Conjugation Reactions

Question 1

What are the phases of metabolism?

Answer 1

Phase 1 = Oxidation –> metabolites –> renal elimination

Phase 2 = Conjugation–> renal elimination or blliary elimination.

The drug doesn’t necessarily have to follow phase 1

Question 2

Phase II reactions characteristics:

Answer 2

• Require functional group as a site for conjugation

- Conjugating molecules are endogenous - glucose or amino acid derivatives

Question 3

Resulting conjugates are extremely ____ (pKa<3) – TERMINAL metabolites

Answer 3

Polar

Question 4

Biliary and renal excretion of conjugates – facilitated via ____ _____.

Answer 4

efflux transporters (e.g., MRP2, MRP4)

Question 5

Phase II reactions – general principles=

Answer 5

Drug with a certain
functional group e.g. COOH
+ Endogenous compound e.g., glucuronic acid = Conjugate e.g., glucuronide

Question 6

Type of conjugation reactions:

Answer 6

a) Direct conjugation of the drug – molecules already have the required functional group
- -> Often parallel reaction to CYP metabolism - diclofenac, propofol

b) Conjugation of the phase I metabolite

Question 7

OH phase 2 reaction:

Answer 7

Glucuronidation or Sulphation

Question 8

COOH phase 2 reaction:

Answer 8

Glucuronidation or Glycine conjugation

Question 9

-NH2 phase 2 reaction:

Answer 9

Glucuronidation or Acetylation

Question 10

Common mechanism of conjugation:

Answer 10

A. ACTIVATION STEP

1) Conjugating agent as a coenzyme (for glucuronides, sulphates and acetyl conjugates) or
2) Drug (for amino acid conjugates)

B. SYNTHETIC STEP – simple, transferase enzymes

Question 11

UDP-glucuronosyltransferases (UGT) are located:

Answer 11

on the luminal side of the endoplasmic reticulum (membrane bound)

Question 12

UDP-glucuronosyltransferases (UGT) function:

Answer 12

Glycoproteins that catalyse the addition of glucuronic acid to a substrate

Functional groups: -OH, -COOH, -NH2, -SH

Question 13

How many human UGT’s?

Answer 13

• Superfamily of enzymes – 22 human UGTs

- UGT1A and UGT2B subfamilies the most relevant - - UGT1A1, UGT1A9, UGT2B7

Question 14

Where are the enzymes present:

Answer 14

Enzymes present mainly in the liver, but also in the intestine and kidney

Question 15

Process to make glucuronide metabolite?

Answer 15

UDP-Glucuronic acid (glucose derivative)* + ROH —–UGT—->B glucuronide metabolite

Question 16

Importance of conjugation metabolic pathways in drug development:

Answer 16

Glucuronidation is the most common.

Glucuronidation important for:

a) Drugs - lamotrigine, mycophenolic acid, valproic acid
b) endogenous compounds

Question 17

Conjugates generally pharmacologically inactive exceptions:

Answer 17

• Morphine 6 glucuronide
• Renal toxicity – acyl glucuronides of NSAIDs
• Contribute to DDIs - gemfibrozil glucuronide (inhibitor of OATP1B1 and CYP2C8)

Question 18

UGTs conjugate multiple endogenous compounds:

Answer 18

bilirubin, steroid hormones, thyroxine and bile acids

Question 19

Ontogeny of UGT and reduced activity compared to adults1:

Answer 19

• UGT1A1 activity and protein expression reach adult levels at 3-6 months of age
• Congenital jaundice - Increase in bilirubin levels (UGT1A1)
• Morphine glucuronidation (UGT2B7) deficient in young infants

Question 20

What is Gilberts syndrome?

Answer 20

• Unconjugated hyperbilirubinemia, deficiency in UGT1A1 in adults (UGT1A1*28)
• Occurs in 2-13 % of Caucasian

Question 21

Patients with UGT1A1*28 variant associated with increased risk of ________ when receiving irinotecan.

Answer 21

neutropenia

Question 22

_____, _____ and ____ the most abundant glucuronidation enzymes in both healthy kidneys and tumour tissue.

Answer 22

UGT1A9, UGT2B7, and UGT1A6

• Reduced renal glucuronidation in tumours
  In vitro evidence for inhibition of UGT1A9 and 2B7 by uremic toxins accumulating in CKD2
• Increased glucuronidation reported in morbidly obese patients3

Question 23

Other phase II reactions – sulphation (SULT).

Characteristics:

Answer 23

• Mainly cytosolic enzymes in contrast to membrane-bound UGTs
• Need different co factors e.g. P450’s = NADPH, UGTs = glucornic acid, SULTs + Activation step required - via ATP, cofactor PAPS
• More readily saturable than UGTs – concentration of PAPs is app. 20 fold < UDPGA
• Overlapping substrate specificity with UGTs. –OH, -COOH
• Enzymes present in the liver and small intestine (SULT1A1) Sulphates linked to hepatoxicity of some drugs (troglitazone) SULT1A3 expressed in the human foetal liver

Question 24

Other phase II reactions. Glycine conjugation:

Answer 24

• Limited to acids

- Drug becomes activated as acyl coenzyme A intermediate

Question 25

Other phase II reactions.

Acetylation:

Answer 25

• Limited to amines – formation of N-acetyl conjugates
• Acetyl CoA is the co-enzyme
  Genetic polymorphisms in N-acetyl transferase (NAT)
  –> rapid and slow acetylators e.g. sulfasalazine

Question 26

What is phase III?

Answer 26

Conjugate hydrolysis and enterohepatic cycling

Phase III = back to parent molecule/recycling
β- Gluronidase (for glucuronide metabolites)
Sulphatase (for sulphate metabolites)

Phase III occurs in the small intestine due to biliary excretion of the metabolites
Important for prolonging drug effect

Question 27

Enterohepatic recirculation after oral drug administration :

Answer 27

• Oral administration
• Drug absorbed in the small intestine
• Portal vein
• Liver
  (UGT)
• Drug glucuronide
  (MRP2)
• Bile
• Small intestine
  (β-glucuronidase)
• Drug
• Drug absorbed in the small intestine