How drugs can cause irreversible effects
All chemicals and of course drugs, interact with each other through three main routes. These range from relatively weak associations, such as van der Waals forces and hydrogen bonds, through to more potent ionic bonds and finally covalent bonds. Perhaps analogies for these interactions could be a child’s toy magnet for van der Waals and hydrogen bonds, an electromagnet for ionic bonds and a spot-weld for a covalent bond. The first two interactions are reversible, varying in strength, but leading to no permanent changes in the participants. A covalent bond is the product of a chemical reaction- rather than just an interaction and is not desirable unless the drug is intended to ‘weld’ itself to its receptor to destroy its functional capacity. This is the case with anticancer drugs as they cross-link DNA or drugs which react with steroid receptors. However, the vast majority of drug action is propagated through reversible bonds, where a receptor is activated and the drug leaves to interact with other receptors, just as their endogenous counterparts do.
Unfortunately, drugs do cause unintended irreversible changes to organelles, cells and tissues, leading ultimately to organ damage. There are several ways that drugs might cause irreversible toxic effects involving three possibly interrelated pathways.
1. Drugs may alter the expression of key genes in cellular homeostasis that may cause irreversible damage.
2. Drugs may act to cause one group of cells to destroy another, such as by eliciting an immune response, which recruits cellular or antibody-mediated attacks on tissues.
3. Drugs might chemically react directly with a variety of cellular structures, changing their structure and thus their function.
These observations lead to several key questions: firstly, how would any given drug cause these undesirable effects, what chemical interactions are involved and is it the parent drug and/or metabolites that are responsible? Starting with pathway 3, reaction with cell structures requires covalent binding and this would only occur in a highly unstable drug. With some exceptions (penicillin and alkylating agents) highly reactive entities are always weeded out in the drug discovery process, so pathway 3 is most likely to be caused by prior biotransformation to a reactive species either in or near to the tissue involved. It is not really possible to be precise about the processes occurring in Pathways 1 and 2, which may be caused by parent drug, stable or reactive metabolites. So biotransformation is probably the major determinate of irreversible drug toxicity, but it is important to recognize that it is not the only factor involved (Figure 8.4).
Figure 8.4 Main consequences of reactive species formation due to xenobiotic metabolism in different organs and tissues
Role of biotransformation in causing drug reactivity
There are many routes whereby reactive species may be formed. As you will have gathered, CYP enzymes can radically rearrange the structure of a molecule to make it more water-soluble and during this process molecular stability is usually reduced. Oxidation can transform some molecules from innocuous agents to highly reactive species that are lethal to cells if formed in sufficient quantity. In general, the less stable a compound is, the more likely it is going to react with cellular structures.
The CYP enzymes function in a similar way that machine tools do, where, say, a robot welds a piece of bodywork onto a car. The metal is subject to an intense, concentrated assault in a specific area that is designed to form a product. You can imagine what would happen if a live grenade was subject to this treatment. That would be the end of the robot. Obviously the robot is pre-programmed to weld anything of the appropriate dimensions that it is presented with, even something that could destroy it. You might feel that this is an ‘Achilles heel’ in drug metabolism; however, 1 billion years of research and development has indicated that there is really no other way to metabolize otherwise stable chemicals and for the occasional reactive by-product, there are sufficient repair and protection systems (GSTs, GSH, etc.). Just as you are fully insulated inside your car from the engine’s noise and emissions, the efficiency of the whole biotransforming system is reflected in the relative rarity of organ damage in most therapeutic drug use.
On a molecular scale, some chemical structures that are subject to the rigours of CYP-mediated oxidation form reactive products because of unique inherent features of their structure. Examples include strained three-membered rings and epoxides. These structures are the chemical equivalent of the old explosive, nitroglycerine, which could be detonated by shock alone. Some reactive species, such as the mechanism-based inhibitors discussed,can bind covalently to the active site and the enzyme is no longer functional. For biological function to continue, more enzymes must be synthesized. Drugs or chemicals that cause this effect are often termed suicide inhibitors and grapefruit juice and norfluoxetine cause this effect. The long period of inhibition of the CYPs that formed the metabolites reflects the time taken for more enzymes to be synthesized. These metabolites are so reactive that they are paradoxically no problem, as they just destroy the enzyme that formed them and cannot reach the rest of the cell. Indeed, after all the available CYP has been inactivated by these metabolites, no more metabolite will be formed until more CYP is synthesized.The rest of the parent drug may well leave the cell by other pathways. At the other extreme are metabolites such as hydroxylamines, which are no immediate threat if they are stabilized by cellular thiols or antioxidants such as ascorbate. These metabolites are so stable they can travel through cells and leave the organ in which they were formed and enter the circulation. Erythrocytes can thus detoxify them as described previously. In certain conditions, they can spontaneously oxidize forming nitroso derivatives, which are tissue reactive and cytotoxic. Further metabolism is necessary before they can be detoxified. In between these extremes are metabolites that can react with any cell structures that are short of electrons and seek electron-rich structures. Potent electrophiles such as nitrenium ions (N+) will react with nucleophiles, which are electron rich. If a CYP or any other enzyme forms a species that is missing electrons and seeks them, or is too electronegative, the net effect is a reactive species which has the potential to attack cellular proteins, DNA and membrane structures, forming covalent bonds, which can do sufficient damage to necessitate the resynthesis of that structure. It is important to consider also that reductive metabolism can form equally reactive species which are capable of causing similar cellular damage to oxidatively produced species. Whatever process forms reactive species, the likely result will be some form of irreversible binding to cellular macromolecules.
Cellular consequences of CYP-mediated covalent binding
The obvious question is how covalent binding is linked to cell damage; indeed, what are the processes that lead to cell damage? When a reactive species is formed in a cell, it can react with cellular organelles, enzymes, nuclear membranes, DNA, and the structure of the cell membrane. However, the hypothesis that a high rate of protein binding leads directly to cell death is an oversimplification. When animals have been treated with antioxidants prior to exposure to a reactive species, the animals survive, despite their levels of covalent protein binding, which are as high as untreated animals that have developed fatal organ toxicity. So the fate of the cell is subject to a competition between various factors:
• the rate, quantity and reactivity of the toxin formed;
• the extent of reactive ‘secondary’ toxins (superoxide, various free radicals) formed from the initial reactive species;
• the extent the cell can defend itself from the reactive toxin by rendering it harmless;
• the period of time elapsed before cellular defensive resources are overcome;
• which specific molecules are damaged in the cell where irreversible damage occurs;
• the extent of possible repair and restoration;
• whether the intra- and extracellular damage attracts the attention of the immune system.
These competing processes might lead to three main cellular outcomes; necrosis, where destructive forces overtake the cell, or apoptosis may be triggered, leading to an orderly dismantling of the cell and its contents, or the damage may be attenuated and repaired leading to survival.
Sites of biotransformational-mediated injury
Barrier tissues
It is usually the case that although biotransformational capability exists in all tissues to varying degrees, a specific xenobiotic will show metabolism-mediated toxicity in a particular organ, tissue or group of tissues. These are often ‘barrier tissues’ that represent the front line against exposure to environmental toxins; these include the lung, gut, liver and the skin. These tissues are very rich in detoxification enzyme systems and have at their disposal the full apparatus for the control and expression of these enzymes. Although these organs are well defended, reactive species generation can still cause irreversible damage, either locally or distant from the site of formation. This is partly due to the nature of our acute and chronic exposure to environmental and dietary xenobiotics that overtaxes the capability of our defences and partly due to our genetically differing capabilities to resist these toxins over our lifespans. The skin does possess considerable local bio-transformational activity, but lacks the in- depth protection of high hepatic GSH levels and it is subject to many local torsional movements, which may influence tissue stress. The following sections give some examples of agents that can cause irreversible damage to a number of different cells, tissues and organs, although the liver features most prominently.
Hepatotoxicity
Ironically, although the liver boasts extremely comprehensive detoxification defences and astonishing powers of recovery, hepatotoxicity is one of the commonest reasons for the failure of a candidate drug in clinical trial and even the withdrawal of a new drug from the market. This is mainly because the liver contains the largest concentration of biotrans-formational enzymes in the body and bears the greatest burden in clearing drugs and other xenobiotics. This means that it is more likely to form reactive metabolites in quantities sufficient to cause cellular injury or to trigger an immune response. In addition, the central position of the liver in homeostasis makes severe hepatic injury a life-threatening event that may only be remedied by a transplant. Unlike toxicity in many other tissues,hepatotoxicity has been studied extensively in detail over the last few decades and there are accurate diagnosis criteria for drug induced liver injury. These validated criteria are applied to existing or newer drugs by regulatory organizations. They are termed ‘Hy’s Rule’ after their originator, the renowned hepatologist Dr Hyman J. Zimmerman. The rule states that if ALT levels equal or exceed three times the upper limit of normal (ULN) and serum bilirubin equal or exceed twice the ULN, then mortality can range from 10-50 per cent, depending on the drug and patient pathology. Hepatotoxicity may have many causes and be the result of different drug or dosage levels, but the commonest clinical manifestation of liver failure remains necrosis due to drugs, either by overdose or idiosyncratic causes.
