Many diseases involve unwanted oxidation of cells and molecules in the tissues and lead to formation of extremely reactive free radicals, which in turn may lead to tissue damage. Drugs with anti-oxidant properties have been developed during the last decades, but there is still a need for developing safe drugs with a broad therapeutic potential for the treatment or prophylaxis of diseases or conditions that have an oxidative stress element.
Oxidative Stress
Oxidation is a chemical process which involves loss of electrons, i.e. a compound is oxidized when one or more electrons are removed from it. The opposite chemical process is called reduction. Oxidative stress in the human organism is defined as an increased, unwanted oxidation of cells and molecules in the tissues (reviewed in 1). It arises from an imbalance between oxidants, mediators of oxidative stress, and antioxidants, agents that can either prevent oxidation, detoxify oxidants or repair oxidized molecules (FIG. 1). The most important oxidants in humans and animals are reactive oxygen species (ROS) which include hydrogen peroxide, superoxide and the hydroxyl radical. The latter two belong to a group of compounds called free radicals. Free radicals are extremely reactive compounds due to the presence of unpaired electrons in their outer electron shells. ROS- and free radical-formation can be induced by, for example, metals and the oxygen-binding organic compound heme. Heme is an iron-containing component of haemoglobin and cytochromes, which are proteins that participate in the utilization of oxygen (see below). ROS, oxidants, and free radicals react with proteins, DNA and other molecular cell- and tissue components, which leads to unwanted modifications of the target molecules and ultimately loss of cellular functions.
Free radicals and oxidants are constantly introduced to the human body, both exogenously via the environment (food, air, smoke, etc) and endogenously as by-products of normal metabolism (FIG. 1). Endogenous free radicals and oxidants are important components of the metabolism in the animal organism. A certain amount is necessary for “house-keeping” cellular processes. For example, physiological cell-signalling is dependent on a continuous production of cellular free radicals (reviewed in 2), controlled by an intricate system of cellular antioxidants. Thus, cells need to maintain a normal, well-controlled reduction/oxidation (redox)-balance both intra- and extracellularly. Oxidative stress will result when the redox balance is upset. Free radicals and the strong oxidant hypochlorite (HOCl) are also produced in white blood cells during bacterial and fungal infections as weapons to kill the pathogens (reviewed in 3). This also leads to oxidative stress.
Haemoglobin and Other Heme-Containing Proteins
Haemoglobin is one of the most common proteins in the human body. It is found in enormous quantities in the red blood cells and its function is to carry oxygen from the lungs to all cells. The oxygen is bound to the iron-containing heme-group, which gives the haemoglobin molecule its red colour. All haemoglobin is normally kept inside the red blood cells and thus prevented from contact with other cells and extracellular components. This is important because haemoglobin is toxic due to strong oxidant properties. When the red blood cells break (haemolysis) in diseases like autoimmune haemolytic anemia, sickle cell anemia and malaria or in iatrogen situations including mismatched blood transfusion, stem cell and solid organ transplantation and major surgery, oxy-haemoglobin (haemoglobin plus oxygen) is released from the red blood cells. Oxy-haemoglobin spontaneously reacts with itself by rearranging electrons in a process called auto-oxidation, forming the free radical superoxide and methaemoglobin, an oxidized form of the protein. Methaemoglobin continues to decompose, ultimately forming free globin, heme and iron. The products are oxidative as described above. Free heme, being a hydrophobic molecule, can enter cells by diffusion over the cell membrane or dissolving the membranes. Free haemoglobin (located outside the red blood cells) is therefore an inducer of tissue damage during many diseases and other pathological conditions. In addition, free oxy-haemoglobin is indirectly a vasoconstrictor because it binds nitric oxide (NO) strongly, one of the most important dilators of small blood vessels and capillaries. NO-scavenging by free oxy-haemoglobin leads to consumption of NO and subsequent constriction of the capillaries resulting in high blood pressure.
Other heme-containing proteins include NADPH-oxidase, myeloperoxidase (MPO) and mitochondrial cytochromes. The enzymes NADPH-oxidase and myeloperoxidase are found in monocytes and neutrophil granulocytes, two subsets of white blood cells. In a process called oxidative burst, these enzymes produce superoxide radicals and hypochlorite, respectively, both of which are involved in the defense against microbial infection. The most important of the mitochondrial cytochromes are cytochrome c and NADH-dehydrogenase. These enzymes are components of the respiratory complexes I-IV which convert oxygen to water by using electrons from nutrients, stored fats, etc. In this process, large amounts of free radicals, mostly superoxide anions, are produced as intermediary metabolites by the mitochondrial enzymes.
Antioxidants
Normally, oxidant activity is balanced by the activity of antioxidants, protective factors that eliminate oxidants or prevent their oxidation reactions. During conditions of extreme oxidative stress, however, the antioxidants may be overwhelmed, leading to oxidative damage to molecules and/or cells and tissues.
Both endogenous and exogenous antioxidants are described. Twenty years ago, the prevailing view was that human homeostasis was dependent on externally added antioxidants, for instance via food intake. Today, an increasing number of human antioxidants have been discovered and shown to be produced constitutively within the body, i.e. under normal, unstressed conditions. Antioxidants operate by elimination of free radicals and oxidants. They can achieve this by three major mechanisms (see FIG. 2A and figure legends for details): 1) enzymatic addition of electrons derived from cellular aerobic metabolism or other sources to the oxidants, 2) non-enzymatic addition of electrons from the antioxidant molecule itself to the oxidant, and 3) binding (scavenging) of the radicals/oxidants to the antioxidant. Examples of the first category are the enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase and heme oxygenase. Examples of the second category are thioredoxin, glutathione and alpha-lipoic acid. Vitamins C and E, unsaturated fatty acids and plant flavonoids are exogeneous category 2 antioxidants that are not produced in the body but can be found in food. Some of the antioxidants of the second category, for example thioredoxin and glutathione, can be re-generated by reduction of electrons from other sources (FIG. 2A). Most antioxidants in the food are poorly re-generated after reacting with their targets. Thus, the consumed (=Oxidized) vitamins C and E, etc, present oxidative stress to the tissues unless quickly removed.
Electrons which are produced by cellular aerobic metabolism (ultimately derived from nutrients, e.g. glucose, fat, proteins via the electron-carrier NADH) provide the reducing equivalents to the antioxidants of category 1, when re-generating antioxidants of category 2 and in the scavenging process (category 3) (FIG. 2A). Therefore, most antioxidants are dependent on an intact cell metabolism and only operate intra-cellularly. In fact, most antioxidants, being intracellular, are part of the normal cellular “house-keeping” machinery.
Several antioxidants are specialized against haemoglobin-induced oxidative stress. The plasma proteins haptoglobin, hemopexin and transferrin bind free, extracellular haemoglobin, heme and iron, respectively, in the blood. The cellular protein ferritin binds and stores free, cellular iron. Heme oxygenase-1 (HO-1) is produced in most cells in response to increased concentrations of haemoglobin, heme and free radicals and eliminates heme by degradation into bilirubin, carbon monoxide and free iron.
However, none of the above-mentioned antioxidants act by all three mechanisms and, accordingly, a general therapeutic use of such an antioxidant is limited. An antioxidant having all mechanisms of action would be advantageous as it will have a much more general use and be less dependent on the cellular homeostasis for functioning.