results are mean±SEM for 5 rats in each group. ^*P<0.05 vs vehicle for SUL and NOSH-SUL from 2–5 h.Panel C: SUL and NOSH-SUL were equally effective in inhibiting human platelet aggregation. Results are mean±range for two individuals.

Fig. 7

Effect of NOSH-sulindac on colon cancer cell kinetics. NOSH-SUL inhibits proliferation by altering cell cycle progression and inducing apoptosis. HT-29 cells were treated with vehicle, 0.5x IC₅₀ (50 nM), 1×IC₅₀ (100 nM) or 2×IC₅₀ (200 nM) NOSH-SUL for 24 h and analyzed for (A) proliferation by PCNA antigen expression; (B) cell cycle phases by PI staining and flow cytometry; (C) apoptosis by Annexin V staining and flow cytometry. In (A) and (C), results are mean±SEM for 3 different experiments performed in duplicate, *P<0.05 compared to control. In (B), results are representative of two different experiments.