Abstract
There is a large published literature presenting information regarding the toxicology of CO in humans. There is information showing that CO-Hb levels below 15% are not associated with clinical adverse signs or symptoms in healthy individuals and smokers have CO-Hb levels around 6%, but may be above 10% on occasion.
It has also been found that CO at low levels may have positive effects in SCD. It has been reported that non-toxic levels of CO extend the red cell life span in SCD patients (Beutler E, 1975.). Further, CO might play a role in preventing sickle cell formation by preventing the polymerization of hemoglobin (Sirs JA, 1963), and it has been reported that CO might have a preventative effect on the occurrence of clinical symptoms of SCD (Yallop D et al, 2007). Inhaled CO administered in low concentrations to transgenic sickle mice for one hour per day, 3 days per week for 10 weeks, was non-toxic and substantially decreased white cell counts and cellular inflammatory signals (Beckman J et al, 2009).
This research program hypothesizes that CO, when administered orally at non-toxic levels, results in CO-Hb levels that will result in benefit to patients with SCD through two mechanisms: reducing the sickling phenomenon of red cells and by reducing the inflammatory response in these patients. It also hypothesizes that the safest and most efficient mode of administration of CO is by the oral route via a solution with a known dose of CO.
The carbon monoxide (CO) is obtained from a commercial source. The liquid formulation (LF) is composed of a mixture of the following components: water, proteins, lipids and carbohydrates ( PLC Mixture). The CO is instilled into this LF and tested for CO concentration. The principle of the method used for CO measurement is to allow the CO and added Hb to interact and to measure the CO-Hb formed by using the dithionite reduction method employing a spectrophotometer. (A.H. Chalmers 1991).The container is weighed before and after CO instillation to determine the occurrence of CO gas leakage.
The container for the CO-LF is of low gas permeability. Once CO instillation in the LF is complete, the containers of CO-LF are sealed and labeled. The prepared containers are stored at 2-8°C for no longer than 72 hours.
The CO content in blood is expressed as ‘%’ of total hemoglobin (CO-Hb) and mg/L in the CO-LF.
CO-LF was administered via esophageal-placed gavage using Sprague-Dawley rats with jugular venous catheters.
In the first study, there were 5 groups of 4 non-anemic rats studied, with each group comprising of a different LF made with the following percent of PLC Mixture combined with water: 50% PLC Mixture, 20% PLC Mixture, 10% PLC Mixture, 5% PLC Mixture, and 0% PLC Mixture (water only).
Two 3 mL doses of CO-LF were administered to each rat one hour apart. Data generated showed levels of CO-Hb at baseline varying between 0 and 1.1 %. After the administration of the test article, the highest CO-Hb levels were 6.1% in one animal at 1 hour after the first dose, and 9.6% one hour after the second dose in one animal.
The levels of CO-Hb fell at different rates depending upon the dose and formulation. The PK data for the first study is shown in Table 1. Differences among the test articles were not statistically different.
PK Data for the CO-LF Non-Anemic Rat Studies.
| Experiment . | Terminal half-life (hours) . | AUC 0-inf (h-%) . |
|---|---|---|
| 0% (water only) | 0.51 | 0.085 |
| 5% PLC Mixture | 0.65 | 0.076 |
| 10% PLC Mixture | 0.81 | 0.07 |
| 20% PLC Mixture | 0.5 | 0.09 |
| 50% PLC Mixture | 0.64 | 0.11 |
| Experiment . | Terminal half-life (hours) . | AUC 0-inf (h-%) . |
|---|---|---|
| 0% (water only) | 0.51 | 0.085 |
| 5% PLC Mixture | 0.65 | 0.076 |
| 10% PLC Mixture | 0.81 | 0.07 |
| 20% PLC Mixture | 0.5 | 0.09 |
| 50% PLC Mixture | 0.64 | 0.11 |
The PK parameters evaluated over 24 hours were not impacted by the administered PLC Mixture, but animals receiving CO-50% PLC Mixture showed an initial fall to baseline levels, then a second rise in CO-Hb levels peaking in one animal at 3.1% at 48 hours. Such late increments were not seen with other CO-LF’s.
The second PK study was comprised of 2 groups of 4 anemic rates, and employed CO-0% and CO-10% PLC Mixture. This study was carried out on rats made anemic by blood draw to an estimated 50% blood volume. The PK characteristics show data similar to that seen in non-anemic rats.
This research has documented the feasibility in a proof-of-concept rat model that orally administered CO via a CO liquid formulation can produce CO-Hb levels that would be expected to impact the pathophysiology of the Sickle Cell Disease-associated micro-vasculature. The terminal half-life of orally administered CO in the rat is similar to that reported via inhaled CO.
Gomperts:Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties. Forman:Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties.
Author notes
Asterisk with author names denotes non-ASH members.
