Drug Metabolism – Part 1 Comprehensive Study Notes

Pharmacokinetic Background and Rationale for Drug Metabolism

• Goal of the lecture: explain how the body chemically alters (biotransforms) xenobiotics to facilitate elimination.

• Xenobiotic = any foreign chemical entering the body (≈ 99.9 % of marketed drugs).
  • Exception: endogenous replacements such as thyroid hormone.

• Why metabolism is essential – ‘octane on the skin’ thought-experiment:
  • Octane ($C<em>8H</em>{18}$, highly lipophilic hydrocarbon) is absorbed → circulates → filtered at the glomerulus.
  • In the renal tubule lipophilic molecules are readily re-absorbed.
  • Continuous filtration/re-absorption loop ⇒ compound would never leave the body without chemical modification.
  • Metabolic conversion increases hydrophilicity → metabolite stays in aqueous urine and is excreted.

Renal Elimination vs. Metabolic Elimination

• ≈ 99 % of small-molecule drugs are freely filtered.

• Molecules too large (e.g., albumin-bound drugs, monoclonal antibodies) are not filtered.

• Polar/lipophobic molecules (e.g., penicillins) may leave unchanged via:
  • Glomerular filtration.
  • Active tubular secretion via an anion transporter ("acid secretor").

• Take-home: lipophilicity ↔ re-absorption; polarity ↔ renal secretion.

Major Patterns of Drug Metabolism

1. Active drug → inactive/excretable metabolite (most common).

2. Active drug → active (sometimes more potent) metabolite.

3. Inactive pro-drug → active drug (requires metabolism).

4. Un-excretable → excretable metabolite (umbrella concept).

Functional Groups & Organic Chemistry Refresher

• Students must visually recognize: ketone, aldehyde, ester, ether vs. thio-ether, nitro, carbamate, urea, azo ($N{=}N$), etc.

• Functional group identity predicts metabolic site (handles for Phase I/II).

Anatomic Sites of Biotransformation

• Liver = dominant organ (smooth ER of hepatocytes).

• Extra-hepatic: kidney, GI wall (e.g., $L$-dopa), skin, lung; all tissues possess some capacity.

Phase I vs. Phase II—Core Concepts

Phase I ("functionalisation")

• Reaction classes: Oxidation, Reduction, Hydrolysis.

• Primary aims:
  • Introduce or unmask nucleophilic "handles" – \ce{OH}, \ce{NH}, \ce{SH}.
  • Only new hetero-atom that can be added de-novo is oxygen (e.g., hydroxylation).
  • Creates slight polarity, may inactivate/activate drug, or create toxic intermediates.

Phase II ("conjugation")

• Reactions: glucuronidation, sulfation, acetylation, methylation, glutathione conjugation, amino-acid conjugation, etc.

• A nucleophilic handle attacks an endogenous, highly polar donor → overall polarity skyrockets → urinary or biliary excretion.

• $\approx 99\%$ of Phase II conjugates are pharmacologically inactive.

• A drug already containing \ce{OH}/\ce{NH}/\ce{SH} can skip Phase I (e.g., acetaminophen) yet may still undergo extra Phase I steps—“the liver has no brain.”

Key Enzymes in Phase I

• Cytochrome $\text{P450}$ (CYP) family (heme-containing mono-oxygenases).

• Flavin-containing mono-oxygenases (FMO) – e.g., monoamine oxidase (MAO).

• Epoxide hydrolase.

• Alcohol dehydrogenase (ADH) & aldehyde dehydrogenase (ALDH).

Cytochrome P450 System in Depth

• Located in smooth ER; historically called "microsomal mixed-function oxidases (MFO)."

• Each CYP is a distinct isoform ("cousins") with its own substrate spectrum.

• Prosthetic group: heme-Fe.

• Electron source: $\text{NADPH}$ → CYP-redutase (contains FAD & FMN) → CYP-heme.

• Catalytic cycle (simplified):

  1. Drug binds near heme.

  2. \ce{O2} binds Fe(II).

  3. Two e⁻ from $\text{NADPH}$ (as hydride) reduce complex.

  4. Forms highly electrophilic "oxene" (Fe$^{IV}{=}O$), denoted $[O]$.

  5. One O atom inserted into substrate (mono-oxygenation); the second reduced to \ce{H2O}.
     \ce{RH + O2 + NADPH + H+ -> ROH + H2O + NADP+}.

• Naming/importance of isoforms (drug metabolism share):
  • $\text{CYP3A4/5}$ ≈ 30 % of CYP-mediated clearance.
  • $\text{CYP2E1}$ – ethanol & small solvents; inducible by chronic alcohol.
  • $\text{CYP2C9}$ – phenytoin, warfarin, barbiturates.

• Genetic/inducible variability → major source of drug–drug interactions and idiosyncratic toxicity.

Oxidative Phase I Reactions—Focus on Epoxides

Aromatic Hydroxylation via Arene Oxide Pathway

1. CYP forms an arene epoxide (aka arene oxide) across an aromatic $\pi$-bond.

2. Four possible fates (must memorise):
   a. NIH Shift → rearrangement → phenol (most frequent).
   b. Epoxide hydrolase + \ce{H2O} → trans-1,2-diol (detoxification).
   c. Covalent binding to macromolecules (DNA, RNA, proteins) → mutagenicity, hypersensitivity (hapten formation).
   d. Conjugation with glutathione (GSH) via nucleophilic \ce{S^-} of cysteine → detox.

• Glutathione structure: $\text{γ-Glu-Cys-Gly}$ tripeptide; cysteinyl \ce{-SH} is the reactive site.

Alkene Epoxidation (Non-aromatic)

• CYP attacks isolated $C=C$ bonds yielding aliphatic epoxides; same four fates apply.

Clinically Relevant Examples of Epoxide-Mediated Toxicity

1. Benzo[a]pyrene (polycyclic aromatic hydrocarbon)

• Sources: coal tar, cigarette & marijuana smoke, car exhaust, flame-grilled meat.

• Sequential CYP oxidation → diol-epoxide that intercalates DNA; guanine N7 opens epoxide → covalent adduct → mutations → lung & other cancers.

• Demonstrates cumulative risk (e.g., lifelong smoker).

2. Carbamazepine

• Antiepileptic; CYP produces arene oxide metabolite.

• Usually detoxified by epoxide hydrolase → diol.

• Rarely, epoxide binds macromolecules → teratogenicity (cleft palate), aplastic anaemia, hepatotoxicity.

3. Aflatoxin B₁

• Produced by Aspergillus species on moldy peanuts/grains.

• CYP creates 8,9-epoxide → guanine adducts in hepatocyte DNA → hepatocellular carcinoma.

• Competing detox:
  • Epoxide hydrolase → dihydrodiol.
  • GSH conjugation via cysteinyl \ce{SH}.

Additional High-Yield Bullet Points & Numerical Facts

• $\approx 80\%$ of clinically important drug–drug interactions arise from CYP inhibition or induction.

• Phase I: memorize three reaction categories (Oxidation, Reduction, Hydrolysis).

• Phase II: memorize every conjugation class; most common = \ce{O}-glucuronidation and \ce{O}-sulfation.

• Induction example: chronic ethanol ↑ $\text{CYP2E1}$ levels → altered acetaminophen toxicity profile.

• Absorption vs. filtration rule-of-thumb:
  • Lipophilic, \log P > 2 ⇒ likely to be re-absorbed.
  • Polar, MW < $\approx 500\text{ Da}$ ⇒ filtered & excreted.

• "Microsomal fraction" = vesiculated smooth ER obtained after ultracentrifugation; experimental source of CYPs.

Ethical & Clinical Implications

• Risk–benefit of drugs producing reactive intermediates (e.g., carbamazepine in pregnancy).

• Lifestyle factors (smoking, charred diet) amplify xenobiotic load and cancer risk.

• Occupational exposure (coal miners) demands protective equipment to minimise PAH inhalation.

Study Checklist (Star System Recap)

• Double ★★ / Triple ★★★ items in slides = examinable:
  • Phase I vs. Phase II definitions & goals.
  • Arene oxide four-pathway schema (NIH shift, hydrolase, macromolecule adduct, GSH).
  • CYP catalytic cycle & electron flow (NADPH → FAD/FMN → heme Fe).
  • Major CYP isoforms and inducibility.
  • Mechanistic basis of benzo[a]pyrene, carbamazepine, aflatoxin B₁ toxicity.

Quick-Reference Equations & Structures

• Octane: \ce{C8H18}.

• Overall CYP reaction: \ce{RH + O2 + NADPH + H+ -> ROH + H2O + NADP+}.

• GSH: $\gamma\text{-Glu-Cys-Gly}$ (cysteine \ce{-SH} = nucleophile).

• Epoxide opening (generic): \ce{R1-CH-CH2 (epoxide) + H2O -> R1-CH(OH)-CH2OH} (trans).

(End of Part I notes – next lecture continues Phase I oxidation subclasses and all Phase II conjugations)