Peptide-based delivery to bone

Peptides are attractive as novel therapeutic reagents, since they are flexible in adopting and mimicking the local structural features of proteins. Versatile capabilities to perform organic synthetic manipulations are another unique feature of peptides compared to protein-based medicines, such as antibodies. On the other hand, a disadvantage of using a peptide for a therapeutic purpose is its low stability and/or high level of aggregation. During the past two decades, numerous peptides were developed for the treatment of bone diseases, and some peptides have already been used for local applications to repair bone defects in the clinic. However, very few peptides have the ability to form bone themselves. We herein summarize the effects of the therapeutic peptides on bone loss and/or local bone defects, including the results from basic studies. We also herein describe some possible methods for overcoming the obstacles associated with using therapeutic peptide candidates.     

Peptide-based molecules in angiogenesis

Angiogenesis refers to the process of remodeling the vascular tissue characterized by the branching out of a new blood vessel from a pre-existing vessel. Angiogenesis is particularly active during embryogenesis, while during adult life it is quiescent and limited to particular physiologic phenomena. Recently, the study of molecular mechanisms of angiogenesis has stirred renewed interest due to the recognition of the role played by angiogenesis in several pathologies of significant medical impact, such as cancer and cardiovascular disease, and due to the pharmacologic interest rising from the possibility of modulating these phenomena. Antibodies, peptides and small molecules targeting active endothelial cells represent an innovative tool in therapeutic and diagnostic fields. In this study, we reviewed the literature of peptide and peptidomimetics in angiogenesis and their potential applications. Two specific protein systems, namely the vascular endothelial growth factor and its receptor and integrins, will be discussed in detail.     

Peptide-based cationic liposome-mediated gene delivery

INTRODUCTION: As an important type of nonviral gene delivery vector, peptide-based cationic liposomes have shown many advantages over other cationic liposomes, such as good biodegradability, excellent biocompatibility and targeting ability to cells, and great potential application in improving the delivery of gene therapeutics. AREAS COVERED: This article reviews the research progress on peptide-based cationic liposomes for gene delivery, including the structure characteristics of peptide lipids, the development of peptide cationic liposomes and three special types of peptide cationic liposomes: peptide-based Gemini lipids, lipitoids and lipids with cholesterol hydrophobic group. This review hopes to provide some suggestions on the design of peptide cationic lipids and insight into their development trend in the field of gene delivery. EXPERT OPINION: As peptide-based cationic liposomes still hold some limitations, future research needs to select suitable peptide heads and investigate the surface modification of peptide cationic liposomes, in order to facilitate targeting and reduce cytotoxicity.     

Recent progress in application of nanovaccines for enhancing mucosal immune responses

Mucosal vaccines that stimulate both mucosal and systemic immune responses are desirable, as they could prevent the invading pathogens at their initial infection sites in a convenient and user-friendly way. Nanovaccines are receiving increasing attention for mucosal vaccination due to their merits in overcoming mucosal immune barriers and in enhancing immunogenicity of the encapsulated antigens. Herein, we summarized several nanovaccine strategies that have been reported for enhancing mucosal immune responses, including designing nanovaccines that have superior mucoadhesion and mucus penetration capacity, designing nanovaccines with better targeting efficiency to M cells or antigen-presenting cells, and co-delivering adjuvants by using nanovaccines. The reported applications of mucosal nanovaccines were also briefly discussed, including prevention of infectious diseases, and treatment of tumors and autoimmune diseases. Future research progresses in mucosal nanovaccines may promote the clinical translation and application of mucosal vaccines.     

Peptide-based vaccines in the treatment of specific allergy

The efficacy of conventional allergen-specific immunotherapy (SIT) for allergic conditions and venom hypersensitivity is well documented. However it's use is limited due allergic side effects including anaphylaxis and the difficulty of standardising proteins in complex allergenic mixtures. The aim of new therapeutic strategies is to circumvent these limitations and approaches include allergen non-specific therapy, such as anti-IgE and anti-cytokine therapy and other allergen specific techniques including the peptide based vaccines (PBV), modified allergens (allergoids) and DNA vaccines. PBV are small linear peptide fragments containing T cell epitopes which are designed to reduce the ability to cross link antigen-specific IgE. Studies in animal models have confirmed proof of principle demonstrating the induction of hyporesponsiveness using high doses of peptides. However, the principle limitation to clinical use of PBV is the polymorphism of HLA class II molecules. There are ongoing clinical studies using peptide-based vaccines for cat, bee and grass allergies--looking at both immunological mechanisms and clinical outcome measures. The mechanisms underlying the efficacy of PBV appear to be similar to those described for classical immunological tolerance. Thus, the peptides may induce anergy due to absence of co-stimulation, activation-induced cell death, a switch from a Th2 to a Th1 cytokine profile, the induction of regulatory T cells or combinations of these mechanisms. Successful immunotherapy, in bee sensitive individuals, is associated with the elaboration of IL-10. Clonal deletion is unlikely as an overall mechanism as there is evidence that the subsequent in vitro response to associated, non-injected, peptides can be suppressed. Mechanistic studies continue to provide insight into the mode of action of whole allergen and peptide-based immunotherapy. Clinical studies designed on the basis of these observations hold the promise of safer vaccines with improved efficacy. Whether this strategy can be used for allergy to complex allergen mixtures such as dust mites will need further evaluation.     

Peptide-based inhibitors of the phagocyte NADPH oxidase

Phagocytes such as neutrophils, monocytes and macrophages play an essential role in host defenses against pathogens. To kill these pathogens, phagocytes produce and release large quantities of antimicrobial molecules such as reactive oxygen species (ROS), microbicidal peptides, and proteases. The enzyme responsible for ROS generation is called NADPH oxidase, or respiratory burst oxidase, and is composed of six proteins: gp91phox, p22phox, p47phox, p67phox, p40phox and Rac1/2. The vital importance of this enzyme in host defenses is illustrated by a genetic disorder called chronic granulomatous disease (CGD), in which the phagocyte NADPH oxidase is dysfunctional, leading to life-threatening recurrent bacterial and fungal infections. However, excessive NADPH oxidase activation and ROS over-production can damage surrounding tissues and participate in exaggerated inflammatory processes. As ROS production is believed to be involved in several inflammatory diseases, specific phagocyte NADPH oxidase inhibitors might have therapeutic value. In this commentary, we summarize the structure and activation of the phagocyte NADPH oxidase, and describe pharmacological inhibitors of this enzyme, with particular emphasis on peptide-based inhibitors derived from gp91phox, p22phox and p47phox.     

Advances in Peptide-based Human Papillomavirus Therapeutic Vaccines

Cervical cancer is the second leading cause of cancer in women worldwide. Human papillomavirus (HPV) is responsible for all cases of cervical cancer. Commercial prophylactic HPV vaccines are now available, but unfortunately these vaccines have no therapeutic effect against established HPV infections. In order to accelerate the control of cervical cancer and treat established HPV infections, it is necessary to develop therapeutic vaccines to eradicate HPV by generating cell-mediated immunity against HPV infected cells. Two HPV-encoded early proteins, the E6 and E7 oncoproteins, are the preferred targets because they are consistently expressed in virtually all cervical cancer cells and are necessary for the induction and maintenance of HPV-associated disease. A variety of vaccine strategies have been employed targeting immune responses to these proteins. Peptide-based vaccines are a promising strategy for the development of therapeutic HPV vaccines because of their safety, stability, and ease of production. This review summarizes the prospects of peptidebased vaccines for the treatment of established HPV infections. We address the challenges that scientists currently face for developing peptide-based vaccines and explore feasible strategies for improving the potency of the induced immune response with the aim of treating established HPV infections.     

Rational Peptide-based vaccine design for cancer immunotherapeutic applications

Immune responses to cancer cells can be elicited in vivo by administering synthetic peptides derived from proteins uniquely or overexpressed on tumor cells (tumor associated antigens--TAAs). Peptides derived from TAAs are presented in the context of major histocompatibility complex (MHC) molecules to cytotoxic T cells (CTL), which can recognize and lyze tumor cells. In contrast to peptides derived from an exogenous source (viral or bacterial), tumor peptides bind weakly to MHC class I molecules. The low binding affinity of these peptides makes them poor candidates for vaccination due to the poor immunogenic response produced. In order to enhance antigen recognition and hence immunogenicity, peptide binding affinity for MHC can be initially improved by modifying the "anchor" residues. However, the task at hand is highly unpredictable and minor changes in peptide sequence can alter/abolish the T cell response. Furthermore, despite the wealth of information obtained over the last decade from high resolution X-ray structures of MHC class I in complex with peptides (pMHC) as well as pMHC in complex with T cell receptor (TcR), prediction remains difficult. Nonetheless, peptides represent convenient chemical entities that can be rapidly synthesized in clinical grade for therapeutic applications. Herein, the rationale behind modifying TAAs will be discussed including the synthesis/use of proteolytically tolerant peptides (and peptide mimetics) which incorporate non-natural amino acids, retro-inversion and cyclization to improve bioavailability.     

Peptide-based drug discovery: Current status and recent advances

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Peptide-based in vitro assay for the detection of reactive metabolites

We describe a novel peptide-based in vitro method for the detection of reactive metabolites that is amenable for use with microsomal or purified enzyme systems. Covalently bound adducts are detected by mass spectrometry using a surface-enhanced laser desorption ionizationtime of flight detector. The trapping molecule is an 11 amino acid peptide (ECGHDRKAHYK) that contains cysteine and other nucleophilic amino acid residues, as well as charged residues to enhance binding to a weak cation exchange chip surface used with the detection system. The assay concept was initially tested using rat or human liver microsomes with a series of benzodioxolanes. The assay was refined using human recombinant cytochrome P450 3A4 as the bioactivation system and validated with a series of positive and negative reference compounds. Alternative individual human recombinant P450 enzymes (e.g., 1A1, 2C9, or 2D6) may be used in place of 3A4 as the bioactivation system, or several P450 enzymes can be combined together into a single bioactivation system. We found that a mixture of P450s 3A4, 2C9, and 2D6 was suitable as a rapid general screen for the detection of reactive metabolites that covalently bind to proteins. Combining results from assays of individual P450 enzymes with microsomal systems allows the rapid profiling of metabolic pathways involved in reactive metabolite generation and provides valuable information that can be used to guide structural modifications to minimize the potential for metabolic bioactivation. In addition, non-P450 enzymes may be used as activation systems, such as peroxidases or alcohol dehydrogenase. In summary, this peptide-based assay system is able to detect reactive metabolites generated from a structurally diverse set of drugs and xenobiotics using a variety of microsomal or purified enzyme activation systems.     

Peptide-based imaging agents targeting phosphatidylserine for the detection of apoptosis

A 14-residue phosphatidylserine (PS)-binding peptide FNFRLKAGQKIRFG (PSBP-0) was scanned with Ala. In addition, a radiometal chelator (SAAC) was introduced at selected sites of the lead peptides. Substitution of the Gln(6) residue in PSBP-0 with Ala resulted in a significant increase in binding affinity to PS as determined by surface plasmon resonance sensorgrams. The binding affinity of the resulting peptide FNFRLKAGAKIRFG (PSBP-6, molecular mass = 1623 Da) to PS (K(d) ～ 100 nM) increased 10-fold as compared to PSBP-0 (K(d) ～ 1.38 μM). Introduction of SAAC-Re to the N terminus of PSBP-6 further increased the binding affinity of the resulting peptide SAAC(Re)-PSBP-6 (K(d) ～ 26 nM). SAAC(Re)-PSBP-6 shows specific binding to apoptotic cells in cell-based assays. Biodistribution studies showed significantly higher uptake of SAAC((99 m)Tc)-PSBP-6 in B16/F10 melanoma treated with poly(L-glutamic acid)-paclitaxel than untreated tumors (4.06 ± 0.55% ID/g vs 1.61 ± 0.33% ID/g, P = 0.00011). SAAC((99 m)Tc)-PSBP-6 is a promising probe for noninvasive imaging of apoptotic cells.     

Hybrid nanoparticle-based nicotine nanovaccines: Boosting the immunological efficacy by conjugation of potent carrier proteins

A series of hybrid nanoparticle-based nicotine nanovaccines (NanoNicVac) were engineered in this work by conjugating potent carrier protein candidates (Keyhole limpet hemocyanin (KLH) multimer, KLH subunit, cross-reactive material 197 (CRM₁₉₇), or tetanus toxoid (TT)) for enhanced immunological efficacy. NanoNicVac with CRM₁₉₇ or TT were processed by dendritic cells more efficiently than that with KLH multimer or subunit. NanoNicVac carrying CRM₁₉₇ or TT exhibited a significantly higher immunogenicity against nicotine and a considerably lower immunogenicity against carrier proteins than NanoNicVac carrying KLH multimer or subunit in mice. The in vivo results revealed that NanoNicVac with CRM₁₉₇ or TT resulted in lower levels of nicotine in the brain of mice after nicotine challenge. All findings suggest that an enhanced immunological efficacy of NanoNicVac can be achieved by using CRM₁₉₇ or TT instead of KLH or KLH subunit as carrier proteins, making NanoNicVac a promising next-generation immunotherapeutic candidate against nicotine addiction.     

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8-30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 +/- molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10-17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.     

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8–30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4?±?molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10–17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.     

Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position

The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.     

Tamoxifen alters gating of the BK alpha subunit and mediates enhanced interactions with the avian beta subunit

Mammalian BK channels are modulated by estrogen and non-steroidal estrogen-like compounds (i.e. xenoestrogens), but the effects are dependent on channel composition. (Xeno)estrogens preferentially activate BK channels through accessory beta subunits, but reduce single-channel conductance by interaction with alpha subunits. In this report, the xenoestrogen tamoxifen was applied to chicken BK channels, in order to asses the mechanism behind drug interaction and to determine the extent to which (xeno)estrogen interaction is extended to avian BK homologs. As with mammalian isoforms, the properties of chicken BK channels were modulated by tamoxifen in a subunit-dependent manner. Tamoxifen reduced single-channel conductance through interaction with the alpha subunit. However, if the expression construct included the beta subunit, tamoxifen increased the channel's open probability and shifted the voltage-activation range to more negative potentials. This effect on channel gating was concentration-dependent, with an EC(50) of about 0.2 microM. Tamoxifen-mediated reductions in gating charge and in the intrinsic energetics that govern channel equilibrium. The relative contribution of these two effects on channel gating was altered by beta co-expression. Modulation by (xeno)estrogens may be an evolutionarily conserved mechanism for non-genomic hormonal actions, and the limited conservation between avian and mammalian beta subunits may suggest potential binding motifs. Alternatively, the data are consistent with a tamoxifen-mediated conformation change in the alpha subunit that alters the way alpha and beta subunits interact, resulting in enhanced gating without direct binding to beta.