Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury

Abstract

Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-d (PKCd) as a critical regulator of the acute inflammatory response and demonstrated that PKCd inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCd is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)-conjugated PKCd inhibitory peptide (PKCd-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [,1% initial dose (ID)], whereas IT administration resulted in lung retention of .65% ID with minimal uptake in liver or kidney (,2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCd-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCd-TAT at the time of CLP surgery significantly reduced PKCd activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide.

A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil–endothelium interaction

“…Previously, our group has introduced a novel therapeutic paradigm that uses a peptide antagonist to inhibit selectively PKCd activity and neutrophilendothelial interactions to protect vascular endothelial integrity and attenuate sepsis-induced tissue damage [6][7][8]. The role of PKCd in rodent sepsis models and in neutrophils or endothelial is shown to be stimulus dependent and organ specific [7,8,36,37]. The role of PKCd in rodent sepsis models and in neutrophils or endothelial is shown to be stimulus dependent and organ specific [7,8,36,37].…”

“…A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium [4,5]. Our previous studies indicated a role for PKCd in regulating neutrophil migration but did not address specific mechanisms or identify specific steps by which PKCd impacts the interaction of neutrophils and endothelial cells during inflammation [6][7][8]. Our previous studies indicated a role for PKCd in regulating neutrophil migration but did not address specific mechanisms or identify specific steps by which PKCd impacts the interaction of neutrophils and endothelial cells during inflammation [6][7][8].…”

Neutrophil‐endothelial interactions of murine cells is not a good predictor of their interactions in human cells

“…In this study, we use bMFA to investigate whether mouse neutrophil‐endothelial cell interactions and microvascular endothelium barrier characteristics, as well as response to a novel anti‐inflammatory agent, are predictive of human cell responses. Previously, we demonstrated that protein kinase C‐delta (PKCδ) is an important regulator of neutrophil‐endothelial cells interaction during inflammation and that a PKCδ‐TAT peptide inhibitor (PKCδ‐ i ) may serve as a potential novel anti‐inflammatory therapeutic . In this study, we tested the hypothesis that mouse and human cells do not respond similarly to activation and investigated whether the response of mouse cells to an anti‐inflammatory agent is likely to provide data that would be predictive of its efficacy in human cells.…”

Protein kinase C‐delta inhibition is organ‐protective, enhances pathogen clearance, and improves survival in sepsis

“…Sepsis was induced by the cecal ligation and puncture (CLP) method as described previously . Sham controls underwent a laparotomy without cecal ligation or puncture.…”

“…At 24 post‐surgery, rats were euthanized and the lungs were gravity‐fixed with 10% neutral buffered formalin instillation into the airways; the trachea was then tied off to maintain inflation during fixation. After fixation, lungs were paraffin‐embedded, sectioned (5‐10 μm thick), and stained with hematoxylin‐eosin (H&E) as we described previously …”