Biotransformation and drug discovery

Question 1

Why is drug biotransformation necessary?

Answer 1

• Xenobiotic: pharmacological, endocrinal, toxicological substance foreign to an organism
• Metabolism (eg most lipophilic xenobiotics are metabolized to ionized form for clearance)
  > renal excretion common pathways for most drugs with barriers including: larger molecules, less ionized, strongly bound to plasma proteins, lipophilic
• Increase xenobiotic effect (also potentially toxic)
• Active inactive drugs

Question 1

Phase 1 reaction

Answer 1

• Oxidation, Reduction, Hydrolysis
• Convert parent drug to a more polar metabolite by introducing or unmasking a functional group (-OH, -NH2, -SH)
• Often these metabolites are inactive
• Many Phase 1 products are not eliminated rapidly and undergo a subsequent (Phase 2) reaction

Question 2

Where do drug biotransformations occur?

Answer 2

• Liver (principal organ of drug metabolism)
• Other tissues : GIT, Lungs, Skin, Kidneys, Brain
• GIT > Liver: clonazepam, chlorpromazine, cyclosporine
• Low gut has microorganisms capable of biotransformation
• Other phase 1 catalysts: gastric acid, digestive enzymes, enzymes in GI wall

Question 3

Explain the mechanism of oxidation

Answer 3

1. Oxidized P450 [Fe3+] binds to drug, forming a binary complex
2. Transfer of electron: from NADPH -> P450 reductase -> P45-drug complex.
   => reduced P450-drug complex [Fe2+]
3. O2 binds: covalently to haem iron
   - second electron is transferred from NADPH via P450 reductase (or another source) to the O2 –> reduces molecular O2 to a peroxide group
   - forms an (activated O2)-P450-drug complex
4. Complex transfers activated O2 to the drug substrate
   - 2H+ react with the peroxide group -> H2O + (oxidized drug) -P450
   - Oxidized drug product dissociates from P450
   - P450 has returned to oxidized form, to re-enter cycle

Question 4

Enzyme induction

Answer 4

• Some of the drugs substrates, on repeated administration, induce P450 by enhancing the rate of its synthesis or reducing the rate of its degradation
• Results in accelerated substrate metabolism and decrease in pharmacologic action of inducer
• Enzyme induction may exacerbates metabolite-mediate toxicity
• Various substrates appears to induce various P450 isoforms with different molecular masses and different substrate specificities

Question 5

Enzyme inhibitor

Answer 5

• Certain drug substrates inhibit CYP450 enzyme activity
• Imidazole-containing drugs (eg: Cimetidine, Ketoconazole) bind tightly to P450 haem iron -> inactivate P450
• Erythromycin derivatives -> metabolized to compounds that bind to P450 haem iron -> inactive
• Chloramphenicol -> metabolized to product which modifies P450 proteins -> inactive
• Suicide inhibitors (eg: Spironolactone and certain steroids) -> attacks the haem or the protein moiety

Question 6

Phase II reaction

Answer 6

• An endogenous substrate combines with the newly incorporated functional group to form a highly polar conjugate (eg: Glucuronic acid, sulfuric acid, acetic acid, or an amino acid)
• May precede Phase I reactions (if parent drug already possesses functional group), can occur to parent drug or Phase 1 metabolite
• Involves conjugation with an endogenous substance to yield drug conjugates
• Requires specific transferases
  > most important is UDP-glucuronosyl transferases (UGTs): microsomal enzyme couples glucuronic acid) or similar) to a drug
  > Also: Sulfotransferases (SULTs), glutathione transferases (GCSTs), methyltransferases (MTs), N-acetyltransferases (NATs)
• Reactions tend to be relatively faster than P450-mediated metabolism , thus accelerating drug biotransformation

Question 7

What is “tolerance”?

Answer 7

Progressively reduced therapeutic effectiveness due to enhancement of their own metabolism

Question 8

Most common P450 enzymes

Answer 8

CYP 1A2 - 15%
CYP 2A6 - 4%
CYP 2B6 - 1%
CYP 2C9 - 20%
CYP 2D6 - 5%
CYP 2E1 - 10%
CRP 3A4 - 30%

Question 9

Metabolism of drugs to toxic products

Answer 9

• Most commonly seen when resources exhausted
• Eg: Paracetamol [95% metabolized via phase 1 (glucuronidation, sulfation), 5% phase 2 (glutathione conjugation pathway)]
  > Overdose = glucoronidation/ sulfation saturated = glutathione pathway becomes more important = more dependent on P450 (ie phase 1 metabolites accumulate) = hepatic glutathione is depleted faster than it is regenerated = accumulation of hepatoxic metabolites
  > Antidote: N-acetylcysteine -> converted to glutathione

Question 10

Clinical relevance of drug metabolism

Answer 10

“Individual differences”
1. Genetic factors
2. Diet and environmental factors
3. Age and sex
4. Drug-drug interactions during metabolism
5. Interactions between drugs and endogenous compounds
6. Diseases