Myeloperoxidase leads the way toward safe and efficient antiseptics

Myeloperoxidase (MPO) is a vital enzyme in the immune system, with the potential to revolutionise antiseptics and cancer therapy.

Professor Robert C. Allen has developed MPO-based antiseptics effective in blood which are systemically safe, while also demonstrating selective anticancer properties. Together with Jackson T. Stephens, the work overcomes obstacles to bring these therapies into widespread use, offering promising solutions for infection control and bladder cancer treatment.

Read more in Research Features

Read the original research: doi.org/10.1158/1538-7445.AM2024-2029

Transcript

Hello and welcome to Research Pod! Thank you for listening and joining us today. 

In this episode we will be learning more about antiseptics. There is a vital need for antiseptics that work well in physiological conditions. All currently known antiseptics, however, are inhibited by blood.

Through decades of research, Professor Robert C Allen shows how myeloperoxidase, a key enzyme in the human immune system, kills pathogens in the body through generation of highly reactive singlet oxygen species.  Based on these findings, he has developed a myeloperoxidase-based antiseptic which is effective in blood and systemically safe. This new microbicidal agent also demonstrates remarkably selective anticancer properties. Together with Jackson T Stephens, the challenge now is to overcome obstacles restricting the widespread use of myeloperoxidase antiseptics in therapy.  

The immune system is vital for survival and health, protecting the body from pathogens and preventing serious infections. Neutrophils, a type of white blood cell, are essential contributors in this defence. They act as the first line of defence by quickly responding to infections and inflammation, engulfing and destroying pathogens.

The importance of oxygen

Robert C Allen has devoted over 50 years of research to understanding how neutrophils act in the body. Through a series of elegant experimental studies and quantum mechanical considerations, he has identified the key player in the extraordinarily complex and efficient physiological processes carried out by the neutrophils. 

He has shown that, in the presence of pathogens, neutrophils generate a highly oxidative environment that destroys bacteria and other microorganisms. This is achieved through the conversion of normally inert triplet oxygen molecules from the atmosphere into their highly reactive singlet form, which rapidly attacks and decomposes the pathogens. 

Singlet oxygen is a short-lived species and its lifetime is in the order of microseconds. It has powerful antiseptic activity targeted to the proximity of its nascence, avoiding collateral damage to healthy body cells.

According to Allen, the processes through which neutrophils kill bacteria are analogous to combustion, and they are, therefore, highly exergonic. As singlet oxygen reacts with a pathogen’s organic matter, it generates carbonyl products in electronically excited states, which decay to their ground state, emitting visible light. 

Allen has shown that these oxygenation activities can be measured accurately and with high sensitivity using chemiluminigenic probes, small organic molecules that react with the singlet oxygen generated by neutrophils resulting in enhanced light emission. This has led him to create an exquisitely sensitive method to monitor, in real time, the functionality of the immune system during an infection.

A vital enzyme

Within the body, cells involved in specific functions, like the immune response, are equipped with granules.  Granules are small membrane-bound compartments containing enzymes, proteins, or other molecules. In neutrophils, the primary granules contain enzymes that are involved in the initial response to an infection. 

Myeloperoxidase, or MPO, is one of the most important of such enzymes for the immune system’s ability to destroy pathogens. Allen has been studying the physiological role of MPO since 1971. Using chemiluminescence and metabolic studies he has been able to study the complex and finely regulated mechanism of NADPH oxidase driven MPO action in microbicidal activity.

When neutrophils reach an infected area of the body, they engulf external pathogens in membrane-bound vesicles, known as phagosomes. The neutrophil granules, which in addition to MPO also contain lysosomal enzymes that promote the degradation of large macromolecules, fuse with the phagosomes, producing a phagolysosomal vacuole. It is inside these vacuoles that the microbicidal action of MPO occurs. MPO catalyses the conversion of hydrogen peroxide into hypochlorous acid, which reacts with another hydrogen peroxide generating singlet oxygen. 

This aggressive chemical species promptly reacts with the pathogen’s molecular structure, triggering its oxidation and eventual destruction. 

Allen explains:  ‘Phagocytosis is linked to the activation of NADPH oxidase resulting in respiratory burst metabolism and reduction of oxygen to radical products, such as hydroperoxyl.  Disproportionation of these intermediates yields singlet oxygen and the hydrogen peroxide that drives MPO oxidation of chloride to hypochlorite. Reaction of the hypochlorite ion with an additional hydrogen peroxide yields the singlet oxygen that is directed to the combustive elimination of infectious microbes.’  

Neutrophils circulate in the blood for less than one day. After leaving the bloodstream, they migrate to body cavities, such as the mouth, the gastrointestinal tract, transporting MPO with them. In these environments, MPO can act as a powerful microbicidal agent, killing potentially harmful bacteria. 

However, MPO is also highly selective: it does not bind to and thus does not damage the lactic acid bacteria that constitute the natural flora. Allen and Stephens have shown that the targeted microbicidal action of MPO is related to its ability to bind selectively to very specific classes of microorganism. When the concentration of MPO is limiting, only microbes or cells that bind MPO are susceptible to combustive oxidation driven by singlet oxygen, whereas cells that do not bind MPO experience no or minimal damage.

MPO binds to all gram-negative, endotoxin-positive microbes tested. Allen and collaborators have reported that MPO, and to a lesser degree eosinophil peroxidase, inhibit lipopolysaccharide 

and lipid A. Such inhibition does not require haloperoxidase action. Likewise, endotoxin lethal dose 90 studies in mice demonstrate that MPO and EPO increase survival. 

MPO is a very stable enzyme, which can be coated onto surfaces or maintained in solution for extended periods of time. Allen discovered that combining MPO with a suitable source of hydrogen peroxide provides a powerful antimicrobial, which is as efficient and selective as the MPO naturally occurring in the human body. 

The E-101 solution developed at Exoxemis, Inc makes use of glucose oxidase, an enzyme that catalyses the oxidation of glucose, to produce hydrogen peroxide, which, in the presence of MPO and chlorite ion, produces singlet oxygen. 

E-101, trade named Zempia, can be prepared in the absence of oxygen, for instance under a controlled nitrogen atmosphere, and can be activated simply by exposing it to atmospheric oxygen, which initiates the generation of singlet oxygen.

E-101 is the first antiseptic of its class that retains its activity in blood and can be used to clean and disinfect wounds with minimal systemic toxicity and no adverse effects on the human body. 

MPO has been found to act not only as a powerful antiseptic, but also as an anticancer agent. In collaboration with Mayo Clinic, C-202, a solution with the same pharmaceutical ingredients of E-101, showed focused cytotoxicity against bladder cancer cells, with no damage for healthy cells. 

The results demonstrate selective toxicity of MPO for cancer cells that is related to their anomalous physiology, that is, Warberg effect, which causes their membrane to acquire an anionic (negative) charge, unlike healthy cells, which are charge neutral. 

Since MPO is a positively charged macromolecule, it binds preferentially to cancer cells and, in the presence of hydrogen peroxide, generates singlet oxygen, which oxidises and destroys cell membrane components essential for their survival. 

In a letter of recommendation to the Food and Drug Administration, C-202 has been put forward as a potential treatment for non-muscle invasive bladder cancer patients. Not only does C-202 have potential to improve treatment outcomes, it could also mitigate the supply–demand issue facing current FDA-recommended therapy.

We were lucky enough to speak with Allen and Stephens about their work. They described their   experiences in getting E-101 to the point where it can be used by patients.

Their journey began in 1987 with the purification of sufficient quantities of MPO for in-depth study. A significant breakthrough was identifying the selective surface binding mechanisms of MPO, allowing the enzyme to specifically target and kill pathogens in the presence of blood while sparing normal flora and human tissue. Subsequent work involved resource-intensive formulation development and scaling up production.

MPO represents a significant advancement due to its combined benefits: focused antiseptic action, systemic and wound safety, targeted endotoxin inactivation. In general, cationic MPO shows targeted anti-cancer action based on the increased negative surface charge of cancer cells.

The researchers also detailed the obstacles and challenges that restrict the widespread use of MPO antiseptic or antineoplastic formulations in therapy.  They explained that the primary challenges are bureaucratic. The FDA has imposed standards on E-101 that other antiseptics have never met, creating an uneven playing field and prohibiting its market introduction in the U.S. Existing antiseptics were grandfathered by the FDA in 1976 without the rigorous safety and efficacy studies required for MPO/E-101. 

Additionally, the investigation of MPO/C-202 cancer therapy is restricted by the FDA to human trials in BCG-unresponsive patients only complicating the situation further. BCG, an FDA-approved cancer therapy, is in limited supply and is expected to remain so at least through 2025. Given these challenges, regulatory authorities in other countries are being asked to review the data and consider the benefits of MPO. 

The researchers are continuing their efforts via the available FDA avenues for E-101 and C-202.

That’s all for this episode – thanks for listening, and stay subscribed to Research Pod for more of the latest science. 

See you again soon.