Lecture 5 - Drug Metabolism

Question 1

Why are mechanisms for drug metabolism needed?

Answer 1

Drugs tend to be lipophilic which are not easily excreted. They must be metabolised so they become more polar and thus more water soluble, they are then more easily excreted in the kidneys. The metabolism and subsequent excretion tends to eliminate or reduce the pharmacological, and importantly, the toxicological activity of the drug.

Question 2

What is the major site of drug metabolism in the body?

Answer 2

The liver

Question 3

What is meant by “first pass metabolism”? What is this termed if it occurs in the liver?

Answer 3

Any alteration of the drug via metabolism before it enters the systemic circulation.
Becomes “hepatic first pass metabolism”.

Question 4

Why is first pass metabolism important when producing a drug and what is the ideal amount of first pass metabolism?

Answer 4

If the drug is metabolised extensively before it can enter the systemic circulation, very little of the drug is now pharmacologically active.
The ideal amount would be none and can be almost attained with IV administration.

Question 5

What does the term “Bioavailability” mean?

Answer 5

This constitutes the proportion of the drug administered that enters the systemic circulation and becomes “bioavailable” to the body.
e.g. low bioavailability = extensively metabolised before systemic entry and vice versa

Question 6

What is the alternative administration method if a drug is extensively first pass metabolised?

Answer 6

Intravenous administration (only problem is it’s invasive)

Question 7

What are the 3 Phase I metabolic reaction types and which is the most common?

Answer 7

Oxidation (most common)
Reduction
Hydrolysis

Question 8

What are the 6 Phase II metabolic reaction types and which is the most common?

Answer 8

Glucuronidation (most common)
Acetylation
Amino acid conjugation
Sulphonation
Methylation
Glutathione conjugation

Question 9

What is the main aim of Phase I reactions and which do which?

Answer 9

To release or create new functional groups

e.g. oxidation/reduction creates new functional groups while hydrolysis unmasks functional groups

Question 10

Is there large or little change in polarity with Phase I reactions and do they tend to inactivate or activate drugs?

Answer 10

Little change in polarity

Often inactivate but can also activate (prodrugs)

Question 11

What is a prodrug and how does it become a pharmacologically active drug?

Answer 11

A prodrug is an inactive substance that requires metabolism in order to become pharmacologically active.
Metabolism of the prodrug in the liver converts it to something pharmacologically active and it enters systemic circulation.

Question 12

What is the main site of Phase I reactions and what enzymes does this organ contain that mainly carry out these reactions?

Answer 12

Liver

Cytochrome P450 enzyme system - 57 enzyme system, variety allows for metabolism for huge number of xenobiotics

Question 13

What other substances do the P450 enzymes metabolise in the body?

Answer 13

Endogenous substances such as steroids and oestrogens

Question 14

What is the danger of drugs that can inhibit or induce CYP450 and some examples?

Answer 14

Changing the system’s ability to metabolism and process certain drugs
e.g. drinking grapefruit juice before taking TERFENADINE renders drug useless, careful what drugs taken with WARFARIN as can cause excess bleeding, PHENOBARBITAL reduces P450 100x

Question 15

What is the general reaction equation of CYP450 mediated oxidation a drug and what is needed for the reaction?

Answer 15

RH + NADPH + O2 + H+ --> ROH + NADP+ + H2O (R is drug, ROH is oxidised drug)
Needed:
- drug
- NADPH (reducing agent)
- Molecular oxygen
- Source of protons (H+)

Question 16

CYP450 enzymes catalyse a hydroxylation step to begin the process of oxidation, what are the steps in the cycle of reactions involved in this? (draw on paper if poss)

Answer 16

1) Active site of CYP450 enzyme contains a catalytic heme iron centre (fe3+)
2) Substrate binds to this active site and induces a conformational change in the enzyme
3) Electron is then transferred from NADPH to the P450 reducing Fe3+ to Fe2+
4) Molecular oxygen binds to the P450 active site and picks up the electron thus oxidising the Fe2+ to Fe3+ and the O2 becoming unstable (O2-)
5) Another electron is donated by another molecule of NADPH reducing the Fe3+ to Fe2+ again
6) Electron is moved onto the O2- again oxidising the Fe2+ back to Fe3+ and creating agitated O2(2-). Drug has still not changed by this point.
7) Agitated oxygen is then used to oxidise the drug to its hydroxylated derivative.
8) Oxidised drug detaches, the oxygen is cleaved from the enzyme and reacts with two protons to form water and P450 is recycled with its heme centre in the Fe3+ state.

Question 17

What is the total number of different molecules required and released from one P450 oxidation cycle?

Answer 17

Required:

• Drug
• 2xNADPH
• O2
• P450

Released:

• Oxidised drug
• H2O
• P450

Question 18

What are the 5 subtypes of metabolic Phase I OXIDATIVE reactions and explain what they are? Which of these are NOT catalysed by P450 enzymes?

Answer 18

• Hydroxylation (both aliphatic and aromatic), addition of OH group
• N-demethylation, removal of a methyl group attached to N atom usually replaced by H
• O-demethylation, removal of a methyl group attached to O atom usually replaced by H
• N-oxidation, addition of O to tertiary amine forming a dative bond from N to O (NOT P450 catalysed)
• Alcohol oxidation, alcohol (usually ethanol) converted to acetaldehyde then acetic acid which is removed (NOT P450 catalysed)

Question 19

Why is N-demethylation a very common reaction and why is important regarding drug metabolism?

Answer 19

Very common due to 80-90% drugs having amine functional groups on them
Very effective way of REMOVING pharmacological activity

Question 20

Which enzymes catalyse N-oxidation and alcohol oxidation reactions? (hint: it’s not P450)

Answer 20

N-oxidation = Flavin-containing monooxygenase

Alcohol oxidation = alcohol dehydrogenase

Question 21

Reactions involving P450, flavin containing monooxygenase, and alcohol dehydrogenase are each which order reactions? (one of them is different)

Answer 21

P450 = first order
FC monooxygenase = first order
Alcohol dehydrogenase = ZERO order

Question 22

Which enzymes are involved in reduction and hydrolysis (ester and amide) Phase I reactions?

Answer 22

Reduction - Mainly bacterial REDUCTASES in the GI tract

Hydrolysis - ESTERASES (for ester hydrolysis) and AMIDASES (for amide hydrolysis)

Question 23

Are reduction and hydrolysis reactions more or less common than oxidative reactions and where do they occur?

Answer 23

Much less common

Occur in specific parts of the body e.g. the GI tract (low oxygen environment)

Question 24

What are sometimes problematic products of Phase I reactions? (one of the reasons for Phase II reactions)

Answer 24

Can sometimes produce toxic metabolites

e.g. paracetamol metabolism

Question 25

What are the enzymes involved in the 6 Phase II reactions?

Answer 25

• Glucuronidation = Glucuronyl transferase
• Acetylation = Acetyl transferase
• Amino acid conjugation = acyl transferase
• Sulphonation = Sulphotransferase
• Methylation = Methyl transferase
• Glutathione = Glutathione-S-transferase

Question 26

There are a discrete number of Phase II reactions and they alter the drug molecule using a conjugating agent. Is the conjugate formed usually active or inactive, how is its polarity affected, and what does this mean for ease of excretion of the molecule?

Answer 26

The conjugate formed is almost always INACTIVE
It becomes more polar and therefore more water soluble/less lipid soluble
Far easier to excrete

Question 27

What is a conjugating agent and what are their usual features?

Answer 27

Large, polar, endogenous compounds that are used in Phase II reactions

Question 28

Which is one of the most important Phase II processes regarding toxicology and why?

Answer 28

Glutathione conjugation

• Glutathione reacts with electrophiles, these are damaging species often generated during metabolism
• Removed because they can damage DNA and proteins

Question 29

Glucuronidation can be thought of as the addition of sugar to a foreign compound. Which enzyme catalyses the reaction, what type of xenobiotic derivative is formed, is the derivative polar or non-polar, are the products small or large molecular weight, and how are they excreted?

Answer 29

• Glucuronyl transferase
• Sugar derivative formed
• Polar
• Products are large molecular weight
• Makes them unsuitable for glomerular filtration so usually excreted in the bile rather than urine (like most large molecular weight molecules)

Question 30

What happens in acetylation, which functional group does it occur on, and which enzyme catalyses this?

Answer 30

• Addition of acetyl group to N/O/S group (electron rich) producing acetylated derivative of the drug
• Acetyl transferase
• Produced CoA recycled to form more Acetyl CoA

Question 31

What happens in methylation, which functional group does it occur on, and which enzyme catalyses this? How does this reaction affect the drug’s polarity and give an example of a natural use of this?

Answer 31

• Addition of methyl group from S-adenosylmethionine to N/O/S (electron rich) to create the methylated derivative
• Methyl transferase
• Decreases polarity
• Used naturally in conversion of noradrenaline to adrenaline for adrenaline to be reabsorbed instead of excreted

Question 32

What happens in sulphation, what derivative is formed, what is the polarity of this derivative and what enzyme is this catalysed by?

Answer 32

• PAPS donates the sulphate. Produces the sulphuric acid derivative of the molecule
• Derivative is very polar and water soluble
• Catalysed by sulfotransferases

Question 33

What are the three components of glutathione, which is the most important, what happens in glutathione conjugation, and which enzyme catalyses this?

Answer 33

• Tripeptide = Glycine + glutamine + cysteine
• Cysteine most important as it has the thiol (SH) group that reacts
• Glutathione added to the molecule
• Glutathione transferase

Question 34

As a summary of Phase II reactions, what type of reactions are these, what functional groups do they utilise, and what do they require?

Answer 34

• Conjugation reactions
• Utilise -OH, -NH2, -SH, -COOH
• Require their own enzyme and a high energy intermediate (conjugating agent) such as UDGPA for glucuronidation or PAPS for sulphation

Question 35

What is the overall important of drug metabolism? 
Think here of 
- Drug half-life
- Duration of exposure
- Drug accumulation 
- Potency or duration of activity
- Pharmacology/toxicology of the drug

Answer 35

• Biological half-life of the drug is reduced due to Phase I/II reactions
• Duration of exposure reduced
• Accumulation of drug in the body is avoided
• Potency/duration of activity can be altered by metabolism
• Pharmacology/toxicology of the drug governed by its metabolism

Question 36

What is an example of a disorder brought about by having defective flavin containing monooxygenase (FCM)?

Answer 36

Fish odour syndrome (FCM deficiency)

• body produces trimethylamine which smells terrible
• in the liver, trimethylamine is converted by FCM to trimethylamine N-oxide which is odourless and polar thus excreted in the urine
• a small subset of the population has defective FCM so they sweat and breathe out the un-metabolised trimethylamine and smell terrible
• serious mental effects