Research Article: Oral carbon monoxide therapy in murine sickle cell disease: Beneficial effects on vaso-occlusion, inflammation and anemia

Date Published: October 11, 2018

Publisher: Public Library of Science

Author(s): John D. Belcher, Edward Gomperts, Julia Nguyen, Chunsheng Chen, Fuad Abdulla, Zachary M. Kiser, David Gallo, Howard Levy, Leo E. Otterbein, Gregory M. Vercellotti, Ming Dao.

http://doi.org/10.1371/journal.pone.0205194

Abstract

Carbon monoxide (CO) at low, non-toxic concentrations has been previously demonstrated to exert anti-inflammatory protection in murine models of sickle cell disease (SCD). However CO delivery by inhalation, CO-hemoglobin infusion or CO-releasing molecules presents problems for daily CO administration. Oral administration of a CO-saturated liquid avoids many of these issues and potentially provides a platform for self-administration to SCD patients. To test if orally-delivered CO could modulate SCD vaso-occlusion and inflammation, a liquid CO formulation (HBI-002) was administered by gavage (10 ml/kg) once-daily to NY1DD and Townes-SS transgenic mouse models of SCD. Baseline CO-hemoglobin (CO-Hb) levels were 1.6% and 1.8% in NY1DD and Townes-SS sickle mice and 0.6% in Townes-AS control mice. CO-Hb levels reached 5.4%, 4.7% and 3.0% within 5 minutes in NY1DD, SS and AS mice respectively after gavage with HBI-002. After ten treatments, each once-daily, hemoglobin levels rose from 5.3g/dL in vehicle-treated Townes-SS mice to 6.3g/dL in HBI-002-treated. Similarly, red blood cell (RBC) counts rose from 2.36 x 106/μL in vehicle-treated SS mice to 2.89 x 106/μL in HBI-002-treated mice. In concordance with these findings, hematocrits rose from 26.3% in vehicle-treated mice to 30.0% in HBI-002-treated mice. Reticulocyte counts were not significantly different between vehicle and HBI-002-treated SS mice implying less hemolysis and not an increase in RBC production. White blood cell counts decreased from 29.1 x 103/μL in vehicle-treated versus 20.3 x 103/μL in HBI-002-treated SS mice. Townes-SS mice treated with HBI-002 had markedly increased Nrf2 and HO-1 expression and decreased NF-κB activation compared to vehicle-treated mice. These anti-inflammatory effects were examined for the ability of HBI-002 (administered orally once-daily for up to 5 days) to inhibit vaso-occlusion induced by hypoxia-reoxygenation. In NY1DD and Townes-SS sickle mice, HBI-002 decreased microvascular stasis in a duration-dependent manner. Collectively, these findings support HBI-002 as a useful anti-inflammatory agent to treat SCD and warrants further development as a therapeutic.

Partial Text

Carbon monoxide (CO) at low, non-toxic concentrations exerts key physiological functions in various models of tissue inflammation and injury, providing potent cytoprotection in models of inflammation including SCD [1–3], organ transplantation [4], and acute lung injury [5], among others [6–8]. The protection observed, both prophylactically and therapeutically, is associated with an inhibition in the inflammatory response and restoration of tissue function, including abrogating ischemia reperfusion injury [9, 10]. CO may also inhibit polymerization of hemoglobin (Hb) S and increase RBC life span [11, 12]. However, delivery systems that include inhaled CO, metallic CO-releasing molecules (CORMs) and CO conjugated to a PEGylated Hb, may not be suitable for the chronic administration of CO in humans that will be necessary to prevent vaso-occlusive crises. Inhaled CO is challenging to precisely dose given the variability in patient ventilation and has environmental safety concerns for patients and bystanders, as it requires the presence of large amounts of compressed CO gas in cylinders. Metal-containing CORMs present potential long-term health concerns [13]. PEGylated CO-Hb is also not appropriate for chronic home use as it would require daily intravenous infusions and could present toxicology challenges [14, 15]. Oral administration of CO avoids many of the challenges associated with inhaled CO, CORMs and PEGylated CO-Hb and additionally provides a platform for outpatient administration and compliance. An oral drug product that would deliver a predictable low, non-toxic dose of CO, might be a better alternative for treating SCD patients. To test the efficacy of oral administration of CO and its anti-inflammatory, anti-vaso-occlusive properties, a liquid CO formulation (HBI-002) was administered by gavage to NY1DD and Townes-SS transgenic mouse models of SCD.

In preliminary studies to examine HBI-002 bioavailability, after a single gavage (10 ml/kg), CO-Hb was cleared from circulation in approximately 3 h in outbred CD-1 mice and peaked between 0–10 min after gavage in Townes-AS mice. Subsequent blood samples were collected 5 min after gavage. In NY1DD sickle mice, CO-Hb levels were 1.6 ± 0.2% (mean ± SD) after treatment with vehicle and increased significantly to 5.4 ± 0.8% after treatment with HBI-002 (p<0.01) (Fig 1). In Townes-AS mice, CO-Hb levels were 0.6 ± 0.2% after gavage with vehicle and increased significantly to 3.0 ± 0.6% after gavage with HBI-002 (p<0.01). Similarly, in Townes-SS mice, CO-Hb levels were 1.8 ± 0.2% (mean ± SD) after treatment with vehicle and increased significantly to 4.7 ± 0.4% after treatment with HBI-002 (p<0.01). Vehicle-treated Townes-AS mice had significantly lower CO-Hb than vehicle-treated Townes-SS mice (p<0.05), likely because of a markedly reduced hemolytic rate in AS mice. This is reflected in significantly shorter RBC half-lives and 6-fold higher expired CO levels in SS versus AS mice [18]. These data document that following oral administration of HBI-002, CO is rapidly absorbed from the gut into the vasculature and is bioavailable in the circulation as measurable CO-Hb within minutes.   Source: http://doi.org/10.1371/journal.pone.0205194

 

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