Evaluation of brain delivery of Evans Blue (EB) by various peptides (A) and by different concentration of peptide K16ApoE (B).

<p>67.5 picomoles of each of the peptides (K16, ApoE and K16ApoE) was either injected first followed in 10 min by injection of EB (40 ul of a 2% solution) or the dye and the peptides were mixed together and then injected. Mice were perfused with saline 2 h after injection and then brains were collected for visualization.</p

Brain imaging and quantification (via microSPECT) of brain-uptake of I-125 via insulin injection.

<p>I-125 was injected 10 min after injection of 250 ug, 500 ug and 1000 ug of insulin, respectively, and 200 ug of K16ApoE. Quantification of I-125 in the brain was done after cardiac perfusion. Six mice were evaluated for each group.</p

Peptide Carrier-Mediated Non-Covalent Delivery of Unmodified Cisplatin, Methotrexate and Other Agents via Intravenous Route to the Brain

<div><p>Background</p><p>Rapid pre-clinical evaluation of chemotherapeutic agents against brain cancers and other neurological disorders remains largely unattained due to the presence of the blood-brain barrier (BBB), which limits transport of most therapeutic compounds to the brain. A synthetic peptide carrier, K16ApoE, was previously developed that enabled transport of target proteins to the brain by mimicking a ligand-receptor system. The peptide carrier was found to generate transient BBB permeability, which was utilized for non-covalent delivery of cisplatin, methotrexate and other compounds to the brain.</p><p>Approach</p><p>Brain delivery of the chemotherapeutics and other agents was achieved either by injecting the carrier peptide and the drugs separately or as a mixture, to the femoral vein. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a ‘small-molecule’ drug.</p><p>Principal findings</p><p>Seven-of-seven different small molecules were successfully delivered to the brain via K16ApoE. Depending on the method, brain uptake with K16ApoE was 0.72–1.1% for cisplatin and 0.58–0.92% for methotrexate (34-50-fold and 54–92 fold greater for cisplatin and methotrexate, respectively, with K16ApoE than without). Visually intense brain-uptake of Evans Blue, Light Green SF and Crocein scarlet was also achieved. Direct intracranial injection of EB show locally restricted distribution of the dye in the brain, whereas K16ApoE-mediated intravenous injection of EB resulted in the distribution of the dye throughout the brain. Experiments with insulin suggest that ligand-receptor signaling intrinsic to the BBB provides a natural means for passive transport of some molecules across the BBB.</p><p>Significance</p><p>The results suggest that the carrier peptide can non-covalently transport various chemotherapeutic agents to the brain. Thus, the method offers an avenue for pre-clinical evaluation of various small and large therapeutic molecules against brain tumors and other neurological disorders.</p></div

K16ApoE-mediated brain delivery of blue (EB), red (Crocein Scarlet) and green (Light Green SF) dyes to the brain.

<p>Three different approaches were assessed for dye delivery: 1. K16ApoE was injected first then a given dye was injected 10 min after (second columns of brain specimens); 2. K16ApoE was mixed with 300 ug of cetuximab and injected followed by injection of a given dye 10 min after 3^rd column of brain specimens), and 3. K16ApoE and the dyes were mixed and injected (fourth column of brain specimens). The first column of brain specimens represents animals receiving injection of a given dye alone. Mice were perfused with saline 2 h after injection and then brains were collected for visualization. 67.5 picomole of K16ApoE was used in each experiment. 40 ul of a 2% solution of each of the dyes were used for injection into a 20 g mouse (amount of dye injected varied accordingly with weight of mice).</p

Quantification of K16ApoE-mediated brain-uptake of cisplatin (Cp) and methotrexate (MTX).

<p>300 ug of the carrier peptide K16ApoE, 300 ug of cetuximab and 300 ug of cisplatin (Cp) were used in this experiment. Group 1- these animals received only Cp or MTX. Group 2- these animals received injection of K16ApoE then injection of either Cp or MTX. Group 3- these animals received an injection of K16ApoE mixed with cetuximab, followed by an injection of Cp or MTX. Group 4- these animals received an injection of K16ApoE mixed with Cp or MTX. Post-perfused brains were collected after 1 h of final injection and processed for respective assays. Fold change for Group 2 has been obtained by dividing the mean value for Group 2 by the mean value for group 1; fold change for Group 3 has been obtained by dividing the mean value for this group by the mean value of Group 1, and so on. ‘% delivery’ indicates the fraction of Cp or MTX in brain compared to the injected dose. Six animals in each group have been used.</p

Qualitative comparison of brain uptake of Evans Blue (EB) via direct intracranial and K16ApoE-mediated intravenous injections.

<p>Intravenous injection through femoral vein involved delivery of 1mg of EB with or without K16ApoE. Assuming (based on several experiments and results from delivery of cisplatin and methotrexate presented in this paper) 1% of the intravenously-injected molecules reaches into the brain (when delivered via K16ApoE), 10 ug (1X in the Figure) and 20 ug (2X in the Figure) of EB were used for intracranial injection. All animals used in the experiment underwent cardiac perfusion 1 h after delivery of the dye by either method, after which brains were collected and photographed before and after coronal sections were made.</p

Evaluation of time-frame of BBB permeabilization by K16ApoE and retention-time of EB in the brain delivered by K16ApoE.

<p>A- Stains of EB from brain specimens from mice in which K16ApoE was injected first and then EB was injected 5 min, 10 min, 30 min, 1 h, 2 h and 4 h after. Cardiac perfusion was done 1 h after injection of the dye followed by collection of brains. B - Stains of EB from brain specimens from mice in which K16ApoE was injected first and then EB was injected 10 min after. Perfusion was done at indicated times followed by collection of brains.</p

Brain imaging and quantification (via microSPECT) of brain-uptake of Y8 delivered via K16ApoE.

<p>^I-125labeled Y8 was delivered to the brain by first injecting K16ApoE then injecting Y8 as described earlier. Brain delivery of Y8 was also assessed after injection of a mixture of K16ApoE and cetuximab (300 ug of each). A. The bars represent brain uptake of ^I-125labeled Y8 when injected by itself (blue), injected after injection of K16ApoE (majenta) or injected after injection of a mixture of K16ApoE and cetuximab (yellow). Six mice were evaluated for each group. Images of brains by microSPECT representing delivery of ^I-125labeled Y8 at different conditions are presented on top of the bars. Brain images were taken after cardiac perfusion with saline. T- thyroid gland; S – salivary gland; B- brain.</p

Semimechanistic Population Pharmacokinetic Modeling to Investigate Amyloid Beta Trafficking and Accumulation at the BBB Endothelium

Elevated exposure to toxic amyloid beta (Aβ) peptides and consequent blood–brain barrier (BBB) dysfunction are believed to promote vasculopathy in Alzheimer’s disease (AD). However, the accumulation kinetics of different Aβ isoforms within the BBB endothelium and how it drives BBB dysfunction are not clearly characterized. Using single positron emission computed tomography (SPECT)-computed tomography (CT) dynamic imaging coupled with population pharmacokinetic modeling, we investigated the accumulation kinetics of Aβ40 and Aβ42 in the BBB endothelium. Brain clearance was quantified after intracerebral administration of 125I-Aβ, and BBB-mediated transport was shown to account for 54% of 125I-Aβ40 total clearance. A brain influx study demonstrated lower values of both maximal rate (Vmax) and Michaelis constant (Km) for 125I-Aβ42 compared to 125I-Aβ40. Validated by a transcytosis study in polarized human BBB endothelial cell (hCMEC/D3) monolayers, model simulations demonstrated impaired exocytosis was responsible for inefficient permeability and enhanced accumulation of Aβ42 in the BBB endothelium. Further, both isoforms were shown to disrupt the exocytosis machinery of BBB endothelial cells so that a vicious cycle could be generated. The validated model was able to capture changes in Aβ steady-state levels in plasma as well as the brain during AD progression and allowed us to predict the kinetics of Aβ accumulation in the BBB endothelium

Quantitative evaluation of histological tau burden in brain samples in AD and non-AD groups.

A—Quantitative Evaluation of immature histopathological tau (clone AT8) in control and AD group. Note a progressive intraindividual increase in AT8 (above the red dotted line) B—Quantitative analysis of the non-AD group showing a more heterogeneous increase in AT8 across different brain regions. Representative pictures from comparative frontal, temporal, and occipital regions, show the neuropathological variability of AT8 distribution.</p