Kyung Dall Lee

Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities

The first disulfide linkage-employing drug conjugate that exploits the reversible nature of this unique covalent bond was recently approved for human use. Increasing numbers of drug formulations that incorporate disulfide bonds have been reported, particularly in the next generation macromolecular pharmaceuticals. These are designed to exploit differences in the reduction potential at different locations within and upon cells. The recent characterization of a novel redox enzyme in endosomes and lysosomes adds more excitement to this approach. This review focuses on understanding where and how the disulfide bond in the bioconjugate is reduced upon contact with biological milieu, which affects delivery design and the interpretation of the delivery strategies.

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-α and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-α production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-α production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1–positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-α from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1–positive endosomes accompanied by the loss of IFN-α production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-α and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-α response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.

Comparison of Human Duodenum and Caco-2 Gene Expression Profiles for 12,000 Gene Sequences Tags and Correlation with Permeability of 26 Drugs

AbstractPurpose. To compare gene expression profiles and drug permeability differences in Caco-2 cell culture and human duodenum. Methods. Gene expression profiles in Caco-2 cells and human duodenum were determined by GeneChip® analysis. In vivo drug permeability measurements were obtained through single-pass intestinal perfusion in human subjects, and correlated with in vitro Caco-2 transport permeability. Results. GeneChip® analysis determined that 37, 47, and 44 percent of the 12,559 gene sequences were expressed in 4-day and16-day Caco-2 cells and human duodenum, respectively. Comparing human duodenum with Caco-2 cells, more than 1000 sequences were determined to have at least a 5-fold difference in expression. There were 26, 38, and 44 percent of the 443 transporters, channels, and metabolizing enzymes detected in 4-day, 16-day Caco-2 cells, and human duodenum, respectively. More than 70 transporters and metabolizing enzymes exhibited at least a 3-fold difference. The overall coefficient of variability of the 10 human duodenal samples for all expressed sequences was 31% (range 3% to 294%) while that of the expressed transporters and metabolizing enzymes was 33% (range 3% to 87%). The in vivo / in vitro drug permeability measurements correlated well for passively absorbed drugs (R2 = 85%). The permeability correlation for carrier-mediated drugs showed 3- 35-fold higher in human above the correlation of passively absorbed drugs. The 2- 595-fold differences in gene expression levels between the Caco-2 cells and human duodenum correlated with the observed 3- 35-fold difference in permeability correlation between carrier-mediated drugs and passively absorbed drugs. Conclusions. Significant differences in gene expression levels in Caco-2 cells and human duodenum were observed. The observed differences of gene expression levels were consistent with observed differences in carrier mediated drug permeabilities. Gene expression profiling is a valuable new tool for investigating in vitro and in vivo permeability correlation.

Innate recognition of bacteria by a macrophage cytosolic surveillance pathway

Host recognition of bacterial pathogens is a critical component of the immune response. Intracellular bacterial pathogens are able to evade the humoral immune system by residing within the host cell. Here we show the existence of an innate host surveillance mechanism in macrophages that specifically distinguishes bacteria in the cytosol from bacteria in the vacuole. Recognition of Gram-positive and Gram-negative bacterial products by this surveillance system results in transcription of the ifnb gene. The activation of cytosol-specific signaling is associated with translocation of NF-κB into the nucleus and phosphorylation of the p38 mitogen-activated protein (MAP) kinase. Activation of the p38 kinase is required for the induction of gene expression by the cytosolic surveillance pathway. Our studies suggest that infection by intracellular bacterial pathogens results in an immune response distinct from that of infection by extracellular bacterial pathogens.

Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency, activity, and tumor protection.

Cytotoxic T lymphocytes (CTLs) are primed by peptide antigens that are endogenously processed in the cytosol and presented in the context of major histocompatibility complex I (MHC I) molecules of antigen-presenting cells (APCs). Exogenous soluble protein antigens do not gain efficient entry into the cytosol of APCs, and therefore requires a special cytosolic delivery method. We have developed such a delivery strategy adopting the well-elucidated cytosol-invading listerial endosomal escape mechanism, and report here an efficient delivery of exogenous whole protein antigen into the cytosol in a mouse model. Co-encapsulation of listeriolysin O (LLO) inside liposome (LLO-liposome) was required for delivery of ovalbumin (OVA) into the cytosol of APCs in primary cultures. LLO-liposome-mediated OVA immunization in mice engendered significantly higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor (CTLp) frequency as compared to non-LLO-liposome or soluble OVA immunizations. Interferon-gamma (IFN-gamma) production upon specific stimulation by MHC I-restricted peptide was also significantly stronger by the inclusion of LLO in the liposomes. Rerouting of antigen into the cytosol by LLO-liposomes, however, did not reduce the extent of anti-OVA antibody responses. Moreover, LLO-liposome-antigen vaccination was robust in conferring protection to mice from lethal challenges with antigen-expressing tumor cells. Our study demonstrates a novel delivery system for efficient introduction of exogenous protein into the cytosol in vivo, priming cellular immune responses, which are protective in nature.

Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate

One of the most important requirements for non-viral gene delivery systems is the ability to mediate high levels of gene expression with low toxicity. After the DNA/vector complexes are taken up by cells through endocytosis, DNA is typically contained within the endocytic compartments and rapidly degraded due to the low pH and hydrolytic enzymes within endosomes and lysosomes, limiting its accessibility to the cytosol and ultimately to the nucleus. In this study, the endosomolytic protein listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes was conjugated with polyethylenimine (PEI) of average molecular weight 25 kDa (PEI25) via a reversible disulfide bond (LLO-s-s-PEI), and incorporated into plasmid DNA condensed with disulfide-crosslinked low molecular weight PEI 1.8 kDa (PEI1.8). We have investigated and demonstrated that high gene transfection efficiency, which is comparable to that by the most commonly used PEI25, can be achieved by reversibly crosslinking low molecular weight PEI (PEI1.8) using disulfide bonds, with greatly reduced cytotoxicity of the PEI. The reversible incorporation of LLO into the DNA condensates of PEI, through the use of the synthesized LLO-s-s-PEI conjugate, further enhances the transfection efficiency beyond that of DNA condensates with disulfide-crosslinked PEI1.8 alone.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of α-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific α-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Enhancing the intestinal membrane permeability of zanamivir: A carrier mediated prodrug approach

The purpose of this study was to improve the membrane permeability and oral absorption of the poorly permeable anti-influenza agent, zanamivir. The poor oral bioavailability is attributed to the high polarity (cLogP ∼ -5) resulting from the polar and zwitterionic nature of zanamivir. In order to improve the permeability of zanamivir, prodrugs with amino acids were developed to target the intestinal membrane transporter, hPepT1. Several acyloxy ester prodrugs of zanamivir conjugated with amino acids were synthesized and characterized. The prodrugs were evaluated for their chemical stability in buffers at various pHs and for their transport and tissue activation by enzymes. The acyloxy ester prodrugs of zanamivir were shown to competitively inhibit [(3)H]Gly-Sar uptake in Caco-2 cells (IC(50): 1.19 ± 0.33 mM for L-valyl prodrug of zanamivir). The L-valyl prodrug of zanamivir exhibited ∼3-fold higher uptake in transfected HeLa/hPepT1 cells compared to wild type HeLa cells, suggesting, at least in part, carrier mediated transport by the hPepT1 transporter. Further, enhanced transcellular permeability of prodrugs across Caco-2 monolayer compared to the parent drug (P(app) = 2.24 × 10(-6) ± 1.33 × 10(-7) cm/s for L-valyl prodrug of zanamivir), with only parent zanamivir appearing in the receiver compartment, indicates that the prodrugs exhibited both enhanced transport and activation in intestinal mucosal cells. Most significantly, several of these prodrugs exhibited high intestinal jejunal membrane permeability, similar to metoprolol, in the in situ rat intestinal perfusion system, a system highly correlated with human jejunal permeability. In summary, this mechanistic targeted prodrug strategy, to enhance oral absorption via intestinal membrane carriers such as hPepT1, followed by activation to parent drug (active pharmaceutical ingredient or API) in the mucosal cell, significantly improves the intestinal epithelial cell permeability of zanamivir and has the potential to provide the high oral bioavailability necessary for oral zanamivir therapy.

Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules.

Advances in our understanding of fundamental cell biological processes have facilitated an expansion of therapeutic approaches to altering cellular physiology and phenotype. As many of these methods involve macromolecular agents that act on targets within the nucleus or cytoplasm, achieving their full potential ultimately requires the efficient delivery of these agents across the cell membrane barrier into the cytosol. Various strategies have been employed to enhance cytosolic delivery. These include either directly penetrating the plasma membrane, or avoiding degradation within the hydrolytic environment of the endosomal/lysosomal pathway after endocytic uptake. Some of the more promising methods in this regard have exploited the mechanisms utilized by certain viruses and bacteria for escaping into their host cells cytosol. In this review, we will discuss some of these methods with an emphasis on the use of pore-forming proteins from bacteria. Particular attention will be drawn to the pH-sensitive endosomolytic bacterial hemolysins, such as listeriolysin O, and the potentiol for their use in cytosolic drug delivery systems.

Increasing Oral Absorption of Polar Neuraminidase Inhibitors: A Prodrug Transporter Approach Applied to Oseltamivir Analogue

Poor oral absorption is one of the limiting factors in utilizing the full potential of polar antiviral agents. The neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing prodrug to target the apical brush border peptide transporter 1 (PEPT1). Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl) ester based amino acid linked prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1. Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected prodrugs and the parent drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model. Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) <1 mM) was observed for l-valyl and l-isoleucyl amino acid prodrugs in competition experiments with [(3)H]Gly-Sar, indicating a 3-6 times higher affinity for PEPT1 compared to valacyclovir, a well-known PEPT1 substrate and >30-fold increase in affinity compared to GOCarb. The l-valyl prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent drug and showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.