Mass Balance / ADME studies

Published

March 18, 2026

One of the most informative studies for a small molecule – determines routes and rates of elimination, clearance mechanisms, and circulating metabolites.

Study design

  • Nonrandomized, open-label, ≥6 healthy adult male subjects, generally single-dose.
  • Small, radiolabeled dose (typically 14C) to accurately track drug disposition in biological matrices (blood, plasma, urine, feces).
    • Standard radio dose: ~50–100 µCi (LC-MS profiling).
    • Alternatively: <1 µCi analyzed by AMS (accelerator mass spectrometry) – avoids dosimetry requirement.
  • Dose: Final intended dose, or at least in the anticipated therapeutic dose range (linear PK range).
  • Route: Oral at final intended route. Fit-for-purpose formulation (solution, suspension, or capsule; uniform distribution of unlabeled/radiolabeled drug).

Timing

Common in Phase 2b; at the latest before initiating Phase 3 (FDA/EMA).

WarningLead time

Must be started ≥12 months ahead of planned dosing date due to radiolabel synthesis, pre-study activities, and limited CRO availability. Only a few CROs worldwide can perform this study.

Pre-study activities

  • Synthesis and manufacture of the radioactive drug; stability and purity (>98%) assessment for at least 6 months.
  • Radiolabel position: Must be chemically and metabolically stable so the radionuclide is not lost during metabolism. Two separate labeling positions can be used if needed.
  • QWBA (quantitative whole-body autoradiography) study in a rodent species for human dosimetry report (required for standard radio doses; not needed for AMS-level doses).
  • Nonclinical mass balance in at least one species (preferably bile-duct cannulated) – not required but useful to learn about metabolic profiles and radiolabel stability before the human study.

Sample collection

  • Urine and feces collected continuously. Aim to recover >90% of radioactivity.
  • Consider that the radioactivity half-life is often much longer than the parent drug half-life.
  • Stop collection when cumulative radioactivity in urine and feces is <1% of administered dose over a 24-hour period on 2 consecutive days.
  • Record the volume of urine, feces’ weight, and collection duration for accurate excretion calculations.

What to measure and characterize

  • Characterize excreted radioactivity (urine and feces): Identify parent compound and metabolites. Aim to characterize >80% of excreted radioactivity.
  • Metabolites in Safety Testing (MIST): Identify metabolites contributing to >10% of total plasma radioactivity AUC. Metabolites with significant plasma exposure or pharmacological activity – consider DDI liability, plasma protein binding, and safety testing coverage (ICH M12).
  • Construct a quantitative mass balance diagram and metabolic scheme showing enzymes/transporters involved in pathways accounting for >25% of elimination.
  • If parent drug is found in feces, distinguish between nonabsorbed drug and biliary excretion – may require an IV arm to resolve.

Key outputs

The mass balance study informs:

  • Elimination pathways (renal vs. metabolic vs. biliary).
  • Whether hepatic and/or renal impairment studies are needed.
  • Which enzymes and transporters to investigate for DDI.
  • The regulatory submission (CTD Module 2.7) and SmPC/labeling (sections 4.2 and 5.2).
WarningDon’t perform this study too late

Discovering a unique human metabolite late in development can force expensive repeat of toxicology studies.