IDM - Safety Pharmacology and Toxicology

What is Safety Pharmacology? Safety Pharmacology sets out to predict whether a drug, if administered to human (or animal) populations, is likely to be found unsafe, and its professional mandate is to prevent such an occurrence. What is post marketing surveillance? After drug is on the market the manufacturer and regulatory authorities must keep and report various things such as adverse drug reactions. How do regulatory authorities monitor adverse drug effects? Regulatory authorities use tools such as drug experience reports, medical literature (clinical trial data) and multiple agency data sources and spontaneous reporting system (SRS) to monitor adverse drug effect patterns potentially indicative of a public health concern. What is terfenadine? Terfenadine was an antihistamine. It was used to treat allergies, hives, and other allergic inflammatory conditions. The antihistamine, terfenadine, was withdrawn following a growing awareness that the drug could evoke the potentially life-threatening cardiac syndrome, torsades de pointes (TdP), in otherwise healthy patients. Why was terfenadine removed from the Terfenadine had a low efficacy to induce market? TdP making it so rare and event that it required several million prescriptions before its liability became suspected. Terfenadine was used for hay fever so the risk (death) outweighed the benefit (a runny nose). What is preclinical toxicology testing? Preclinical toxicology testing is an approach that involved determining the high-dose adverse event profile of a compound given at chronic, toxic doses. 1 / 17 It would not have detected a rare lethal event liability at a therapeutic dosage. This problem could have been avoided if, instead of routine toxicology, a programme of specific high throughput screening (HTS) for TdP liability had been utilised in the early drug discovery How could the delayed reaction of TdP at the time. been avoided? However, The consideration of biomarkers for rare adverse event liability was not part of the toxicology agenda in the early 1990s. The structure of a safety pharmacology program is to determine the potential undesirable pharmacodynamic effects of a drug on the central nervous, cardiovasWhat is the structure of a Safety Phar- cular and respiratory systems, as well macology program? as to implement supplementary tests to evaluate other organ systems. It is primarily designed to take account of regulatory requirements: scientific issues are secondary. - Preclinical safety pharmacology models require better validation. - Validation requires a quantitative and accurate human template of liabilities of positive and negative controls with which to compare model data sets. - Validation is not possible for models What are the main challenges to effecscreening for liabilities that are rare or tive Safety Pharmacology Programs? imprecise with current drugs in humans. - Validation is also not possible for methods for evaluating human-specific biologics (that are antigenic in animals). - It must be understood that interpretation of surrogate biomarker data sets is unavoidably subjective. 2 / 17 - Preclinical safety testing in a non-validated setting must therefore be regarded as non-scientific whereby yes/no judgments will remain subjective in the absence of true validation of the models available. - Scientific validation of safety testing methods remains the goal, however elusive this may seem. - Scientific validation requires blinded randomised testing of drugs known to have and known to not have a liability foe What are the main challenges to efthe specific adverse effect in humans. fective Safety Pharmacology Programs? - A rank order of liable drugs in humans (Continued...) ('gold standard') is the best template. - It must be acknowledged that a gold standard does not exist for most adverse effect liabilities. This poses a problem. - In the absence of validation it is better to live with false positives than risk the chance of a false negative. What is toxicity? Toxicity is the degree to which a substance is poisonous or can cause injury. Unlike pharmacological effects, the onset of which is usually relatively rapid, toxic effects can surface after months or Why are many toxicities identified after a even years of drug treatment. drug has been launched? Because of this, many toxicities are identified after the drug has been launched and is in widespread use. Why is toxicity so important? The toxicity of drugs in widespread therapeutic use is of major concern. Furthermore, approximately 10% of accident and emergency workload is attributed to toxicity as a result of drug overdosing (often accidental). 3 / 17 The side effects associated with the use of a particular drug are referred to as adverse effects or adverse drug reactions (ADRs). These terms incorporate any effect that What are the side effects associated with is not a beneficial therapeutic effect. A the use of a particular drug referred to? particular drug can cause one (or multiple) adverse effects. These effects can themselves be brought about by single (or multiple) mechanisms. What can toxic effects include changes in? Toxic effects are diverse in nature and can include changes in: - Biochemistry - Growth - Morphology - Development - Physiology - Lifespan How long do clinical manifestations of toxicity take to appear? Clinical manifestations of toxicity can appear rapidly (within minutes of dosing) or after many years of treatment. The latter makes identifying certain toxicities very difficult since they can only become evident after long-term dosing extending beyond the time frame assessed during clinical trials. What is a target organ? When a drug is toxic, the organ it affects is described as the target organ. ADRs are widespread and can affect many different body tissues and systems. However, toxicity to the liver and kidney are commonly observed. 4 / 17 The liver is a metabolic powerhouse, designed to convert potentially toxic substances into safer (more soluble) forms for rapid excretion. What is hepatoxicity? Unfortunately, in some cases, reactive metabolites can be formed which are more toxic than the parent drug. Hepatocytes are therefore exposed to high concentrations of those reactive metabolites making toxicity to the liver a commonly encountered ADR. It is found that a reactive metabolite is formed and this leads to toxicity, it is often possible to block certain metabolic How can certain metabolic pathways be pathways by replacing the responsible blocked? functional groups(s) with atoms such as fluorine. However, this may have a knock-on effect on drug efficacy. What is nephrotoxicity? Two major functions of the kidney are to remove waste products from the blood and to reabsorb water. However, drugs (and their polar metabolites) therefore concentrate in renal tubular fluid as water is reabsorbed, meaning the renal tubules are potentially exposed to high concentrations of toxins. What is exposure? The exposure of a patient to a drug is a key element of toxicity and is dependent upon the route of administration. E.g. oral administration renders a drug susceptible to first pass metabolism and possible metabolic activation. How can exposure to drugs happen? Exposure to drugs can be via a single dose or by multiple doses over differing durations. Typically, medications are pre5 / 17 scribed as multiple doses over a period of a few days to many months/years. Acute exposure - for a duration of <24 hours - single dose Subacute exposure - for a duration of 1 month - repeated doses How is exposure to drugs categorised? Subchronic - for a duration of 1-3 months - repeated doses Chronic - for a duration of >3 months repeated doses Why is duration important? Differences in the duration of exposure can lead to different toxic effect (for the same compound). Acute exposure - Central nervous system (CNS) narcosis. Exposure to Benzene Chronic exposure - Bone marrow damage and leukaemia. Acute exposure - CNS stimulation (nicotine). Exposure to cigarette smoke Chronic exposure - Cancer of the mouth, larynx, lung, oesophagus, pancreas, bladder; emphysema, etc... - Very common or very rarely seen. - Minor or life-threatening. - Easily identified during animal testing and clinical trials. Adverse Drug Reactions (ADRs) can be: - Very difficult to identify and may only become evident following years of use in humans and after millions of doses have been dispensed. 6 / 17 ADRs are of great concern to regulatory agencies that are responsible for both drug safety and efficacy. Such agencies include the European Medicines What regulatory agencies are responsiAgency (EMA), the Food and Drugs Adble for both drug safety and efficacy? ministration (FDA), the Medicines and Healthcare products Regulatory Agency (MHRA) and the World Health Organisation (WHO). What are the two types of ADRs? Related and Unrelated. What are Related ADRs? Related ADRs are: Related to the drugs main pharmacological action. - Referred to as 'Type A' adverse reactions or 'augmented'. - Commonly known as 'on-target toxicity'. - Related to the dose and susceptibility of the patient. - These are often predictable if the mechanism of pharmacological action is well understood, e.g. postural hypotension with alpha1-adrenoreceptor antagonists and bleeding with anticoagulants. - Usually reversible in nature (often avoided by reducing dosage). - Sometimes very minor but can be very serious (e.g. hypoglycaemic coma with insulin). What are Unrelated ADRs? Unrelated ADRs are: Unrelated to the drugs main pharmacological action. - Referred to as 'Type B' adverse reactions or 'bizarre'. - Commonly known as 'off-target toxicity'. - Can be sometimes predicted, e.g. when taken in overdose or when susceptibility is increased (e.g. pregnancy or predisposing disorder). 7 / 17 - Usually unpredictable idiosyncratic reactions initiated by a reactive metabolite. - Uncommon, but severe idiosyncratic ADRs include aplastic anaemia from chloramphenicol and anaphylaxis in response to pencillin. Idiosyncratic drug reactions (IDRs) are not related to the dose of the drug and are 'particular to an individual'. Different individuals may have differing susceptibilities to toxicants due to numerous factors, including genetic traits (as a result What are Idiosyncratic drug reactions? there is sometimes a 10-30 fold difference between patients). Due to the idiosyncratic nature of these effects, they are unfortunately, difficult to detect and almost impossible to predict. Toxic concentration of substances (or commonly, their reactive metabolites) What can toxic concentrations of sub- can cause necrosis (cell death). Reacstances do? tive metabolites can form covalent bonds with target molecules and/or cause damage via non-covalent mechanisms. Reactive metabolites can potentially be cytotoxic by one of four common covalent processes: What are non-covalent interactions? - Lipid Peroxidation - Reactive Oxygen Species (ROS) - Depletion of Glutathione - Modification of Sulfhydryl Groups 8 / 17 Peroxidation (oxidative degradation) of unsaturated lipids can be initiated by reactive metabolites or by reactive oxygen species (ROS). Lipid Peroxidation Lipid peroxyradicals (ROO.) can produce hydroperoxides (ROOH), which can produce more ROO. - This initiates a chain reaction - known as peroxidative cascade. Cell damage results from alteration of membrane permeability and/or by products of lipid peroxidation reacting with proteins. Reactive Oxygen Species (ROS) Involves the reduction of molecular oxygen (O2) to form a superoxide anion (O2-). This can then be enzymatically converted to form: - Hydrogen peroxide (H2O2) - Hydroperoxy radicals (HOO.) - Hydroxyl radicals (OH.) - Singlet oxygen All of which are cytotoxic directly or via lipid peroxidation. Glutathione (GSH) is the primary antioxidant in cells. It is a tripeptide consisting of cysteine, glycine and glutamate units. The function of the GSH redox cycle is to protect from oxidative stress caused by: Depletion of Glutathione - Normal oxidative products of cell metabolism - Toxins (including drugs) - GSH Iso has a role in the defence from the viruses, bacteria, heavy metal toxicity, radiation, etc. 9 / 17 Importantly, GSH can be depleted. When cellular GSH falls to 20-30% of normal, cellular defence is compromised and cell death can occur leading to severe toxicity. Modification of Sulfhydryl Groups Free sulfhydryl groups are critical in the catalytic activity of many enzymes. However, they can be altered reversibly by oxidising species or by covalent interactions with toxicants. Important sites for sulfhydryl groups include the cytoskeletal protein actin and the Ca2+ transporting ATPases. What can drug substances capable of covalent interactions do? Any drug substance capable of covalent interactions can bind to numerous biological targets including DNA, proteins, peptides, lipids and carbohydrates. Covalent interaction with DNA is the primary mechanism of mutagenic chemicals. However, non-mutagenic chemicals can also bind covalently to macromolecules. What is Paraoxon? Paraoxon is an active metabolite of pesticide (parathion). It is used as an ophthalmic drug against glaucoma. It acts as a cholinesterase inhibitor and binds to acetylcholinesterase at the neuromuscular junction and causes necrosis of skeletal muscle. What chemicals are toxic? All chemicals can be toxic at the right dose. Even water can be toxic. In other words, the toxic response is a function of the concentration of the substance at the site of action. Some compounds are highly toxic and 10 / 17 can be lethal if ingested in minute quantities (e.g. botulinum toxin). Others are only toxic in very high doses (e.g. saccharin). How is a dose-response relationship generated? The toxicity of a compound (percentage response) can be plotted against dose (usually in mg/kg) to generate a dose-response relationship. What is NOAEL? The NOAEL is defined as the No Observable Adverse Effect Level. This is the maximum dose of a compound a laboratory animal or human receives leading to no observable toxicity (adverse effect). The NOAEL highlights if there is a dose-threshold limit that must be exceeded in order for a toxicity to be observed. Sometimes no dose-threshold is present (or it is very small) and any dosing results in toxicity. What is LOAEL? The LOAEL is defined as the Lowest Observable Adverse Effect Level. This is the minimum concentration of a substance needed to elicit an adverse (toxic) effect. How is toxicity measured? When measuring a desired therapeutic response, it is common to express this using an EC50 value. This is the effective concentration (in mg/kg) necessary to produce the desired effect in 50% of test subjects. Sometimes this is shown as ED50 for the effective dose in place of concentration. What is TD50? The TD50 is the toxic dose (in mg/kg) that produces toxicity in 50% of test subjects. 11 / 17 What is LD50? The LD50 is the lethal dose (in mg/kg) that produces lethality in 50% of test subjects. The therapeutic Index (TI) relates the pharmacologically effective dose to the toxic (or lethal) dose and is expressed as: TI = TD50/ED50 or LD50/ED50 What is the therapeutic index? How to test for toxicity? The higher the TI value the higher the relative safety. In order to identify and screen for ADRs, numerous toxicity tests are performed to identify possible hazards. Toxicity testing effectively begins at the pre-clinical stage when safety pharmacology screening is being conducted. The more general assessments made at this stage are usually sufficient to identify serios and commonly occurring ADRs. Toxicity tests: How do toxicity tests work? - Are performed in different (at least two) animal species. - Consist of short and long term studies (depending on the endpoint). - Involve regular monitoring for numerous physiological and biochemical changes. - Are often performed at higher dose ranges than the expected therapeutic range. 12 / 17 - Are used to identify the likely target organs. - Include recovery studies to assess whether the toxicity is reversible or irreversible. The basic assumption of any test utilising animals (or animal tissue) is the the animal models used mimic what will happen in humans. This is sensible given the simWhat is assumed when animal testing? ilarities at the cellular and molecular level. However, interspecies variations do exist (e.g. differences in metabolism capability). What is Pronethalol? Pronethalol was the first beta-adrenoreceptor antagonist (beta blocker) developed at ICI but was not developed further as it caused carcinogenicity in mice. Subsequently, it was shown only to occur un the ICI strain of mouse, nut by this point other beta-blockers were already in use and in development. What are In Vitro tests? These are generally rapid screening assays for large numbers of compounds outside of the body in a laboratory environment. These can generate numerous false positive and negatives since they are simplistic models of a vastly more complex system. What is the Ames Test? One of the most commonly used in vitro assays is an assay for mutagenicity known as the Ames Test. This assay uses Salmonella strains of bacteria that have histidine synthesis gene mutations (they cannot synthesis histidine which they require to grow). 13 / 17 A negative result is seen when there is very limited growth - with just very small number of bacteria naturally mutatWhat can be seen from the Ames Test? ing back (revertant) to their original form. A positive test sees ab increase in the rate of revertants near the site where the test chemical is applied. Liver metabolism in vivo generates reactive metabolites which are mutagenic. This metabolic step is missing from the standard Ames test. How to combat the generation of reactive In order to combat this, the efficiency metabolites? of the Ames Test can be increased by adding fresh 'liver extracts' containing metabolic enzymes to mimic mammalian metabolism. Advantages of the Ames Test The Ames test is a simple, cheap and widely used toxicological test for mutagenic potential. Drugs that test positive in the Ames test are usually rejected for further study. Commonly used assays in in vitro include: What other in vitro tests are there? In vitro mammalian chromosomal aberration test In vitro cell micronucleus test What are In Vivo tests? These tests utilise animal models or human volunteers. Advantages of In Vivo testing These are much more reliable since they are whole-animal models complete with complex metabolic processes, etc. Disadvantages of In Vivo testing In vivo tests are time-consuming and very expensive, there are interspecies differences to consider, but they are usu14 / 17 ally required by regulatory authorities in order to get marketing approval. What are some examples of in vivo tests? - COMET assay to identify substances that cause DNA damage - Reproductive/developmental toxicity screening test - Carcinogenicity studies. How is toxic potential assessed? Due to the possibility of drug-induced toxicity, comprehensive assessment of toxic potential is required for all new pharmaceuticals. However, there is no single toxicity test which is adequate in isolation, since they are all focused towards particular toxicity endpoints. A battery of in vivo and in vitro tests is used to generate a robust toxicity profile. This battery of tests typically includes: - A test for gene mutation in bacteria (e.g. Ames). How is a robust toxicity profile generat- An in vitro test with cytogenetic evaluaed? tion of chromosomal damage. - An in vivo test for chromosomal damage using rodent haemopoietic cells. - Reproductive/developmental toxicity testing. - Carcinogenicity testing. How can toxicity be predicted? It is sometimes possible to predict the toxicity of certain drugs if the mechanism of pharmacological action is well understood. In addition, toxicity may be predicted as a result of changes in drug formulation, as a result of renal/liver impairment or as a result of drug-drug interactions. 15 / 17 It is possible to utilise computational models and tools to predict drug toxicity from knowledge of chemical structure alone. The area of predictive toxicology has received a lot of interest in recent years since it has the potential to reduce or even replace the use of in vivo animal tests. Commercial or freely accessible models How can toxicity be predicted using com- now exist enabling the prediction of: putational methods? - Carcinogenicity and mutagenic potential - Hepatoxicity - Cardiotoxicity - Nephrotoxicity - Binding to specific receptors known to be associated with toxicity issues (e.g. hERG) - Drug-induced phospholipidosis What is a pharmacophore? A pharmacophore is the atoms/functional groups required for a specific activity, and their relative positions in 3-dimensional space. These groups are often responsible for the molecular interactions (e.g. H-bonding, Pi stacking) between a drug and target receptor. Other functional groups in the molecule may be required to hold the pharmacophore groups in optimal positions or may contribute to other desired properties such as solubility. What is a toxicophore? A toxicophore is a feature/functional group within a chemical structure which is responsible for the toxic properties of the molecule, either directly or after metabolic processes. 16 / 17 Bioisosteres are groups that produce compounds with similar biological activity What are bioisosteres? Example of bioisosteres: when you replace a fluorine with a hydrogen, you generate molecules with similar biological activity. However, this is not a rule—it doesn't mean that every time you substitute a molecule with a bioisostere or an isostere, the result will be similar biological activity. 17 / 17