TE3 - Samenvatting colleges en opdrachten TE3 PDF

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Thema Effecten van Geneesmiddelen 3Introductiecollege TE3 is about the safety of medication. It is primarily about the T in ADMET (absorption, distribution, metabolism, excretion and toxicity). Toxicity of drugs is often discovered too late, in phase 3 of the research or even when the drug is alread...

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Thema Effecten van Geneesmiddelen 3 Introductiecollege TE3 is about the safety of medication. It is primarily about the T in ADMET (absorption, distribution, metabolism, excretion and toxicity). Toxicity of drugs is often discovered too late, in phase 3 of the research or even when the drug is already on the market. We will look at the targets where toxicity occurs, with a focus on the three organs where toxicity happens most: the liver, the heart and the kidney. We will also look at the mechanisms of toxicity, through examining the biochemical systems in cell. Our goal is protecting the human against the toxic effects of drugs. Therefore we need better predictive systems, that can improve our risk and hazard assessment. College 1 - Introduction drug safety Goal as toxicologist is protecting the population for exposure to dangerous substances. Every substance is toxic, toxicity depends on the dose. There is a difference between acute and chronic toxicity. For example, for paracetamol there are the most emergency room visits per year. “All things are poison and nothing is without poison, only the dose permits something not to be poisonous.” - Paracelsus Toxicity in drugs is exemplified by adverse drug reactions, or unwanted side effects. Risk-benefit analysis needs to be done to determine whether a drug can be put on the market. Toxicity of drug is usually discovered very late in drug discovery process, sometimes only in Phase III or after the drug has been put on the market. Long term safety is unpredictable, can be solved by better understanding of toxicology. Mechanisms of toxicity:

Every mechanism starts with a drug, that can be converted through Phase I mechanism into a metabolite and then converted to another metabolite through the Phase II mechanism. The drug and its metabolites can initiate acute (organ failure/cytotoxicity), medium term (inflammation/ cholestasis) and long term (cancer/allergy/aging) effects. Chemical exposure leads to cellular

toxicity, eventually leading to organ toxicity. Cellular toxicity can be pathologically examined through molecular initiation events of cell state changes leading to a breaking point in cell fate. Severity of drug reaction dependent on dose of drug, the chemistry of the drug and the biology of the individual.

Measures used to understand causal relationship between drug exposure and effect:

The dosage-response effect is normally distributed, therefore a cumulative dose-response graph is usually used to determine dosage. The dose is often presented in log, to make the graph easier to read. The probit scale is used to assess dose-effect relationships. The duration of exposure also effects the ADR. Drug chemistry is very important as well in the severity of toxic reactions. Therapeutic drug development optimizes structure for certain receptor, specificity is the aim. During adverse drug reaction every receptor or metabolic pathway can be compromised if the structure happens to fit, the problem is non-specific interaction. There is a relationship between drug selectivity and drug attrition, more promiscuous drugs tend to be less safe. The understanding of the biology is tried to be understood through in vitro and in vivo models. Pre-clinical studies are required per international guidelines. These include single-dose and sub-acute toxicity studies, repeat-dose toxicity studies, development and reproduction toxicity studies, genotoxicity studies, carcinogenicity studies, local tissue tolerance, neurotoxicology, safety pharmacology studies and additional specialization studies (e.g. immunotoxicology, phototoxicity). College 2 - Organ toxicity: liver Liver is most common target organ of toxicity in preclinical toxicology studies. Drug-induced hepatotoxicity is a major cause of failure in drug development and of adverse drug reactions (Phase I: 43%, Phase II: 25%, Phase III: 27%). The physiology is an important cause of the high prevalence of toxicity as the blood first passes the liver. The hepatic vein provides the liver with oxygen. The liver lobule is the smallest functional unit in the liver. Blood enters the liver through the portal vein, joins with the hepatic artery leading to the central lobular vein and eventually leaves through the hepatic vein.

Hepatocytes have a very important function, the processing of xenobiotics. Waste substances are carried off through the bile canaliculi into the bile duct. There is a difference between the hepatocytes depending on the distance to the oxygen-rich blood (zone 1, 2 and 3) leading to a different expression of enzymes. Between the blood in the sinusoid and the hepatocytes there are epithelial cells with fenestra that allow efficient exchange in fluids. Stellate cell is important in the formation of fibrosis. Kuppfer cells are macrophages take take away waste, for example bacteria. Hepatocytes are very rich in mitochondria and rough endoplasmic reticulum to mediate metabolism. The liver is a target for toxicity due to high blood supply and first pass effect and its high phase 1, 2 and 3 drug metabolism capacity. The high oxygen consumption by the mitochondria makes it a sensitive target cell and as its hepatocytes are damaged it can easily become damaged fully through stacking of endogenous toxic substances as excretion functions may fail. There are many types of drug-induced liver injury (DILI) and these can be divided into three categories; acute hepatotoxicity, chronic hepatotoxicity and idiosyncratic reactions. These reactions can lead to certain pathologies, the most important ones being necrosis, cholestasis (obstruction of bile flow), sinusoidal damage, steatosis (fatty liver), fibrosis and cirrhosis (scarring and collagen), enzyme induction, carcinogenesis, inflammation and idiosyncratic hepatotoxicity. First pass effect is only observed after oral uptake, blood with substances that are absorbed in intestines goes to liver. In the liver these substances are metabolized and excreted through the bile duct, or reabsorbed into the blood then going to the kidney. Drugs optimally will not be metabolized in the liver and be spread through the blood in intended form. The metabolism that happens in the liver is divided into three phases. The phase 1 metabolism is mediated by cytochrome P450 enzymes to oxidize the molecules, making them more hydrofile and thereby more easily excreted. The phase 2 metabolism causes the molecule to become even more hydrophilic, through glutathione transferases, glucorodinases and sulphatases. The goal of these metabolism steps is detoxification but sometimes biotransformation can lead to bioactivation, when the metabolites are more toxic than the parent substance. Reactive metabolites can interact with proteins and DNA and these modification can lead to cytotoxicity, hypersensitivity (through formation of hapten) or cancer. When acute liver toxicity happens there are two forms of cell death. Apoptosis is regulated cell death while necrosis is unregulated cell death. One example of DILI necrosis is tetrachloride, this leads to zone 3 necrosis through membrane damage. Another example is the accumulation of reactive metabolite (NAPQI) formed from paracetamol after metabolism by Cyp2E leading to centrilobular necrosis. Regeneration is critical to restore liver after damage, and co-exposure to factors that inhibit proliferation therefore increases toxicity of many compounds. An example of DILI apoptosis is the AAF induced p53 accumulation leading to apoptosis in zone 1 hepatocytes. Another type of acute liver toxicity is steatosis. This injury is not necessarily just induced by drugs but most often by diet. Liver vacuoles fill with fat, through the mechanism of action of valproic acid, leading to the stacking of fatty acids. Chronic steatosis can lead to cirrhosis, the formation of scar tissue to such a state that the liver cannot function properly anymore. Cholestasis is another form of acute liver toxicity observed as jaundice, where disturbances in bile transport cause accumulation of bile acids in liver cells. This can have a hepatic or non-hepatic origin. Drugs can cause inhibition of bile acid transporters or hypoplasmia in bile ducts. Development of cholestasis can stop development of certain drugs.

Chronic hepatotoxicity also is observed in different ways. Fibrosis is when dying healthy cells are replaced with fibrous material made out of fibroblast and extracellular matrix components. Excessive accumulation of ECM leads to loss of fenestra and microvili, inhibiting function of hepatocytes. Fibrosis is caused by the overproduction of ECM proteins which is stimulated by stellate cells which are in turn activated by cytokines. Drugs that contribute to fibrosis are methotrexate, alcohol etc. Another chronic effect is hepatocellular carcinoma. Reactive metabolites that are hard electrophiles interact with DNA leading to damage and mutations. Mutations can lead to cancer development. Inflammation reactions in the liver can also be chronic. Immune cells such as macrophages (Kuppfer cells) and NK cells are already present in the liver, and neutrophils and lymphocytes can migrate to the damaged region of the liver. When these cells are activated their initial function is to remove dead and damaged cells, however when hepatocytes are damaged interaction between these cells and Kuppfer cells can induce a cycle of continued immune reaction, leading to damage. Idiosyncratic DILI population can be divided into three: tolerators (no injury), adaptors (transient injury) and susceptibles (serious toxicity). Only one in ten thousand develops serious adverse reactions due to individuals mixture of characteristics (nature and nurture). These adverse effects are not necessarily dosage, duration or prior-disease related therefore making it unpredictable and the risk factors not well known. To understand the factors of idiosyncrasy we need to understand the effects of genetic factors such as gender, HLA type and lifestyle such as diet, infections, chemical exposures. The other important factor is the chemical structure of the drug. Immune system can be involved in idiosyncratic DILI. Metabolism of drug to form hapten activating the immune system to kill hepatocytes. Toxicity can either be intrinsic or idiosyncratic. Intrinsic toxicity is predictable, dosage-related, similar in animals, high incidence, short interval and can be directly destructive, indirect or metabolic or cholestatic. Idiosyncratic drugs are the opposite and can be typically due to hypersensitivity or metabolic. Substances can be put on a scale of intrinsic/idiosyncratic effect. Drugs have been brought on the market with idosyncraticity, but more mechanistic understanding, in vitro testing methods and translational biomarkers are necessary to predict idiosyncratic effect. College 3 - Organ toxicity: kidney Kidney is also an important organ in toxicity. 25% of acute renal failure is caused by drug induced kidney injury. Toxicity in the kidney is difficult to screen pre-clinically, as the methods available are not sensitive enough. Knowledge about mechanisms and biomarkers are necessary for better screening. The kidneys are vulnerable for toxicity because they receive 25% of cardiac output. The kidney filters this blood and eliminates substances in urine to keep balance in body. This makes dosage important, as the concentration of drugs in the kidney is usually high. The blood is filtered in the glomerulus. The pre-urine contains all components that are in the serum, including glucose. Glucose is reabsorbed for 100%, and bicarbonate, sodium and chloride are almost reabsorbed 100% as well. This requires transport, and this requires energy. Therefore the kidney requires a lot of oxygen-rich blood to keep transport going. In kidney cells there is a high amount of mitochondria. Outside area of the kidney is the cortex, and on the inside are the medulla. These are specific areas where oxygen concentration is different. The smallest unit of the kidneys are

the nephrons. The kidney therefore has two important systems; the vascular system of the kidney, and the intrinsic kidney cells forming the nephron. The nephron is made up out of the glomerulus and the proximal tubule. The proximal tubule is made up out of the convoluted tubule (bochtjes) and the straight tubule. All glomeruli lie within the cortex. After the proximal tubule there is the descending thin limb, and the ascending thin and thick limb which enter into the distal convoluted tubule. This ends in the collecting tubule where the urine gathers and eventually ends up in the bladder. Depending on its position in the intrinsic kidney, every cell type has a different organization and thereby its own composition. Nephrotoxicants can target different sites in the kidney, as drugs that disrupt renal blood flow can cause ischemia, reduce filtration and cause cell death, while drugs that disrupt nephron cells can alter filtering, transport and concentrating properties. Drugs can induce acute kidney injury (AKI). This is when a decrease in the kidney function happens in the span of a few days, leading to the kidney not being able to eliminate harmful substances. This is linked to damage to the cells on the proximal tubule. These cells are needed for most of the transport, and need more energy. Hypo-perfusion therefore leads to lowering of ATP access due to low access to oxygen, ultimately leading to cell death. Nephrotoxic injury is increased in people with chronic kidney injury and circulatory problems. AKI mortality ranges from 30-70% depending on cause, age and severity and often leads to permanent effects in the kidney. There are three classes of AKI: pre-renal, intrinsic renal and post-renal. Pre-renal means that something happens in the body that has an effect on the kidney. An example of pre-renal is when there is heart failure and the kidney does not get enough oxygen. Intrinsic renal means that there is a problem in the kidney itself, for example nephrotoxins killing proximal tubuli cells. Post-renal means there is a problem in the urine tube, leading to pressure that kills cells in the kidney. Chronic kidney failure is when the kidney does not function optimally for a longer period. This can be measured by excretion of proteins (~1g/24hours). This can be caused by an irreversible AKI or prolonged use of drug. The disfunction of the kidney can also effect the rest of the body. The kidney function is measured through the glomerular filtration rate (GFR). Markers of GFR are the amount of serum creatanine in the blood or blood urea nitrogen, as this accumulates when the kidney doesn’t function properly. Causes of decreased GFR include afferent arteriolar constriction, tubular obstruction or tubular back-leak. Vasoconstriction leads to through decrease of the glomerular hydrostatic pressure. Obstruction means that crystals precipitate in pre-urine, not allowing the pre-urine to pass. Tubular back-leak is when cells in the walls of the arteriole die, allowing the pre-urine to flow back. In blue, factors that can contribute to GFR decrease. Damage is also an important factor in the GFR, this can lead to obstruction or back-leak. Measure of lower GFR can have a lot of different causes, making it difficult to determine mechanisms of kidney damage.

Physicochemical properties of drug that can contribute to kidney damage include charge, protein binding, solubility and molecular size. Charge impacts the kidney as it disrupts charge selectivity in the glomerular basement membrane, charge-selective transports in pro-urine uptake. Protein binding leads to disruption of kidney function. An interesting component is the binding of metals to methallothionine in liver, which has a lot of cysteine residues. Zinc usually binds to these residues but can be displaced by cadmium, which then induces methalliothionine production. Cadmium-methalliothionine re-uptake and metabolization leads to high levels of cadmium in liver, which is toxic. This is a very complex mechanism that can contribute to toxicity levels. Solubility is important, as drugs easily precipitate due to their lipophilic characteristics. High concentration of the drug in the pre-urine can lead to obstruction of the tubules. Large size molecules accumulate in the glomerulus as they cannot be filtrated. This is a problem when concentration of toxic (antibody) conjugates can cause kidney injury. There are many types of kidney injury, thrombotic microangiopathy, hemodynamic alterations, proximal tubular injury, distal tubular injury, tubular obstruction and interstitial nephritis. These are varying forms of injuries caused by toxicity so to discover which form is caused by the drug biomarkers are necessary. The proximal tubule is a major target for drug toxicity. The main reason for this is the transport function of the tubule, as its function is to eliminate xenobiotics. Another function is the high ATP turnover, and thereby the high number of mitochondria. The proximal tubule cells also have a high drug metabolism capacity. The transporters are important for kidney function. They are membrane proteins that transport drugs into and out of the cell, evolved to transport natural solutes but can also be used to transport xenobiotics. Transporters are expressed in many organs with barrier functions, including the liver, kidney, brain, intestine and placenta but differ in expression on apical and basal surfaces. These transporters regulate the bioavailability, efficacy and pharmacokinetics of the drug. There are many classes of transporters, influx/efflux, secretory/absorptive, passive/active/secondary active transporters. Passive transporters allow transport down an electrochemical gradient and do not consume energy, active transporters move molecules against gradient and do require hydrolysis of ATP for energy, secondary active transporters utilize ion gradients produced by primary active transporters (co-transport) to move molecules against gradient. Classes of transporters and example substrates include:

Drug transporters are found on the renal proximal tubule epithelium. Charged substances that are recognized as xenobiotic need transporters on the blood side of the cell and on the urine side of the cell to be excreted. Peptide transporters are only on the apical side to provide reuptake amino acids in blood. Localization of transporters is logical based on function of molecules in body.

Mechanism of cysteine-conjugate mediated proximal tubular cell toxicity Cysteine conjugates have certain transport pathways leading to uptake by the proximal tubule cells. Glutathione (GSH) conjugates are excreted in the blood, ending up in the ultra-filtrate. On the membrane of the proximal tubule cells there are enzymes that break down the glutathione, causing uptake of the amino acids and cysteine conjugate. The cysteine conjugate is metabolized to cysteine to be excreted, but a certain enzyme in the mitochondria metabolizes cysteine into a reactive metabolite leading to cytotoxicity. Mechanism of aminoglycoside nephrotoxicity Aminoglycoside is an antibiotic which is filtrated by the proximal tubular cells and ends up in the pre-urine. In the proximal tubule there are many vili with a certain charge, and aminoglycoside likes to bind to the lipids in those vili. Usually membrane vesicle intake through pinocytosis to lysosomes leads to break down of lipids. Lipids cannot be broken down when bound to aminoglycoside, leading to lipid accumulation which causes the lysosomes to burst and expel their contents into the cell. Lysosome contents include enzymes that damage the cytosol and eventually cause cell death. When tubular cell death happens, the cells release and end up in pre-urine forming clusters that can obstruct the proximal tubulus. This also causes back-leak when the cells are not yet replaced. Cellular repair depends on the extend of disruption and damage. Uninjured cells exposed to stress leads to adaptation of the cell for example through cellular proliferation, leading to refilling of the cell membrane. The immune response is important for cellular repair and recovery. Cytokines are released when the cell is damaged by activating macrophages, that allow repair and transmit proliferation signals to other cells. Adaptive immune system can also c...