99 SMALL PROBLEMS / NO. 03
Washout Check
Did the drug leave, or did the protocol merely stop adding it?
Useful models for assumptions with expensive ambitions.
THE ASSUMPTION UNDER TEST
Removing medium is not removing exposure.
| Mechanism | Occupancy | Free (nM) | Free AUC (nM·h) |
|---|
THE LOADING ASSUMPTION AUDIT
Binding can be ready before the reservoir.
| Quantity | Selected loading | Equilibrium reference |
|---|
95% is a mathematical filling reference, not an experimentally validated acceptance threshold. Postwash AUC begins at the first wash.
What changed? Not the off-rate.
The useful next experiment is a perturbation, not a more confident adjective: change wash timing, residual volume, or accessible target capacity, then ask which explanation survives.
FOLLOW THE INVENTORY
The wash touches only one pool.
All quantities are expressed per the same assay volume. An apparent concentration in the retention pool is an inventory unit, not an intracellular concentration.
A persistence audit, not a residence-time assay.
These are competing synthetic scenarios, not parameter estimates. The model contains no receptor turnover, trafficking, spatial diffusion, covalent binding, active transport, metabolism, or downstream pharmacodynamics. Occupancy is not functional recovery. Fixed-free comparisons can contain different total drug inventories; finite-dose comparisons conserve the same starting dose but allow prewash occupancy to differ.
Rebinding and reversible partitioning complicate kinetic interpretation, as discussed by Vauquelin (2012). The mass-balance and equilibrium-binding foundations are discussed by Hulme & Trevethick (2010). This implementation is an original, deliberately simplified teaching and experimental-design model, not a reproduction of either paper.
Read the equations, assumptions & numerical methods ↗Versioned source and release notes ↗