Julie A. Kelly

RNAi-mediated reversible opening of the blood-brain barrier

The blood‐brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier.

Discovery of a dual action first-in-class peptide that mimics and enhances CNS-mediated actions of thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, L-pyroglutamyl-L-asparaginyl-L-prolyl-D-tyrosyl-D-tryptophan amide (Glp-Asn-Pro-D-Tyr-D-TrpNH(2)), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K(i) 151 nM) with high affinity binding to central TRH receptors (K(i) 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.

Pharmacologically distinct binding sites in rat brain for [3H]thyrotropin-releasing hormone (TRH) and [3H][3-methyl-histidine2]TRH

We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [3H][3-Me-His(2)]TRH or [3H]TRH binding to rat cortical homogenates. [3H][3-Me-His(2)]TRH was observed to bind to a single population of high-affinity, low-capacity sites (K(d): 4.54+/-0.62 nM, N=5; B(max): 4.38+/-0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K(i) of 22 nM. K(i) values for the library peptides at [3H][3-Me-His(2)]TRH-labelled sites varied from 10(-3) to 10(-9)M; the potency order was: [3-Me-His(2)]>His>Thi>Leu,Phe,Asn>Gln, Arg, Thr, Ala, HomoPhe. All other replacements had K(i) values >10(-4)M. [3H]TRH was observed to label a single population of low-affinity, high-capacity sites (K(d): 7.55+/-1.23 microM, N=6; B(max): 3.40+/-0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [3H]TRH-labelled sites did not correlate with their affinities for [3H][3-Me-His(2)]TRH-labelled sites (r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme (r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [3H]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2.

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Clinical Trials in Neurodegeneration

Significant progress made over the last few decades in the management and treatment of many neurodegenerative diseases. However, successful translation of effective therapeutic compounds from animal models to humans has been disappointing. This chapter provides an overview of the current models of drug development and clinical trial design along with a summary of the advantages and limitations of each model. The chapter also discusses the challenges of performing clinical trials in neurodegenerative disorders, including the selection of appropriate biomarkers of disease activity and the appropriate definition of meaningful clinical outcomes, and identifies promising new approaches towards the development of efficacious disease modifying therapeutics.