Aeolus Technology Base

The findings of research on free radical biology support the use of catalytic antioxidants as a broad new class of pharmaceuticals. Aeolus was founded to establish a research and development program to exploit the therapeutic potential of small molecule catalytic antioxidants. Aeolus’ catalytic antioxidant drug discovery program has been guided by its scientific founders, who are among the creators of the field of reactive species biology (free radicals, i.e., reactive oxygen and reactive nitrogen species) and remain leading investigators:

• Irwin Fridovich, Ph.D. of Duke University School of Medicine, whose co-discovery of superoxide dismutase launched free radical biology;
• James D. Crapo, M.D., a pulmonologist and former Chairman of the Department of Medicine at the National Jewish Medical and Research Center; and
• Brian Day, Ph.D., the Company’s Chief Science Officer, is a pharmacologist at National Jewish Medical and Research Center who was the first to show that a manganoporphyrin was an effective antioxidant in a mammalian model of disease.

Based upon these efforts, the Company has created a proprietary class of small molecules that catalytically consume reactive species, that is, they are not themselves consumed in the reaction. The small molecules developed by Aeolus all have the same core structure. This class of compounds is a patent protected group of manganoporphyrins that retain the positive benefits of antioxidant enzymes, are active in animal models of disease and, unlike the body’s own enzymes, have properties that make them suitable drug development candidates.

All of the Company’s manganoporphyrins contain a positively-charged manganese metal ion, Mn+, that is able to accept and give electrons to and from reactive oxygen species (“ROS”) and reactive nitrogen species (“RNS”). The manganese ion (Mn+) is stably held within the central region of the larger porphoryin molecule by four nitrogen (N) compounds. The Company believes that this structure is essential to maintaining the metal ion within the catalytic antioxidant, and thus prevents leakage or release of the manganese ion

In addition, new research indicates that ROS and RNS have important cell signaling roles, and beneficially, based upon continuing research on the Aeolus compounds, the compounds have multiple mechanisms of action whereby they can provide the following activities:

• anti-oxidant
• anti-inflammatory
• anti-angiogenic
• anti-oncogeni

It has been experimentally demonstrated in tissue culture and animal models that oxidative stress, caused by reactive species, plays a critical role in neuronal cell death, and is apparent in a plethora of both acute and chronic diseases and disorders. While the body’s natural antioxidants have demonstrated some efficacy in models of neurodegeneration, delivery and stability issues have reduced enthusiasm to clinically develop these natural antioxidants. The Company’s catalytic antioxidants were designed to create stable small molecule antioxidants without the limitations of the body’s natural antioxidants.

Because the Company’s compounds have the same core structure and because these compounds do not focus on a specific receptor or enzyme system or pathway, nor focus on specific aspects of a disease or disorder, but rather address a common causality to multiple diseases and disorders (i.e., ROS / RNS damage), Aeolus’ catalytic antioxidants are believed to have a broad range of potential therapeutic uses.

Free Radicals, Oxidative Stress and Nitrosative Stress

Oxidative stress is caused by the body’s inability to inactivate reactive species including free radicals, resulting in an imbalance of too many oxidants and not enough antioxidants. This imbalance plays a central role in a number of diseases and disorders, including ALS, stroke, cancer, exposure to radioactivity, diabetes, Parkinson’s disease, chronic pulmonary obstructive disorder, arthritis and ulcerative colitis. Free radical biology is one of the most widely studied areas in modern science; over 50,000 papers on the subject have been published in the past 30 years.

A free radical is an atom, or group of atoms, that contains one or more unpaired electrons in the outer orbit of that atom, forming an unstable molecule that is “free” to react with the rest of the molecules that compose all of the cells and tissues of the body. In most atoms, the outer orbits are filled with paired electrons that move in opposite directions to balance their spins. Having an unpaired electron makes free radicals unstable and therefore highly reactive. Free radicals react with the molecules that make up the cells of our bodies by a process called oxidation. This process results in the removal of an electron from the target molecule, turning this originally stable molecule into a free radical. This new free radical then goes on to oxidize other molecules. Thus, a single free radical can start an electron-snatching chain reaction that continues to produce more and more free radicals. This uncontrolled free radical production and the resulting damage can lead to abnormal cellular function and contribute to some disease states. By doing so, free radicals break apart many important molecules in the body, causing damage to the cells and tissues. Therefore, free radicals can damage DNA, proteins and lipids resulting in inflammation and both acute and delayed cell death.

It is important to note that both free radicals and other reactive species are generated as byproducts of many naturally occurring processes in the body. For example, when the body uses oxygen to generate energy, either through aerobic metabolism or cellular respiration, reactive species are created as metabolic waste. Accordingly, the levels of these reactive species in the body increase during exercise. However, natural and essential biological processes produce reactive species as well, such as tissue repair, the normal turnover of cells and tissues, and inflammation, the body’s protective mechanism to fight invading pathogens and other threats. Reactive species are also taken up by the body from a variety of external sources, including tobacco smoke, medical X-rays, radiotherapy for certain types of cancer and radiation poisoning due to radioactivity.

Many of the most common reactive species include at least one oxygen atom, and these are generally referred to as reactive oxygen species, or ROS. Thus, ROS are oxygen-containing compounds that have paired electrons, but they behave similarly to free radicals because of the unstable conformation of their electrons. These include the, hydrogen peroxide, various lipid peroxides, and the hypochlorite anion. Reactive oxygen species that are free radicals include superoxide, hydroxyl radical and numerous peroxyl radicals that form during lipid peroxidation. The actions of free radicals and ROS often alter the function of cellular macromolecules, such as proteins, lipids and DNA. Proteins that are involved in critical cellular reactions, for example enzymes, that are altered by free radicals and ROS can disrupt vital processes throughout the cell, causing injury and cell death. The main free radical that causes damage to DNA is the hydroxyl radical, which specifically targets nucleic acids. Damage can include single-strand breaks, modification of base pairs, and cross-links between strands. If the DNA repair mechanisms are unable to fix the damage, the damaged DNA may contain various mutations that can affect cellular function, and in some cases this leads to carcinogenesis.

Some free radicals contain a reactive nitrogen molecule, and these nitrogen-based molecules are referred to as reactive nitrogen species, or RNS. The most common forms of RNS are the nitric oxide radical and peroxynitrite. In the same manner that ROS facilitate oxidation, RNS facilitate nitrosylation and nitration. Nitration reactions preferentially damage enzymes and proteins, which are composed of aromatic amino acids, resulting in inhibition of normal functions.

Regardless of how the ROS or RNS are generated, these molecules are ubiquitously linked to a variety of diseases and disorders. Ideally, then, a therapeutic molecule designed to address the deleterious effects of ROS or RNS should have the following characteristics:

• Retain the catalytic mechanism and high antioxidant efficiency of the natural enzymes,
• Broad antioxidant activity, particularly the following attributes:

  o attenuate superoxide radical mediated injury,
  o attenuate hydrogen peroxide mediated injury,
  o prevent formation of lipid peroxides, and
  o scavenge peroxynitrite,

• Ability to penetrate tissues and cells, and
• Move away from non-protein based molecules, which are more difficult and expensive to manufacture, to small chemical molecules.

The Aeolus compounds exhibit these and other characteristics that provide beneficial opportunities with respect to drug development.

Natural Defense Against Free Radicals: Antioxidants

The body protects itself from the harmful effects of free radicals through multiple antioxidant enzyme systems such as superoxide dismutases, or SOD. These endogenous antioxidants convert the reactive molecules into compounds suitable for normal metabolism. For example, and with respect to ROS, SOD catalyzes the conversion of two molecules of the superoxide radical into hydrogen peroxide and molecular oxygen. Hydrogen peroxide formed by this reaction is neutralized by the enzyme catalase, which combines two molecules of hydrogen peroxide to form water and molecular oxygen. Glutathione peroxidase also can neutralize hydrogen peroxide, but also plays an additional role in neutralizing lipid peroxides. Dietary antioxidants also exist, such as vitamins C and E, and beta-carotene. Antioxidants can effectively neutralize RNS in the same manner that they neutralize ROS.

When too many reactive species are produced for the body’s normal defenses to convert these into less reactive molecules, or when the body’s normal defenses against reactive species are compromised or defective, oxidative stress and / or nitrosative stress occurs with a cumulative result of reduced cellular function and, ultimately, disease.

Because of the role that reactive species play in disease, scientists are actively exploring the possible role of antioxidants as a treatment for related diseases and disorders. Preclinical and clinical studies involving treatment with the body’s endogenous antioxidant enzyme, SOD, or more recently, studies involving over-expression of SOD in transgenic animals, have shown promise of therapeutic benefit in a broad range of animal models of human disease. Increased SOD function improves outcome in animal models of conditions including stroke, ischemia-reperfusion injury to various organs, harmful effects of radiation and chemotherapy for the treatment of cancer, and in neurological and pulmonary diseases. Clinical studies with bovine SOD, under the brand Orgotein, or recombinant human SOD in several conditions including arthritis and protection from limiting side effects of cancer radiation or chemotherapy treatment, have also shown promise of benefit. The major limitations of enzymatic SOD as a therapeutic are those found with many proteins, most importantly limited cell penetration and allergic reactions; the latter resulted in withdrawal of Orgotein from the market in all but Spain.

Not All Antioxidant Therapeutics Are the Same

From a functional perspective, antioxidant therapeutics can be divided into two broad categories, (1) scavengers and (2) catalysts. Antioxidant scavengers are compounds where one antioxidant molecule combines with one reactive species and both are consumed, or destroyed, in the reaction. There is a one-to-one ratio of the antioxidant scavenger and the reactive molecule. With catalytic antioxidants, in contrast, the antioxidant molecule can repeatedly inactivate reactive species, thus a many-to-one ratio exists between ROS and the antioxidant. Vitamin derivatives that are antioxidants are scavengers – it is primarily because of this aspect of vitamins that they are poor candidates for use as therapeutics – far too much vitamin is required to destroy the reactive species for vitamins to be effective therapeutics. The SOD enzymes produced by the body are catalytic antioxidants. Catalytic antioxidants are typically much more potent than antioxidant scavengers, in some instances up to 10,000 times more potent. Use of antioxidant scavengers, such as thiols or vitamin derivatives, has shown some promise of benefit in preclinical and clinical studies. Ethyol, a thiol-containing antioxidant, is approved for reducing radiation and chemotherapy toxicity during cancer treatment, and clinical studies have suggested benefit of other antioxidants in kidney and neurodegenerative diseases. Again, large sustained doses of the compounds are required as each antioxidant scavenger molecule is consumed by its reaction with the reactive species. Toxicities and the inefficiency of scavengers have limited the utility of antioxidant scavengers to very specific circumstances.

Prior attempts to develop catalytic antioxidant therapeutics have had mixed results. Most of these approaches have been based upon development of chemical compounds that contain metal ions that are naturally found in the body and are essential to maintaining proper health. This is because some metal ions such as manganese, iron, copper, and zinc have the ability to donate or accept electrons during certain cellular reactions. For example, SOD enzymes contain copper and manganese. This ability makes such metal ions key components of many enzymes that are essential to the fundamental metabolic pathways of the body. However, in certain conditions these metals can promote the formation of free radicals. For example, the iron found in blood hemoglobin plays a critical role in oxygen transport from the lungs to the rest of the body, but this iron can also mediate the conversion of hydrogen peroxide into the more damaging hydroxyl radical. Catalytic antioxidants developed as potential therapeutics therefore include as a portion therefore a metal ion to accept free electrons from free radicals to neutralize the radicals.

However, catalytic antioxidants developed as potential therapeutics that have not had success in human clinical studies almost always have failed due to safety and toxicity issues, most often as the result of the metal ion being released, or leaking, from the compound – the released metal ions, as noted above, can themselves lead to the formation of free radicals.

Mechanistically, ROS tissue damage is essentially the same across animal species. While absolute predictability is never possible when animal models of human diseases and disorders are used, in many cases a more robust understanding of the efficacy of a potential therapeutic catalytic antioxidant molecule can be derived from animal studies then with other therapeutics that are directed to a particular receptor or enzyme. Safety issues are therefore of importance in the human testing of such molecules because this is the area where most often such compounds have experienced clinical issues.