GW BGZ2026 Case3 - Therapeutic and toxic effects of drugs - a matter of individuality?!

What is the role of DNA in higher organisms?

• Primary genetic material in animals, plants, fungi

• Functions:

  • Stores genetic information (A, T, C, G sequence)

  • Organized into chromosomes in the nucleus

  • Inherited from parents → offspring

  • Template for RNA and protein production

  • Replicates before cell division

• Key property:

  • Stable, double-stranded structure → long-term information storage

  • Complementary strands enable repair mechanisms

What is the role of RNA in higher organisms?

• RNA is not long-term genetic storage (unlike DNA)

• It functions in gene expression and regulation

• Types:

  • mRNA: carries genetic code to ribosome

  • tRNA: delivers amino acids during translation

  • rRNA: structural + catalytic part of ribosome

  • Regulatory RNA (miRNA): controls gene expression

• Key idea:

  • RNA = temporary working copy + functional/regulatory molecule

What are the main differences between DNA and RNA?

• DNA:

  • Long-term storage

  • Stable, double-stranded

  • Located mainly in nucleus

• RNA:

  • Short-term functional copy

  • Single-stranded, less stable

  • Works in nucleus + cytoplasm

What is DNA replication?

• Process of copying DNA before cell division

• Ensures each daughter cell receives identical genetic information

Each new DNA molecule contains:

• one original (parental) strand

• one newly synthesized strand → semiconservative replication

Main steps

1. Unwinding

   • The DNA double helix opens.

   • Hydrogen bonds between complementary bases break.

   • Helicase helps separate the two strands.

2. Primer formation

   • DNA polymerase cannot start a new strand by itself.

   • Primase produces a short RNA primer.

3. Elongation

   • DNA polymerase adds nucleotides complementary to the template strand.

   • Base-pairing rules:

     • A pairs with T

     • C pairs with G

   • DNA is synthesized only in the 5′ → 3′ direction.

4. Leading and lagging strands

   • The leading strand is made continuously.

   • The lagging strand is made discontinuously as short pieces called Okazaki fragments.

   • DNA ligase joins the fragments.

5. Proofreading and repair

   • DNA polymerases can detect and correct many copying errors.

   • This keeps mutation rates relatively low.

Why is DNA replication accurate?

• Complementary base pairing (A–T, C–G)

• DNA polymerase proofreading activity

• Additional DNA repair systems

What is the central dogma of molecular biology?

• Flow of genetic information:

  • DNA → RNA → Protein

• Steps:

  • Transcription (DNA → mRNA)

  • Translation (mRNA → protein)

How is DNA translated into a protein?

Step 1: Transcription

A gene’s DNA sequence is copied into mRNA.

• RNA polymerase reads one DNA strand as a template.

• RNA uses uracil (U) instead of thymine (T).

• RNA base pairing:

  • DNA A → RNA U

  • DNA T → RNA A

  • DNA C → RNA G

  • DNA G → RNA C

In eukaryotes, transcription occurs in the nucleus.

Step 2: RNA processing in eukaryotes

Before leaving the nucleus, pre-mRNA is processed:

• introns are removed

• exons are joined by splicing

• a 5′ cap and poly-A tail are added

This produces mature mRNA.

Step 3: Translation

Translation occurs at ribosomes in the cytoplasm or on the rough endoplasmic reticulum.

Key components

• mRNA: contains codons, groups of three bases.

• Ribosome: reads the mRNA.

• tRNA: carries amino acids and has an anticodon complementary to an mRNA codon.

• Amino acids: building blocks of proteins.

Translation stages

1. Initiation

   • Ribosome binds mRNA.

   • Translation usually begins at the start codon, AUG.

   • AUG codes for methionine.

2. Elongation

   • The ribosome reads codons one at a time.

   • Matching tRNAs bring amino acids.

   • Peptide bonds join amino acids into a growing polypeptide chain.

3. Termination

   • Translation stops at a stop codon: UAA, UAG, or UGA.

   • The completed polypeptide is released.

The polypeptide then folds and may be modified to become a functional protein.

What is a DNA mutation?

• Permanent change in DNA sequence

• Causes:

  • DNA replication errors

  • Radiation (UV, ionizing)

  • Chemicals (mutagens)

  • DNA repair failure

What are the main types of mutations?

Mutation type	DNA-level change	Possible effect
Substitution	One base is replaced by another	May be silent, missense, or nonsense
Insertion	One or more bases added	May cause a frameshift
Deletion	One or more bases removed	May cause a frameshift
Duplication	DNA segment copied extra times	Can alter gene dosage
Inversion	DNA segment reversed	May disrupt a gene or regulation
Translocation	DNA segment moves to another chromosome	Can disrupt genes or create abnormal gene regulation

What are effects of substitution mutations?

• Silent mutation: no amino acid change

• Missense mutation: different amino acid

• Nonsense mutation: stop codon → shortened protein

What is a frameshift mutation?

• Caused by:

  • Insertion or deletion (not divisible by 3)

• Effects:

  • Shifts reading frame

  • Changes all downstream codons

  • Often produces nonfunctional protein

When does a mutation affect phenotype?

A mutation affects phenotype if it alters:

• Protein structure

• Protein amount

• Gene regulation (timing/location)

• RNA processing

• Chromosome structure

• Outcomes:

  • Neutral

  • Harmful

  • Beneficial

  • Environment-dependent

What is a gene?

• DNA sequence encoding a functional product (usually protein)

• Example:

  • CYP2D6 → enzyme metabolizing many drugs

What is an allele?

• Variant form of a gene

• Example CYP2D6 alleles:

  • *1 = normal

  • *4 = no function

  • *10 = reduced function

  • gene duplication = increased function

What is drug metabolism and why does it occur?

Drug metabolism is the biochemical conversion of drugs into more water-soluble compounds to enable elimination.

• Purpose of metabolism:

  • Convert lipophilic drugs → hydrophilic metabolites

  • Facilitate excretion via urine or bile

• Main site:

  • Liver (primary organ)

• Other sites:

  • Kidney

  • Intestine

  • Lung

  • Plasma

• Overall outcome:

  • Drug is inactivated OR activated OR converted to toxic metabolites

What are Phase I reactions in drug metabolism?

Phase I reactions are functionalization reactions that introduce or expose functional groups.

• Main reactions:

  • Oxidation

  • Reduction

  • Hydrolysis

• Main enzyme system:

  • Cytochrome P450 system (CYP450)

• Effects on drugs:

  • Increase polarity slightly

  • Create reactive intermediates

  • May activate or inactivate drugs

  • May produce toxic metabolites

What are Phase II reactions in drug metabolism?

Phase II reactions are conjugation reactions that attach endogenous molecules to drugs.

• Main processes:

  • Glucuronidation

  • Sulfation

  • Acetylation

  • Methylation

  • Glutathione conjugation

• Main effect:

  • Strongly increases water solubility

  • Usually terminates pharmacological activity

• Outcome:

  • Easier renal or biliary excretion

What is the overall pathway of drug metabolism?

Drug metabolism converts lipophilic drugs into hydrophilic metabolites for elimination.

• Stepwise pathway:

  • Drug (lipophilic)

  • → Phase I (functionalization)

  • → Phase II (conjugation)

  • → Water-soluble metabolite

  • → Excretion (urine/bile)

• Key goal:

  • Reduce biological activity + increase elimination

What are CYP enzymes and why are they important?

CYP enzymes are heme-containing monooxygenases responsible for most Phase I drug metabolism.

• Key features:

  • Located in smooth endoplasmic reticulum of hepatocytes

  • Responsible for ~70–80% of drug metabolism

• Major families:

  • CYP3A4/5 (largest contribution)

  • CYP2D6

  • CYP2C9

  • CYP2C19

  • CYP1A2

• Clinical importance:

  • Major source of drug–drug interactions

  • Major cause of genetic variability in drug response

What is CYP2D6 and why is it clinically important?

CYP2D6 is a highly polymorphic enzyme that metabolizes many important drugs.

• Metabolizes ~25% of drugs:

  • Antidepressants

  • Antipsychotics

  • β-blockers

  • Opioids

• Genetic variability:

  100 alleles identified

• Phenotypes:

  • Poor, intermediate, normal, ultrarapid metabolizers

• Key risk:

  • Large differences in drug efficacy and toxicity

How is codeine metabolized in the body?

Codeine is a prodrug, meaning it has limited activity until it is metabolized in the liver into active compounds.

It undergoes three main metabolic pathways:

• 1. CYP2D6 (O-demethylation, ~5–10%)

  • Converts codeine → morphine

  • Morphine is the main active analgesic metabolite

  • Responsible for most pain relief effects

• 2. CYP3A4 (N-demethylation, ~70–80%)

  • Converts codeine → norcodeine

  • Has minimal to no analgesic activity

• 3. UGT2B7 (glucuronidation, ~10–15%)

  • Converts codeine → codeine-6-glucuronide

  • Has modest analgesic contribution

Further metabolism of morphine:

• Morphine → M3G (inactive, may be neurotoxic at high levels)

• Morphine → M6G (active, potent analgesic metabolite)

Key idea:

• Codeine = inactive prodrug

• Morphine = primary active drug

What is the pharmacodynamics of codeine?

Codeine’s effects are mainly mediated through its metabolite morphine, which acts on the:

• μ-opioid receptor (MOR)

  • A Gi/Go protein-coupled receptor (GPCR)

Mechanism of action:

When activated, MOR causes:

• ↓ Adenylyl cyclase activity

  • ↓ cAMP → ↓ neuronal excitability

• ↓ Presynaptic Ca²⁺ influx

  • ↓ release of neurotransmitters like:

    • Substance P

    • Glutamate

• ↑ K⁺ efflux (postsynaptic)

  • Hyperpolarization

  • Neurons become less excitable

What factors influence drug metabolism in general?

Drug metabolism is influenced by genetic, environmental, physiological, and disease-related factors.

1. Genetics

• CYP polymorphisms (e.g., CYP2D6, CYP2C19)

• Determines metabolizer phenotype

2. Age

• Neonates:

  • Immature liver enzymes → slow metabolism

• Elderly:

  • ↓ liver blood flow + ↓ enzyme activity

3. Liver disease

• ↓ CYP activity

• ↓ first-pass metabolism

• ↑ drug levels

. Kidney disease

• Reduced excretion of metabolites

• Indirect effect on metabolism

5. Lifestyle

• Smoking → induces CYP1A2

• Alcohol:

  • Acute → inhibits metabolism

  • Chronic → induces CYP2E1

6. Diet

• Grapefruit juice → inhibits CYP3A4

• Cruciferous vegetables → induce CYP1A2

• Malnutrition → ↓ enzyme synthesis

7. Drug interactions

• Inhibitors → ↓ metabolism → toxicity risk

• Inducers → ↑ metabolism → reduced effect

Examples:

• Fluoxetine → CYP2D6 inhibitor

• Rifampicin → strong inducer

8. Disease/inflammation

• Cytokines suppress CYP expression

• ↓ metabolic capacity during illness

9. Epigenetics

• DNA methylation / histone changes

• Alters enzyme expression without DNA mutation

How do drugs enter breast milk?

Most drugs enter breast milk via passive diffusion, driven by concentration gradients.

Only the free (unbound) drug fraction crosses membranes.

Key determinants:

• Lipid solubility

• Molecular size

• Plasma concentration

• Protein binding

• Ionization state

What is ion trapping in breast milk?

Ion trapping occurs because breast milk is slightly more acidic than blood.

• Blood pH ≈ 7.4

• Breast milk pH ≈ ~6.8–7.0

For weak bases (e.g., morphine):

• In blood:

  • more non-ionized → crosses membranes

• In milk:

  • becomes ionized (charged)

  • cannot diffuse back

Result:

• Drug becomes trapped in breast milk

• Higher infant exposure

Why can codeine be dangerous during breastfeeding?

Risk comes from combined pharmacogenetics + milk transfer.

Mechanism:

1. Codeine → metabolized by CYP2D6 → morphine

2. Morphine enters breast milk via passive diffusion

3. Ion trapping increases milk concentration

4. Infant ingests morphine

Risk scenario: ultrarapid metabolizer mother

• ↑ CYP2D6 activity

• ↑ morphine production

• ↑ breast milk morphine levels

• Infant exposure increases

Possible infant effects:

• Excessive sleepiness

• Poor feeding

• Hypotonia

• Respiratory depression

• Potential death (rare but documented)

How does CYP2D6 genotype affect the response to codeine?

• CYP2D6 determines how efficiently codeine is converted into morphine.

• Important phenotypes include:

  • Poor metabolizer (PM)

    • Very little CYP2D6 activity.

    • Produces little morphine.

    • May have poor or absent analgesia.

  • Intermediate metabolizer (IM)

    • Reduced enzyme activity.

    • May have reduced analgesic response.

  • Normal metabolizer (NM)

    • Expected morphine formation and clinical response.

  • Ultrarapid metabolizer (UM)

    • Very high CYP2D6 activity, often due to extra functional gene copies.

    • Produces excessive morphine.

    • Has increased risk of opioid toxicity and respiratory depression.

What different types of metabolizers are there?

An ultrarapid metabolizer (UM) is an individual who possesses significantly increased activity of a drug-metabolizing enzyme due to inherited genetic variants.

Metabolizer Phenotypes

Based on total activity score:

• Poor metabolizer (PM): Activity score = 0

  • Little or no enzyme activity

  • Drug accumulation and toxicity or failure to activate prodrugs

• Intermediate metabolizer (IM):

  • Reduced enzyme activity

  • Slower metabolism than normal

• Extensive metabolizer (EM):

  • Normal enzyme activity

  • Expected drug response

• Ultrarapid metabolizer (UM):

  • Increased enzyme activity due to gene duplication

  • Drugs eliminated rapidly or excessive activation of prodrugs

  • High doses → toxic for the person itself or the baby

What is polymorphism?

A genetic polymorphism is a DNA variation that is relatively common in a population. Traditionally, “polymorphism” meant a variant present in at least 1% of a population, but modern genetics often uses the term more broadly for common inherited variation. It does not automatically mean disease.

• CYP2D6 is highly variable and affects metabolism of drugs such as codeine.

• Different alleles can produce:

  • A normal enzyme

  • Reduced enzyme activity

  • No enzyme activity

  • Extra copies of a functional enzyme

Examples:

• *CYP2D6 1: usually normal function.

• **CYP2D6 3 or 4: usually no function.

• *CYP2D6 5: gene deletion, so no CYP2D6 enzyme from that copy.

• Duplicated functional CYP2D6 genes: can cause ultrarapid metabolism.

The combination of inherited CYP2D6 alleles determines whether someone is a poor, intermediate, normal, or ultrarapid metabolizer, which can affect drug effectiveness and toxicity.