Patent Description:
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 as Treponema (causative agent of syphilis) and Borellia (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 <CIT>, <CIT>, and<CIT>, 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 (<NUM>O<NUM>) at the surface of the target microorganism. Singlet molecular oxygen has a microsecond lifetime and a reactive radius of about <NUM> 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 <CIT> <CIT>, and <CIT>, 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., <NPL>; <NPL>; <NPL> (tetanus toxin); and<NPL>.

Prior art document <CIT> describes eosinophil peroxidase compositions and methods of their use for preventing and/or inhibiting the growth <NUM> of susceptible microorganisms. The compositions include an eosinophil peroxidase, a peroxide or peroxide source, and at least two amino acids.

Document <CIT> describes that haloperoxidases are used to selectively bind to and, in the presence of peroxide and halide, inhibit the growth of target microbes without eliminating desirable microbes or significantly damaging other components, such as host cells, in the environment of the target microbe. When a target microbe, e.g., a pathogenic microbe, has a binding capacity for haloperoxidase greater than that of a desired microbe, e.g., members of the normal flora, the target microbe selectively binds the haloperoxidase with little or no binding of the haloperoxidase by the desired microbe. In the presence of peroxide and halide, the target bound haloperoxidase catalyzes halide oxidation and facilitates the disproportionation of peroxide to singlet molecular oxygen at the surface of the target microbe. The lifetime of singlet molecular oxygen restricts damage to the surface resulting in selective killing of the target microbe with a minimum of collateral damage to the desired microbe or physiological medium.

The methods and compositions as described in this document are used in the therapeutic or prophylactic antiseptic treatment of human or animal subjects, since their use can be effective in combatting bacterial or fungal infections without significant damage to normal flora or host cells. Suitable haloperoxidases include myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO) and derivatives thereof capable of selective binding to target microbes.

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.

The references to methods of treatment in the subsequent paragraphs of this description are to be interpreted as references to the compounds, pharmaceutical composition and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

The present disclosure relates to methods for the treatment of gram-negative microbial infections using compositions consisting essentially of 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 relates to 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 a subject a composition consisting essentially of myeloperoxidase, wherein the composition acts to detoxify lipopolysaccharides and lipid A present at the site of the infection.

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 one embodiment, the compositions of the present disclosure comprise from <NUM> to <NUM>,<NUM>µg/ml of myeloperoxidase.

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.

The present disclosure is broadly directed to methods of treating a human or animal subject having a gram-negative bacterial infection consisting essentially of 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. Myeloperoxidase compositions are capable of binding to and detoxifying lipopolysaccharide (endotoxin) and lipid A (the purified component of endotoxin responsible for toxicity).

In one aspect, the methods of the present disclosure are highly suitable for the topical treatment of susceptible infections in a human or non-human mammalian subject at sites permitting direct contact of the myeloperoxidase compositions of the present disclosure with the microbial infection, such as, for example, gram-negative bacterial infections of the skin, eyes, ears, mouth, nasal and sinus passages, traumatic injury sites, surgical sites and the like. When in contact with host tissue, the myeloperoxidase compositions of the present disclosure can inactivate lipid A and lipopolysaccharides without associated host tissue destruction or disruption of normal flora.

In some embodiments, the compositions of the present disclosure will comprise from about <NUM> to about <NUM>,<NUM>µg/ml of myeloperoxidase.

Myeloperoxidase inhibits both lipopolysaccharide and lipid A endotoxin activities through a direct binding independent of haloperoxidase enzymatic action.

Compositions of the present disclosure consisting essentially of myeloperoxidase, inhibit endotoxins in the absence of substrate, and therefore, in the absence of haloperoxidase enzyme activity.

The compositions may additionally comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions may be conveniently provided in a liquid carrier. Any liquid carrier may be generally used for this purpose, provided that the carrier does not significantly interfere with the selective binding capabilities of the myeloperoxidase or with enzyme activity (if microbicidal action is desired). Alternatively, the compositions may be provided in solid form with activation on solubilization in liquid.

For topical applications, the detoxifying compositions can be administered in any effective pharmaceutically acceptable form to warm blooded animals, including human and animal subjects, e.g., in topical, lavage, oral, vaginal or rectal suppository dosage forms, as a topical, buccal, nasal spray, aerosol for inhalation or in any other manner effective to deliver active myeloperoxidase to a site of bacterial infection. The route of administration will preferably be designed to obtain direct contact of the compositions with the toxins produced by or associated with the infecting bacteria. In one aspect of the present disclosure, the compositions of the present disclosure are delivered or administered topically to areas of a human or animal subject that are infected or susceptible to infection, such as, for example, to the gums, eyes, ears, skin, wounds, vaginal areas, groin areas, bed sores, burns, areas under medical dressings, diapers or other coverings which are likely to be moist, and the like.

For topical applications, the pharmaceutically acceptable carrier may take the form of liquids, creams, foams, lotions, ointments, suspensions, suppositories or gels, and may additionally comprise aqueous or organic solvents, buffering agents, emulsifiers, gelling agents, moisturizers, stabilizers, surfactants, wetting agents, preservatives, time release agents, and minor amounts of humectants, sequestering agents, dyes, perfumes, and other components commonly employed in pharmaceutical compositions for topical administration. In addition, the compositions of the present disclosure may be impregnated in dressings or coverings for application to a subject.

The inhibition of bacterial endotoxin lipopolysaccharides and lipid A by various agents was studied using the Limulus Amebocyte Lysate Endosafe® Endochrome-K™ kit ("LAL") available from Charles River Endosafe, Charleston, South Carolina. The LAL assay is a means to detect and measure bacterial endotoxin activity in a sample. The LAL assay used in the following examples is a kinetic colorimetric assay that detects and measures the onset of color. The time of onset of color is inversely related to the amount of endotoxin in a sample. Endotoxin levels in an unknown sample are determined by comparison to a standard curve. The amount of endotoxin present in a sample is designated by endotoxin units ("EU"). The Endosafe® LAL assay can detect from <NUM>-<NUM> EU/ml.

This example demonstrates the inhibition of lipopolysaccharide endotoxin by compositions comprising myeloperoxidase. The following four agents, or combinations of agents, were tested: (<NUM>) myeloperoxidase; (<NUM>) glucose oxidase; (<NUM>) myeloperoxidase, glucose oxidase and amino acids; and (<NUM>) myeloperoxidase, glucose oxidase, glucose, and amino acids.

The myeloperoxidase ("MPO") used in this example was porcine myeloperoxidase (Exoxemis, Inc. , Little Rock, Ark. The glucose oxidase ("GO") was from Aspergillus niger and was purchased from Biozyme, Inc. The myeloperoxidase and glucose oxidase were further purified by passing the MPO or GO through a polymyxin b column twice to remove LPS present in the test additives using the Toxin Eraser Endotoxin Removal Kit available from Genscript, cat # L00338.

Stock solutions were made of the following test additives: myeloperoxidase (<NUM>/ml); glucose oxidase (<NUM>/ml); E-<NUM> (enzyme) (<NUM>/ml MPO; <NUM>/ml GO); E-<NUM> (substrate) (<NUM>/ml glucose final); E-<NUM> (complete) (<NUM>/ml MPO; <NUM>/ml GO; <NUM>/ml glucose).

As used herein, the term "E-<NUM> (enzyme)" refers to a solution comprised of myeloperoxidase, glucose oxidase and amino acids glycine, alanine, and praline in an aqueous vehicle comprising <NUM> sodium chloride and <NUM>% w/v polysorbate <NUM> in <NUM> sodium phosphate buffer pH <NUM>.

As used herein, the term "E-<NUM> (substrate)" refers to a solution of <NUM>/ml glucose in an aqueous vehicle comprising <NUM> sodium chloride and <NUM>% w/v polysorbate <NUM> in <NUM> sodium phosphate buffer pH <NUM>.

As used here, the term "E-<NUM> (complete)" refers to a formulation formed by combining one part E-<NUM> (enzyme) and two parts E-<NUM> (substrate).

Testing was performed on <NUM>-well microtiter plates using a final volume of <NUM>µL per well. Reagents were prepared at twice the final desired concentration, and <NUM>µL volumes were added per well. <NUM>µL of LPS endotoxin was added per well. In wells where a reagent was omitted, its volume was replaced with an equal volume of low endotoxin reagent water ("LRW') supplied with the Endosafe® assay kit.

The ability of myeloperoxidase compositions to inhibit lipopolysaccharide was determined by measuring endotoxin inhibition at increasing amounts of endotoxin and decreasing amounts of myeloperoxidase in the test solutions, i.e., over a range of endotoxin:myeloperoxidase ratios. This approach allowed estimation of maximum inhibitory activity of one milligram of myeloperoxidase where endotoxin availability was limiting, and also enabled the comparison of inhibitory activity of myeloperoxidase to glucose oxidase and in combination with glucose oxidase, with and without substrate.

Eight activities (concentrations) of LPS were tested: <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, and <NUM> EU per test solution. The concentration of myeloperoxidase varied from <NUM>/ml to <NUM>/ml test solution. The concentration of glucose oxidase when tested alone varied from <NUM>/ml to <NUM>/ml. The concentration of glucose oxidase tested in combination with myeloperoxidase varied from <NUM>/ml to <NUM>/ml. (The ratio of MPO to GO in E <NUM> enzyme and E <NUM> complete was <NUM>:<NUM>. ) Serial dilutions were made using the low endotoxin reagent water (LRW) supplied with the Endosafe® assay kit.

Lipopolysaccharide was pre-incubated with the various test additives for <NUM> minutes before the LAL assay was run to determine the amount of endotoxin activity present in the test samples. After the pre-incubation period, <NUM>µL of chromogenic Limulus Amebocyte Lysate (LAL) solution (Charles River Kit R1708K) was added and the change in absorbance was measured using a Tecan Sunrise microplate spectrophotometer at a wavelength of <NUM>. The amount of endotoxin inhibited at the various concentrations of test agents was determined and then the value was extrapolated to determine the number of endotoxin units inhibited per milligram of myeloperoxidase. For example, if a sample containing <NUM> EU LPS and <NUM>/ml of MPO resulted in complete inhibition of the <NUM> EU, then the number of EU inhibited by <NUM> of MPO was calculated to be <NUM> EU.

The data in Table <NUM> reports the amount of endotoxin units inhibited by <NUM> MPO.

The above data illustrate that myeloperoxidase is a potent inhibitor of lipopolysaccharide endotoxin. The data illustrate that one milligram of myeloperoxidase can inhibit more than <NUM>,<NUM> EU/mL of LPS. Glucose oxidase exhibits about half the inhibitory effect of myeloperoxidase when LPS is tested at low concentrations, but at higher LPS doses (e.g., <NUM>,<NUM> EU/mL), GO also inhibits greater than <NUM>,<NUM> EU/mL.

In sharp contrast to MPO and GO individually, the combination of one milligram of myeloperoxidase and <NUM> milligrams of glucose oxidase exhibits an inhibitory effect several fold greater than the effect achieved with myeloperoxidase without glucose oxidase. Similar results were found for myeloperoxidase in combination with glucose oxidase and glucose (substrate for the glucose oxidase).

This example demonstrates the inhibition of lipid A, the toxic component of lipopolysaccharide, by compositions comprising myeloperoxidase. In this example, the ability of myeloperoxidase compositions to inhibit lipid A was determined by measuring endotoxin inhibition at increasing amounts of lipid A and decreasing amounts of myeloperoxidase in the test solutions, i.e., over a range of endotoxin:myeloperoxidase ratios. The test solutions had the same components and concentrations as in Example <NUM> and the procedure was the same. Nine activities (concentrations) of lipid A were tested: <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, <NUM> EU, and <NUM> EU per test solution. Calculations were performed as in Example <NUM> to determine the number of lipid A endotoxin units inhibited per milligram of myeloperoxidase. The results are reported in Table <NUM> below.

The data in Table <NUM> illustrate that the inhibitory effect of myeloperoxidase alone, and myeloperoxidase in combination with glucose oxidase, on lipid A activity is even more dramatic than the data in Table <NUM> illustrating the inhibitory effect of myeloperoxidase on lipopolysaccharide activity. The data in Table <NUM> show that glucose oxidase alone has little inhibitory effect on lipid A, but the inhibitory effect of myeloperoxidase alone on lipid A is greater than <NUM>,<NUM> EU per mg MPO (extrapolated from the inhibition of <NUM>,<NUM> EU of lipid A). At the same <NUM>,<NUM> EU lipid A concentration, the combination of MPO and GO exhibits an inhibitory effect on lipid A of greater than <NUM>,<NUM> EU per mg myeloperoxidase.

Moreover, as in Example <NUM>, the data show that the endotoxin inhibitory action is independent of haloperoxidase microbicidal activity. It is notable that inhibitory action with regard to lipid A appears to be mildly decreased in the enzymatically active preparation.

The data in Tables <NUM> and <NUM> illustrate that the myeloperoxidase and myeloperoxidase:glucose oxidase inhibition of endotoxin is proportional to the concentration of endotoxin tested (LPS or lipid A); i.e., the reaction is essentially first order with respect to increasing endotoxin over the range of endotoxin tested.

To test the limit of myeloperoxidase inhibition of endotoxin, high concentrations of LPS and lipid A were tested by using a constant concentration of MPO, E101 enzyme (MPO:GO) or E101 complete (MPO:GO:glucose) equivalent to <NUM>/mL MPO, and varying the concentration of LPS and lipid A by <NUM>n dilutions from <NUM>,<NUM>,<NUM> EU/mL (<NUM>/mL of LPS, and <NUM>/mL lipid A) to <NUM>,<NUM> EU/mL. The results are shown in Tables <NUM> and <NUM> below.

The data in Tables <NUM> and <NUM> illustrate that at the highest concentration (<NUM>,<NUM>,<NUM> EU/mL), LPS and lipid A were completely inhibited by <NUM>/mL MPO in combination with GO, i.e. E101 enzyme (MPO:GO), or E <NUM> complete (MPO:GO:glucose), and MPO alone showed partial inhibition (delayed EU kinetic), but could not be further quantified.

Claim 1:
A composition consisting essentially of myeloperoxidase, for use in a method of treating a gram-negative bacterial infection in a human or animal subject, wherein the activity of a lipopolysaccharide endotoxin present at a site in the subject is inhibited, and wherein the method comprises administering said composition to the site where the lipopolysaccharide endotoxin is present in the subject, wherein the myeloperoxidase directly contacts, binds to, and inhibits the activity of the lipopolysaccharide endotoxin.