Drug Metabolism

Question 1

What are the important ways in which biotransformation can alter drugs? [4]

Answer 1

1. An active drug may be converted into an inactive drug.
2. An active drug may be converted to an active or toxic metabolite.
3. An inactive drug may be converted to an active drug.
4. An unexcretable drug may be converted into an excretable metabolite.

Question 2

The _____ is the main organ of drug metabolism. This fact figures prominently in the phenomenon known as _____-____ metabolism. Orally administered drugs are often absorbed in the GI tract and transported directly to the liver via the ______ circulation. In this manner, the liver has the opportunity to metabolise drugs before they reach the ________ circulation and, therefore, before they reach their target organs. The _____-____ effects must be taken into account when designing dosing regimens because, if hepatic metabolism is extensive, the amount of drug that reaches the target tissue it much ____ than the dose that is administered orally. Certain drugs are inactivated so efficiently upon their first pass through the liver that they cannot be administered orally and must be given ____________, e.g. antiarrhythmic lidocaine.

Answer 2

The liver is the main organ of drug metabolism. This fact figures prominently in the phenomenon known as first-pass metabolism. Orally administered drugs are often absorbed in the GI tract and transported directly to the liver via the portal circulation. In this manner, the liver has the opportunity to metabolise drugs before they reach the systemic circulation and, therefore, before they reach their target organs. The first-pass effects must be taken into account when designing dosing regimens because, if hepatic metabolism is extensive, the amount of drug that reaches the target tissue it much less than the dose that is administered orally. Certain drugs are inactivated so efficiently upon their first pass through the liver that they cannot be administered orally and must be given parenterally, e.g. antiarrhythmic lidocaine.

Question 3

What are the two main classifications of biotransformation?

Answer 3

Biotransformation is also known as drug metabolism.

1. Phase I (oxidation/reduction)
2. Phase II (conjugation/hydrolysis)

Question 4

What are the main enzymes involved in phase I metabolism?

Answer 4

• Cytochrome P450 (CYP450) enzymes
• Flavin mono-oxygenase (FMO) enzymes
• Prostaglandin synthetase enzymes (less commonly used)

Question 5

Outline CYP450 enzymes and their involvement in drug metabolism.

Answer 5

Cytochrome P450 enzyme (haem protein mono-oxygenases) catalyse phase I reactions. The result of a CYP450-dependent oxidation reaction is:

Drug + O₂ + NADPH +H⁺ → Drug-OH +H₂O + NADP⁺

• CYP450 enzymes are holoenzymes, meaning they need a prosthetic group in order to function, in this case that group is haem.
• They are one the largest group of enzymes we know about. 50 out of 1900 are expressed in the human body, with each enzyme having the capacity to metabolise more than one substrate.
• CYP450s comprise a series of alpha helices and beta folds. Two of the alpha helices need to contact the haem group (not enough haem = affects metabolism). There is a 6-substrate recognition sequences within the structure key to finding the substrate, but not all substrates need 100% homology.
• CYP450s add groups to a drugs/toxins to make them more reactive. This is done in a nine-step catalytic cycle.
• Cytochrome P450 catalyses different types of reactions: Aromatic hydroxylation, Aliphatic hydroxylation, De-alkylation, Oxidation, Alcohol oxidation, Oxidative de-amination, and Dehalogenation. Different members of the CYP family mediate different reactions, and can lead to different toxicities.

Question 6

CYP1, CYP2, CYP3A

Name some examples of CYP450-mediated toxicity.

Answer 6

• CYP1 mediated toxicity: CYP1 is mostly associated with toxicity, and they activate a large number of pro-carcinogens (pro-carcinogen → carcinogen), including poly-cyclic aromatic hydrocarbons and heterocyclic aromatic amines (HCAs).
  
  • HCAs are formed during the cooking of proteinaceous food, e.g. meat and fish, and cause tumors in organs such as liver, colon, breast, and prostate in experimental animals. These compounds tend to get metabolised quickly in phase II, but if they are left to build up after phase I they can cause toxic effects. Bioactivation of HCAs requires N-hydroxylation by CYP1A enzymes, particularly CYP1A2. 4-Aminobiphenyl (ABP), a prototypical aromatic amine carcinogen in humans, requires bioactivation to manifest its toxic effects. The first step in the bioactivation of ABP is its N-hydroxylation in the liver. In vitro enzyme kinetic studies using human liver microsomes identified the cytochrome P450 isoform CYP1A2 as the main enzyme that carries out this reaction. Persons exposed to ABP in industrial manufacturing are 90x more likely to develop bladder cancer. Also in tobacco smoke, but smokers are only 7x more likely to develop bladder cancer. Metabolism by CYP can lead to formation of ABP-Hb which is highly reactive and capable of forming DNA adducts, which interferes with normal cellular processes, increasing risk of cancer development.
• CYP2 mediated toxicity: paracetamol is metabolised this way, and can be metabolised by CYP2E1 which can result in a number of toxic effects. At therapeutic dosage, about 60% is conjugated directly into phase II (glucuronidation), around 20% is conjugated into another compound (sulphation). However, CYP450 enzymes, including CYP2E1, CYP1A2, and CYP3A4, convert 5–9% of acetaminophen to a highly reactive metabolite, N-acetyl-p-benzoquinonimine (NAPQI). Hydroxylation by CYP2E1 forms NAPQI, which can either undergo glutathione conjugation, or lead to toxicity if there is a build-up of NAPQI. It is thought that NAPQI causes hepatotoxicity by binding to cellular macromolecules. Detoxification occurs primarily by glutathione (GSH) conjugation, after GSH depletion toxicity occurs (overdose).
• CYP3A mediated toxicity: aflatoxin B1 (AB1) is mediated this way, which is produced by aspergillus flavus. AB1 is most commonly contaminates peanut crops, and peanut workers have particularly high levels of AB1 inside them. In rats, AB1 has been shown to be highly carcinogenic, and in humans there is an increased rate of cancer formation upon exposure to AB1. AB1 is conjugated by CYP3A4 into a compound containing an epoxide group, which is very open to nucleophilic attack. This can then directly interact with albumin and guanine, forming protein and DNA adducts. If the phase I product goes through phase II metabolism, AB1 is converted into a safe excretable product.

Question 7

Outline the steps in the CYP catalytic cycle. [9]

Answer 7

1. Complex of drug bound to the iron (Fe³⁺) in the haem.
2. Incorporation of an electron (e^-).
3. Incorporation of molecular oxygen (O₂).
4. Rearrangement of the complex/redistribution of electrons.
5. Second electron binding (e^-).
6. Release of water (2H⁺→H₂O).
7. Another rearrangement/redistribution of electrons.
8. Substrate is oxidised.
9. Release of oxidised substrate.

Question 8

Name an example of flavin mono-oxygenase (FMO)-mediated toxicity.

Answer 8

There are fewer examples of FMO mediated toxicity than for CYP, which could be because FMO-metabolism has evolved to deal with detoxification, and opposed to toxification-style reactions. It acts as a backup for CYP-mediated reactions. There are some examples of metabolism of compounds that produces toxic effects, however these are few and far between.

FMO3-mediated toxicity: Thiocarbamates (e.g. thiourea) are found as a basis in therapeutic drugs, and in industrial chemicals, which can cause formation liver and thyroid tumours. Thiourea can be oxidised by NADPH to form a sulphenic intermediate, which can be further oxidised to form sulphinic acid intermediate. This sulphinic acid intermediate can undergo conjugation to glutathione and then convert it into cyanamide/urea (excretable) and sulphonic acid. The sulphonic acid can form DNA adducts because it contains double bonds that can bond to DNA.

Question 9

Outline the main steps in the Flavin mono-oxygenase-(FMO) mediated catalyic cycle. [5]

Answer 9

1. Oxidised flavin is reduced by NADPH
2. Molecular oxygen is added
3. Substrate (S) binds, gets oxidised and is released in one step
4. Water is released
5. Complex gets reformed ready to start cycle again

Question 10

Define xenobiotics.

Answer 10

Foreign substances that are not naturally found in the body.

Question 11

What class of reactions occur during Phase I metabolism?

Answer 11

Groups are aded to a drug/toxin to make it more reactive. This can be done by: Aromatic hydroxylation, Aliphatic hydroxylation, De-alkylation, Oxidation, Desulphuration, Alcohol oxidation, Oxidative de-amination, Decarboxylation and Dehalogenation.

Question 12

What groups are exposed from oxidation reactions in Phase I metabolism?

Answer 12

Oxidation reactions typically transform the drug into more hydrophilic metabolites by adding or exposing polar functional groups such as:

• Hydroxyl (-OH)
• Amine (-NH₂)

Question 13

What types of conjugation can occur in Phase II metabolism?

Answer 13

• Glucuronidation
• Acetylation
• Glycine conjugation
• Sulphate conjugation
• Glutathione conjugation
• ​N-, O-, and S-Methylation

Question 14

What is the net result of a CYP450-dependent oxidation reaction?

Answer 14

Drug + O₂ +NADPH + H⁺ → Drug-OH + H₂O + NADP⁺

Question 15

Outline Cytochrome P450 (CYP450) enzymes.

Answer 15

Cytochrome P450s (CYP) are one the largest group of enzymes we know about, and they function to metabolise potentially toxic compounds, including drugs and products of endogenous metabolism such as bilirubin, principally in the liver. CYP450 enzymes add groups to a drug during Phase I metabolism to make it more reactive. This is done in a nine-step catalytic cycle. 50 out of 1900 are expressed in the human body. They are systematically named based on amino acid homology. CYP 1, 2, 3 =family (40% amino acid homology), A, B, C =subfamily (70% amino acid homology), 1, 2, 3 etc. =individual enzymes. Examples of CYP450 enzymes include CYP1A1, CYP1A2, CYP1B1, CYP3A4, CYP2E1 etc.

• Holoenzymes: CYP450 enzymes are holoenzymes, meaning they have a prosthetic group that is required to function. In cytochrome p450 this is haem. CYP450 enzymes are a class of haem protein mono-oxygenases.
• Structure: CYP450 enzymes comprise a series of alpha helices and beta folds, and are important in both anabolism (synthesis of molecules) and catabolism (breakdown of complex molecules). Two of the alpha helices need to contact the haem group (lack of haem affects metabolism). There is a 6-substrate recognition sequence within the structure key to finding the substrate, but not all substrates are needed to be recognised by the whole sequence.

Question 16

Describe the substrate range of CYP450 enzymes.

Answer 16

Most liver CYP450 oxidases exhibit broad substrate specificity. This is due in part to the activated oxygen of the complex, which is powerful oxidising agent that can react with a variety of substrates. Many of the P450 enzymes have partially overlapping specificities that together allow the liver to recognise and metabolise a wide array of xenobiotics.

Question 17

What is the purpose of Phase II metabolism?

Answer 17

Phase II metabolism adds a large conjugate group to a drug/toxin to make it more water soluble for secretion. The larger the compound is, generally the less lipid soluble it is, making it more water soluble.

Question 18

When does toxicity arise from drug metabolism? Give an example.

Answer 18

Toxic responses from phase I metabolism result from a build up of reactive intermediate products, therefore it is important for phase II to happen quickly after phase I. Phase II needs to happen at the same speed, or faster, than phase I metabolism.

e.g. NAPQI, salicylic acid

Question 19

What are the three major enzyme families involved in Phase II metabolism? What form of conjugation do they metabolise?

Answer 19

• UDP-glucuronosyltransferase enzymes (UGTs): Glucuronide conjugation.
• Sulphotransferases (SULTs): Sulphate conjugation.
• Glutathione S-transferase enzymes (GSTs): Glutathione conjugation.

Question 20

How much homology is shared in enzyme families and subfamilies?

Answer 20

Family: ~45%

Subfamilies: ~60%

Question 21

Outline glucuronide conjugation.

Answer 21

Glucuronidation consists of transfer of the glucuronic acid component of uridine diphosphate glucuronic acid to a substrate –OH, -COOH, SH, or -NH2 by any of the 16 types of UDP-glucuronosyltransferase (UGTs).

Question 22

UDP-glucuronic acid (UDPGA) is a key substrate in glucuronide conjugation. How is UDPGA made? How is this advantageous?

Answer 22

Formation of the UDPGA is key in toxicity, and issues arise when its progenitor is depleted. Glucose-6-P is the entry molecule of glycolysis, which is made through the phosphorylation of glucose. This is favourable as there is an abundance of glucose-6-P to use in the conjugation of drugs. This means that the phase II reaction can fulfil the need to be faster than phase I. There is an abundance of glucose in the body, therefore under normal metabolic conditions there is an almost unlimited supply of conjugate. The image below demonstrates that UDP-glucuronate is obtained through the conversion of glucodse-1-P.

Question 23

Outline how morphine is biotransformed in Phase II.

Answer 23

Morphine is biotransformed via glucuronide conjugation. This makes the metabolite more soluble for excretion in the urine or bile, and it detoxifies the morphine intermediate. This ensures there are no toxic events in the body.

Question 24

Give an example of UGT-mediated toxicity.

Answer 24

An example of UGT-mediated toxicity is acyl migration between carbons in an unstable glucose ring, which can break open leave an oxygen open to nucleophilic attack, leading to the formation of protein adducts that could lead to damaging protein activity and/or DNA damage. Under normal physiological conditions this is not a major issue, but if we absorb something that drastically shifts the pH (drugs/toxins affecting pH), then this is much more likely to lead to adduct formation.

Question 25

Outline sulphate conjugation.

Answer 25

Sulphate conjugation is catalysed by sulhotransferases (SULTs), which are in two distinct classes: membrane bound, and cytosolic.

Cytosolic SULTs are grouped into SULT1 (phenol sulphotransferases/group 1) and SULT2 (steroid sulphotransferases/group 2). Group one SULTs tend to catalyse the addition of sulphate to phenol groups, whereas group 2 SULTs predominantly catalyses the addition of sulphate groups to steroids. These are loose groups because there are exceptions within each group.

As the name suggests, the reactions involve the transfer of sulphur. Again, transfer is to -OH, -NH₂ and -SO₂NH₂ groups. And the sulphur is donated by a molecule called PAPS. This creates a sulphated conjugate, releasing PAP. This is a straightforward enzymatic reaction, with no need for specific orders for binding of substrates, unlike the catalytic cycle. As long as the donor and the reactive group are in the active site of the enzyme at the same time, this reaction can occur.

Question 26

Give an example of SULT-mediated toxicity.

Answer 26

SULT can catalyse the formation of products which can create NH₂^+​ groups (nitrenium ion), which can cause DNA adducts. For example, 2-nitropropane was a widely used industrial solvent, which is less widely used now. It has been shown to cause liver cancer in rats, but there is a smaller association in humans. This is due to species variation. In rats liver cancer occurs more frequently as the sulphate conjugation metabolic process is more relied on, but in humans there is a different metabolic pathways (i.e. CYP), meaning the effects are not as severe as in humans.

Question 27

Outline glutathione conjugation

Answer 27

Glutathione conjugation is catalysed by glutathione S-transferase enzymes (GSTs), which are made up of two distinct classes: cytoslic and microsomal. These enzymes function as dimers, and two units are needed in order for it to act as a functional unit.

• GSTα1, GSTα2, GSTα3, GSTα4
• GSTμ1,2,3,4or 5
• GSTπ1
• GSTτ1,2
  
  • Dimers e.g., GSTM1-1, GSTA1-1,

The glutathioine conjugate is a very large innert molecule. As a result, it is restricted to highly reactive intermediates. This form of conjugation is referred to as the molecular hoover.

Question 28

Give an example of GST-mediated toxicity.

Answer 28

Epoxide is a triangle shape, which is a highly unstable group. The molecules are under strain making it very reactive. Toxicity arises from GST enzymes that create ring formations, which can create DNA/protein adducts.

• Halogenated alkenes can interfere with cells and create toxic compounds. They used to be prevalent in dry cleaning products, but have now been removed.