Section 3 Pharmacology

Biotransformation and Drug Discovery

Drug Biotransformation
 Phase I reactions
o Usually convert the parent drug to a more
polar metabolite by introducing or
unmasking a functional group
(Examples: -OH, -NH2, -SH)
o Often these metabolites are inactive
(sometimes only modified activity,
sometimes enhanced activity)
o Many phase I products are not eliminated
rapidly and undergo a subsequent (phase
II) reaction
 Phase II reactions
o An endogenous substrate combines with
the newly incorporated functional group
to form a highly polar conjugate
(Examples: Glucuronic acid, sulfuric acid,
acetic acid, or an amino acid)
o Phase II reactions may precede phase I reactions (if parent drug already possesses functional group)
 These events are not always innocuous biochemical events, leading to detoxification and elimination of the compound.
Several compounds have been shown to be metabolically
transformed to reactive intermediates that are toxic to various
organs. Such toxic reactions may not be apparent at low levels of
exposure when alternative detoxification mechanisms are not yet
overwhelmed or compromised and the availability of endogenous
detoxifying co-substrates (examples: glutathione (GSH),
glucuronic acid, sulfate) is not limited. However, when these
resources are exhausted, the toxic pathway may prevail → Overt
organ toxicity or carcinogenesis.
o Example: Paracetamol induced hepatotoxicity
 Normally → Glucuronidation, sulfation → 95%
of excreted metabolites
 Alternative (P450-dependent) glutathione
conjugation pathway → Remaining 5%
 High doses of paracetamol → Glucuronidation and
sulfation pathways are saturated → Glutathione
pathway becomes more important
 If hepatic glutathione is depleted faster than it is
regenerated → Accumulation of hepatotoxic
metabolites
 Antidote: N-acetylcysteine → Converted to
glutathione

Phase I Reactions
 Cytochrome P450 mixed function oxidases (MFOs) = Monooxygenases
o Class of enzymes located on smooth endoplasmic reticulum (in vitro: microsomes)
o Adds a hydroxyl group to the substrate
o Activity requires, NADPH (a reducing agent), O2, and 2 microsomal enzymes:
 NADPH-cytochrome P450 reductase (= P450 reductase)
 A flavoprotein
 Cytochrome P450 (= CYP = P450)
o Mechanism of oxidation:
1. Oxidized P450 (with Fe3+ , not Fe2+) binds to drug, forming a binary complex
2. Transfer of electron-: from NADPH → P450 reductase → P450-drug complex
 Reduced P450-drug complex (Fe2+)
 3. O2 binds: covalently to the haem iron
 Second electron is transferred from NADPH via P450 reductase (or another source) to the O2 →
Reduces molecular oxygen to a peroxide group
 Forms an (activated oxygen)-P450-drug complex
4. Complex transfers activated oxygen to the drug substrate
 2H+ react with the peroxide group → H2O + (oxidised drug)-P450
 Oxidized drug product dissociates from P450
 P450 has returned to oxidised form, to re-enter cycle
o Substrate specificity is very low for this enzyme complex (only high lipid solubility is necessary)
o Many different iso-enzymes (most important is CYP3A4 → responsible for >50% of the clinically prescribed drugs
metabolised by the liver)

 Enzyme induction: Some of these drug substrates, on repeated administration, induce P450 by enhancing the rate of its
synthesis or reducing the rate of its degradation
o The result is acceleration of substrate metabolism, and usually a decrease in the pharmacologic action of the
inducer
o Enzyme induction may exacerbate metabolite-mediated toxicity
o Various substrates appear to induce various P450 isoforms with different molecular masses and different substrate
specificities.
 Enzyme inhibition: Certain drug substrates may inhibit cytochrome P450.
o Imidazole-containing drugs (Examples: Cimetidine, ketoconazole) → Bind tightly to P450 haem iron → Inactivate
P450
o Erythromycin derivatives → Metabolised to compounds that bind to P450 haem iron → Inactive
o Chloramphenicol → Metabolised to product which modifies P450 protein → Inactive
o Suicide inhibitors (Example: Spironolactone) → Attacks the haem or the protein moiety
 Important P450 inhibitors, inducers and substrates:
o CYP1A2
 Substrates: TCAs, olanzapine, verapamil, warfarin
 Inducers: Tobacco smoke, chargrilled meat (polycyclic aromatic hydrocarbons), omeprazole
 Inhibitors: Fluoroquinolones, verapamil
o CYP2A6
 Substrates: Nicotine, valproate, paracetamol
 Inducers: Phenobarbital
 Inhibitors: Ketoconazole
o CYP2D6:
 Substrates: TCAs, SSRIs, haloperidol, β-blockers, promethazine
 Inducers: Rifampicin, dexamethasone
 Inhibitors: SSRIs (self-inhibition), quinidine
o CYP2E1:
 Substrates: Halothane, ethanol, paracetamol
 Inducers: Isoniazid, ethanol
 Inhibitors: Disulfiram
o CYP3A4:
 Substrates: Amiodarone, Macrolides, opiates, benzodiazepines
 Inducers: Glucocorticoids, macrolide antibiotics, phenytoin, carbamazepine, St John’s Wort
 Inhibitors: Macrolides (erythromycin), chloramphenicol
Phase II Reactions
 Can occur to parent drug, or a phase I metabolite
 Involves conjugation with an endogenous substance to yield drug conjugates.
 Requires specific transferases
o Most important is UDP-glucuronosyl transferases (UGTs): microsomal enzyme couples glucuronic acid (or
similar) to a drug
o Also: Sulfotransferases (SULTs), glutathione transferases (GSTs), methyltransferases (MTs), N-acetyltransferases
(NATs)
 Reactions tend to be relatively faster than P450-mediated metabolism

Clinical Relevance of Drug Metabolism

 This is mainly the reasons for individual differences in drug metabolism
o Genetic factors:
 Gene polymorphisms
 Example: Pseudocholinesterase defect leading to halved rate of succinylcholine metabolism
o Diet and environmental factors:
 Example: Grapefruit juice inhibiting CYP3A4 → ↑ Substrate concentrations (Example: Felodipine)
o Age and sex:
 Increased susceptibility to pharmacologic and toxic effects in the elderly or the very young
 Androgenic hormones alter drug metabolism in rats
 Not just related to differences in absorption, distribution and metabolism
 Example: Sex-dependent difference in alcohol metabolism
o Drug-drug interactions during metabolism
 Enzyme induction → tolerance
 Can affect metabolism of other drugs, but induction or inhibition of enzymes
 Example: Cimetidine → Competitive inhibition of metabolic pathways (diazepam, warfarin, and others)
o Interactions between drugs and endogenous compounds:
 Two drugs may rely on the same endogenous substrate (e.g. glutathione) for their inactivation
o Diseases affecting drug metabolism
 Example: Liver disease greatly increases half-life of diazepam

Drug Development
 Discovery
1. Drug screening (tests at molecular, cellular, organ system, whole animal levels) → Define (mechanism of) activity
and selectivity
 Rational design of drugs based on receptors
 Genetic methods of determining agonist compounds from genetic structure
 Modification of existing compounds
 Searching for new targets for drugs
2. Define pharmacologic profile (pharmacokinetic and pharmacodynamic properties) → Cardiovascular and renal
function studies → Determine need for further chemical modification of compound
3. Results in development of a lead compound (= leading candidate for a successful new drug) → Patent application
may be filed
 Preclinical safety and toxicity testing: Testing acute/subacute/chronic toxicity, effect on reproductive performance,
carcinogenic/mutagenic potential, investigative toxicology
o Determination of ‘no-effect’ dose (= maximum dose at which a specified toxic effect is not seen), minimum lethal
dose, median lethal dose (LD50)
 Evaluation in humans (clinical trials)
Phase 1: Effects of drug as a function of dosage; tested in small number of healthy volunteers (sometimes in volunteer
patients); non-blinded
Phase 2: Small number of patients is studied in great detail; often single-blinded design (vs. placebo or older active
drug)
Phase 3: Evaluation in much larger numbers of patients to further establish safety and efficacy; double-blind and
crossover techniques → New drug application (FDA) may be submitted
Phase 4: Monitoring the safety of the new drug under actual conditions of use in large numbers of patients