PHAR 9: Drug Metabolism and Toxicology Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
What is toxicology?
A
- Toxicology is commonly called the “science of poisons”.
- More specifically, it is a field of science where an understanding of the potential harm that can come from exposure to chemical, physical, and biological agents is sought.
3
Q
What are toxicants and toxins?
A
- Substances that cause toxicity are collectively called toxicants, with those produced naturally (e.g., in a plant) also known as toxins.
- You will find that these terms are used flexibly/interchangeably so you may encounter both.
4
Q
How can we classify toxicants?
A
- We can classify toxicants in multiple ways, such as their intrinsic nature, origin, target, etc.
- For example, toxic responses/effects may be observed for both chemical and non-chemical agents that interact with components of biological systems:
- chemical: e.g. ethanol
- physical: e.g. ionising radiation
- biological: e.g. botulinum toxin
5
Q
Match the terms to the most appropriate definition of toxicity
A
6
Q
What is systemic toxicity?
A
- This affects the whole organism or a large number of different tissues/organs.
- Toxicants that affect very common biochemical processes typically give rise to systemic toxicity
- e.g., hydrogen cyanide inhibits cytochrome oxidase and the oxygen utilization in any relevant tissue and therefore its effects are widespread
7
Q
What is target organ toxicity?
A
- this affects only one or more target organs.
- Toxicity may be organ-specific due to accumulation, function, metabolism, etc.
- e.g., asbestos causes inflammation specifically in the respiratory system and subsequent pleural mesothelioma
8
Q
Describe how toxicants can be classified by duration or time of the original exposure
A
- acute
- subchronic
- chronic
- carcinogenicity
- developmental
9
Q
Describe acute toxicity
A
- acute toxicity occurs almost immediately after exposure.
- Acute exposure is usually a single dose or a series of doses received within a 24-hour period.
10
Q
Describe subchronic toxicity
A
- Subchronic toxicity results from repeated exposure for several weeks or months.
- This is a common human exposure pattern for some pharmaceuticals and environmental agents.
- Examples include:
i) ingestion of warfarin (anticoagulant) for several weeks as a treatment for venous thrombosis, can cause internal bleeding;
ii) workplace exposure to lead over a period of several weeks can result in anemia.
11
Q
Describe chronic toxicity
A
- Chronic toxicity represents cumulative damage to an organism - many months or years to develop a disease.
- With repeated exposures or long-term continual exposure, the damage from these subclinical exposures slowly builds up until it exceeds the threshold for chronic toxicity.
- Ultimately, the damage becomes so severe that the organ can no longer function normally and a variety of chronic toxic effects may result.
- Examples include
i) hepatic cirrhosis in alcoholics who have ingested ethanol for several years;
ii) chronic kidney disease in workmen with several years of exposure to lead;
iii) chronic bronchitis in long-term cigarette smokers;
iv) pulmonary fibrosis in coal miners (black lung disease)
12
Q
Describe carcinogenicity
A
- Carcinogenicity is a complex multistage process of abnormal cell growth and differentiation.
- The process normally takes many years and cancer is commonly, but not exclusively, a disease of old age.
13
Q
Describe developmental toxicity
A
- Adverse toxic effects to the developing embryo or foetus.
- Can result from toxicant exposure to either parent before conception or to the mother and her developing embryo-foetus.
- The three basic types of developmental toxicity are:
- embryolethality
- embryotoxicity
- teratogenicity.
14
Q
List some factors that influence the toxicity of agents
A
- Form(ulation) and innate chemical (re)activity
- Dose
- Exposure route and duration of exposure
- Species
- Sex
- Age
- Physicochemical properties
- Extent, type, and site of metabolism
- Route of excretion
- Presence of other chemicals - additive/synergistic/antagonistic effects
15
Q
What is arguably the most straightforward and clear endpoint that can be measured for a toxin?
A
- death
16
Q
What is the median lethal dose (LD50 / MLD)?
How useful is this measurement?
A
- the median lethal dose describes the dose required to kill half of the population under study
- Therefore if the toxicological endpoint is death, then the LD50 is equivalent to the TD50.
- This was a common endpoint used in the early days of toxicology/pharmacology, but it is not a particularly useful endpoint to use, and therefore more specific measures are commonly used to assess toxicity.
- Clearly, if we are establishing the toxic effects of a toxicant or a drug in humans, then it would be both inappropriate (to say the very least) to use death as the toxicological endpoint!!!
- Note that although some regulatory agencies involved in the classification of toxicants often include LD50 measures, it is not considered to be very useful in assessing the safety of drug candidates in drug development.
- As above, more mechanistically-relevant and/or nuanced measures of toxicity are preferred, such as biomarkers of inflammation, DNA damage, cellular aberrations, etc.
17
Q
Why is it that in some cases it is better to describe drug effects as on-target or off-target instead of non-toxic and toxic?
A
- in some cases we may deliberately be seeking to cause toxicity:
- In PHAR 7 – Antimicrobials, we introduced the concept of using differences between species to be able to differentially affect biological functions
- e.g., specific inhibition of bacterial ribosomal function vs human ribosomal function, often with the intention of deliberately inhibiting/disrupting/dysregulating function to prevent infection.
- In this instance, administration of the drug is to deliberately cause toxicity in the target cell, and response measures of this would reflect the efficacy of the drug (on-target); antimicrobials are known to cause toxic effects in humans (off-target)
- In PHAR 8 – Cancer Drug Therapy, we studied the related idea that we can target specific cell sub-populations based on biological differences
- e.g., preferentially targeting rapidly proliferating cancer cells with DNA alkylating agents
- In this instance, the administration of the drug is to deliberately cause cellular toxicity in those specific cell sub-populations (on-target) but the drug may affect other cells (off-target).
18
Q
What is the dose-response assessment?
A
- The dose-response assessment quantitates toxicological hazards that have been identified;
- it determines the relationship between dose and incidence of (adverse) effects (usually in humans).
19
Q
What are the two major extrapolations required in the dose-response assessment?
A
- The first is an extrapolation from high experimental doses to low environmental doses.
- Non-carcinogenic effects (e.g., neurotoxicity) are considered to have dose thresholds below which the effect does not occur.
- Carcinogenic effects are generally not considered to have a threshold and mathematical models are generally used to provide estimates of carcinogenic risk at very low dose levels.
- The second is an extrapolation from preclinical models to humans; safety in experimental animals does not necessarily indicate safety in humans, and toxicity in experimental animals does not necessarily predict toxicity in man.
20
Q
What is acceptable daily intake (ADI)?
A
- The ADI procedure is used to calculate permissible chronic exposure levels for humans.
- For this purpose, it is assumed (for convenience) that humans are as sensitive as animal test species.
- The ADI is the amount of a substance to which a person can be exposed each day for a long time (usually a lifetime) without suffering harmful effects.
- It is determined by applying safety factors to the highest dose in human or animal studies that has been demonstrated not to cause toxicity.
21
Q
What is NOAEL and LOAEL?
What do they help us understand?
A
- NOAEL: No observable adverse effect level.
- The highest dose level at which no adverse response is detected.
- LOAEL: Lowest observable adverse effect level.
- The lowest dose level at which an adverse response is detected.
- Knowing these doses helps us establish a point of departure in the toxicological dose-response curve where the response increases from no effect.
22
Q
Why do we want to know the NOAEL and LOAEL scores?
A
- The main reason is to calculate how much of a substance someone can be exposed to on a regular basis (e.g., daily) without experiencing adverse/toxic responses.
- Doing this is useful in many situations, particularly in those where it is possible to regulate exposures to substances (e.g., substances in food products, occupational exposures to chemicals, environmental exposures arising from known sources).
- Knowing the range of doses that elicit a response allows an acceptable daily intake (ADI) to be calculated and used to help ensure humans are not exposed above this level.
- Clearly, it would not be a good idea to set the at the same as the NOEAL, as this value may have been determined in an animal model, and therefore there is some model-to-human extrapolation to account for.
- The ADI calculation takes into account the NOAEL value that has been determined and then applies a number of uncertainty factors (or safety factors).
23
Q
What is the ADI calculation?
A
- ADI = NOAEL / Uncertainty Factors
24
Q
Study these images of a dose-response curve showing the NOAEL and LOAEL
A
27
Q
Describe the liver, a major site of xenobiotic metabolism
- why
- what drugs are metabolised there
A
- the liver is important because of its size, position, blood supply, and function.
- To recap, orally-administered drugs that are absorbed via the GI tract are transported by the hepatic portal vein into the liver, where they perfuse between and into liver cells (most important for drug metabolism are hepatocytes).
- Liver cells exhibit very high expression of genes encoding drug-metabolizing enzymes in comparison to other organs/tissues, and consequently, there is a high capacity for drug metabolism in the liver.
- First pass metabolism of drugs by the liver may mean low systemic exposure; some drugs are well absorbed but extensive metabolism by the liver results in little reaching the site of action.
28
Q
Describe the probablistic ‘Pachinko’ mechanism of xenobiotic metabolism
A
- The biotransformations that are involved in normal/core endogenous biological functions (e.g., catalysis of the steps in glycolysis) tend to be tightly regulated, astonishingly specific, and very efficient.
- One might expect this to be a result of evolutionary pressure, where every refinement of these catalytic steps helps confer an advantage to the overall ability of the organism to function normally and survive.
- By contrast, the ability of living systems to metabolize xenobiotics is arguably driven by a different pressure; the ability to detoxify substances that are not part of normal/core endogenous functions, and that may potentially cause harm.
- This may include substances that are commonly present in the environment, but also can include those that are completely novel (e.g., a synthetic compound not found in nature, made by a chemist).
- The result of this is that organisms often have genes that encode proteins able to catalyze the biotransformation of xenobiotics, more commonly known as drug-metabolizing enzymes (DMEs).
- Higher organisms typically express a wide range of DMEs that catalyze a range of different biotransformations.
- The variety and broad substrate specificity of DMEs means that one compound may be metabolized into a large number of metabolites.
- In addition to the above, the expression of DMEs is different in different tissues/compartments, DME expression is often modulated by xenobiotic exposures, and multiple biotransformations are possible for a single compound;
- together this means that xenobiotic metabolism arguably resembles combinatorial chemistry, as opposed to a tightly-regulated set of highly efficient steps in a metabolic pathway.
- Some biotransformations are going to be more common than others, and all depend on the relevant chemical functional groups being present, and therefore the idea of xenobiotic metabolism being more like a pachinko machine provides a nice analogy
- figure legend: Pachinko model diagram of xenobiotic metabolism and interactions with endogenous, sym-endogenous, and other elements over time.
- In this diagram, the pins represent key metabolizing enzymes or transporter molecules, some of which are arranged in sequence to indicate probable pathways a hole represents an exit point from the system of an excreted metabolite.
- Some pathways through the machine are more probable than others, dictated by a combination of the chemistry and enzyme-substrate interactions.
- A mutation or single-nucleotide polymorphism (SNP) variation at any point is the equivalent of moving or changing the size of a pin that alters the probabilities of routes through the machine (metabolic fate).
- The outcome is conditional on the sequence and sites of the previous metabolic events.
- Highly probable events lead to macro-metabolites (the most familiar in drug metabolism), the number and type of these being highly compound specific.
- Low-probability events lead to the formation of micrometabolites
32
Q
Describe the cytochrome P450 (CYP450) enzymes
A
- Multi-enzyme membrane protein superfamily
- 12 CYP gene families have been identified in humans (~57 CYPs), and the categories are based upon protein sequence homology; most of the drug metabolising enzymes are located in CYP 1, 2, & 3 families
- Some P450 enzymes catalyze essential intermediary metabolic processes (e.g., steroid hormone metabolism and bile salt biosynthesis); others mediate metabolic clearance of drugs and other foreign compounds
- Frequently, two or more enzymes can catalyse the same type of oxidation, indicating broad substrate specificity
- Mostly located in the smooth endoplasmic reticulum (the microsomal fraction)
- Mainly liver but present in most tissues, particularly GI; CYP3A4 is important in the metabolism of many drugs and its presence in the GI tract can result in poor oral bioavailability
33
Q
Which isoforms of CYPs metabolise over half the orally effective drugs?
A
- in humans, CYP2D6 and CYP3A4 metabolize over half the orally effective drugs currently in use.
34
Q
How do cytochrome P450 enzymes catalyse the oxidation of their substrates?
A
- Cytochrome P450 enzymes catalyze the oxidation of their substrates through a series of redox steps, where the central heme (iron) group at the centre of the enzyme undergoes successive oxidation and reduction steps and ultimately introduces an oxygen atom to form the metabolic product.
Note that this redox cycling is made possible by another enzyme system, the NADPH-P450 reductase (POR) that supplies electrons during two steps; the absence / deletion / inhibition of this enzyme effectively stops all CYP-related metabolism.
35
Q
What are some substrates of cytochrome P450 isoforms?
A
39
Q
Study this table of the types of conjugation reactions and their target functional groups
A
40
Q
Describe the conjugation reaction: glucuronidation
A
- The glucuronidation of substrates is catalyzed by DMEs called uridine 5’-diphospho-glucuronosyltransferases (UDP-glucuronosyltransferases; UGTs).
- There is a superfamily of UGTs that collectively have a very wide substrate specificity.
- They all catalyze the conjugation of a substrate containing a nucleophilic function group (typically -OH, -COOH, -NH2, -SH), with glucuronic acid, which is provided by uridine 5’-diphosphate glucuronic acid (UDP-glucuronic acid; UDPGA).
- UDP-GA is synthesized from UTP and glucose-1-phosphate, and is highly abundant (particularly in the liver); humans make ~5g/day.
- Nucleophilic drugs react with UDP-GA as it contains a good leaving group for a nucleophilic substitution reaction.
41
Q
Describe the conjugation reaction: sulfonation/sulfation
A
- Another quantitatively important conjugation reaction is the sulfation (or, more correctly, sulfonation - you will see both terms used interchangeably in the literature).
- Sulfonation is typically less extensive than glucuronidation but there is a similar preference for functional groups (e.g., -OH, -NH2).
- Sulfonation reactions are catalyzed by sulfotransferases (SULTs), which fall into two broad classes:
i) membrane-bound SULTs that are located in the Golgi apparatus and are responsible for the metabolism of peptides, proteins, lipids (less relevant to drugs and xenobiotics)
ii) cytosolic SULTs that are responsible for the metabolism of xenobiotics and low molecular weight endogenous substrates such as bile acids. - In humans, three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members.
- These SULTs catalyze the conjugation of a substrate containing a nucleophilic functional group with a sulfonate group provided by 3′-phosphoadenosine-5′-phosphosulfate (PAPS).
- PAPS is synthesized endogenously from inorganic sulfate and ATP.
- Sulfonation is often a high affinity, low capacity pathway, while glucuronidation is frequently low affinity, high capacity.
- Therefore, at a low dose, sulfation may dominate, but as the dose increases, glucuronidation can become the major route.
42
Q
Describe the conjugation reaction: acetylation
A
- N-acetyltransferases (NATs) are cytosolic enzymes present in the liver and require the co-factor acetyl coenzyme A to acetylate their substrates.
- In humans, two genes encode NATs, with NAT1 being monomorphic, and NAT2 being polymorphic and facilitating considerable inter-individual variability in the ability to N-acetylate drugs (slow/intermediate/fast acetylators).
- Both N-acetylation and O-acetylation are possible.
43
Q
Describe the conjugation reaction: methylation
A
- Methylation is catalysed by methyltransferase enzymes; there are many types including catechol-O-methyltransferase (COMT), and phenol-O-methyltransferase (POMT).
- These enzymes catalyze the reaction of a nucleophilic functional group in a substrate (-OH, -NH2, -SH) with S-adenosylmethionine (SAM) to form a methylated product; the leaving group in this reaction is S-adenosylhomocysteine (SAH).
- Methylation is a common but generally minor pathway; it slightly decreases water solubility and often masks functional groups that can affect both pharmacological and toxicological responses.
44
Q
Describe the conjugation reaction: amino acid conjugation
A
- Amino acid (AA) conjugation is commonly observed for drugs containing a carboxylic acid group.
- The reaction of -COOH in the drug with -NH2 of AA forms an amide.
- The reaction requires initial activation of the -COOH group with acyl-CoA to produce a thioether, which subsequently reacts with the -NH2 of an AA.
45
Q
Describe the conjugation reaction: glutathione conjugation and mercapturic acid formation
A
- All the previous examples of conjugation reactions we have explored have been for drugs containing nucleophilic functional groups, and largely follow the same mechanism, in which the nucleophile reacts with a specific donor molecule that contains a good leaving group in order for it to become conjugated.
- Glutathione conjugation occurs between the tripeptide glutathione (GSH) and electrophilic substrates.
- These reactions are catalyzed by glutathione-S-transferases (GSTs), but can occur without catalysis. GSTs are cytosolic enzymes that are highly expressed in the liver, kidney, gut, testis, and adrenal gland, with at least six isoenzymes known, each exhibiting some substrate specificity.
- The nucleophilic thiol in GSH readily reacts with electrophiles to form the GSH-conjugate.
- An associated series of reactions can also result in the formation of the mercapturic acid (mercapturate; N-acetylcysteinyl conjugate) from the GSH-conjugate;
- cleavage of the glycine and glutamic acid residues from the GSH-conjugate is followed by N-acetylation of amino group on the remaining cysteine.
- The mercapturic acid is readily excreted in urine.
50
Q
The main pathways involved in paracetamol metabolism and the transport of metabolic intermediates between various compartments are shown below.
Why do you think an elevated dose of paracetamol leads to toxicity?
A
- You can see from the figure that some of the drug is metabolized via the CYP450 pathway into NAPQI, which is a highly reactive and toxic intermediate.
- NAPQI is removed from the system by conjugation with GSH in the liver.
- If left to accumulate, NAPQI binds to proteins and subcellular structures and induces rapid cell death and necrosis, which can lead to liver failure.
- Therefore, overdosing on paracetamol, or consumption of paracetamol by individuals who are more susceptible to adverse effects to due to reduced glutathione levels, leads to elevated NAPQI production and toxicity.
- In the previous section, we have established that NAPQI is the key reactive metabolite that is responsible for the toxicity observed in paracetamol overdose.
- Inspecting the structure of NAPQI, we can see that the reactivity comes from the presence of the quinone imine that is highly electrophilic, and can therefore react readily (with or without a catalyst) with nucleophiles such as hydroxyl or sulfhydryl groups (-SH).
- As discussed in the previous section (Conjugation reactions), one of the most abundant small molecules in the liver is glutathione, a tripeptide that contains a cysteine residue.
- The nucleophilic sulfhydryl group on the cysteine side chain is very well suited to react with the NAPQI electrophile, resulting in the formation of the paracetamol-GSH conjugate.
51
Q
What may be a likely explanation of NAPQI accumulation in paracetamol overdose?
A
55
Q
Describe how reactive metabolites are assessed during drug safety testing
A
- As we established in the previous section, knowing whether or not a drug candidate produces reactive metabolites (e.g., the highly electrophilic NAPQI intermediate metabolite produced from CYP2E1 metabolism of paracetamol) is important as it can form part of a comprehensive drug safety profile.
- RMs are often the result of biotransformation by CYPs, and one relatively simple way in which electrophilic intermediate metabolite formation can be assessed is by conducting an in vitro ‘trapping experiment’ in which a drug candidate is subjected to a hepatic microsomal incubation (both with, and without additional GSH added).
- The presence of the parent and GSH-conjugated metabolites is assessed (e.g., using liquid chromatography-mass spectrometry; LC-MS), and reveals the amount/proportion of drug that has formed an RM and subsequently reacted (been ‘trapped’) with GSH.
60
Q
Describe some models used to study drug metabolism
- focusing on cell lines
A
