1. Detoxification by the Liver
Phase I and II reactions

2. Xenobiotics Foreign chemical substance
Can be absorbed across lungs, skin or ingested
Drugs are considered xenobiotics
Excreted in bile, urine, sweat, breath
Foreign chemical substance not normally found or produced within a body

3. Pharmacologically Active Compounds
Lipophilic
To pass through plasma membranes to reach metabolising enzymes
Non-ionised at pH7.4
Bound to plasma proteins
To be transported in the blood

4. Enzymes Microsomal and Non-microsomal enzymes
BOTH be involved in phase I and II reactions
Microsomal enzymes mainly Phase I
Non-microsomal mainly Phase II
Microsomal enzymes generally just mean that they can be found in microsomes which are sedimented ER in the lab

5. Microsomal enzymes Located on smooth endoplasmic reticulum
Phase I reactions – biotransform substances
Phase II – glucuronidation
Activity can be induced or inhibited
Drugs, food, age, bacteria, alcohol
Examples: Cytochrome P450 (CYPs), Flavin monooxygenase (FMOs), UDP- glucuronosyltransferase (UGT)
Involved in oxidative, reductive and hydrolytic reactions
Can be induced or inhibited by various compounds
An inducer causes the increased metabolism of the drug metabolised by CYP thus reducing the efficacy of the drug
Microsomal enzymes are mostly found in the liver hepatocytes, but can be found in the kidneys and lungs as well

6. Non-microsomal enzymes
Located in cytoplasm and mitochondria
Non-specific so Phase I and Phase II reactions
All conjugation reactions EXCEPT GLUCURONIDATION
Non-inducible
Genetic polymorphic – affects metabolism
Examples: protein oxidases, esterases, amidases, conjugases (transferases), alcohol dehydrogenase, aldehyde dehydrogenase
Does oxidative, reductive and hydrolytic reactions

7. Drug Metabolism Why? Most drugs excreted by kidneys
Lipophilic drugs not effectively removed
AIM: To make drugs more polar
Mostly occur in liver
2 mechanisms – phase I and II reactions
Usually sequentially
Mostly occur in liver – where enzymes are

8. Phase I Non-synthetic catabolic reactions
Oxidation, Reduction, Hydrolysis
Introduces reactive group to drug – attack point for conjugation
Hydrophilic molecules usually do not reach the metabolising enzymes

9. Phase I reactions Oxidation Reduction Hydrolysis
Hydroxylation (add –OH)
Dealkylation (remove –CH side chains)
Deamination (remove –NH)
Hydrogen removal
Reduction
Add hydrogen (saturate unsaturated bonds)
Hydrolysis
Split amide and ester bonds
C-N-C (amide) and C-O-C (ester)

10. Phase I Non-synthetic catabolic reactions Known as ‘functionalisation’
Oxidation, Reduction, Hydrolysis
Known as ‘functionalisation’
Introduces reactive group to drug
Includes adding or exposing –OH, -SH, -NH2, -COOH
Product usually more reactive
Small increase in hydrophilicity
Mainly occur in the liver
Mainly catalysed by Cytochrome P450
Drug has to get into cell – more lipophilic
Introduces reactive group to drug – attack point for conjugation
Hydrophilic molecules usually do not reach the metabolising enzymes

11. Cytochrome P450 Enzymes Type of microsomal enzyme Phase I reaction
Haem group to oxidise substances
Products more water soluble

12. Cytochrome P450
Large family with prefix CYP – known as isoforms/ isozymes
1st number – indicates the family the enzyme belongs to
Letter – to indicate subfamilies
2nd number – individual genes involved
Isoforms catalyse different reactions (specificity)
Some important isozymes – CYP1A2, CYP2C9, CYP2C19, CYP2D6,CYP2E1, CYP3A4

13. Cytochrome P450 Reductase
Flavoprotein
Contains both Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN)
FAD – accepts electrons from NADPH
FMN – electron donor to CYPs
NADPH – Nicotinamide adenine dinucleotide phosphate
3 electrons to 1 electron

14. Cytochrome P450 Reactions (oxidation)
Drug H
NADP+
NADPH
P450[Fe3+]
Flavoprotein (oxidised)
P450[Fe3+]
TIME TO PUT IT TOGETHER!
NADPH + H+ + O2 + RH  NADP+ + H2O + R-OH
Drug H
Drug OH
Flavoprotein (reduced) e-
P450[Fe3+]
In general this is what they do
2nd electron may also come from the P450 reductase O2
P450[Fe2+]
Drug OH
P450[Fe2+] e-
Drug H O2
H2O
Drug H
2H+

15. Remember! Non-microsomal enzymes Phase I reactions can:
Alcohol dehydrogenase
Aldehyde dehydrogenase
Reduction
Hydrolysis
Phase I reactions can:
Inactivate drug
Further activate drug
Activate drug from pro-drug (inactive form)
Make a drug into a reactive intermediate (could be carcinogenic or toxic)

16. Phase II Synthetic anabolic reactions Known as ‘conjugation’ reactions
Glucuronidation, sulfation, Glutathione conjugation, amino acid conjugation, acetylation, methylation, water conjugation
Known as ‘conjugation’ reactions
Attachment of substituent groups (endogenous molecules)
Usually inactivate products
Catalysed by transferases
Significantly increase hydrophilicity for renal excretion
Also mainly in the liver
Note can occur in other tissues – like the lungs and kidneys

17. Glucuronidation
Glucuronosyltransferase (UGT) – microsomal enzyme, phase II reaction.
Uridine diphospho-glucuronic acid (UDPGA) needed to conjugate glucuronic acid.
Pathway for bilirubin conjugation and drugs including corticosteroids & paracetamol.
UGT – Uridine 5’-diphopho-glucuronosyltransferase (enzyme that catalyses the reaction)
Substances resulting from this process is known as glucuronides
Forms covalent bonds

18. Glucuronidation Reaction
UDPGA
Drug
UGT
If you don’t believe me go look at glucuronic acid, it has so many –OH groups.
Uridine diphosphate
Drug
Glucuronide
MORE HYDROPHILIC!

19. Remember! Most phase II reactions involved non-microsomal enzymes
Mostly found in the cytoplasm or mitochondria
Donor compounds:
Acetylation – Acetyl CoA
Methylation – S-adenodyl methionine

20. Elimination (usually polar drug, excreted unchanged)
Phase II
Elimination (functionalised without Phase I)
Phase I
Phase II
Elimination

21. Aspirin Analgesic NSAID Antiplatelet
Non steroidal anti-inflammatory drug
Antiplatelet
Irreversibly inhibits cyclooxygenase (COX)

22. Aspirin Phase 1 metabolism
Prodrug so it is activated upon metabolization
Hydrolysis reaction
Aspirin (+H20) → Salcylic acid + Ethanoic acid
Salcyclic acid is the active anti-inflammatory and analgesic

23. Aspirin Phase 2 metabolism
Conjugated with glycine or glucuronic acid
Forms a range of ionised metabolites
Excreted in the urine

24. Paracetamol
Also known as Acetaminophen
Analgesic
Antipyretic agent

25. Paracetamol Metabolism
Predominantly PHASE 2 metabolism
Conjugation with glucuronic acid and sulphate

26. Paracetamol toxicity
If stores of glucuronic acid and sulphate are running low…
Undergoes PHASE 1 metabolism (oxidation) to produce toxic NAPQI
This is removed by conjugation with glutathione
In overdose stores of glutathione can run low leading to toxicity
Treated with N-Acetyl Cysteine

27. Alcohol metabolism Ethanol → Acetaldehyde → Acetate (ADH) (ALDH)
Acetate  CO2 + H2O
ADH – Alcohol Dehydrogenase
ALDH – Aldehyde Dehydrogenase
Operate at different speeds in different people
Acetaldehyde
Carcinogenic
High levels: Facial flushing, rapid heartbeat, nausea

28. Any Questions?