Tatyana Yakovleva

ICAM-1 Targeting of Doxorubicin-Loaded PLGA Nanoparticles to Lung Epithelial Cells

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphocytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been identified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic surfactant on poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1-mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system.

Characterization of Binding Properties of ICAM‐1 Peptides to LFA‐1: Inhibitors of T‐cell Adhesion

In the present study, we characterized the binding site of two intercellular adhesion molecule‐1‐derived cyclic peptides, cIBC and cIBR, to the LFA‐1 on the surface of T cells. These peptides had been able to inhibit LFA‐1/intercellular adhesion molecule‐1 signal by blocking the signal‐2 of immune synapse. Both peptides prefer to bind to the closed form of LFA‐1 I‐domain, indicating that two peptides act as allosteric inhibitors against intercellular adhesion molecule‐1. Binding site mapping using monoclonal antibodies proposes that cIBC binds to around residues 266–272 of LFA‐1 I‐domain where this site is adjacent to the metal ion‐dependent adhesion site. On the other hand, cIBR binds to the pocket called L‐site where is distant from metal ion‐dependent adhesion site. Cross‐inhibition mapping between two peptides show that cIBR could inhibit the binding of cIBC but not vice versa, suggesting that cIBR has some properties that allow this peptide bind to more than one site. Structural comparison between cIBC and cIBR reveals that cIBR is more flexible than cIBC, allowing this peptide bind to exposed region, such as cIBC‐binding site as well as cramped pocket like L‐site. Our findings are important for understanding the selectivity of cIBC and cIBR peptides; thus, they can be conjugated with drugs and transported specifically to the target.

Sequence Recognition of α-LFA-1-derived Peptides by ICAM-1 Cell Receptors: Inhibitors of T-cell Adhesion

Blocking the T‐cell adhesion signal from intercellular adhesion molecule‐1/leukocyte function‐associated antigen‐1 interactions (Signal‐2) can suppress the progression of autoimmune diseases (i.e. type‐1 diabetes, psoriasis) and prevent allograph rejection. In this study, we determined the active region(s) of cLAB.L peptide [cyclo(1,12)Pen‐ITDGEATDSGC] by synthesizing and evaluating the biologic activity of hexapeptides in inhibiting T‐cell adhesion. A new heterotypic T‐cell adhesion assay was also developed to provide a model for the T‐cell adhesion process during lung inflammation. Two hexapeptides, ITDGEA and DGEATD, were found to be more active than the other linear hexapeptides. The cyclic derivative of ITDGEA [i.e. cyclo(1,6)ITDGEA] has similar activity than the parent linear peptide and has lower activity than cLAB.L peptide. Computational‐binding experiments were carried out to explain the possible mechanism of binding of these peptides to intercellular adhesion molecule‐1. Both ITDGEA and DGEATD bind the same site on intercellular adhesion molecule‐1 and they interact with the Gln34 and Gln73 residues on D1 of intercellular adhesion molecule‐1. In the future, more potent derivatives of cyclo(1,6)ITDGEA will be designed by utilizing structural and binding studies of the peptide to intercellular adhesion molecule‐1. The heterotypic T‐cell adhesion to Calu‐3 will also be used as another assay to evaluate the selectivity of the designed peptides.

Methotrexate (MTX)-cIBR Conjugate for Targeting MTX to Leukocytes: Conjugate Stability and In Vivo Efficacy in Suppressing Rheumatoid Arthritis

Methotrexate (MTX) has been used to treat rheumatoid arthritis at low doses and leukemia at high doses; however, this drug can produce severe side effects. Our hypothesis is that MTX side effects can be attenuated by directing the drug to the target cells (i.e., leukocytes) using (cyclo(1,12)PenPRGGSVLVTGC) peptide (cIBR). To test this hypothesis, MTX was conjugated to the N-terminus of cIBR peptide to give MTX-cIBR conjugate. MTX-cIBR (5.0 mg/kg) suppressed joint arthritis in adjuvant arthritis rats and prevented periarticular inflammation and bone resorption of the limb joints. In vitro, the toxicity of MTX-cIBR peptide against Molt-3 T cells was inhibited by anti-lymphocyte function-associated antigen-1 (LFA-1) antibody and cIBR peptide in a concentration-dependent manner, suggesting that the uptake of MTX-cIBR was partially mediated by LFA-1. Chemical stability studies indicated that MTX-cIBR was most stable at pH 6.0. The MTX portion of MTX-cIBR was unstable under acidic conditions, whereas the cIBR portion was unstable under basic conditions. In biological media, MTX-cIBR had short half lives in rat plasma (44 min) and homogenized rat heart tissue (38 min). This low plasma stability may contribute to the low in vivo efficacy of MTX-cIBR; therefore, there is a need to design a more stable conjugate to improve the in vivo efficacy.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

Peptide C‐terminal N‐alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C‐terminal N‐alkyl amides using the well‐known Fukuyama N‐alkylation reaction on a standard resin commonly used for the synthesis of peptide C‐terminal primary amides, the peptide amide linker‐polyethylene glycol‐polystyrene (PAL‐PEG‐PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N‐alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N‐alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N‐alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N‐alkyl amides.

Two short-acting kappa opioid receptor antagonists (zyklophin and LY2444296) exhibited different behavioral effects from the long-acting antagonist norbinaltorphimine in mouse anxiety tests

Prototypical long-acting kappa opioid receptor (KOPR) antagonists [e.g., norbinaltorphimine (norBNI)] have been reported to exert anxiolytic-like effects in several commonly used anxiety tests in rodents including the novelty-induced hypophagia (NIH) and elevated plus maze (EPM) tests. It remains unknown if the short-acting KOPR antagonists (e.g., zyklophin and LY2444296) have similar effects. In this study effects of zyklophin and LY2444296 (s.c.) were investigated in the NIH and EPM tests in mice 1h post-injection and compared with norBNI (i.p.) 48h post-administration. In the NIH test, zyklophin at 3 and 1mg/kg, but not 0.3mg/kg, or LY2444296 at 30mg/kg decreased the latency of palatable food consumption in novel cages, but had no effect in training cages, similar to norBNI (10mg/kg). Zyklophin at 3 or 1mg/kg increased or had a trend of increasing the amount of palatable food consumption in novel cages, with no effects in training cages, further indicating its anxiolytic-like effect, but norBNI (10mg/kg) and LY2444296 (30mg/kg) did not. In the EPM test, norBNI (10mg/kg) increased open arm time and % open arm entries or time, but zyklophin at all three doses and LY2444296 (30mg/kg) had no effects. In addition, zyklophin at 3mg/kg increased numbers of close and total arm entries on EPM, suggesting increased activity; however, norBNI and LY2444296 had no effects on close and total arm entries. Thus, all three KOPR antagonists had anxiolytic-like effects in the NIH test. However, only the long-acting one (norBNI), but not the short-acting ones (zyklophin and LY2444296), demonstrated anti-anxiety like effects in the EPM test. It remains to be investigated if the differences are due to the differences in their durations of action and/or pharmacodynamic properties.

Optimizing a Larger Scale Synthesis of Zyklophin, a Highly Selective Peptide Kappa Opioid Receptor Antagonist

We are interested in developing metabolically stable peptidic ligands for kappa opioid receptors (KOR) as potential treatments for drug abuse and pain. In particular KOR antagonists have recently demonstrated potential therapeutic applications in the treatment of drug addiction, depression and anxiety [1]. Our research group has designed the KOR antagonist zyklophin ([N-benzylTyr,cyclo-(DAsp,Dap)]dynorphin A-(1-11) amide, Figure 1) [2] based on the endogenous KOR peptide dynorphin A. Zyklophin is a selective KOR antagonist that exhibits activity in the CNS after peripheral administration [3]. Utilization of this important peptide in vivo requires the synthesis and purification of the peptide on a larger scale, but structural modifications (cyclization and N-terminal modification) make its larger scale synthesis and purification more challenging. Here we describe the optimization of the solid phase synthesis of zyklophin, in particular the selective deprotection [4,5] and cyclization of the side chains of D-Asp and Dap (2,3-diaminopropionic acid), and the use of microwave irradiation to facilitate the rapid cyclization of the side chains. To substantially increase the amount of peptide purified per chromatographic run we utilized a modified reversed phase HPLC purification method [6]. These modified procedures permitted preparation of the larger quantities of pure peptide (>200 mg) necessary for extensive studies in vivo. Results and Discussion

Utilization of ICAM-1 Peptide Conjugates for Drug Targeting to T-Cells

The objective of this work is to demonstrate that ICAM-1 peptides can be used to target drugs (e.g. methotrexate = MTX) to T-cells. Previously, we have shown that cIBR peptide derived from the ICAM-1 sequence can bind to and be internalized by the LFA-1 receptor on the surface of T-cells [1–3]. Therefore, we conjugated MTX to the N-terminus of cIBR for targeting MTX into T-cells. MTX has been used to treat leukocyte-related diseases such as leukemia and rheumatoid arthritis. Unfortunately, MTX produces side effects, and its prolonged administration may produce drug resistance. Therefore, the MTX-cIBR conjugate may have improved selectivity and lower toxicity than MTX alone.