Teruna J. Siahaan

The role of thiols and disulfides on protein stability.

There has been a tremendous increase in the number of approved drugs derived from recombinant proteins; however, their development as potential drugs has been hampered by their instability that causes difficulty to formulate them as therapeutic agents. It has been shown that the reactivity of thiol and disulfide functional groups could catalyze chemical (i.e., oxidation and beta-elimination reactions) and physical (i.e., aggregation and precipitation) degradations of proteins. Because most proteins contain a free Cys residue or/and a disulfide bond, this review is focused on their roles in the physical and chemical stability of proteins. The effect of introducing a disulfide bond to improve physical stability of proteins and the mechanisms of degradation of disulfide bond were discussed. The qualitative/quantitative methods to determine the presence of thiol in the Cys residue and various methods to derivatize thiol group for improving protein stability were also illustrated.

Improvement of oral peptide bioavailability : Peptidomimetics and prodrug strategies

Clinical development of orally active peptide drugs has been restricted by their unfavorable physicochemical properties, which limit their intestinal mucosal permeation and their lack of stability against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical characteristics of these potential drugs, without changing their biological activity, in order to overcome the physical and biochemical barrier properties of the intestinal cells. This manuscript will focus on the physiological limitations for oral peptide delivery and on various strategies using chemical modifications to improve oral bioavailability of peptide-based drugs.

Structural requirements for intestinal absorption of peptide drugs

The clinical development of orally active peptide drugs has been restricted by their unfavorable physicochemical characteristics, which limit their membrane permeation, and their lack of stability against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical properties of these potential drugs, without changing their biological activity, in order to circumvent the barrier properties of the intestinal epithelial cells. This manuscript will focus on the physical and metabolic barrier functions of the intestinal mucosa, the structural features of peptides which influence their passive diffusion and carrier-mediated transport, including efflux mechanisms, and various approaches used to prevent enzymatic degradation of the peptides and increase their permeability across the intestinal mucosa.

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.

Peptide-Mediated Targeted Drug Delivery

Targeted drug delivery to specific group of cells offers an attractive strategy to minimize the undesirable side effects and achieve the therapeutic effect with a lower dose. Both linear and cyclic peptides have been explored as trafficking moiety due to ease of synthesis, structural simplicity, and low probability of undesirable immunogenicity. Peptides derived from sequence of cell surface proteins, such as intercellular adhesion molecule‐1 (ICAM‐1), LHRH, Bombesin, and LFA‐1, have shown potent binding affinity to the target cell surface receptors. Moreover, peptides derived from ICAM‐1 receptor can be internalized by the leukemic T‐cells along with the conjugated moiety offering the promise to selectively treat cancers and autoimmune diseases. Systematic analyses have revealed that physicochemical properties of the drug–peptide conjugates and their mechanism of receptor‐mediated cellular internalization are important controlling factors for developing a successful targeting system. This review is focused on understanding the factors involved in the development of an effective drug–peptide conjugate with an emphasis on the chemistry and biology of the conjugates. Reported results on several promising drug–peptide conjugates have been critically evaluated. The approaches and results presented here will serve as a guide to systematically approach targeted delivery of cytotoxic drug molecules using peptides for treatment of several diseases.

Controlling ligand surface density optimizes nanoparticle binding to ICAM-1

During infection, pathogens utilize surface receptors to gain entry into intracellular compartments. Multiple receptor-ligand interactions that lead to pathogen internalization have been identified and the importance of multivalent ligand binding as a means to facilitate internalization has emerged. The effect of ligand density, however, is less well known. In this study, ligand density was examined using poly(DL-lactic-co-glycolic acid) nanoparticles (PLGA NPs). A cyclic peptide, cLABL, was used as a targeting moiety, as it is a known ligand for intercellular cell adhesion molecule-1 (ICAM-1). To modulate the number of reactive sites on the surface of PLGA NPs, modified Pluronic with carboxyl groups and Pluronic with hydroxyl groups were combined in different ratios and the particle properties were examined. Utilizing a surfactant mixture directly affected the particle charge and the number of reactive sites for cLABL conjugation. The surface density of cLABL peptide increased as the relative amount of reactive Pluronic was increased. Studies using carcinomic human alveolar basal epithelial cells (A549) showed that cLABL density might be optimized to improve cellular uptake. These results complement other studies, suggesting that surface density of the targeting moiety on the NP surface should be considered to enhance the effect of ligands used for cell targeting.

Prodrug strategies to enhance the intestinal absorption of peptides

Clinical development of orally active peptide drugs has been restricted by the unfavorable physicochemical properties of these molecules limiting intestinal mucosal permeation and the lack of stability of peptides against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical characteristics of these potential drugs, without changing their biological activity, in order to increase the permeation across intestinal cells. This manuscript will focus on the biological barrier properties that limit oral peptide bioavailability and on prodrug strategies designed to overcome these barriers.

The effect of β-turn structure on the passive diffusion of peptides across Caco-2 cell monolayers

AbstractPurpose. To investigate the relationships between the β-turn structure of a peptide and its passive diffusion across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. Methods. Linear hydrophilic peptides (Ac-TyrProXaaZaaVal-NH2; Xaa = Gly, Ile and Zaa = Asp, Asn) and hydrophobic (Ac-YaaPro-XaaIleVal-NH2; Yaa = Tyr, Phe and Xaa = Gly, Ile: and Ac-PhePro-XaaIle-NH2; Xaa = Gly, He) peptides were synthesized and their effective permeability coefficients (Peff) were determined across Caco-2 cell monolayers. The lipophilicities of the peptides were estimated by measuring their partition coefficients (Po/w) between 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the solution structures of these model peptides. Results. Using 2D-NMR spectroscopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac-TyrProGlyZaaVal-NH2; Zaa = Asp, Asn) were shown to exhibit a higher degree of β-turn structure in solution than the Ile-containing peptides (Ac-TyrProIleZaaVal-NH2; Zaa = Asp, Asn). CD spectroscopy was used to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIleVal-NH2; Yaa = Tyr, Phe: and Ac-PheProGlyIle-NH2) exhibited a higher degree of β-turn structure in solution than the Ile-containing hydrophobic peptides. The Peff values of all four hydrophilic peptides across unperturbed Caco-2 cell monolayers were very low and no statistically significant differences were observed between the Gly- and Ile-containing penta-peptides within either the Asp or Asn series. The Peff values for the hydrophobic Gly-containing peptides were significantly greater than the Peff values determined for their Ile-containing counterparts. The Gly-containing penta- and tetrapeptides in the Phe series, which exhibited high permeation, were shown to be metabolically unstable. In contrast, the Gly- and Ile-containing pentapeptides in the Tyr series and the Ile-containing penta- and tetrapeptides in the Phe series, which exhibited low permeation, were metabolically stable. Conclusions. Hydrophobic peptides that exhibit significant β-turn structure in solution are more lipophilic as measured by log Po/w and more readily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ability of these peptides to permeate Caco-2 cell monolayers via the transcellular route also exposed them to metabolism, presumably by cytosolic endopeptidases. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties fo the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.

Effect of restricted conformational flexibility on the permeation of model hexapeptides across Caco-2 cell monolayers

AbstractPurpose. To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics. Methods. Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH2; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide. Results. The cyclic hexapeptides used in this study were 2−3 times more able to permeate (e.g., Papp = 9.3 ± 0.3 × 10−8 cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., Papp = 3.2 ± 0.3 × 10−8 cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and β-turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two β-turns. Conclusions. Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two β-turns, explain its greater lipophilicity.

Vaccine-like Controlled-Release Delivery of an Immunomodulating Peptide to Treat Experimental Autoimmune Encephalomyelitis

The objective of this work is to use colloidal gel from alginate-chitosan-PLGA complex to deliver Ac-PLP-BPI-NH₂-2 peptide in a controlled-release manner as a vaccine-like therapeutic to suppress experimental autoimmune encephalomyelitis (EAE) in the mouse model. Oppositely charged PLGA nanoparticles were prepared by a solvent diffusion method. The carboxyl group of the alginate and the amine group of the chitosan coated the nanoparticles with negative and positive charges, respectively. The peptide (Ac-PLP-BPI-NH₂-2), designed to bind to MHC-II and ICAM-1 simultaneously, was formulated into the colloidal gel by physical mixture. Vaccine-like administration of the peptide-loaded colloidal gel (Ac-PLP-BPI-NH₂-2-NP) was achieved by subcutaneous (sc) injection to EAE mice. Disease severity was measured using clinical scoring and percent change in body weight. Cytokine production was determined using the splenocytes from Ac-PLP-BPI-NH₂-2-NP-treated mice and compared to that of controls. Ac-PLP-BPI-NH₂-2-NP suppressed and delayed the onset of EAE as well as Ac-PLP-BPI-NH₂-2 when delivered in a vaccine-like manner. IL-6 and IL-17 levels were significantly lower in the Ac-PLP-BPI-NH₂-2-NP-treated mice compared to the mouse group treated with blank colloidal gel, suggesting that the mechanism of suppression of EAE is due to a shift in the immune response away from Th17 production. The results of this study suggest that a one-time sc administration of Ac-PLP-BPI-NH₂-2 formulated in a colloidal gel can produce long-term suppression of EAE by reducing Th17 proliferation.