7.3 Phase I Biotransformation: Cytochrome P450 Monooxygenases & Non-CYP Oxidation

Why Phase I is a susceptibility topic

Handbook Domain II.2 wants species and pathway differences in biotransformation; II.3 adds genetic polymorphisms as a reason two humans (or two strains) do not share the same internal dose of metabolite. Phase I reactions—oxidation, reduction, hydrolysis—usually insert or expose a functional group. That group may be conjugated in Phase II, or the product may be the toxic species. Independent OpenExamPrep material in this section covers cytochrome P450 (CYP) families most often cited in human toxicology, how induction differs from inhibition, non-CYP oxidases, and a species acetylation difference you must not mix up with feline glucuronidation. It is not an ABT product and does not claim official approval, review, or partnership with ABT.

CYP enzymes are heme-thiolate monooxygenases in the endoplasmic reticulum. They split molecular oxygen: one atom goes to substrate (as hydroxyl, epoxide, or related product), the other to water. NADPH-CYP reductase supplies electrons. Activity in vitro is often measured in microsomes or with recombinant isoforms. S9 is the post-mitochondrial supernatant used in genotoxicity batteries because it contains microsomal CYPs plus some cytosolic enzymes.

Human CYP families you must be able to assign

CYP3A4 (with CYP3A5 in some people) oxidizes a large fraction of drug-like xenobiotics and many steroids. It is abundant in liver and small-intestinal epithelium. Midazolam 1′-hydroxylation is a classic probe. Ketoconazole, itraconazole, clarithromycin, and ritonavir inhibit CYP3A. Rifampin, carbamazepine, phenytoin, and St. John’s wort induce it via PXR (and related nuclear-receptor programs). Grapefruit furanocoumarins inactivate intestinal CYP3A and can raise oral AUC of substrates that rely on gut first-pass.

CYP2D6 oxidizes many psychotropics, beta-blockers, and codeine (O-demethylation to morphine). It is highly polymorphic (poor, intermediate, extensive, ultrarapid metabolizers) and is not readily inducible by the PXR/CAR ligands that raise CYP3A4. Inhibitors include quinidine, fluoxetine, and paroxetine. An ultrarapid metabolizer can form excess morphine from codeine; a poor metabolizer gets little analgesia and more parent.

CYP2C9 oxidizes S-warfarin, phenytoin, and several NSAIDs. **CYP2C92 and 3 reduce function and increase bleeding risk on standard warfarin doses. Inhibitors include fluconazole and amiodarone.

CYP2C19 activates clopidogrel and oxidizes some proton-pump inhibitors and antidepressants. Loss-of-function alleles (*2, *3) are common in some East Asian populations; *17 increases function. Poor metabolizers activate less clopidogrel.

CYP1A2 oxidizes caffeine, theophylline, and some aromatic amines. Smoking and charbroiled food (PAHs) induce CYP1A enzymes through the aryl hydrocarbon receptor (AhR), the same receptor classically cited for TCDD-like induction of CYP1A1/1B1 extrahepatically. Fluvoxamine inhibits CYP1A2.

CYP2E1 oxidizes low-molecular-weight solvents and alcohols: ethanol, benzene, acetaminophen (a fraction to NAPQI), carbon tetrachloride, chloroform, and vinyl chloride. It is induced by chronic ethanol, fasting/acetone, and isoniazid, and it sits preferentially in centrilobular hepatocytes—the zone that dies first in CCl4 and APAP overdose. Competitive inhibition by ethanol can spare APAP bioactivation during drinking and then unmask a large induced CYP2E1 pool after ethanol is gone; that timing question is a classic interpretation trap.

Induction versus inhibition

Inhibition can begin with the first dose: reversible competition at the active site, or mechanism-based (suicide) inactivation that destroys enzyme until new protein is made (some macrolides, grapefruit furanocoumarins, a subset of HIV protease inhibitors). Induction requires transcription and new enzyme (PXR → CYP3A4/2B6/2C; CAR → CYP2B6 and some 2C/3A; AhR → CYP1A). Clinical onset is days, not minutes. Autoinduction (carbamazepine) lowers parent over the first weeks. Do not call a same-day AUC drop “induction.” Do not call CYP2D6 “induced by rifampin” as if it were CYP3A4.

Polymorphisms as susceptibility (II.3)

A poor metabolizer of a detoxifying CYP accumulates parent (phenytoin and CYP2C9). A poor metabolizer of a bioactivating CYP is relatively protected (less NAPQI if CYP2E1 activity is low; less morphine from codeine if CYP2D6 is absent). An ultrarapid CYP2D6 genotype does the reverse for codeine. NAT2 slow acetylation is a Phase II polymorphism, but exam items mix it with CYP stories: slow acetylators accumulate isoniazid and have more peripheral neuropathy if pyridoxine is inadequate, and they handle some aromatic amines differently. Record the enzyme, the direction (gain or loss), and whether the metabolite is toxic or therapeutic.

Non-CYP oxidation and hydrolysis

Flavin-containing monooxygenases (FMOs) use FAD and NADPH to N- and S-oxidize many soft nucleophiles (including trimethylamine via FMO3; FMO3 deficiency is trimethylaminuria). FMOs are not heme CYPs, are relatively heat-labile in vitro, and are not induced by the same PXR/AhR programs.

Alcohol dehydrogenase (ADH) in cytosol oxidizes ethanol to acetaldehyde (NAD+). Aldehyde dehydrogenase (ALDH), largely mitochondrial ALDH2, oxidizes acetaldehyde to acetate. Disulfiram inhibits ALDH. ALDH2*2 (common in some East Asian populations) raises acetaldehyde and flushing—another II.3 susceptibility example.

Monoamine oxidases (MAO-A, MAO-B) on the outer mitochondrial membrane oxidatively deaminate amines. MAO-B converts the protoxin MPTP to MPP+, which kills nigrostriatal neurons; MAO-B inhibitors can block that bioactivation in experimental systems.

Microsomal epoxide hydrolase (mEH, EPHX1) adds water to epoxides, yielding trans-dihydrodiols. That is detoxification for many arene oxides, but for benzo[a]pyrene the diol is a stepping-stone to the diol-epoxide (taught with Phase II in 7.4). Esterases include carboxylesterases (CES) that hydrolyze many esters and carbamates, plasma butyrylcholinesterase, acetylcholinesterase (nerve-agent target), and paraoxonase (PON1) that hydrolyzes paraoxon. PON1 status is another susceptibility modifier for some organophosphates.

Species difference: dogs as poor acetylators

N-acetylation is a Phase II reaction (acetyl-CoA, cytosolic NAT). It is taught here because species ranking is a standard II.2 trap next to CYP tables. Dogs (and foxes) have functionally deficient NAT activity and are poor N-acetylators of many aromatic amines and hydrazines. Humans express NAT1 (wide tissue distribution) and hepatic/intestinal NAT2, and NAT2 is polymorphic (slow versus rapid acetylators). Do not say dogs are rapid NAT2 metabolizers. Do not use the dog acetylation defect as a stand-in for cat phenolic glucuronidation deficiency (UGT1A6)—that conjugation gap is a 7.4 topic. A dog TK package can therefore miss an acetylated metabolite that humans form, or accumulate an aromatic amine that humans would acetylate.

CYP3A first-pass: ketoconazole and midazolam, rewritten

Oral midazolam undergoes substantial first-pass extraction by CYP3A in enterocytes and hepatocytes; oral bioavailability is often on the order of ~30% because both gut and liver extract parent. Ketoconazole is a strong CYP3A inhibitor. When oral ketoconazole is given for a few days and then oral midazolam is repeated, midazolam AUC and Cmax rise several-fold to more than ten-fold in published interaction studies, because both intestinal and hepatic extraction fall. If midazolam is given intravenously, the intestinal first-pass is already bypassed, so the fold-increase is typically smaller than after oral dosing, though hepatic CYP3A inhibition still slows systemic clearance. This is an extraction-and-route lesson, not a claim that ketoconazole “binds midazolam in the gut as an insoluble salt.” Sort route, which CYP, and inhibition versus induction before you interpret an AUC change.

Realistic scenario

A first-in-human protocol uses oral midazolam as a CYP3A probe on day −1, starts a strong azole antifungal on day 1, and repeats oral midazolam on day 5. AUC rises about an order of magnitude. The team then claims “the azole induced a new CYP” because a metabolite peak appeared earlier. That is backwards: ketoconazole-class azoles inhibit CYP3A; an earlier metabolite peak would need a different explanation (assay interference, another isoform). Switching the second midazolam dose to IV should shrink the fold-change if gut CYP3A first-pass was a large part of the oral interaction. A parallel dog study that fails to form an acetylated aromatic-amine metabolite that humans make is a poor-acetylator species finding, not proof that canine CYP2D6 is missing.

Traps

• Treating CYP2D6 as rifampin-inducible like CYP3A4.
• Calling same-day AUC collapse “induction.”
• Equating dog NAT deficiency with cat UGT1A6 deficiency.
• Forgetting intestinal CYP3A when only hepatic microsomes were measured.
• Assuming every solvent is a CYP3A4 substrate; small halogenated aliphatics often run through CYP2E1.