Hypochlorous acid as a potential wound care agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity

Abstract

Objective: Hypochlorous acid (HOCl), a major inorganic bactericidal compound of innate immunity, is effective against a broad range of microorganisms. Owing to its chemical nature, HOCl has never been used as a pharmaceutical drug for treating infection. In this article, we describe the chemical production, stabilization, and biological activity of a pharmaceutically useful formulation of HOCl.

Methods: Stabilized HOCl is in the form of a physiologically balanced solution in 0.9% saline at a pH range of 3.5 to 4.0. Chlorine species distribution in solution is a function of pH. In aqueous solution, HOCl is the predominant species at the pH range of 3 to 6. At pH values less than 3.5, the solution exists as a mixture of chlorine in aqueous phase, chlorine gas, trichloride (Cl(3) (-)), and HOCl. At pH greater than 5.5, sodium hypochlorite (NaOCl) starts to form and becomes the predominant species in the alkaline pH. To maintain HOCl solution in a stable form, maximize its antimicrobial activities, and minimize undesirable side products, the pH must be maintained at 3.5 to 5.

Results: Using this stabilized form of HOCl, the potent antimicrobial activities of HOCl are demonstrated against a wide range of microorganisms. The in vitro cytotoxicity profile in L929 cells and the in vivo safety profile of HOCl in various animal models are described.

Conclusion: On the basis of the antimicrobial activity and the lack of animal toxicity, it is predicted that stabilized HOCl has potential pharmaceutical applications in the control of soft tissue infection.

Figures

Figure 1

A schematic representation of hypochlorous acid (HOCl) production during the oxidative burst process. During this process, cells utilize O2 and convert it to hydrogen peroxide (H2O2) using a mitochondrial-membrane–bound enzyme NADPHase. Then, myeloperoxidase catalyzes the reaction between H2O2 and Cl− to generate HOCl. As deregulations take place, the lumen of the phagasome progressively becomes more acidic and leaves the bacterium within a vacuole (phagolysosome) containing MPOse and H2O2 in a medium containing 0.1 M Cl− at estimated pH 4 to 6. During this process, conditions are optimal for MPOse-catalyzed generation of HOCl as depicted in this figure. On the basis of these principles, we set out to establish the conditions of generating the stable form of HOCl (NVC-101).

Figure 2

Chlorine speciation profile as a function of pH.

Figure 3

Comparative time kill studies of HOCl, NaOCl, and H2O2 against 3 test organisms—Escherichia coli 25922, Pseudomonas aeruginosa 27853, and Staphylococcus aureus 29213—at room temperature for total of 90 miniutes.

Figure 4

Relative cell toxicity of hypochlorous acid (HOCl; pH 4.0), hypochlorite (OCl−; pH 10.5), and hydrogen peroxide (H2O2; pH 7.0) on L929 cells. Cytotoxicity measured in a cell proliferation assay is expressed as the concentration (μg/mL) that reduces the cell number by 50% of vehicle-treated control. CT50 is shown as the average of 7, 3, and 5 independent experiments (consisting of 10 different concentrations of each test article plus/minus the standard deviation), respectively.

Figure 5

Relative therapeutic index of hypochlorous acid (HOCl; pH 4.0), hypochlorite (OCl−; pH 10.5), and hydrogen peroxide (H2O2; pH 7.0). Therapeutic index is expressed as a ratio of the CT50 concentration (μg/mL) on L929 cells divided by the minimum bactericidal concentration (μg/mL) for Staphylococcus aureus 29213, Pseudomonas aeruginosa 27853 and Escherichia coli 2592. The higher the therapeutic index, the safer the test article will be.