Page 71 - AN-4-2

Advanced Neurology Brain bioavailability of targeted protein degraders

low cellular permeability, and diminished plasma and 14. Diffusion
hepatocyte stability.
The impact of efflux is potentially low for small molecules
13. TPD bidirectional permeability with a high propensity for passive diffusion. For P-gp
assessment in MDCK-MDR1 and Caco-2 cell substrates, the K p,uu,brain typically ranges from moderate to
lines high (≥0.3). For TPDs, both uptake and efflux transporters
play a significant role in BBB permeability, and K
MDCK-MDR1 and Caco-2 cell lines are widely used values range from 0.3 – 0.05. p,uu,brain
to assess membrane permeability and efflux liability of High-molecular-weight oligonucleotides, peptides,
TPDs. Despite the canine origin of MDCK cells, MDCK- monoclonal antibodies, and proteins primarily rely on
MDR1 is preferred over Caco-2 due to its high expression transcytosis, with passive diffusion being negligible. The
of human P-gp and faster turnaround time (7 days versus tissue concentrations of these macromolecules are very low
21 days). 138,139 Endogenous (canine) Mdr1 and Mrp2 compared to plasma, with K values being extremely
(Abcc2) mRNA expression is significantly lower in MDCK- low (<0.01). p,uu,brain
MDR1 cells compared to MDCK wild type (WT). Both
140
The mechanisms of intra-tissue penetration remain
MDCK-MDR1 cells and Caco-2 cells express comparable largely unknown. For small molecules, diffusion in brain
levels of the 170 kDa isoform of P-gp. However, the regions is effective only over short distances, spanning
141
150 kDa isoform is overexpressed in MDCK-MDR1 cells a few cell bodies (~10 – 100 uM), but is extremely low
compared to Caco-2 and MDCK-WT cells. The readouts and limiting over larger distances (≥1 mm). This slow
generated from the Caco-2 cell line may differ from the distribution is a limiting factor for the effective distribution
apparent kinetic constants and affinities of substrates of drugs into the brain’s affected regions. For TPDs, due to
determined in MDCK-MDR1 cells. 139 their large size, intra-tissue diffusion is expected to be even

In MDCK-MDR1 bidirectional permeability assays, more restricted.
TPDs often exhibit a high efflux ratio and low permeability,
which may not be relevant to BBB permeability. The 15. Wider pharmacokinetic/
addition of 0.5% bovine serum albumin in the donor well pharmacodynamic disconnect
and 2% in the receiver well improves solubility slightly For small molecule non-covalent inhibitors, the therapeutic
and reduces non-specific binding. However, this does effects often depend on maintaining adequate trough
not significantly improve permeability in the absorptive concentrations. In contrast, for TPDs, the concentration-
direction. Regardless, TPDs still exhibit quantifiable time profile has less relevance to the observed therapeutic
concentrations in the brain. This raises questions regarding effects over time. This disconnect is primarily due to the
the suitability of MDCK-MDR1 cell line permeability time lag between target engagement and degradation,
indices for assessing BBB penetration. The efflux liability coupled with an event-driven, catalytic mechanism.
of TPDs may be overestimated in the MDCK-MDR1 assay. The therapeutic effects of TPD are driven by the
The human MDR1-transfected porcine-kidney-derived degradation of disease-causing proteins by cellular
LLC-PK1 cell line, with a similar turnaround time (7 days), proteosomal machinery. The TPD transiently binds to
is a relatively better indicator of TPDs’ BBB permeability. a target protein and directs it for enzymatic degradation
LLC-PK1 cells express multiple endogenous transporters, through ubiquitination. The PD effect persists even after
145
including P-gp and members of the MRP/ABCC family of the TPD has been cleared off from systemic circulation and
efflux transporters. MDR1-transfected LLC-PK1 cells its levels fall below the lower limit of quantitation. Thus,
142
exhibit comparable Mdr1 levels but significantly higher the systemic exposure of TPDs exhibits a pronounced
Mrp2 mRNA levels than WT cells. Alternatively, disconnect with the therapeutic response. A high
140
BBB permeability can be assessed using human brain maximum concentration therefore is not necessary for
microvascular endothelial cells in astrocytes co-culture. TPDs and may instead contribute to unwanted toxicities.
Another option is to utilize primary porcine brain Consequently, infrequent dosing regimens at very low
endothelial cell-astrocyte co-culture or porcine endothelial doses may still provide sustained efficacy, particularly in
cells, pericytes, and astrocytes culture for BBB permeability cases where the protein synthesis rate is low. 146,147
assessment. 143,144 However, the validation, repeatability, This PK/PD disconnect is expected to be more
and reproducibility of the primary co-culture approach pronounced for CNS therapeutics due to the presence of
can be challenging, and high inter-experiment variation is the BBB, low diffusion propensity in the CNS, difficulties
difficult to address. with neuronal cell transfection, and altered proteasomal

Volume 4 Issue 2 (2025) 65 doi: 10.36922/an.5140