Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.

Noninvasive Brain Delivery and Efficacy of BDNF to Stimulate Neuroregeneration and Suppression of Disease Relapse in EAE Mice.

The number of FDA-approved protein drugs (biologics), such as antibodies, antibody-drug conjugates, hormones, and enzymes, continues to grow at a rapid rate; most of these drugs are used to treat diseases of the peripheral body. Unfortunately, most of these biologics cannot be used to treat brain diseases such as Alzheimer's disease (AD), multiple sclerosis (MS), and brain tumors in a noninvasive manner due to their inability to permeate the blood-brain barrier (BBB). Therefore, there is a need to develop an effective method to deliver protein drugs into the brain. Here, we report a proof of concept to deliver a recombinant brain-derived neurotrophic factor (BDNF) to the brains of healthy and experimental autoimmune encephalomyelitis (EAE) mice via intravenous (iv) injections by co-administering BDNF with a BBB modulator (BBBM) peptide ADTC5. Western blot evaluations indicated that ADTC5 enhanced the brain delivery of BDNF in healthy SJL/elite mice compared to BDNF alone and triggered the phosphorylation of TrkB receptors in the brain. The EAE mice treated with BDNF + ADTC5 suppressed EAE relapse compared to those treated with BDNF alone, ADTC5 alone, or vehicle. We further demonstrated that brain delivery of BDNF induced neuroregeneration via visible activation of oligodendrocytes, remyelination, and ARC and EGR1 mRNA transcript upregulation. In summary, we have demonstrated that ADTC5 peptide modulates the BBB to permit noninvasive delivery of BDNF to exert its neuroregeneration activity in the brains of EAE mice.

In Vivo Brain Delivery and Brain Deposition of Proteins with Various Sizes.

It is very challenging to develop protein drugs for the treatment of brain diseases; this is due to the difficulty in delivering them into the brain because of the blood-brain barrier (BBB). Thus, alternative delivery methods need further exploration for brain delivery of proteins to diagnose and treat brain diseases. Previously, ADTC5 and HAV6 peptides have been shown to enhance the in vivo brain delivery of small- and medium-size molecules across the BBB. This study was carried out to evaluate the ability of ADTC5 and HAV6 peptides to enhance delivery of proteins of various sizes, such as 15 kDa lysozyme, 65 kDa albumin, 150 kDa IgG mAb, and 220 kDa fibronectin, into the brains of C57BL/6 mice. Each protein was labeled with IRdye800CW, and a quantitative method using near IR fluorescence (NIRF) imaging was developed to determine the amount of protein delivered into the brain. ADTC5 peptide significantly enhanced brain delivery of lysozyme, albumin, and IgG mAb but not fibronectin compared to controls. In contrast, HAV6 peptide significantly enhanced the brain delivery of lysozyme but not albumin and IgG mAb. Thus, there is a cutoff size of proteins that can be delivered by each peptide. The distribution of delivered protein in other organs such as liver, spleen, lung, kidney, and heart could be influenced by HAV6 and ADTC5. In summary, ADTC5 is a better BBB modulator than HAV6 in delivering various sizes of proteins into the brain, and the size of the protein affects its brain delivery.

Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological evaluation against Trypanosoma brucei.

In 2014, a published report of the high-throughput screen of>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.

Peptides and Drug Delivery.

Peptides have been used as drugs to treat various health conditions, and they are also being developed as diagnostic agents. Due to their receptor selectivity, peptides have recently been utilized for drug delivery to target drug molecules to specific types of cells (i.e. cancer cells, immune cells) to lower the side effects of the drugs. In this case, the drug is conjugated to the carrier peptide for directing the drug to the target cells (e.g. cancer cells) with higher expression of a specific receptor that recognizes the carrier peptide. As a result, the drug is directed to the target diseased cells without affecting the normal cells. Peptides are also being developed for improving drug delivery through the intestinal mucosa barrier (IMB) and the blood-brain barrier (BBB). These peptides were derived from intercellular junction proteins such as occludins, claudins, and cadherins and improve drug delivery through the IMB and BBB via the paracellular pathways. It is hypothesized that the peptides modulate protein-protein interactions in the intercellular junctions of the IMB and BBB to increase the porosity of paracellular pathways of the barriers. These modulator peptides have been shown to enhance brain delivery of small molecules and medium-sized peptides as well as a large protein such as 65 kDa albumin. In the future, this method has the potential to improve oral and brain delivery of therapeutic and diagnostic peptides and proteins.

Non-invasive Brain Delivery and Efficacy of BDNF in APP/PS1 Transgenic Mice as a Model of Alzheimer's Disease.

Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) have been demonstrated for their potential as a neuroregenerative treatment of Alzheimer's disease (AD). Unfortunately, most proteins cannot be effectively delivered into the brain from the blood stream due to the presence of the blood-brain barrier (BBB). In this study, we delivered BDNF using ADTC5 as BBB modulator (BBBM) into the brains of transgenic APP/PS1 mice, a mouse model for AD. As controls, two groups of APP/PS1 mice were treated with BDNF alone and vehicle, respectively. All three groups were subjected to behavioral/cognitive assessments in Y-maze and novel object recognition (NOR) tests as well as evaluation of the brain markers activated by BDNF. The results showed that BDNF + ADTC5 group performed significantly better in both the Y-maze and NOR assessments compared to mice that received BDNF alone or vehicle. In addition, significant upregulations of NG2 receptors as well as EGR1 and ARC mRNA transcripts were observed in the brain cortex of mice treated with BDNF + ADTC5, further indicating the efficacy of delivered BDNF in the brain. There were high plaque loads in all groups of mice, suggesting no influence of BDNF on the plaque formation. In summary, ADTC5 can deliver BDNF into the brains of APP/PS1 mice and the activity of BDNF in improving cognitive function was likely due to improvement in synaptic plasticity via NG2 glia cells and not by reducing the plaque load.

Doxorubicin-loaded iron oxide nanoparticles for glioblastoma therapy: a combinational approach for enhanced delivery of nanoparticles.

Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood-brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin-Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX's uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.

Improving In Vivo Brain Delivery of Monoclonal Antibody Using Novel Cyclic Peptides.

Many proteins can be used to treat brain diseases; however, the presence of the blood-brain barrier (BBB) creates an obstacle to delivering them into the brain. Previously, various molecules were delivered through the paracellular pathway of the BBB via its modulation, using ADTC5 and HAV6 peptides. This study goal was to design new cyclic peptides with N-to-C terminal cyclization for better plasma stability and modulation of the BBB. Cyclic HAVN1 and HAVN2 peptides were derived from a linear HAV6 peptide. Linear and N-to-C terminal cyclic ADTHAV peptides were designed by combining the sequences of ADTC5 and HAV6. These novel cyclic peptides were used to deliver an IRdye800CW-labeled IgG monoclonal antibody into the brain. Cyclic HAVN1 and HAVN2 peptides deliver IgG into the brain, while the parent linear HAV6 peptide does not. Cyclic and linear ADTHAV and ADTC5 peptides enhanced brain delivery of IgG mAb, in which cyclic ADTHAV peptide was better than linear ADTHAV (p = 0.07). Cyclic ADTHAV and ADTC5 influenced the distribution of IgG mAb in other organs while HAV6, HAVN1 and HAVN2 did not. In summary, the novel cyclic peptides are generally better BBB modulators than their linear counterparts for delivering IgG mAb into the brain.

Validation of Cadherin HAV6 Peptide in the Transient Modulation of the Blood-Brain Barrier for the Treatment of Brain Tumors.

The blood-brain barrier (BBB) poses a major obstacle by preventing potential therapeutic agents from reaching their intended brain targets at sufficient concentrations. While transient disruption of the BBB has been used to enhance chemotherapeutic efficacy in treating brain tumors, limitations in terms of magnitude and duration of BBB disruption exist. In the present study, the preliminary safety and efficacy profile of HAV6, a peptide that binds to the external domains of cadherin, to transiently open the BBB and improve the delivery of a therapeutic agent, was evaluated in a murine brain tumor model. Transient opening of the BBB in response to HAV6 peptide administration was quantitatively characterized using both a gadolinium magnetic resonance imaging (MRI) contrast agent and adenanthin (Ade), the intended therapeutic agent. The effects of HAV6 peptide on BBB integrity and the efficacy of concurrent administration of HAV6 peptide and the small molecule inhibitor, Ade, in the growth and progression of an orthotopic medulloblastoma mouse model using human D425 tumor cells was examined. Systemic administration of HAV6 peptide caused transient, reversible disruption of BBB in mice. Increases in BBB permeability produced by HAV6 were rapid in onset and observed in all regions of the brain examined. Concurrent administration of HAV6 peptide with Ade, a BBB impermeable inhibitor of Peroxiredoxin-1, caused reduced tumor growth and increased survival in mice bearing medulloblastoma. The rapid onset and transient nature of the BBB modulation produced with the HAV6 peptide along with its uniform disruption and biocompatibility is well-suited for CNS drug delivery applications, especially in the treatment of brain tumors.