Christine R. Xu

Synthesis of a novel cyclic prodrug of RGD peptidomimetic to improve its cell membrane permeation

The objective of this work was to synthesize cyclic prodrug 2 derived from the parent RGD peptidomimetic 1 and to evaluate its chemical and enzymatic stabilities and antithrombic activity. Cyclic prodrug 2 was formed to improve the cell membrane permeation of RGD peptidomimetic 1 by transiently masking the unfavorable physicochemical properties of compound 1. Cyclic prodrug 2 was synthesized by linking the amino and carboxylic acid groups of parent 1 via the (acyloxy)alkoxy promoiety. The prodrug-to-drug conversion of cyclic prodrug 2 was evaluated in isolated esterase and human plasma in the absence and presence of the esterase inhibitor paraoxon. The rate of hydrolysis of cyclic prodrug 2 was significantly faster in plasma (t(1/2)=33.5+/-0.6 min) than in PBS (t(1/2)=314+/-11 min). Cyclic prodrug 2 was converted by esterase to the parent compound 1 and this conversion was inhibited by an esterase inhibitor, paraoxon. The IC50 (4 micro M) of cyclic prodrug 2 was higher than the IC50 (1.9 micro M) of parent drug 1. The antithrombic activity of cyclic prodrug 2 depends on the incubation time in platelet-rich plasma; the activity increases with incubation time, suggesting that the prodrug-to-drug conversion is time-dependent and mediated by esterase. Cyclic prodrug 2 was more stable under acidic and neutral conditions than under basic conditions, suggesting that handling and formulation of this prodrug should be undertaken under acidic conditions.

Molecular dynamics simulations of conformational behavior of linear RGD peptidomimetics and cyclic prodrugs in aqueous and octane solutions.

Abstract Conformations available to a class of cyclic prodrugs and corresponding linear RGD peptidomimetics were explored using 1 ns length molecular dynamics simulations performed with the program CHARMM. Water and octane, modeled explicitly, were used as solvents to mimic the change of the environment experienced by the solutes upon partition from water to membrane in the trans-cellular transport process. In water, the linear peptidomimetics tended to populate extended-like structures, characterized by strong favorable interactions with solvent and low intrinsic stability. In these extended conformations the charged termini are able to assume large distances, above 15 Å for the longest systems. These linear peptidomimetics have been found to exhibit the highest potency in experimental studies, in accord with the trends experimentally observed for RGD peptides. In contrast, in octane compact conformers of the linear peptidomimetics were favored, with all charged groups aggregated and shielded from solvent, exhibiting high intrinsic stability and weak solute-solvent interactions. Our calculations predict a large unfavorable energy change for transferring the linear systems from water to octane, in agreement with experimental findings that these compounds are not transported via the trans-cellular pathway. The cyclic pro- drugs did not exhibit major structural differences between the simulations in water and octane, adopting turn-like conformations in both solvents. The limited response of the cyclic structures to changes in the environment leads to energies of transfer from water to octane that are also unfavorable, but much less so than for the linear molecules. This effect is in accord with the observed enhanced passive trans-cellular transport of the cyclic prodrugs.

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.

Synthesis and comparison of physicochemical, transport, and antithrombic properties of a cyclic prodrug and the parent RGD peptidomimetic

Cyclic prodrug 1 was derived from RGD peptidomimetic 2 by linking the amino and carboxylic acid groups via an (acyloxy)alkoxy linker. The formation of a cyclic prodrug can transiently alter the physicochemical properties of the RGD peptidomimetic. Cyclic prodrug 1 was synthesized via the key intermediate 8, and the synthesis of this key intermediate was accomplished using two different routes. Cyclic prodrug 1 has a smaller hydrodynamic radius and higher membrane interaction potential than those of the parent RGD peptidomimetic 2. The cell membrane permeation of cyclic prodrug 1 is twice that of the parent peptidomimetic 2. The prodrug-to-drug conversion can be carried out in isolated porcine esterase as well as human plasma. The cyclic prodrug is more stable at pH 4 than pH 7, and is very unstable at pH 10. The cyclic prodrug has antithrombic activity, suggesting that it can be converted to the RGD peptidomimetic in platelet-rich plasma (PRP).

Regulation of Cadherin-Cadherin Interaction: Secondary Structure of the HAV and ADT Peptides Derived from Human E-Cadherin Sequence

Tight junctions present a major obstacle for delivery of drugs through the intestinal and blood brain barriers. These tight junctions are regulated in part by E-cadherins, which are calcium(dependent cell-cell adhesion molecules. E-cadherins are type-1 glycoproteins with a single transmembrane domain, a tandem repeat at the extracelluar domain (EC1-EC5) and a short cytoplasmic domain. E-cadherins are also calcium(de-pendent molecules that are mediators of cell adhesion through homophilic interactions [1]. Peptides derived from this binding region have been shown to increase the opening of the intercellular tight junctions in a reversible fashion, and may be used to improve paracellular drug transport by altering the intercellular junctions [1,2].

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.