conjugation reactions

Question 1

what are the phases of metabolism?

Answer 1

some drugs needs conjugation reactions after oxidation, these can then be eliminated.
They can be eliminated after phase I reactions but many will form conjugates and get excreted.
there is also a chance of direct conjugation of the parent molecule

Question 2

what are phase II reactions?

Answer 2

• Require functional group as a site for conjugation- often introduced by the phase I metabolite
• Drug is transported into cells where it can be metabolised
• Conjugating molecules are endogenous- glucose or amino acid derivatives often come from other metabolic reactions,
• Resulting conjugates are extremely polar (pKa<3) – TERMINAL metabolites- these are the ones that are eliminated
• Biliary and renal excretion of conjugates – especially for glucuronides facilitated via efflux transporters (e.g., MRP2, MRP4)
• Some gluconides get into blood: other transporters, basolateral influx into systemic circulation (MRP3 and MRP4) then these will get excreted via the kidneys

Question 3

what are the general principles for phase II reactions?

Answer 3

drug with a certain functional group + endogenous compound ==> conjugate

Question 4

what are the two typws of conjugation reactions?

Answer 4

A) Direct conjugation of the drug- as they already have the particular groups in the structure
Functional group Phase II reaction
-OH Glucuronidation or Sulphation
-COOH Glucuronidation or Glycine conjugation
-NH2 Glucuronidation or Acetylation
 Often parallel reaction to CYP metabolism - diclofenac, propofol
B) Conjugation of the phase I metabolite

Question 5

what are the functional groups that can take part in phase II reactions?

Answer 5

OH = glucuronidation or sulphation
COOH = glucuronidation or glycine conjugation
NH2 = glucuronidation of acetylation

Question 6

what are examples of conjugation reaction?

Answer 6

midazolma CYP3A4 via oxidation reaction produces 1 and 4 hydroxy midazolam

Question 7

what are the common mechanisms of conjugation?

Answer 7

A. ACTIVATION STEP- 2 different things can be activated

B. SYNTHETIC STEP

Question 8

what is the activation step?

Answer 8

1. Most common: Conjugating agent (gets activated) as a coenzyme (for glucuronides, sulphates and acetyl conjugates)- this is a multi step process that we don’t need to know or
2. Drug (for amino acid conjugates)

Question 9

what is the synthetic step?

Answer 9

simple, transferase enzymes that transfer the conjugate to the molecule and forms the metabolite

Question 10

where are the enzymes located for glucuronidation via uDP?

Answer 10

 Enzymes located on the luminal side of the endoplasmic reticulum- membrane bound but face the luminal side- this affects the molecules access to the active site

Question 11

what are the common functional groups that are likely to glucuronosyltransferase?

Answer 11

OH, -COOH, -NH2, -SH

Question 12

what are the super families of enzymes?

Answer 12

 UGT1A and UGT2B subfamilies the most relevant - UGT1A1, UGT1A9, UGT2B7: clinically relevant- metabolise a lot of drugs on the market

Question 13

where are the enzymes mainly present for UGT?

Answer 13

mainly in liver but also in the intestine and kidney

Question 14

what are the importance of conjugation metabolic pathways in drug development?

Answer 14

• glucuronidation is the most common mainly for lamotrigin, mycophenolic acid, valproic
• conjugates are often pharmacologically inactive
• renal toxicity
• contribute to drug-drug interactions

Question 15

what happens to bilirubin?

Answer 15

extensively Glucuronidated, steroid hormones, thyroxine and bile acids

Question 16

what is the ontogeny of UGT?

Answer 16

 UGT1A1 activity and protein expression reach adult levels at 3-6 months of age (vary between different enzymes)
 Congenital jaundice - Increase in bilirubin levels (UGT1A1) that doesn’t get conjugated due to reduced activity- does pass as enzymes develop
 Morphine glucuronidation (UGT2B7) deficient in young infants

Question 17

what is gilberts syndrome?

Answer 17

unconjugated hyperbilirubinemia, deficiency in UGT1A1 in adults (UGT1A1*28)- don’t have this enzyme which causes higher levels of bilirubin
 Occurs in 2-13 % of Caucasian

Question 18

what is irinotecan disposition/

Answer 18

Does undergo CYP metabolism
UGT1A1 is needed for metabolism (right)
If deficient, this increases neutropenia as the SN-38 metabolite is directly related to neutropenia
Could use the genotype to see if dose adjustments are needed

Question 19

what are the most abundant glucuronidation enzymes in kideny and tumour tissue?

Answer 19

UGT1A9, UGT2B7, and UGT1A6

Question 20

what are the enzymes present in liver and small intestine?

Answer 20

SULT1A1
 Metabolism of paracetamol, salbutamol, troglitazone
 Sulphates linked to hepatoxicity of some drugs (troglitazone)- not always the case with all drugs
 SULT1A3 expressed in the human foetal liver- differs to UGTs

Question 21

what is the parallel metabolism of paracetamol?

Answer 21

• Blue is the dominant metabolism compared to CYPs direct glucuronidation and direct sulfation
• Red is the CYP mediated mechanism
• Purple is linked to toxicity/ overdose linked to paracetamol.

Question 22

what is glycine conjugation?

Answer 22

 Limited to acids

 Drug becomes activated as acyl coenzyme A intermediate

Question 23

what is acetylation?

Answer 23

 Limited to amines – formation of N-acetyl conjugates
 Acetyl CoA is the co-enzyme
 Genetic polymorphisms in N-acetyl transferase (NAT)- rapid or slow (CYP450 lecture)
 rapid and slow acetylators

Question 24

what is enterohepatic recirculation after drug admin?

Answer 24

via the gluronide step, to include a metabolite.
Give the durg and it is absorbed by the small intestine, reaches circulation and enters liver (standard oral absorption process).
In the liver you will get metabolism via UGT, to produce glucuronide, this will get taken into the bile by efflux transporters (MRP2), which goes to the small intestine. Parts of that will be converted by beta glucuronidase back to the drug not all of it has to go this way, there are some elements that will be excreted via bile to the faeces but portion of it can be converted back into drug and will be reabsorbed in the lumen of the small intestine