Delivery of therapeutic carbon monoxide by gas-entrapping materials

Abstract

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Fig. 1.. GRAS materials for GI delivery of CO.

(A) Schematic depicting oral and rectal routes of administration for GEMs. (B) Macroscopic and microscopic images of three GEMs, including foam GEMs, solid GEMS, and hydrogel GEMs. (C) Quantity of CO in each formulation compared to CO-enriched lactated Ringer’s solution (LR). (D) Maximum COHb achieved for each GEM administered through the GI tract. The foam GEMs were rectally administered (5 g/kg), and the solid GEMs (5 g/kg) and hydrogel GEMs (5 g/kg) were surgically placed in the stomach. The CO-enriched LR was administered via oral gavage (5 g/kg). The maximum COHb for solid GEMs was 30 min, hydrogel GEMs at 45 min, and foam GEMs at 15 min. Data are means (n = 3 mice per group). P values were determined by one-way ANOVA with multiple comparisons. NS, not significant.

Fig. 2.. Foam GEMs enable tunable delivery of CO.

(A) Photographs of foam stability in vitro at 0, 6, and 24 hours using different concentrations of xanthan gum (XG). (B) Representative images of foam GEMs with different concentrations of xanthan gum at 0, 6, and 24 hours. (C) Volumetric stability over 24 hours. (D) Pharmacodynamics of a single dose of rectally administered foam GEMs in mice over 45 min. Three foam GEMs with different concentrations of xanthan gum were evaluated and demonstrated different pharmacodynamics profiles (n = 5 mice per time point). (E) Carboxyhemoglobin after rectal administration in mice. (F) Carboxyhemoglobin as a function of canister pressure. Data are means (n = 3). P values were determined by one-way ANOVA with multiple comparisons.

Fig. 3.. CO-GEMs achieve sustained, elevated COHb percentages in small and large animals.

(A) Schematic of rectal administration of foam to swine. (B) COHb percentages in mice, rats, and swine after rectal foam GEM administration (n = 3 animals per group). (C) Organ-specific concentrations of CO at 15 min after foam administration intrarectally in mice (5 g/kg). Data are means (n = 4 animals per arm, with each sample evaluated in triplicate as represented by a circle, square, or triangle). P values were determined by unpaired t test. LI, large intestine. SI, small intestine. SM, skeletal muscle.

Fig. 4.. Rectally administered GEMs reduce inflammation in vivo.

(A) Schematic of experimental timeline. CO-GEMs (5 g/kg) was administered to mice at 1, 2, and 3 hours after injection of APAP (250 mg/kg, i.p.) to cause acute liver failure. ALT was assessed 24 hours after APAP. Controls included no treatment and air-GEM (P < 0.0001). Data represent means (n = 15 to 17 per arm). P values were determined by one-way ANOVA with multiple comparisons. Histopathology of liver sections showing hepatocyte cell death in controls (air-GEM, naïve) as measured by activated caspase 3 and H&E staining compared to liver sections from mice treated with APAP and CO-GEMs showed less activated caspase 3 and normal architecture. (B) In a model of DSS-induced experimental colitis, foam GEMs (5 g/kg) were administered to mice beginning on day 3 after DSS treatment was started and then daily for 10 days. Colon length and histology scores of DSS-treated animals administered CO-GEMs were compared to air-GEM and no treatment. H&E staining of liver tissue is shown below. Data represent means (n = 10 per arm). P values were determined by one-way ANOVA with multiple comparisons. (C) In a model of radiation-induced proctitis, rats were treated with CO-GEMs rectally 1 day prior (5 g/kg), within 1 hour before irradiation (5 g/kg), and once daily for 8 days (5 g/kg) after exposure to 18 Gy of radiation directed to the rectum. H&E staining of rectal tissue depicts the quantity of normal intestinal crypts in animals treated with CO-GEMs was compared to no-treatment and air-GEM–treated control rats. Data represent mean (n = 7 per arm). P values were determined by one-way ANOVA with multiple comparisons.

Fig. 5.. CO-GEMs reduce DSS-induced increases in protein oxidation in large intestine in mice.

(A) In the model of DSS-induced experimental colitis, foam GEMs were administered to mice beginning on day 3 of DSS treatment and daily until day 10, and the tissues were collected, fixed with formalin, paraffin-embedded, and sectioned. Representative images of large intestine stained for glutathione (GSH)–adducted proteins under nonreducing conditions and 3-nitrotyrosine (3NT) are shown as markers of oxidative damage. Arrows point to foci of 3NT within the large intestine. (B) Quantification of GSH-adducted proteins and 3NT-positive areas analyzed from randomly selected 600 μm by 600 μm sections of full thickness tissue (×8 magnification). Data represent means (n = 10 mice per arm, three replicates per mouse). P values were determined by unpaired t test.