Properties of metabolites

Question 1

In most cases metabolites are _____ .

Answer 1

Inert

Question 2

Exceptions of inert metabolites:

Answer 2

1. Pharmacologically active metabolites
   Several examples - mainly phase I metabolites (diazepam, clopidogrel)
   Very few conjugates (ezetimibe)
2. Reactive metabolites
   Paracetamol overdose (N-OH metabolite)
   Adverse Drug Reactions (ADR)
3. Metabolites contributing to drug-drug interactions.
   Inhibition of metabolic enzymes / transporters (gemfibrozil glucuronide)

Question 3

How important are pharmacologically active metabolites? Depends on:

Answer 3

1) Receptor affinity of the metabolite – may be comparable to parent
2) Plasma concentration of the metabolite

Drug –> M1 metabolite -OH (active) –> M2 -O-Gluc (inert)

The balance of formation rate vs. elimination rate is critical

Question 4

Metabolism of diazepam (chemical):

Answer 4

Diazepam –> Nordazepam (M1) active

Diazepam –> Temazepam (M2) inert

Both form M3 Oxazepam –> Glucuronidation

Question 5

Metabolism of diazepam:

Answer 5

All 3 major metabolites are pharmacologically active

• M1 (nordiazepam) is the ONLY important diazepam metabolite – high plasma concentration
• M2 and M3 are rapidly metabolised and not detectable in plasma! NOT important as diazepam metabolites!
• M2 (temazepam) and M3 (oxazepam) marketed as short-acting alternative to diazepam (which is long acting)

Question 6

Chemically reactive metabolites characteristics and examples:

Answer 6

• Short half-life, react quickly
• Result in local toxicity (at the site of formation) unless conjugated to glutathione

Examples:
- Drug induced liver injury (DILI)

• Benzopyrene
• -> Major polycyclic aromatic hydrocarbon in cigarette smoke
• -> Forms reactive metabolites in the lung
• Paracetamol
• -> Causes liver failure in the case of overdose

Question 7

Metabolism – balance between detoxication and toxication pathways

Answer 7

Xenobiotic (foreign molecule)

Reactive metabolites

• -> Toxicity (too unstable to be eliminated)
• -> Protection mechanism*

Stable metabolite
–> Elimination as inert material

Question 8

Glutathione conjugation

Answer 8

Very important protective mechanism.

Phase I metabolite

(e. g., reactive metabolite
- arene oxide) + Glutathione (tripeptide)

(Electrophile + nucleophile)

Question 9

Toxic responses from xenobiotics and reactive metabolites:

Answer 9

• Physiological Response
• Irritant Response
• Developmental damage
• Antigenic conjugate
• Tissue Damage
• DNA damage

Question 10

Adverse Drug Reactions (ADR):

Answer 10

• Common cause of hospital admissions and death of patients
• Yellow card system to monitor ADR* - potential toxicity monitoring continues after drug registration/marketing

Type A – reversible

• 80% of ADR
• Dose related
• May be associated with polypharmacy - exaggerated response

Other types – irreversible - B,C and D

Question 11

Summary of reversible and irreversible drug effects:TYPE A
Reversible,
adverse responses

Answer 11

TYPE A
- Reversible, adverse responses
-> A1 - Effects linked to
pharmacological action
-> A2 - Effects unrelated to drug action	

TYPE B/C/D
Irreversible, toxic 
responses
Non-selective effects
Often reactive metabolites

Question 12

Non-selective toxicities – types B, C and D:

Answer 12

• NOT related to specific enzymes or receptors
• Non-selective effects
• Reactive metabolites are often implicated

Question 13

Type B non selective toxicities:

Answer 13

• Idiosyncratic, non-predictable
• Often have genetic basis – immuno-mediated toxicity
• -> Reactive metabolite formed –> covalent binding to protein –> recognized as antigen –> immune response
• Carbamazepine-induced Stevens–Johnson syndrome – HLA-B*1502
• Commonly associated with LIVER

Question 14

Type C and D toxicities:

Answer 14

Type C

• Toxicity due to chemical reaction between drug or metabolite with tissue macromolecules
• Often leads to cell death and tissue necrosis - necrotic toxicity
• Liver particularly prone to this type
• Paracetamol toxicity as an example

Type D
- Mechanisms similar to B and C but the response is delayed – chronic toxicity

Question 15

Examples of type D toxicities:

Answer 15

TERATOGENIC
- Affects foetal development
- Leads to functional and/or structural abnormality
Thalidomide example

MUTAGENIC

• Damages genetic material
• DNA mutation may be of no consequence or lethal

CARCINOGENIC

• Uncontrolled proliferation of the tissue
• Multistage process with initiation and promotion phases

Question 16

Thalidomide example toxicity:

Answer 16

• Birth defects in babies whose mothers used “anti-sickness” drug
• Forms reactive metabolites which prevent limb development
• Species differences in metabolism
• -> Rodents resistant to teratogenicity of thalidomide
• -> Rabbits and humans are highly susceptible
• Led to improvement in toxicity testing

Question 17

Role of metabolites in DDIs?

Answer 17

• The majority of prescribed drugs have circulating metabolites
  
  • -> 82% of CYP inhibitors reported to have circulating metabolites in plasma
• Plasma concs of metabolites can be > parent compounds
• Metabolites reported to inhibit
• -> metabolic enzymes (N-desmethyl diltiazem)
• -> uptake transporters (cyclosporine AM1)
• 2017 FDA guidance defines criteria when investigation of the effect of metabolite on CYPs/transporters is required

Question 18

Altered DDI profile due to glucuronide metabolite - gemfibrozil story

Answer 18

• Cerivastatin withdrawn because of adverse and sometimes fatal side effects (rhabdomyolysis)
• Cerivastatin mainly metabolised by CYP2C8 and to a lesser extent by CYP3A4
• However, gemfibrozil is NOT a potent inhibitor of CYP2C8 or 3A4
• Gemfibrozil glucuronide, but NOT gemfibrozil itself, is an irreversible inhibitor of CYP2C8 (also of OATP1B1

Question 19

Regulatory requirements:

Answer 19

• Metabolites are potential contributors to toxicity, efficacy, and DDIs depending on relative systemic exposure1
• -> High susceptibility of glucuronides for interactions with transporters - reduced permeability, increased polarity
• FDA DDI guidance recommends evaluation of metabolite’s potential as enzyme/transporter inhibitor if metabolite is:
• -> Less polar and present at ≥25% of parent systemic exposure (AUC parent)
• -> More polar and present at ≥100% of parent systemic exposure (AUC parent)

Question 20

Use of preclinical animal models to investigate drug metabolism and transport:

Answer 20

Pharmaceutical industry

• Safety of new drug candidates
• Humanized transgenic mouse models
• -> Relative levels of enzymes can be modulated to reflect human variability
• -> OATP1B1 humanized mouse1 models for studying the role of this transporter in drug disposition

Toxicology

• -> Cancer research
• -> Disease models

Important to consider species differences

Question 21

Cynomolgus monkey as a preclinical model:

Answer 21

• Increasingly considered for investigation of transporter and metabolic DDIs
• -> High level of homology in gene and amino acid sequences to human
• -> Comparable increase in rosuvastatin AUC by cyclosporine to human
• GI physiology – gastric pH, gastric emptying and intestinal transit time similar to human
• Oral bioavailability
  Significantly lower F in Cyno for CYP3A and Pgp substrates