D10 - Drug metabolism

Question 1

Metabolism

Answer 1

• Greatest number of clinical problems
  
  • Drugs disrupt metabolism of other drugs

Drug metabolism

- The process whereby the chemical structure of an absorbed drug is altered due to enzymatic process by drug metabolising enzyme in the liver or other tissues 
- = biotransformation
- Drug metabolising enzyme - DMEs - has an active site that can perform, through a catalytic step, the conversion of a drug into a metabolite - usually more water soluble

Question 2

DMEs - drug metabolising enzymes

Answer 2

• Drug metabolising enzyme - DMEs - has an active site that can perform, through a catalytic step, the conversion of a drug into a metabolite - usually more water soluble
  ○ Potentially metabolise all xenobiotics (foreign chemicals) - not just drugs
  § Pesticides, pollutants, food additives, solvents
  ○ Drug metabolism usually makes lipophilic drugs more hydrophilic
  § More polar
  § Better substrates for efflux transporters
  § Returned from liver to general circulation
  § Excreted by kidneys (transporters) into urine

Question 3

where does it occur

Answer 3

○ DMEs are most strongly expressed in the liver - also present in other tissues 
			§ Gut wall
			§ Kidneys
			§ Lungs
			§ Skin
			§ Brain
			§ Testes
			§ Heart
			§ Gut microbiome - many microorganisms

Question 4

• In general, hydrophilic drugs undergo minimal metabolism in the liver

Answer 4

○ Excreted unchanged in urine
○ Because they are water soluble, they don’t need to be metabolised to be excreted by the kidneys
- Lipophilic drugs are converted to hydrophilic metabolites that are then removed by kidneys and excreted in the urine
○ Not good substrates for transporters in the kidneys
○ The liver expresses enzymes that convert lipophilic drugs into hydrophobic metabolites so that they can be excreted - otherwise they will accumulate

Question 5

Main classes of metabolism

Answer 5

Phase 1 - oxidative metabolism

Phase 2 - Conjugative metabolism

Question 6

Phase 1 - oxidative metabolism

Answer 6

○ A very lipophilic molecules
○ Functionalisation reactions
○ To make them more water soluble (oxidative reactions - typically add a new hydroxyl group - oxygenation, hydroxylation, N-dealkylation (liberating a free hydroxy group))
○ Gain in water solubility - less lipophilic

Question 7

Phase 2 - Conjugative metabolism

Answer 7

○ Formation of a new chemical bond - attachment of endogenous molecule donated by a cofactor - used as donors of functional groups - groups of atoms attached to metabolites to create more water soluble
○ Formation of conjugate - 2 coupled or connected entities
§ Sulfonation - adds a sulphate group
§ Acetylation - adds an acetyl group
§ Glucuronidation - most prevalent, adds a beta glucuronide
§ Methylation
§ Glutathione conjugation - toxicology
○ Conjugative - drug attached via covalent bond to water soluble molecule
○ More hydrophilic
Some drugs bypass oxidative reactions and only for conjugate metabolites

	Epoxidation 
		§ Alternative route of oxidation 
		§ Generates an epoxide 
		§ Often hydrated by epoxide hydrolases

Question 8

Consequences of metabolism

Answer 8

Quicker elimination

Loss of activity

Question 9

Loss of activity

Answer 9

○ Metabolism often makes drug molecules more polar and water soluble
§ Accelerates rate of removal from body
§ Kidneys are better able to remove water soluble metabolites
○ Usually increases molecular mass and size of foreign molecules
○ Metabolism usually decreases the half life (e.g. plasma T1/2) of drugs
§ Possibly accumulation is decreased
○ Note - medical chemists often alter drug structures to slow down their metabolism in the body
§ Eg. Modifying oxidation prone groups in the drug
§ Once or twice daily dosing more desirable that 3/4 times per day

Question 10

Loss of activity

Answer 10

○ By changing drug structure, metabolism often reduces biological activity
§ Metabolite may not ‘fit’ into drug receptor
○ Some metabolites retain pharmacological activity
§ Active metabolites - contribute to pharmacological profile of parent drug
○ Active metabolites have sometimes been marketed as drugs
§ Nortriptyline (antidepressant) - metabolite of amitriptyline
○ A drug that is inactive until metabolised - a pro-drug
§ Chemists make insoluble drugs into more desirable drugs by pro drugging - adding groups of drugs to make them more soluble that then get removed in metabolism to make the active form

Question 11

pro-drug

Answer 11

○ A drug that is inactive until metabolised - a pro-drug
§ Chemists make insoluble drugs into more desirable drugs by pro drugging - adding groups of drugs to make them more soluble that then get removed in metabolism to make the active form

Question 12

Cytochrome P450 (CYP) - the versatile Catalyst of drug Oxidation

Answer 12

• Cytochrome P450 (CYP): an amazing family of drug metabolising enzymes
  ○ Typically approx. 50 kDa mass
  ○ 57 family members in human genome
  ○ Only a handful are important in human drug metabolism
  ○ Many have been crystallised (X-ray analysis +- substrates, inhibitors
  
  • Wide substrate specificity (c.f. most enzymes)
    ○ Some CYPs metabolise 100’s of drugs
    ○ Metabolise ~ 75% of all drugs in human use

Question 13

P450 structure and function

Answer 13

○ CYPs are Fe-containing haemoproteins
§ Contain protoporphyrin IX in active site
□ Located at base of active site in CYP
□ Size and shape of active site varies between CYP family members
□ Amino acids on surface of active site cavity provide binding options for drugs
□ Contains a haem group
○ Membrane embedded enzyme with a catalytic chamber with protoporferin 9 - drugs diffuse in and reach haem group - undergoes redox chemistry - formation of iron (5) oxide species - oxidising species
○ O2 binding and redox changes in Fe atom during CYP catalytic cycle result in formation of iron 5 oxide species
§ Highly potent oxidising agent
§ Donates O atom to oxidation-prone site in the drug
§ E.g. top example in phenytoin (epilepsy drug) undergoing oxidation

Question 14

CYP depends on NADPH-CYP reductase (redox partner)

Answer 14

CYP depends on NADPH-CYP reductase (redox partner)
Basic CYP-catalysed reaction is
SH + O2 + NADPH + H+ -> SOH + H2O + NADP+
Where
○ SH is the drug substrate
§ Reacts in the presence of oxygen
○ SOH is hydrolysed metabolite
§ result
○ NADPH is essential cofactor
§ Supplies electrons to haem group during CYP catalytic cycle
§ Donates electrons - forms a close multiprotein structure where there is a reductase in close proximity to P450
○ NADPH oxidised by NADPH-CYP reductase
§ Reductase forms close association with CYP in smooth endoplasmic reticulum membrane in liver cells
§ Lipophilic drugs diffuse into CYP active site via lipid bilayer
○ Note: CYP is a monooxygenase ie. Donates 1 O from O2 to drug

Question 15

CYP isoforms - metabolise the majority of drugs

Answer 15

○ Not all are important
○ Small number metabolise drugs
○ Isoform not isoenzymes

Question 16

CYP2D6

Answer 16

○ 2% of liver P450
○ Only metabolises bases
○ Small percentage of total P450 but metabolises large portion of pharmaceuticals

Question 17

First pass metabolism

Answer 17

• Taken by the oral route and absorbed through the gut wall, goes to liver and reaches circulation
  ○ A fraction of drugs eliminated in gut wall and the liver before the drug reaches systemic circulation
  ○ A lot of the administered dose is getting converted to inactive metabolites
  ○ Low bioavailability drugs
  ○ More vulnerable to other drugs that interfere with P450 function
  
  • CYP expression is strong in gut wall and in liver
  • Metabolism of drugs can occur before they reach the systemic circulation

Question 18

Drug transporters and drug metabolism

Answer 18

• Essential roles for transporters
  1. Hepatocellular uptake of drug by influx transporters
  i. Ensures good substrate supply to CYPs
  2. Efflux of metabolites via bile
  3. Efflux of metabolites to blood for eventual removal by urine
  
  • In individuals with polymorphisms in certain transporters involved in bringing drugs to the liver
    ○ Metabolites not formed - drug cant get into the hepatocytes
  • Insure good supply of substrate to cytochrome P540
  • Move metabolites into the bile
  • Removing metabolites across the basolateral membrane and into the blood stream

Question 19

Clinical example - impact of OCT1 polymorphism on sumatriptan pharmacokinetics

Answer 19

• Drug with extensive first pass clearance - 85% get inactivated
  
  • Metabolites aren’t formed as readily in people with OCT1 polymorphism
  • Loss of function in OCT1 meant higher concentration in plasma
  • Area under curve (total exposure) significantly higher with no functional OCT1 transporter
  • Drug is normally extensively metabolised - but if it cannot be accumulated in the liver it wont be metabolised
  • Transporter plays a key role in first pass clearance

Question 20

1. DDI - drug drug interactions

Answer 20

• Few P450 involved in metabolism - high chance of interaction - high chance two drugs use the same P450
  - High risk of drugs interfering by competing with the same P450
  - Inhibition of metabolism
  - Induction of metabolism

Question 21

2. Pharmacokinetic issues

Answer 21

• High frequency of variants - polymorphic P450 genes
  § Vulnerable to side effects of drugs, toxicity, or drug ineffectivenessCYP3A4
  ○ Diazepam - classic substrate
  ○ Some drugs induce it - increase it’s expression eg. dexamethason
  ○ Some inhibit it - grapefruit juice
  All P450 isoforms have things that induce/inhibit it

Question 22

Inhibition of drug metabolism

Answer 22

○ Can occur - competitive inhibition - when two drugs compete for the same enzyme
§ One drug inhibits the metabolism of the other
○ Clinical effects of the inhibited drug may take 3-4 days to appear
○ Common DDI mechanism (DDI)
○ Can involve 2 mechanisms
§ Competitive inhibition
□ Most common, reversible
§ Mechanism based inhibition
□ Less common, irreversible
□ Inhibition causes by chemically unstable metabolite
□ Attacks CYP enzyme to inactivate protein
□ Chemically active unstable metabolise that kills the enzyme leaving it unable to carry out metabolism - takes week for liver to be restored

Question 23

Clinical problems due to CYP inhibition

Answer 23

§ Exaggerated effect if the drug is no longer being cleared by the liver
§ Inhibitor blocks P450 - concentration rises in the plasma and causes toxic result
§ Pro drugs
□ drugs that need an enzyme to convert it into an active metabolite
□ Can block enzyme converting drug into its active metabolite
□ Diminished drug effect

		Codeine 
			□ Pain relieving opioid analgesic 
			□ Is a pro drug 
			□ Needs to be converted into morphine to be active
			□ Morphine needs to be glucuronidated in the liver to form a glucuronide - strongly analgesic and cause the effects on the brain (morphine-6-glucuranide)
			□ A 2D6 inhibitor 
			□ Fluoxetine/Prozac - the patient wont form morphine or morphine-6-glucuranide from codeine if they are on Prozac - wont be any pain relief - inhibited bioactivation

Question 24

§ Pro drugs

Answer 24

□ drugs that need an enzyme to convert it into an active metabolite
□ Can block enzyme converting drug into its active metabolite
□ Diminished drug effect

Question 25

Codeine

Answer 25

□ Pain relieving opioid analgesic
□ Is a pro drug
□ Needs to be converted into morphine to be active
□ Morphine needs to be glucuronidated in the liver to form a glucuronide - strongly analgesic and cause the effects on the brain (morphine-6-glucuranide)
□ A 2D6 inhibitor
□ Fluoxetine/Prozac - the patient wont form morphine or morphine-6-glucuranide from codeine if they are on Prozac - wont be any pain relief - inhibited bioactivation

Question 26

Inhibitors uses

Answer 26

§ Inclusion of cobicistat - cocktail of 4 antiretroviral drugs for HIV
§ One of the drugs is a P450 inhibitor - slows down the metabolism of the other drugs so that it only needs to be taken daily

Question 27

Induction of drug metabolism

Answer 27

○ CYP expression can be increases by drugs eg. Smoking, alcohol
○ Drugs switch on the expression of P450 genes and there is more P450 in the liver - activation of xenosensor proteins - drug responsive transcriptional factors - bind to and induce a ligand - usually a drug - binds to xenosensor - other part of the transcription factor contains DNA binding sites that interact with the promotors
○ Drug acts as inducer, migrates to the nucleus and acts as a transcriptional factor and activates transcriptional activity and the P450 gene is transcribed
○ Inducer increases the quantity of P450 in the liver - able to metabolise drugs more quickly
○ Effects take more time to happen than inhibitors
○ Drugs can also induce their own metabolism
§ Auto-induction
§ Eg. Carbamazepine - used to treat epileptic convulsions
§ Half life goes from 36 hours in the first use to 8 hours if you use the drug regularly for two weeks

Question 28

Clinical problems due to CYP induction

Answer 28

• Inappropriate drug combination involves inducers removing the effects of the drugs
  - Poor drug effect due to accelerated hepatic clearance

Question 29

Genetic variations

Answer 29

○ Polymorphisms
§ Variants in coding
§ Within human populations there is genetic heterogeneity in drug metabolism
○ CPY2D6
§ Variation in the population
§ Normal
§ Ultra
§ Intermediate
§ Some cannot metabolise at all
○ Gene duplications, repeats, point mutations
○ Frequency varies in different populations - sometimes explains why certain drugs have different incidence of side ffects in different populations

Question 30

CYP2D6 - polymorphisms

Answer 30

○ An ultra metaboliser - extra copies - tandem repeats of 2D6 gene - may not get any benefit of regular drug because plasma concentration does not reach therapeutic level
○ Extensive metabolisers - get benefit from recommended dose of the drug - most commonly encountered genotype
○ Intermediate metaboliser - may have a faulty allele - abnormal gene - prone to toxicity because plasma concentration will be too high
○ Poor metabolisers - rare - usually incidence of 1-2 % - dose may be toxic - two inactive alleles of gene - vulnerable to drug mediated toxicity