Conducting BE Studies for NTI Drugs

Here’s a detailed explanation of how bioequivalence (BE) studies are conducted for narrow therapeutic index (NTI) drugs, including regulatory expectations, study design, analytical methods, and statistical analysis:

Detailed Overview: Bioequivalence Study for NTI Drugs

What are NTI Drugs?

NTI (Narrow Therapeutic Index) drugs are those where small differences in dose or blood concentration can lead to serious therapeutic failures or adverse drug reactions.

Common NTI Drugs Include:

  • Warfarin
  • Digoxin
  • Lithium
  • Phenytoin
  • Carbamazepine
  • Tacrolimus
  • Cyclosporine

Regulatory Guidance

FDA Approach:

  • BE limits tightened from standard 80.00–125.00% to 90.00–111.11%
  • Highly recommends replicate design studies (e.g., 4-period or 3-period designs) to account for within-subject variability

EMA Approach:

  • Similar tightening of BE limits, especially for AUC
  • Some flexibility on Cmax based on drug properties, but stricter overall

Study Design for NTI Drugs

Preferred Design:

  • Replicate Crossover Design: Each subject receives both test and reference products multiple times (e.g., TRTR or RTRT sequences)
  • Enables estimation of within-subject variability
  • Required for Reference-Scaled Average BE (RSABE) approach

Why replicate design?

  • Allows statistical scaling of BE limits if intra-subject variability is high
  • Provides more power to detect differences within a tighter range

Pharmacokinetic Parameters

Measured after dosing under controlled conditions (fasted/fed):

  • Cmax (maximum concentration)
  • AUC0–t (area under curve to last measurable concentration)
  • AUC0–∞ (area under curve extrapolated to infinity)
  • Tmax (time to reach Cmax) – not always subjected to statistical testing

Bioanalytical Method Requirements

Use validated LC-MS/MS or HPLC method with:

  • High sensitivity (due to low dosage and critical range)
  • Low LLOQ (lower limit of quantification)
  • Precision: CV < 15%
  • Accuracy: ±15% (except ±20% at LLOQ)

Statistical Analysis

90% Confidence Intervals (CI) for Cmax and AUC should fall within:

  • 90.00–111.11% for NTI drugs (FDA/EMA)

For replicate designs with high variability:

  • RSABE approach may be applied using scaling based on reference variability

RSABE Conditions:

  • If intra-subject variability of reference product (CVwR) > 13.93%
  • BE acceptance limits are scaled, but capped at 75.00–133.00%

Additional Considerations

  • Sample Size: Higher due to tighter CI requirements
  • Inclusion/Exclusion Criteria: Stringent selection to minimize variability
  • Washout Period: Must be long enough to avoid carryover
  • Fasted vs Fed Studies: Both may be required based on label of RLD

Case Example: Tacrolimus

  • Therapeutic Window: 5–15 ng/mL
  • BE Study in healthy volunteers under fasted state
  • 4-period replicate design
  • RSABE used with 90% CI calculated for Cmax and AUC
  • Study approved only when both tight limits and variability assessment criteria were met


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Resource Person: Moinuddin syed. Ph.D, PMP®

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