defensin MiMetics

Using Nature To Keep Us Safer

Despite enormous advances in the evolution of anti-infective treatments, most authorities think we are losing the battle against microorganisms. Resistance to antimicrobial agents has developed in every notable strain of pathogenic bacteria, and to basically every commercially available antibiotic.

The advent of such “superbugs” is one of the most crucial medical dilemmas the world is currently facing. Over 2.5 million residents of the US suffer acute bacterial infections annually, and over 100,000 of these individuals lose their lives, making infections the fourth-highest cause of mortality in the country and the second leading cause of death across the globe. Those who live put up with longer stays in hospital and need to be treated with costly second- and third-line antibiotics that often produce harmful side-effects.

The global market for anti-infective medications, estimated at more than $45 billion annually, is growing due to the increase in bacterial drug resistance. For example, the number of hospitalizations in the US between 1999 and 2005 associated with MRSA infections grew 119 per cent. The most striking rise (approximately 400 per cent) during this period was identified with infections that characteristically occur outside rather than inside hospitals. Previously, MRSA frequently affected hospitalized surgical patients or individuals with compromised immune systems. Recently, it has manifested itself as a community-based infection in otherwise healthy people. Two years ago, about 686,000 hospitalizations for MRSA infections occurred in the US, which resulted in direct healthcare expenses of almost $10 billion and a total bill of approximately $118 billion. The very real prospect of our antibiotic defenses losing their effectiveness or becoming obsolete has produced an urgent need for antimicrobial agents that work via mechanisms that bacteria have not yet recognized.

Ancient defense mechanisms

The path to concocting the antibiotics of the future may involve ancient defense molecules. Molds and other primitive organisms rely on biochemically acting agents to protect against bacteria. Penicillin, created for this purpose by molds, is perhaps the most well-known example. Mammals have a more sophisticated defense against bacteria, the first line of which comprises the host defense peptides (HDPs), known as defensins in humans. These molecules, which make up part of the nonhumoral or innate immune response, quickly eradicate bacteria before the pathogens can dominate.

Biologists have found numerous types of HDPs. Although they and human defensins both have a varied array of amino acid sequences, they have similar physicochemical characteristics. All are amphiphilic, meaning they have an affinity for both charged/polar and uncharged/ nonpolar environments. It is this characteristic, rather than amino acid sequence, that lies behind HDPs’ antimicrobial activity, which has remained robust in spite of hundreds of millions of years of bacterial evolution.

Be this as it may, proteins and peptides have been conspicuously absent from the recent spate of approvals for anti-infective pharmaceutical drugs. Peptide and protein drugs pose many dilemmas for drug discovery and development. Their handling and manufacture takes time and is costly. Since they are virtually always injected rather than taken in pill form, their production faces strict guidelines governing sterility. It is also usually hard to adopt proteins and peptides derived from animals as drugs for humans, due to their toxicity, instability and tendency to induce immune responses in the host.

An approach to anti-infective drug development taken by PolyMedix of Radnor, Pennsylvania replicates the effects of large protein molecules via novel, small-molecule chemistry, and this mimics intrinsic immunity in humans. The PolyMedix lead compounds, known as defensin mimetics, mimic the activity of host defense proteins but in molecules that are much smaller and less complex than defensins.

The defensin mimetic approach possesses multiple potential advantages over existing antibiotics. For example, it has a novel mechanism of action that is unlikely to result in bacterial resistance; a powerful, broad-spectrum activity against more than 150 Grampositive and Gram-negative bacteria; and a fast bactericidal activity versus the bacteristatic activity for many common antibiotics. Additionally, it has been proven to work against drug-resistant bacteria, including multiple MRSA and vancomycin-resistant enterococci (VRE) strains. It also exhibits predictable, powerful systemic activity in animal models of infection; selectivity for bacteria versus human cells of 100 to more than 10,000, compared with 10 to 20-fold for host defence proteins; and it is well tolerated in animals. It exhibits good drug-like characteristics—pharmacokinetics, half-life, serum binding, and tolerability profiles are characteristic of safe, potent drugs; and it is easy to make via chemical synthetic schemes comparable to those for making drugs or polymeric materials. Finally, it possesses an encouraging safety profile in Phase 1 human clinical testing so far.

The defensin mimetics show a distinctive mode of action which, mirroring the natural human defensin proteins, involves directly rupturing the membranes of bacterial cells. Bacteria possess more negatively charged chemical groups on their membranes’ outer surface than do mammalian cells. Bacterial membranes also do not have any cholesterol, which is plentiful in mammalian membranes. Defensin mimetics zero in on cholesterol-free membranes that contain negatively charged phospholipids; thus they are specific and selective for bacterial cell membranes but leave mammalian cells untouched. Since the biophysical, or mechanical, mode of action of the defensin mimetics differs radically from antibiotics’ biochemical mechanisms, it is believed unlikely that antimicrobial resistance to these agents will develop.

The defensin mimetics are one-tenth as large as naturally occurring defensins; up to 100 times more potent killers of bacteria, fungi, and yeast; and up to 1,000 times more selective for bacterial versus mammalian cells. These molecules are intended to mirror the amphiphilic structure of the host defense proteins, but with entirely synthetic, nonpeptide backbones, in small-molecule format. These defensin mimetics directly cause bacterial cell membranes to break open, a mechanism that is unique among known antimicrobial compounds.

To evolve resistance to this attack mechanism, bacteria would need to develop a novel variety of cell membrane to escape the workings of defensin mimetics. This also explains why host defense proteins have stayed effective through hundreds of millions of years of evolution. Figure 1 illustrates this mechanism of action of host defense proteins and defensin mimetics versus conventional antibiotics that work on biochemical targets.

Defensin mimetic compound activity

Figure 1: Learning from Nature’s Defenses Against Bacteria

In research on animal models of bacterial infection, PolyMedix’s lead defensin mimetic compounds reveal a level of safety and efficacy that is significantly higher than that of defensins, not to mention commercially available antibiotics. In December 2008, a Phase 1 clinical trial with PMX-30063, the first defensin mimetic being developed, was successfully completed. This trial revealed that it is possible to safely administer PMX-30063 at doses that result in blood levels associated with full efficacy in animal models of infection, without serious side effects. In vitro studies reveal that defensin mimetics such as PMX-30063 exhibit 1,000-fold or greater selectivity for bacteria versus mammalian cells, including blood erythrocytes, mouse fibroblasts and Mechanism of action of host defense proteins and defensin mimetics versus conventional human liver cells. antibiotics that work on biochemical targets. Some of the early research on PolyMedix’s defensin mimetics involved a general compound structure (Figure 2). These molecules are amphiphilic by virtue of the hydrophobic groups that project from the bottom of the repeat unit, and charged groups at the top of the repeat unit. Employing this chemical scaffold as the foundation for one class of agents, chemists at PolyMedix have created multiple structural analogs: either defined oligomers, small molecules or heterogeneous polymers. They found many compounds that exhibit powerful antimicrobial activity and high killing selectivity for

Figure 2: Defensin mimetics general compound structure.

Food safety

bacterial versus mammalian cells. Subsequently, using molecular modeling and chemical elaboration, the company’s scientists have succeeded in improving these molecules’ activity and selectivity. The product pipeline now includes a small molecule in clinical development as a systemic intravenous antibiotic therapeutic drug, PMX-30063, being developed for the broad treatment of Staphylococcus infections; a variety of next-generation agents that have shown encouraging activity against other infectious agents including tuberculosis, malaria and biowarfare pathogens such as the infectious agents for anthrax and plague; and a range of polymers for antimicrobial biomaterial applications.

Versatile molecular design platform

In addition to its anti-infective program, PolyMedix is actively working on drugs in other therapeutic areas, as well as for nontherapeutic (biomaterial) applications. The other compound now in clinical development is PMX-60056, known as a “heptagonist,” an agent that reverses the anticoagulative properties of heparin and low-molecularweight heparins (LMWHs).

Heparin is an intravenous anticoagulant that is customarily employed following surgery and during cardiothoracic procedures to prevent blood clots that can threaten a patient’s life. Doctors perform about two million cardiothoracic procedures annually. Following the procedure, heparin activity must be counteracted quickly in order to restore normal clotting. Protamine, the only agent currently available for this use, has many drawbacks. It is hard to adjust the dosage, its efficacy is unpredictable, and it exhibits serious toxicities. Additionally, protamine is not approved or reliably effective with LMWHs.

LMWHs are a family of drugs employed for the long-term prevention of clots, such as in deep vein thrombosis, or after heart attack or in conjunction with cancer chemotherapies. About 12 million patients take LMWHs every year for chronic treatment of thrombosis. As many as 20 per cent of these patients may experience bleeding complications. There is an intense need for an effective reversing agent.

PolyMedix’s compounds, including the clinical agent PMX-60056, were designed to bind to the pentasaccharide region on heparin and LMWH, deactivating them and thus allowing blood to clot normally. Animal studies reveal that a single administration of PMX-60056 following heparin treatment completely normalizes blood clotting time. Even a significant overdosage of PMX-60056 causes no apparent deviation of blood clotting time in animal studies. Notably, PMX-60056 also works against LMWHs, and thus may serve as a universal anticoagulant-reversing agent.

In March 2009, PMX-60056 completed a Phase 1 human clinical safety trial. Continued clinical development is planned.

The common use of antibiotics in food animals is a possible source of microbial resistance with immediate effects on public health. According to data from the Centers for Disease Control and Prevention, contaminated food causes 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the US annually. Although contaminated food causes over 200 known diseases, more than 75 per cent of foodborne illnesses are caused by Salmonella, Listeria and Toxoplasma, of which the first two are bacteria (as are eight of the 10 most common food pathogens). Although many of these organisms come from the environment, the carryover of resistant pathogens from slaughtered animals is worrisome.

Foodborne illnesses are not only a cause of worry for meat processors. Recent recalls of spinach, high-end pet foods, peanut butter and baby food, in addition to hamburgers and other meat products, stress the need to reduce contamination sources as much as possible.

Antimicrobial biomaterials based on synthetic mimics of defensins might revolutionize the creation of products that contact foods during production, processing and storage. The same traits that make these molecules work as antimicrobial compounds in vivo could make them effective as germicides.

Although sanitation in food processing is achieved mainly via cleaning, the impact of materials used in food processing surfaces, vessels and implements can be large. Bacteria cling to and persist on rough surfaces better than on smooth surfaces. Thus, many food processing surfaces, particularly metallic ones, are polished to reduce surface roughness.

Yet even ultrapolished metals attract bacteria in varying degrees. A study from the Centre for Applied Microbiology and Research in Salisbury, U.K. concluded that metals commonly used to make foodcontact surfaces harbor bacteria for a length of time that exceeds an average work shift, sometimes almost indefinitely. Stainless steel, the most common food processing surface, retains live bacteria for 34 days; copper, which is known to have antimicrobial properties, retains viable pathogens for up to 14 hours.

PolyMedix believes that substances coated with polymeric defensin mimetics could markedly improve safety at food processing plants and outlets such as butcher shops, delicatessens and restaurants. In proof of principle experiments, bacterial growth was effectively reduced on surfaces made of polymers containing between 0.01 and 1 per cent percent by weight of PolyMedix’s antimicrobial polymer biomaterials. No antimicrobial substance will entirely eradicate pathogenic bacteria from food that contacts a surface. Instead, the benefit of antimicrobial biomaterials is to keep bacteria from surviving and multiplying on work surfaces, thus substantially reducing the chances of crosscontamination or persistent bacterial presence. PolyMedix anticipates the development, most