Selective Delivery of Drugs to the CNS for Treatment of Neurological Diseases

Despite extensive research, the brain barriers remain a major challenge for drug delivery to the brain. Our group recently showed that the blood-cerebrospinal fluid barrier (BCSFB) can facilitate drug delivery to the brain using the Mumps Virus’ (MuV) short-hydrophobic protein as a shuttle peptide. A peptide-drug conjugate (PDC) combining the MuV shuttle peptide (MuV-SP), a drug and a brain-selective cleavable linker enables targeted brain delivery while avoiding off-target effects. This thesis presents work to develop brain-selective cleavable linker strategies.

Chapter 2 covers the development of Acetylcholinesterase (AChE)-cleavable linkers presenting two designs. Design A is based on an electronic-cascade self-immolative scaffold. A library of AChE-cleavable probes is synthesised to investigate the effect of substituents on the aryl dimethylcarbamate choline moiety on the AChE-mediated hydrolysis. Cleavage studies combined with inhibitor studies indicates that the nucleophilic attack of AChE at the carbamate is the rate-determining step. The probes show pseudo-irreversible inhibition of AChE and the release is based on a slow decarbamylation process. To introduce the linker in a PDC, it was found that it was necessary to install an aryl spacer between the linker structure and the MuV-SP handle to maintain AChE-recognition. Design B is based on a cyclization self-immolative scaffold and consists of a MuV-SP handle, an acetylcholine (ACh) mimic and a handle for the drug. The synthesis presented challenges and required major optimization, where especially the introduction of the ACh mimic was extensively investigated.

Chapter 3 covers the development of a reactive oxygen species (ROS)-cleavable linker based on an electronic-cascade self-immolative scaffold. ROS-cleavable probes with varying substituents on the aryl boronic acid moiety are synthesised to study the multi-step ROS-mediated cleavage. Cleavage studies could not alone determine the rate-determining step but show that the release rate is highly affected by the substituents. Potentially, the nature of the substituent may also change the rate-determining step. Initial NMR studies offer a promising method to study the cleavage and release.

Chapter 4 covers the development of soluble epoxide hydrolase inhibitors (sEHI) functionalized for attachment to the AChE-cleavable linker (design A) for use in an AChE-responsive PDC or in a dual drug conjugate. Modifying the sEHI, TPPU and a sulfonyl-based TPPU, introduced a handle for linker attachment, with a phenol proving more potent than an amine. The TPPU with a phenol-handle was attached to the tertiary amine analogue of the linker to obtain dual drug conjugates, that demonstrated blood-brain barrier permeability and AChE inhibition but the sEHI could not be silenced in the conjugate. AChE-mediated cleavage is under evaluation. Future synthesis of a sulfonyl-based TPPU with a phenol-handle could show brain-selective sEH inhibition via an AChE-responsive PDC.