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. 2021 Feb;11(2):456-475.
doi: 10.1016/j.apsb.2020.08.005. Epub 2020 Aug 24.

Organic carbon monoxide prodrug, BW-CO-111, in protection against chemically-induced gastric mucosal damage

Dominik Bakalarz  1   2 , Marcin Surmiak  1   3 , Xiaoxiao Yang  4 , Dagmara Wójcik  1 , Edyta Korbut  1 , Zbigniew Śliwowski  1 , Grzegorz Ginter  1 , Grzegorz Buszewicz  5 , Tomasz Brzozowski  1 , Jakub Cieszkowski  1 , Urszula Głowacka  1 , Katarzyna Magierowska  1 , Zhixiang Pan  4 , Binghe Wang  4 , Marcin Magierowski  1
Affiliations

Organic carbon monoxide prodrug, BW-CO-111, in protection against chemically-induced gastric mucosal damage

Dominik Bakalarz et al. Acta Pharm Sin B. 2021 Feb.

Abstract

Metal-based carbon monoxide (CO)-releasing molecules have been shown to exert anti-inflammatory and anti-oxidative properties maintaining gastric mucosal integrity. We are interested in further development of metal-free CO-based therapeutics for oral administration. Thus, we examine the protective effect of representative CO prodrug, BW-CO-111, in rat models of gastric damage induced by necrotic ethanol or aspirin, a representative non-steroidal anti-inflammatory drug. Treatment effectiveness was assessed by measuring the microscopic/macroscopic gastric damage area and gastric blood flow by laser flowmetry. Gastric mucosal mRNA and/or protein expressions of HMOX1, HMOX2, nuclear factor erythroid 2-related factor 2, COX1, COX2, iNos, Anxa1 and serum contents of TGFB1, TGFB2, IL1B, IL2, IL4, IL5, IL6, IL10, IL12, tumor necrosis factor α, interferon γ, and GM-CSF were determined. CO content in gastric mucosa was assessed by gas chromatography. Pretreatment with BW-CO-111 (0.1 mg/kg, i.g.) increased gastric mucosal content of CO and reduced gastric lesions area in both models followed by increased GBF. These protective effects of the CO prodrug were supported by changes in expressions of molecular biomarkers. However, because the pathomechanisms of gastric damage differ between topical administration of ethanol and aspirin, the possible protective and anti-inflammatory mechanisms of BW-CO-111 may be somewhat different in these models.

Keywords: Anti-inflammation; Carbon monoxide; Ethanol; Gastric mucosal damage; Gastroprotection; NSAID; Prodrug.

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Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Chemical structures of CO prodrug BW-CO-111 and BW-CP-111 (A). Calibration curve for CO release from BW-CO-111 in vitro (B). Percentage of CO release in vitro and half-life at pH 7.4 and 1.2 for BW-CO-111 (C). Results are reported as mean ± SD. Gastric mucosal CO contents in gastric mucosa with or without pretreatment with BW-CO-111 (0.1 mg/kg, i.g.) 45 min prior to sampling (D). Results are mean ± SEM of 5 rats per group. ∗P < 0.05 compared with intact.
Figure 2
Figure 2
Ethanol- (A) and aspirin-induced (B) gastric lesion areas in rats pretreated i.g. with vehicle, CO prodrug BW-CO-111 (0.02–5 mg/kg) or BW-CP-111 (0.1 mg/kg). Results are mean ± SEM of 5 rats per group. ∗P < 0.05 compared with the vehicle-control group.
Figure 3
Figure 3
Macroscopic and microscopic appearance of randomly selected representative gastric mucosa of rats pretreated with vehicle or BW-CO-111 (0.1 mg/kg) and exposed to 75% ethanol (EtOH) (A) or aspirin (ASA) (B). Histological slides were stained with H/E. Yellow arrows indicate the macroscopic and microscopic gastric lesions.
Figure 4
Figure 4
Hmox1 (A), Hmox2 (B) mRNA and HMOX1 (C, F), HMOX2 (D, F), NRF2 (E, F) proteins expression in gastric mucosa of rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg) followed by 75% ethanol administration 30 min later. Results are mean ± SEM of 5 rats per experimental group. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments. ∗P < 0.05 compared with healthy gastric mucosa.
Figure 5
Figure 5
Cox1 (A), Cox2 (B) mRNA, COX1 (C, E), COX2 (D, E) proteins expression and prostaglandin E2 content (PGE2, F) in gastric mucosa of rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg) and administered 30 min later with 75% ethanol. Results are mean ± SEM of 5 rats per experimental group. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments (Intact). ∗P < 0.05 compared with healthy gastric mucosa; ∗∗P < 0.05 compared with vehicle.
Figure 6
Figure 6
Expression of iNos (A), Anxa1 (B) and Tgfb receptor 1 (Tgfbr1) (C), Tgfbr2 (D), Tgfbr3 (E) mRNA in gastric mucosa and alterations in serum concentration of TGFB1 (F), TGFB2 (G), TGFB3 (H) in rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg, i.g.) 30 min before administration of 75% ethanol. Intact refers to healthy rats which were not treated with ethanol. Results are mean ± SEM of 5 samples per each experimental group with statistical significance marked only if 2-fold up- or downregulation was reached. Results are expressed as fold change of normalized gastric mucosal iNos, Anxa1, Tgfbr1, Tgfbr2 and Tgfbr3 mRNA expression. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments. ∗P < 0.05 compared with healthy gastric mucosa; ∗∗P < 0.05 compared with respective values obtained in vehicle-pretreated group.
Figure 7
Figure 7
Changes in serum concentration of interleukin IL1B (A), IL2 (B), IL4 (C), IL5 (D), IL6 (E), IL10 (F), IL12 (G), IL13 (H), tumor necrosis factor TNF (I), interferon IFNG (J), and granulocyte-macrophage colony-stimulating factor GM-CSF (K) in rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg) and administered with 75% ethanol (EtOH). Intact refers to serum concentration of cytokines in rats without any treatments. Results are mean ± SEM of 5 samples per each experimental group. ∗P < 0.05 compared with respective values obtained in intact rats; +P < 0.05 compared with respective values obtained in vehicle-control group.
Figure 8
Figure 8
Hmox1 (A), Hmox2 (B) mRNA and HMOX1 (C, F), HMOX2 (D, F), NRF2 (E, F) proteins expression in gastric mucosa of rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg) and administered 30 min later with aspirin (125 mg/kg, i.g.). Results are mean ± SEM of 5 rats per experimental group. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments. ∗P < 0.05 compared with healthy gastric mucosa.
Figure 9
Figure 9
Effect of pretreatment with vehicle or BW-CO-111 (0.1 mg/kg) administered 30 min prior aspirin (125 mg/kg, i.g.) on alterations in Cox1 (A), Cox2 (B) mRNA, COX1 (C, E), COX2 (D, E) proteins expression and the mucosal concentrations of prostaglandin E2 (PGE2, F). Results are mean ± SEM of 5 rats per experimental group. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments (Intact). ∗P < 0.05 compared with healthy gastric mucosa; ∗∗P < 0.05 compared with respective values obtained in vehicle-pretreated group.
Figure 10
Figure 10
Expression of iNos (A), Anxa1 (B) and Tgfb receptor 1 (Tgfbr1) (C), Tgfbr2 (D), Tgfbr3 (E) mRNA in gastric mucosa and the changes in TGFB1 (F), TGFB2 (G), TGFB3 (H) serum concentrations of rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg, i.g.) 30 min before administration of aspirin (125 mg/kg, i.g.). Intact refers to healthy rats which were not treated with aspirin. Results are mean ± SEM of 5 samples per each experimental group with statistical significance marked only if 2-fold up- or downregulation was reached. Results are expressed as fold change of normalized gastric mucosal iNos, Anxa1 and Tgfbr1, Tgfbr2, Tgfbr3 mRNA expression. Red line indicates baseline value of mRNA expression in healthy gastric mucosa without any treatments (Intact). ∗P < 0.05 compared with intact rats; ∗∗P < 0.05 compared with respective values obtained in vehicle-pretreated group.
Figure 11
Figure 11
Alterations in serum concentration of interleukin IL1B (A), IL2 (B), IL4 (C), IL5 (D), IL6 (E), IL10 (F), IL12 (G), IL13 (H), tumor necrosis factor TNF (I), interferon IFNG (J), and granulocyte-macrophage colony-stimulating factor GM-CSF (K) in rats pretreated i.g. with vehicle or BW-CO-111 (0.1 mg/kg) and administered with aspirin (125 mg/kg, i.g.). Intact refers to serum of healthy rats without any treatments. Results are mean ± SEM of 5 samples per each experimental group. ∗P < 0.05 compared with respective values obtained in intact rats.
Figure 12
Figure 12
Possible mechanisms of BW-CO-111-mediated gastroprotection against ethanol- (A) and aspirin-induced (B) gastric damage.
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