Current Pharmaceutical Design - Volume 11, Issue 28, 2005

This special issue of Current Pharmaceutical Design is dedicated to cell-penetrating peptides, CPPs, their mechanisms and applications. It is divided into seven chapters in which several aspects of cell-penetrating peptides are updated. This issue was compiled in November 2004 and reflects the concise summary of CPP research in recent 10 years since the first CPP, pAntennapedia (later named penetratin) was discovered by Alain Prochiantz' group in Paris in 1994 [1]. The precise definition of a CPP today remains controversial. CPPs are generally defined as short peptides translocating the plasma membrane in an endocytotic fashion as recently reported [2], as well as, in some cases, in a seemingly energy-independent manner[3], thus suggesting additional mechanisms of uptake. These mechanisms of CPP uptake find intensive attention in this special issue. However, alternative definitions and confusing naming of CPPs can be found in the literature, depending on the author and origin of the peptide. CPPs derived from proteins are often referred to as protein transduction domains, PTDs [4, 5]. Membrane-permeable sequences, MPS [6], and model amphipathic peptides, MAP [3], are additional examples of frequently used names for CPPs. In this issue, possible novel classes of CPPs, calcitonin-derived carrier peptides and vascular homing peptides with CPP properties, are described and summarized. Novel classes of CPPs are continually emerging and contribute strongly to the aims of CPP applications such as cell-targeted and in vivo delivery of drugs. An important CPP application is for the improvement of the uptake of genes and gene-silencing agents. Here, chapters addressing the improvement of oligonucleotide uptake by attachment to CPPs, or Bioplex entities, summarize recent achievements in the field. In summary, I hope that this special issue of the Journal will contribute strongly to our understanding and development of cell-penetrating peptides. References [1] Derossi D, Joliot AH, Chassaing G, Prochiantz A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 1994; 269: 10444-50. [2] Drin G, Cottin S, Blanc E, Rees AR, Temsamani J. Studies on the internalization mechanism of cationic cellpenetrating peptides. J Biol Chem 2003; 278: 31192-201. [3] Oehlke J, Scheller A, Wiesner B, Krause E, Beyermann M, Klauschenz E, Melzig M, Bienert M. Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochim Biophys Acta 1998; 1414: 127-39. [4] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999; 285: 1569-72. [5] Mi Z, Mai J, Lu X, Robbins PD. Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo. Mol Ther 2000; 2: 339-47. [6] Chang M, Zhang L, Tam JP, Sanders-Bush E. Dissecting G protein-coupled receptor signalling pathways with membrane-permeable blocking peptides. J Biol Chem 2000; 275: 7021-9.

A major obstacle in the development of new therapeutic agents is the low bioavailability of hydrophilic substances. Drugs that bind to intracellular targets must penetrate the lipid bilayer surrounding the cell in order to exert their effect. A relatively new research area that addresses this problem by introducing novel transport peptides that facilitate the cellular penetration of potential drugs has emerged. These peptides predominantly have a positive net charge and/or an amphipathic nature, but can otherwise have very different characteristics. This group of peptides, although sometimes called protein transduction domains (PTDs), is here referred to as cell-penetrating peptides (CPPs). For many years it was believed that these peptides were internalized into cells via a non-endocytotic, receptorindependent pathway. However, recent publications have suggested that an endocytotic pathway cannot be ruled out, and that earlier results might be based on artifacts associated with fixation of cells and membrane association of peptides. Although the mechanism of cellular uptake remains unclear, there is an increasing amount of reports on biological effects of CPPs and their cargos. Thus, CPPs are an attractive pharmaceutical and biochemical tool that needs more attention. This review will discuss some recent results in this research field with focus on the cell-penetrating peptide transportan.

With the identification of fixation as a major source of artefacts in the cell biological research on cellpenetrating peptides (CPPs), the past two years have witnessed a dramatic development in the CPP field. At least for some of these molecules, endocytosis is now considered to be the major if not the exclusive route of cellular import. However, endocytosis comprises a variety of different pathways with very different implications for the delivery of bioactive molecules to the cytoplasm and nucleus. The endocytosis of CPPs is governed by complex mechanisms similar to those responsible for the internalization of other molecules. Therefore the investigation of uptake and intracellular trafficking of CPPs can benefit enormously from the understanding of the endocytic machinery as well as from the tools that have already been developed for the analysis of endocytosis. This review will introduce aspects of endocytosis relevant to the analysis of CPPs. In addition to the methods, that have already been used for the analysis of CPP trafficking, we will also present other tools and approaches which can be helpful for the research of CPP uptake. Furthermore this review will analyze and summarize recent data providing new insights in endocytosis and intracellular trafficking of CPPs.

This review focuses on two peptides, MPG and Pep-1, which facilitate the transfer of nucleic acids and proteins, respectively, into subcellular compartments. We have investigated the interactions between these two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air - water interface have enabled identification of the nature of the lipid - peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid - peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG it is based on the formation of a β-barrel pore-like structure while for Pep- 1 it is based on association of helices.

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular heterogeneity in the blood vessels of individual normal tissues and shown that pathological lesions put their signature on the vasculature. In tumors, both blood and lymphatic vessels differ from normal vessels. Moreover, the molecular changes in the vasculature parallel progression in tumor development, hence making the vessels in premalignant lesions distinguishable from normal vessels and from the vessels in malignant tumors of the same tissue. Some of the tumor-homing peptides penetrate into tumor endothelial cells (and tumor cells), but not into endothelial cells in normal tissues or other normal cells. Thus, these cell-penetrating peptides are cell type-specific. Peptides that home to tumor vasculature have been shown to be useful in directing therapeutic agents to experimental tumors. The cell penetrating peptides may be particularly useful in drug delivery because they can take their payload inside the target cells and even into a specific subcellular organelle such as the nucleus.

The rescent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.

Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

The development of atherosclerotic lesions in the artery wall is a complex process involving the endothelium, lipid engorged macrophages (foam cells) and smooth muscle cells. In recent years it has become clear that chylomicron remnants, the lipoproteins which carry lipids of dietary origin in the blood, are strongly atherogenic, and there is increasing evidence to indicate that this is due to direct interaction of the remnant particles with cells of the artery wall. Chylomicron remnants have been demonstrated to inhibit endothelium dependent vasorelaxation and to activate signal transduction pathways associated with inflammation in cultured endothelial cells. They have also been shown to be taken up by smooth muscle cells and macrophages, and to cause the extensive lipid accumulation associated with foam cell formation, as well as influencing the expression of key genes regulating macrophage lipid uptake and metabolism. Furthermore, oxidative modification of the remnant particles is not required for many of these effects. Chylomicron remnants, therefore, have mutiple direct effects on three major cell types of the arterial wall which are likely to promote the development of atherosclerotic lesions. These effects may be modulated by various lipids carried by the particles, including the type of fat (saturated or unsaturated or oxidised fat), micronutrients such as vitamins and carotenoids which have antioxidant properties, and orally administered lipophilic drugs. Delayed clearance of chylomicron remnants from the blood occurs in a number of dyslipidemias associated with premature atherosclerosis development, and the potentially atherogenic effects of the particles would clearly be enhanced in these circumstances. Thus, elucidation of the mechanisms involved will aid in the identification of new drug targets which may be particularly useful for these conditions.

The use of highly active antiretroviral therapy (HAART) has significantly slowed the HIV disease progression. However, adverse effects are now a limiting cause of HAART benefit in a substantial proportion of patients. Particularly hepatotoxicity which is a common complication occurring during every HAART regimen. All antiretroviral (ARV) drugs classes, Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), non-Nucleoside reverse transcriptase inhibitors (nNRTI) and Protease Inhibitors (PI) may cause hepatotoxicity but in different pathways. Many risk factors have been identified for developing antiretroviral-related hepatotoxicity, however severe hepatitis remains very uncommon in patients receiving HAART, also if the incidence of hepatotoxicity is rather high. That being the case, means that every new available antiretroviral drug strongly necessities studies which can evaluate its hepatotoxicity and drug-drug interactions, to define the potential risk factors and the outcome of any side effects. This report will review the risk factors, the epidemiology and the pathogenic mechanisms of hepatotoxicity caused in every antiretroviral drug.

Convenient strategies are needed for postmenopausal osteoporosis management. Current oral bisphosphonates are effective and generally well tolerated, but are hampered by strict dosing requirements. Less frequent dosing schedules are expected to improve adherence to therapy and hence outcomes. Ibandronate is a potent, nitrogen-containing bisphosphonate in ongoing clinical development exploring its potential to be administered less frequently than at weekly intervals. Uniquely, ibandronate can be administered either orally or as an intravenous (i.v.) injection, with a betweendose interval of >2 months. In postmenopausal women, oral ibandronate, when administered either daily or intermittently, has demonstrated robust antifracture efficacy at the spine (62% [p=0.0001] and 50% [p=0.0006] risk reduction versus placebo), accompanied by significant increases in bone mineral density (BMD) at the spine and hip, and suppression of bone turnover markers. Studies of intermittent i.v. ibandronate injections in postmenopausal women have shown doserelated BMD gains and bone turnover marker suppression comparable to those obtained after a similar duration of treatment with the proven effective oral ibandronate regimen. Furthermore, in a trial conducted in patients with corticosteroid-induced osteoporosis, intermittent i.v. ibandronate injections reduced vertebral fracture risk by 62% versus an active control (p=0.043). In all these trials, the oral and i.v. ibandronate regimens were well tolerated. Ongoing, large multinational clinical trials are investigating two intermittent ibandronate regimens: once-monthly oral and intermittent i.v. injections. These simple ibandronate regimens are expected to provide the optimal combination of efficacy, tolerability and patient convenience, leading to improved treatment adherence and thus, enhanced outcomes in postmenopausal osteoporosis.