Quantitative Methods to Determine Brain Deposition of Peptides and Proteins after Delivery across the Blood-Brain Barrier
Ulapane, Kavisha Raneendri
University of Kansas
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It is very challenging to develop peptide and protein drugs for treatment of brain diseases because it is difficult to deliver them to the brain due to the presence of the blood-brain barrier (BBB). Therefore, there is an urgent need to develop new and alternative methods to deliver these drugs to the brain for treatment of brain diseases. ADTC5 and HAV6 peptides were derived from the binding sequence of the EC1 domain of E-cadherin protein, and these peptides can enhance the in vivo brain delivery of various molecules through the paracellular pathway of the BBB. Therefore, the overall goal of this project was to evaluate the activity of current and new cadherin cyclic peptides to enhance the in vivo brain delivery of peptides and proteins in rats and mice. The first goal of this project was to evaluate the activity of cadherin peptides (e.g., HAV6, HAV4, cHAVc3, and ADTC5) in delivering peptides (e.g., cIBR and cLABL) and 65 kDa galbumin protein to mouse and rat brains. The brain depositions of peptides and proteins were detected using near IR fluorescence (NIRF) imaging, magnetic resonance imaging (MRI), and mass spectrometry. The brain delivery of unlabeled cIBR7 peptide into rat brains was done to confirm that the intact molecule could be detected in the brain. An efficient extraction method was developed to isolate cIBR7 and ADTC5 from the brain tissue. A novel LC/MS/MS method was developed and validated to quantify cIBR7, an internal standard, and ADTC5 in brain after in vivo delivery. Detection was performed using triple quadrupole tandem mass spectrometry and a multiple reaction monitoring technique. Our results showed a fourfold increase (p = 0.013) in the amount of intact cIBR7 in the brain when it was delivered using ADTC5 compared to cIBR7 alone. The second goal was to compare the activity of ADTC5 and HAV6 peptides in delivering various sized proteins, including IRdye800cw-labeled-lysozyme (15 kDa), albumin (65 kDa), IgG mAb (150 kDa), and fibronectin (220 kDa) into mouse brains. In addition, a quantitative NIRF imaging method was developed to determine brain depositions of these proteins. The results showed that ADTC5 peptide significantly enhanced brain delivery of lysozyme, albumin, and IgG mAb compared to controls; however, no enhancement was observed for fibronectin. HAV6 peptide could enhance the brain delivery of lysozyme, but not the other proteins. The third goal was to design and synthesize new cyclic peptides for better modulation of the BBB. An N-to-C terminal cyclization method was utilized to improve the plasma stability and activity to modulate the BBB of the peptide. Linear and cyclic ADTHAV peptides were designed by combining the sequences of ADTC5 and HAV6. Cyclic HAVN1 and HAVN2 peptides were designed as N-to-C terminal cyclic derivatives of linear HAV6 peptide as new BBB modulator peptides. Binding properties of cyclic ADTHAV and ADTC5 peptides to the EC1 domain of Ecadherin were determined using surface plasmon resonance (SPR), and ADTHAV was found to have higher binding affinity (Kd = 0.114 µM) than ADTC5 (Kd = 26.8 µM). The in vivo activities of these peptides to deliver an IRdye800cw-labeled IgG mAb into the brain were qualitatively and quantitatively determined using NIRF imaging. Cyclic HAVN1 and HAVN2 peptides enhanced brain delivery of IgG mAb compared to HAV6 peptide. Cyclic and linear ADTHAV as well as ADTC5 peptides enhanced brain delivery of IgG mAb. There seems to be a trend that cyclic ADTHAV peptide has better activity than linear ADTHAV under the current conditions (p = 0.07). Overall, these three studies support the potential use of cadherin peptides in transiently modulating the BBB to improve the brain delivery of peptides and proteins
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