BACKGROUND

Bacterial toxins promote infection and disease by directly damaging host tissues and by disabling the immune system. There are two types of bacterial toxins: exotoxins and endotoxins. Exotoxins are secreted by bacteria and cause a variety of symptoms, depending on the bacteria. For example, bacterial exotoxins are responsible for diphtheria, tetanus, botulism, cholera, diarrhea, scarlet fever, toxic shock syndrome, and meningitis. Endotoxins are lipopolysaccharides (“LPS”), and the universal toxic lipid component of the lipopolysaccharide molecule is lipid A. Endotoxins are an integral part of the cell wall of gram-negative bacteria and are liberated on cell death. The presence of endotoxins in the blood can produce fever, diarrhea, septic shock, and loss of function of internal organs such as kidneys, liver, adrenal glands, and lungs.

Gram-negative bacterial infections are challenging to treat because the killing of the bacteria leads to the release of endotoxins. For example, the Jarisch-Herxheimer reaction, a condition with signs and symptoms resembling bacterial sepsis, occurs when endotoxin is released from gram-negative spirochete bacteria, such asTreponema(causative agent of syphilis) andBorellia(causative agent of Lyme's disease) during antibiotic treatment. Therefore, there is a need for a microbicidal agent against gram-negative bacteria that will not only kill the bacteria but also inactivate the endotoxin released on death of the bacteria. As further discussed below, the present disclosure addresses this and other needs.

It is known in the art that a myeloperoxidase/hydrogen peroxide/halide system kills bacterial infectious agents, including gram-negative bacteria. As disclosed in U.S. Pat. Nos. 5,888,505, 6,294,168, and 8,945,540, when the concentration is limiting, myeloperoxidase selectively binds to and, in the presence of peroxide and halide, kills target microorganisms without significantly damaging other components of the medium, such as host cells and normal flora. Due to the selective binding properties of myeloperoxidase, when a target microorganism, such as a pathogenic microorganism, has a binding capacity for myeloperoxidase greater than that of a desired microorganism, such as members of the lactic acid bacteria of the normal flora, the target microorganism selectively binds the myeloperoxidase with little or no binding of the myeloperoxidase by the desired microbes. In this regard, myeloperoxidase demonstrates a high degree of selective binding and selective killing of all gram-negative bacteria tested.

Target bound myeloperoxidase, in the presence of peroxide and halide, catalyzes halide oxidation and facilitates the disproportionation of peroxide to singlet molecular oxygen (1O2) at the surface of the target microorganism. Singlet molecular oxygen has a microsecond lifetime and a reactive radius of about 0.2 micrometer. As such, combustive microbicidal action is limited to myeloperoxidase-bound microbes with a minimum of collateral damage to desired microbes or host cells. Thus, as disclosed in U.S. Pat. Nos. 5,888,505, 6,294,168, and 8,945,540, myeloperoxidase can be employed as an antiseptic in the therapeutic or prophylactic treatment of human or animal subjects to selectively bind to and kill pathogenic microorganisms with a minimum of collateral damage to host cells and normal flora of the host.

It is also known in the art that a myeloperoxidase/hydrogen peroxide/halide system is effective in detoxifying exotoxins. See, e.g., Agner, K., (1950), “Studies On Peroxidative Detoxification of Purified Diptheria Toxin”, JEM, 92 (4) 337-347; Agner, K., (1955), “Peroxidative Detoxification of Diptheria Toxin Studied by Using 1131, Recueil”, 74:373-376; Agner, K., (1947), “Detoxicating Effect of Verdoperoxidase on Toxins”, Nat. 4034:271-272 (tetanus toxin); and Ool et al. (1994), “Inactivation of Clostridium difficile Cytotoxin by the Neutrophil Myeloperoxidase System”, J. Infect. Dis. 149(2):215-219.

All of the foregoing references teach that hydrogen peroxide is necessary for antitoxin activity. The prior art does not teach or remotely suggest that myeloperoxidase alone, in the absence of haloperoxidase activity, is effective in detoxifying endotoxins.

The inventor of the present disclosure has discovered that myeloperoxidase not only binds to gram-negative bacteria, but also binds to gram-negative bacteria endotoxins (lipopolysaccharide) and to lipid A (the component of endotoxin responsible for toxicity), and that such binding inhibits the toxic activity of lipopolysaccharide and lipid A. Moreover, the inventor of the present disclosure has surprisingly discovered that myeloperoxidase inhibition of endotoxin lipopolysaccharide/lipid A does not require haloperoxidase enzymatic generation of hypochlorite or singlet molecular oxygen. Nothing in the prior art discloses that myeloperoxidase would be effective as an anti-lipopolysaccharide (anti-endotoxin) and anti-lipid A agent in the absence of haloperoxidase activity.

The present disclosure demonstrates that myeloperoxidase compositions used to treat bacterial infections have the additional advantage of inhibiting (detoxifying) the lipopolysaccharide and lipid A endotoxin activity of gram-negative bacterial pathogens. The present disclosure thus meets a need to provide an effective treatment for gram-negative bacterial infections that both kills the bacteria and inhibits the endotoxins released on death of the bacteria.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

The present disclosure relates to methods for the treatment of gram-negative microbial infections using compositions comprising myeloperoxidase to bind to and inactivate lipopolysaccharide (endotoxin) and lipid A, the lipid component of endotoxin responsible for the toxicity of gram-negative bacteria. The present disclosure provides methods of treating a human or animal subject having a gram-negative bacterial infection comprising administering to the site of the gram-negative bacterial infection in the subject a composition comprising myeloperoxidase, wherein the composition acts to detoxify lipopolysaccharides and lipid A present at the site of the infection.

In some embodiments, the myeloperoxidase composition further comprises a peroxide-producing oxidase. Examples of peroxide-producing oxidases include glucose oxidase, cholesterol oxidase and galactose oxidase. In some embodiments, the peroxide-producing oxidase is glucose oxidase. In some embodiments, the myeloperoxidase/oxidase composition further comprises halide.

In some embodiments, the methods further comprise contacting the site of infection with the myeloperoxidase/oxidase composition in the presence of a substrate for the oxidase. However, the antitoxin activity of the myeloperoxidase is not dependent on the production of hydrogen peroxide.

In some embodiments the myeloperoxidase/oxidase composition further comprises at least two amino acids. In some embodiments, the at least two amino acids are selected from the group consisting of glycine, l-alanine, d-alanine, l-alanine anhydride, l-glutamine, l-glutamic acid, glycine anhydride, hippuric acid, l-histidine, l-leucine, d-leucine, l-isoleucine, d-isoleucine, l-lysine, l-ornithine, d-phenylalanine, l-phenylalanine, l-proline, l-hydroxyproline, l-serine, taurine, l-threonine, d-threonine, l-tyrosine, l-valine, d-valine, beta amino acids, such as beta alanine, l-beta-homoleucine, d-beta-homoleucine, 3-aminobutanoic acid, l-2,3-diaminopropionic acid monohydrochloride, d-2,3-diaminopropionic acid monohydrochloride, l-3-aminoisobutyric acid, d-3-aminoisobutyric acid, ethyl 3-aminobutyrate, sarcosine methyl ester hydrochloride and nipecotic acid, or an alkyl ester or pharmaceutically acceptable salt thereof.

In other embodiments, the at least two amino acids are selected from the group consisting of glycine, l-alanine, d-alanine, l-alanine anhydride, l-glutamine, l-glutamic acid, glycine anhydride, hippuric acid, l-histidine, l-leucine, d-leucine, l-isoleucine, d-isoleucine, l-lysine, l-ornithine, d-phenylalanine, l-phenylalanine, l-proline, l-hydroxyproline, l-serine, taurine, l-threonine, d-threonine, l-tyrosine, l-valine, and d-valine, or an alkyl ester or pharmaceutically acceptable salt thereof.

In some embodiments the myeloperoxidase/oxidase composition further comprises at least three amino acids. In some embodiments, the at least three amino acids are selected from the group consisting of glycine, l-alanine, d-alanine, l-alanine anhydride, l-glutamine, l-glutamic acid, glycine anhydride, hippuric acid, l-histidine, l-leucine, d-leucine, l-isoleucine, d-isoleucine, l-lysine, l-ornithine, d-phenylalanine, l-phenylalanine, l-proline, l-hydroxyproline, l-serine, taurine, l-threonine, d-threonine, l-tyrosine, l-valine, d-valine, beta amino acids, such as beta alanine, l-beta-homoleucine, d-beta-homoleucine, 3-aminobutanoic acid, l-2,3-diaminopropionic acid monohydrochloride, d-2,3-diaminopropionic acid monohydrochloride, l-3-aminoisobutyric acid, d-3-aminoisobutyric acid, ethyl 3-aminobutyrate, sarcosine methyl ester hydrochloride and nipecotic acid, or an alkyl ester or pharmaceutically acceptable salt thereof.

In other aspects, the at least three amino acids are selected from the group consisting of glycine, l-alanine, d-alanine, l-alanine anhydride, l-glutamine, l-glutamic acid, glycine anhydride, hippuric acid, l-histidine, l-leucine, d-leucine, l-isoleucine, d-isoleucine, l-lysine, l-ornithine, d-phenylalanine, l-phenylalanine, l-proline, l-hydroxyproline, l-serine, taurine, l-threonine, d-threonine, l-tyrosine, l-valine, and d-valine, or an alkyl ester or pharmaceutically acceptable salt thereof.

In some embodiments, the three amino acids are glycine, alanine, and proline.

In one embodiment, the compositions of the present disclosure comprise from 1 to 50,000 μg/ml of myeloperoxidase. In other embodiments, the compositions of the present disclosure comprise 0.1 to about 500 mM of each of the at least two amino acids. In one representative embodiment, the compositions of the present disclosure comprise from 10 to 5,000 μg/ml of myeloperoxidase, from 0.3 to 50 mM of glycine, from 0.3 to 50 mM of l-alanine, from 0.3 to 50 mM of l-proline, and from 1 to 500 U/ml of glucose oxidase.

In some aspects of the present disclosure, the human or animal subject to be treated is suffering from a gram-negative bacterial infection of the gums, eyes, ears, skin, soft tissue, wounds, vaginal areas, groin areas, bed sores or burn areas. In some embodiments, the infection is a polymicrobial infection. In other embodiments, the infection is caused, at least in part, by a multidrug resistant gram-negative bacteria.

