Phase II Drug Metabolism & Conjugation

Phase II Metabolism – What, Why & When

• Goal: attach an endogenous, very polar “handle” to the Phase I metabolite (or to the parent drug if it already owns an $\text{OH}/\text{NH}/\text{SH}$).
• Mechanistic picture ≈ $\text{SN2}$ reaction
  • Drug nucleophile (usually $\text{O}^-$, $\text{N}^-$ or $\text{S}^-$) attacks an activated endogenous co-substrate (RY).
  • Good leaving group Y departs ➜ covalent “conjugate” forms.
• Consequences
  • ~$99.9\%$ probability of complete loss of pharmacological activity.
  • Massive rise in polarity/charge ➜ rapid urinary or biliary excretion.
  • “Liver has no brain” ➜ may still perform Phase I even when OH/NH/SH already present.

Sub-cellular Localisation & Enzyme Nomenclature

• All Phase II enzymes end in Transferase (…T).
• Microsomal (ER membrane) : UGTs share space with CYP450s.
• Cytosolic (soluble) : SULT, NAT, methyl-, amino-acid & glutathione transferases.

Key Endogenous Co-substrates

Glucuronidation – The Work-Horse

• Most common & most versatile Phase II pathway.
• Accepts any $\text{OH},\;\text{NH},\;\text{SH}$ ➜ $\text{Drug–X–GlcA}^{-}$.
• Co-substrate synthesis: glucose → $\text{UDPGA}$ (uridine diphosphate + glucuronic acid).
• Product bears multiple OH plus a COO$^-$ ➜ extremely hydrophilic.
• Neonates: UGT system immature ➜ chloramphenicol toxicity ("grey-baby" syndrome).
• Examples
  • Morphine ➜ morphine-3- and morphine-6-glucuronide.
  • Acetaminophen (minor pathway; major = see below).

Sulfation – Phenol Specialist

• Co-substrate: $\text{PAPS}$; enzyme: SULT.
• High affinity / low capacity (limited inorganic sulfate pool) ➜ dominated by glucuronidation at high doses.
• Prefers phenolic OH > simple alcohol; can also conjugate some amines.
• Classic illustrations
  • Estradiol OH ➜ estradiol sulfate. Conjugated estrogens (Premarin®: pregnant-mare urine) rely on gut bacterial sulfatases to regenerate active estradiol after oral dosing.
  • Acetaminophen OH ➜ APAP-sulfate (major at therapeutic dose, together with glucuronide).

Amino-Acid Conjugation

• Substrates: aryl or aryl-alkyl carboxylic acids $\bigl(\text{Ar-(CH}<em>2)</em>x\,\text{COOH},\; x=0\;\text{or 1–2}\bigr).$
• Sequence
  1. Drug activated to acyl-CoA (good leaving group because of large $\text{S}$ atom).
  2. Amino acid nucleophile (NH$_2$) attacks ➜ $\text{drug–C(=O)–NH–AA}$.
• Species preference
  • Most mammals : glycine conjugation.
  • Primates/humans : glutamine predominates.
• Text-book example : benzoic acid → hippuric acid (benzoylglycine).

Acetylation (N-Acetyltransferase, NAT)

• Only primary amines (–NH$_2$) are substrates.
• Cofactor : $\text{Acetyl-CoA}$; product carries $\text{–NH–C(=O)CH}_3$ (1 H lost).
• Genetic polymorphism (rapid vs slow acetylators) clinically relevant for isoniazid, hydralazine, etc.

Methylation (Methyl-Transferase)

• Universal methyl donor: SAM.
• Targets: phenolic OH, amines, sulfhydryls.
• Usually decreases polarity; function often regulatory (e.g.
  • Catechol-O-methyltransferase (COMT) on dopamine.
  • DNA/RNA or protein methylation).
• Enzymes named O-, N-, or S-methyl-transferase.

Glutathione Conjugation – Cellular Guardian

• Detoxifies electrophiles (epoxides, quinones, free radicals, reactive metal ions).
• GSH = $\gamma$-Glu-Cys-Gly; nucleophile = cysteine $\text{–SH}$.
• Active form : reduced monomer (GSH); oxidised dimer (GSSG) must be reduced back by glutathione reductase.
• After initial conjugation, enzymatic trimming ➜ cysteinyl conjugate, then N-acetylation ➜ mercapturic acid (excreted).

Acetaminophen (APAP) Hepatotoxicity Paradigm

\text{APAP}\xrightarrow[\text{high dose}]{\text{CYP2E1}}\underset{\text{NAPQI}}{\text{Electrophilic quinone imine}}\xrightarrow{+\text{GSH}}\text{Detox conjugate}

• Therapeutic dose : glucuronide (≈60 %) & sulfate (≈35 %) dominate.
• Overdose : UGT/SULT saturated ➜ CYP-mediated NAPQI rises ➜ GSH depleted ➜ covalent binding to hepatic proteins/DNA ➜ massive liver necrosis.
• Antidote : N-acetyl-cysteine (NAC) supplies –SH for conjugation & replenishes GSH.

Additional Nuggets & Clinical Connections

• Phase II almost always inactivates drug; exceptions (e.g., morphine-6-glucuronide retains analgesic activity).
• Neonates lack full UGT capacity ➜ avoid chloramphenicol (grey-baby).
• Pro-drugs often designed as esters; rely on Phase I hydrolysis to liberate active drug (improves taste, permeability, organ-targeting, etc.).
• GI flora harbour de-conjugating enzymes (sulfatase, glucuronidase) ➜ entero-hepatic recycling or oral activation of conjugated estrogens.

Exam-Oriented Cheat Sheet

• Glucuronidation : any $\text{OH}/\text{NH}/\text{SH}$ (UGT, UDPGA, microsome).
• Sulfation : phenols (SULT, PAPS, cytosol).
• Amino-acid conj : aryl(-alkyl) carboxylic acids (glycine→animals, glutamine→humans).
• Acetylation : primary amines (NAT, Acetyl-CoA).
• Methylation : phenolic OH / amines (MT, SAM) – polarity ↓.
• Glutathione : electrophiles, free radicals (GST, GSH) – cell protection.
• Enzyme names = (Functional group) + Transferase; all end with “T”.
• Phase I cofactor patterns (for comparison)
  • CYP450 : $\text{O}_2,\;\text{NADPH}$
  • Alcohol DH : $\text{NAD}^+$, etc.

Wrap-Up

• Identify the new nucleophile created in Phase I (or pre-existing).
• Match functional group to preferred Phase II route.
• Remember enzyme + co-substrate + localisation + clinical example.
• Link mechanistic features to clinical outcomes (toxicity protection, pro-drug strategy, genetic polymorphisms).