Browsing by Subject "Integrins"
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Item Adhesion molecules-potential targets of new anti-inflammatory therapies(1993-11-04) Lipsky, Peter E.Item Development of an αᵥβ₆-Binding Peptide for In Vivo Applications: Modulation of Serum Stability and Biodistribution(2015-06-30) Cupka, Dorothy Lynn; Albanesi, Joseph P.; Brown, Kathlynn C.; Kohler, Jennifer J.; Minna, John D.Targeted delivery of imaging and therapeutic agents to tumors improves detection, characterization, and treatment of many types of cancers. Peptides are capable of efficient and selective delivery of a variety of cargoes to their target cells and have been in development as targeting ligands for a variety of pathologies for several years. The H2009.1 peptide binds to αᵥβ₆, an integrin expressed in 54% of non-small cell lung cancers (NSCLC), 80-100% of head and neck carcinomas, and 37% of colorectal cancers. This peptide binds and is internalized into αᵥβ₆ positive cell lines, and successfully delivers cargo to xenograft tumors. This dissertation presents the optimization of this peptide for in vivo applications (i.e. improvement of serum stability and reduction of renal accumulation). The N-termini of tetrameric H2009.1 peptide were capped by acetylation or by unnatural amino acids. This capping significantly increased the half-life of H2009.1 tetramer in human serum. In vivo near-infrared fluorescence (NIRF) imaging shows that N-terminal capping also decreases kidney accumulation of this peptide at short time periods while maintaining targeting to the tumor for up to 72 hrs. To further decrease off-target accumulation, the valency of the targeting ligands was reduced from a tetramer to a dimer. The dimeric H2009.1 peptide produces similar targeting capabilities compared to the tetrameric peptide, with decreased accumulation in the liver, lung, and kidney. The optimal peptide for long-term targeting of therapeutics to αᵥβ₆-positive NSCLCs is the AcH2009.1 dimer as it gives the highest accumulation and retention in target tumors while avoiding kidney and lung tissue. The best peptide for short-term applications is the AcD-Leu H2009.1 dimer, which differentiates well between αᵥβ₆-positive and negative tumors and clears rapidly from the animal. While further work is necessary to bring the H2009.1 peptide into clinical use, the results and experiences of this thesis project have provided critical insights into the in vivo optimization of this and many other peptide targeting ligands.Item Molecular Imaging of αvβ6–Positive Tumors and Pancreatic β-Cell Mass by Radiolabeled Peptides(2011-02-01) Lin, Mai; Sun, XiankaiConventional diagnostic methodologies of lung cancer and diabetes are limited by sensitivity and specificity. In consequence, patients usually get diagnosed when the symptoms appear and the diseases are at the advanced stages. As the expressions of the avß6 integrin and glucagon-like peptide-1 receptor (GLP-1R) are highly related to aggressive tumor phenotypes and functional pancreatic ß-cells, this work has been set to develop peptide-based radiotracers that can specifically bind to avß6 or GLP-1R for noninvasively monitoring the progression of lung cancer and diabetes. By phage display, a peptide sequence that specific binds to avß6 was identified. In the evaluation of its truncated forms with similar binding affinity, a polyethylene glycol chain (PEG11) was inserted to the C-terminus and a bifunctional chelator (DOTA) was conjugated to either end of the peptides. The conjugates were labeled with 111In (t1/2: 2.8 d) under mild conditions with the highest achievable specific activity of 1.15 × 104 MBq/µmol. The in vivo evaluation was performed in a lung adenocarcinoma xenograft mouse model. Of the six conjugates, 10PD showed the best tissue contrast of the H2009 (avß6+) tumor. However, it also yielded to have the highest renal accumulation. The high kidney uptake of 10PD was found to be alleviated by conjugating DOTA at the N-terminus or reducing the peptide net charges. To evaluate GLP-1-based radiotracers for imaging pancreatic ß-cell mass (BCM), GLP-1, [D-Ala8]GLP-1, two bicyclic GLP-1 analogs (EM2196 and EM2198), and exendin-4 were synthesized and compared for their biological properties. All peptide constructs were tagged with an 6-aminohexanoic linker (Ahx) followed by DOTA conjugation at C-terminus and labeled with 64Cu (t1/2: 12.7 h). The specific activity of the labeled peptide conjugates was up to 1.0 × 106 MBq/µmol with radiochemical purity over 97%. Compared to GLP-1, [D-Ala8]GLP-1 revealed strong resistance against DPP-IV. In addition, EM2198 demonstrated high stability against NEP 24.11 presumably by the shielding effects from the two lactam bridges. All peptide conjugates were highly selective to the GLP-1R with the IC50 values in 0.1-0.4 nM. However, only 64Cu-EM2198 showed clear pancreas area on the microPET/CT studies. The signal of 64Cu-EM2198 from the pancreas was confirmed by the ex vivo imaging scans. The potential of 64Cu-EM2198 for imaging BCM was further supported by co-injecting a blocking dose of unlabeled exendin-4 and performing imaging studies in the STZ-treated diabetic mice.Item Peptide Targeted Drug Delivery to Non-Small Cell Lung Cancer(2012-12-20) Gray, Bethany Powell; Brown, Kathlynn C.Non-small cell lung cancer (NSCLC) is a notoriously deadly disease. The integrin αᵥβ₆ is emerging as a viable target for NSCLC; it is expressed in more than half of NSCLC patient tumor samples and only rarely expressed in normal tissue. Importantly, αᵥβ₆ is turned on early in the disease progression of NSCLC, indicating that it may be a good biomarker for early cancer detection and treatment. The phage display selected H2009.1 peptide exhibits high affinity for αᵥβ₆, specifically binding and internalizing into αᵥβ₆-expressing cells. Tumor targeting therapies that specifically deliver drugs to the tumor, reducing accumulation and toxicity in non-target tissues, are a promising niche of cancer therapeutics, and the H2009.1 peptide is anticipated to have great utility as a targeting ligand for the delivery of therapeutics to alpha(v)beta(6)-positive NSCLC tumors. To examine the ability of the H2009.1 peptide to specifically deliver drugs to NSCLC cells, it was used as a targeting ligand for three different drug platforms: liposomal doxorubicin and direct drug conjugates of both doxorubicin and paclitaxel. Conjugation of the H2009.1 peptide to all three of these drug platforms led to αᵥβ₆-specific targeting and toxicity in vitro. In vitro studies determined the ideal construct for H2009.1 peptide targeting of liposomal doxorubicin. Liposomes displaying the higher affinity multivalent H2009.1 tetrameric peptide demonstrated higher specificity and greater toxicity towards αᵥβ₆-expressing cells than liposomes displaying the lower affinity monomeric H2009.1 peptide. All H2009.1 peptide liposomal doxorubicin formulations exhibited greater toxicity towards αᵥβ₆-expressing cells than control non-targeted liposomes. Both the H2009.1-doxorubicin and paclitaxel conjugates demonstrated alpha(v)beta(6)-specific toxicity in vitro, although they were less toxic than the respective free drugs and exerted their effects on a later time frame. Neither H2009.1 peptide-targeted liposomal doxorubicin nor the H2009.1 direct drug conjugates improved in vivo efficacy compared to the non-targeted drugs. The liposomes suffered from poor tumor penetration and the in vitro studies with the drug conjugates suggest that they suffered from poor intracellular drug release. These results highlight the complexity of drug delivery and targeting in vivo and provide a basis for the design of optimized H2009.1 targeting therapies.