Lipid Bilayer Crossing—The Gate of Symmetry. Water-Soluble Phenylproline-Based Blood-Brain Barrier Shuttles

Abstract

Drug delivery to the brain can be achieved by various means, including blood-brain barrier (BBB) disruption, neurosurgical-based approaches, and molecular design. Recently, passive diffusion BBB shuttles have been developed to transport low-molecular-weight drug candidates to the brain which would not be able to cross unaided. The low water solubility of these BBB shuttles has, however, prevented them from becoming a mainstream tool to deliver cargos across membranes. Here, we describe the design, synthesis, physicochemical characterization, and BBB-transport properties of phenylproline tetrapeptides, (PhPro)4, an improved class of BBB shuttles that operates via passive diffusion. These PhPro-based BBB shuttles showed 3 orders of magnitude improvement in water solubility compared to the gold-standard (N-MePhe)4, while retaining very high transport values. Transport capacity was confirmed when two therapeutically relevant cargos, nipecotic acid and l-3,4-dihydroxyphenylalanine (i.e., l-DOPA), were attached to the shuttle. Additionally, we used the unique chiral and conformationally restricted character of the (PhPro)4 shuttle to probe its chiral interactions with the lipid bilayer of the BBB. We studied the transport properties of 16 (PhPro)4 stereoisomers using the parallel artificial membrane permeability assay and looked at differences in secondary structure. Most stereoisomers displayed excellent transport values, yet this study also revealed pairs of enantiomers with high enantiomeric discrimination and different secondary structure, where one enantiomer maintained its high transport values while the other had significantly lower values, thereby confirming that stereochemistry plays a significant role in passive diffusion. This could open the door to the design of chiral and membrane-specific shuttles with potential applications in cell labeling and oncology.

Overcoming the Blood–Brain Barrier. Challenges and Tricks for CNS Drug Delivery

“…A big advantage of these small shuttles is that they minimize the loss of drug effectivity upon conjugation. Some examples are diketopiperazines [183], N-methylphenylalanines [184,185], and phenylprolines [186].…”

Novel Blood–Brain Barrier Shuttle Peptides Discovered through the Phage Display Method

“…The results from permeability experiments and mass spectrometry analysis showed that according to the Pe coefficient, the VAARTGEIYVPW peptide crossed the BBB with higher efficiency than GLHTSATNLYLH. The permeability obtained for the VAARTGEIYVPW peptide was on the same order of magnitude as other known BBB shuttles such as MiniAp4 with a value of 6.7 × 10 −6 cm/s (human primary cells) and 1.49 × 10 −6 cm/s (bovine primary cells) [52], PhPro4 with a value of 6.88 × 10 −6 cm/s, and NMePhe4 with a value of 6.8 × 10 −6 cm/s-values from a parallel artificial membrane permeability assay (PAMPA) assay [33]. However, because of different primary cell culture models or cell-free systems used in these studied, the exact comparison of our peptides to these shuttles remains to be determined.…”

“…Several CPP peptides have been extensively studied. For delivery of small molecules (<300 Da), short 2-4 amino acid peptides, such as diketopiperazines, methylphenylalanines, and phenylprolines, have been developed [22,23,32,33]. The TAT peptide (GRKKRRQRRRPQ) is the best-studied CPP for brain delivery for proteins and nanoparticles.…”

Possible Roles of Amphiphilic Molecules in the Origin of Biological Homochirality

“…The blood-brain barrier protects our central nervous system against external aggressions, but it also prevents therapeutics from reaching targets in the brain. Teixido and Giralt developed a phenylproline tetrapeptide that can cross the blood-brain barrier by paracellular hydrophilic diffusion [136]. They synthesized a library of the 16 phenylproline tetrapeptides stereoisomers, and revealed the relationship among their stereochemistry, transport properties, and permeability.…”