Gastrointestinal pH Gradient-Induced Phase Transition of Crizotinib: The Significance of pH-Dependent Ionization (Protonation) on Liquid–Liquid Phase Separation of a Weakly Basic Drug

Although weakly basic drugs with specific properties
(e.g., dose
number >1–10, pKa 5–9)
are
known to form supersaturated solutions and exhibit complex phase behavior
in the gastrointestinal tract, key aspects of this process (namely,
the physical stability of nanodroplets and the charge site) remain
poorly characterized. This work employs the dual-pKa anticancer drug crizotinib (CZT; pKa 5.6, 9.4) as a model drug to establish a systematic
methodological framework for investigating the complex behaviors of
weakly basic drugs induced by pH shift. The results revealed that
upon pH shift from 1.0 to above 5.4, a 1 mg/mL CZT solution underwent
liquid–liquid phase separation with nanodroplets forming immediately. 1H nuclear magnetic resonance (NMR), 13C solid-state
NMR, and synchrotron single-crystal X-ray diffraction (SCXRD) data
together confirmed that this phase transition is attributed to the
deprotonation from a dicationic species (with both piperidinium and
pyridinium protonated) to a monocationic species (with only the piperidinium
protonated) in both the drug-rich and drug-lean phases. Ultraviolet–visible
spectroscopy, dynamic light scattering, confocal laser scanning microscopy,
and polarized optical microscopy together revealed that the resulting
additive-free CZT droplets are unstable at pH 6.5 with a low zeta
potential of 5.1 mV, and sedimented into a bulk gel within minutes.
This sedimentation significantly reduces the surface area of the colloidal
particles (drug reservoir) and, consequently, slows the dissolution
of CZT from the drug-rich phasebut interestingly, it has little
effect on transmembrane flux through a cellulose membrane, suggesting
that for CZT, transport across the membrane, not interphase diffusion,
is the rate-limiting step. By establishing the methodology, which
integrates NMR spectroscopy and X-ray crystallography to precisely
locate the charge site of drug in both drug-rich phase (droplets or
gel-like precipitate) and drug-lean phase (bulk solution), this work
bridges the critical gap between macroscopic phase behavior and molecular-level
understanding. These insights provide a mechanistic foundation for
the rational design of oral formulations for weakly basic drugs.