Current Drug Targets (v.14, #9)

Strategies for Developing Tuberculosis Vaccines: Emerging Approaches by Adriano Mollica, Azzurra Stefanucci, Roberto Costante (938-951).
The current vaccine against TB, bacille Calmette-Gu?rin (BCG) fails to protect against the most prevalent diseaseform, the pulmonary TB in adults. Thus, it is not a satisfactory vaccine.</P><P>Given that T cells are central to protection against TB, future vaccine design should focus on T-lymphocyte populations.Most vaccines do not prevent infection but instead disease, that if they allow establishment of the pathogen in the host butprevent its harmful effects.</P><P>The development of synthetic peptide-based immunogens is emerging as a possible approach in human vaccination in thefuture, as a replacement for conventional vaccines that use killed or attenuated whole microorganisms. The advantages ofsuch synthetic vaccines (high potency, low adverse reactions, low cross-reactivity and high stability) are offset somewhatby the poorer inherent immunogenicity of these constructs. There is a greater need therefore to develop adjuvant/carriersystems to increase the immunogenicity of these newer vaccine candidates.

The modern target-based drug discovery process, characterized by the one-drug-one-gene paradigm, has beenof limited success. In contrast, phenotype-based screening produces thousands of active compounds but gives no hint as towhat their molecular targets are or which ones merit further research. This presents a question: What is a suitable targetfor an efficient and safe drug? In this paper, we argue that target selection should take into account the proteome-wide energeticand kinetic landscape of drug-target interactions, as well as their cellular and organismal consequences. We proposea new paradigm of structural systems pharmacology to deconvolute the molecular targets of successful drugs as wellas to identify druggable targets and their drug-like binders. Here we face two major challenges in structural systemspharmacology: How do we characterize and analyze the structural and energetic origins of drug-target interactions on aproteome scale? How do we correlate the dynamic molecular interactions to their in vivo activity? We will review recentadvances in developing new computational tools for biophysics, bioinformatics, chemoinformatics, and systems biologyrelated to the identification of genome-wide target profiles. We believe that the integration of these tools will realize structuralsystems pharmacology, enabling us to both efficiently develop effective therapeutics for complex diseases and combatdrug resistance.

Phytochemistry and Pharmacognosy of Naturally Occurring Prenyloxyanthraquinones by Francesco Epifano, Serena Fiorito, Giuseppe Carlucci, Marcello Locatelli, Salvatore Genovese (959-963).
Several natural compounds containing an anthraquinone core linked to a prenyloxy chain have been reported inthe literature in recent years. The discovery in the plant kingdom of such secondary metabolites is a novel acquisition inthe phytochemistry research field and in many cases led to a re-consideration of the secondary metabolite pool of wellknown anthraquinone-containing plants. In this review article we will focus on the phytochemistry and pharmacognosy ofprenyloxyanthraquinones putting in evidence the natural sources and their biological properties as anti-microbial and anticanceragents.

Yeast as a Biosensor of Detoxification: A Tool for Identifying New Compounds that Revert Multidrug Resistance by Carmen Mart-n-Cordero, Angeles Sanchez-Pico, Antonio J. Leon-Gonzalez, Antonio J. Perez-Pulido, Rafael R. Daga (964-985).
During tumour progression, cells accumulate secondary mutations and/or chromosomal aberrations that generategenetic diversity within the tumour cell population. This may result in the acquisition of new properties that increasetumour malignancy, such as invasiveness or resistance to chemotherapy. One of the important mechanisms of chemotherapyresistance is overexpression or biochemical activation of ABC family transporters. ABC transporters remove antitumourdrugs from the cell, reducing their intracellular concentration and producing resistance against a wide range ofchemically unrelated drugs, known as multidrug resistant phenotype (MDR). During recent decades, much effort has beendevoted to the isolation of compounds able to inhibit the activity of these transporters. However, few such compoundshave reached clinical practice and MDR remains a serious complication in cancer therapy. In an innovative approach tofinding new ABC inhibitors, we propose using fission yeast Schizosaccharomyces pombe as a biosensor of detoxificationthat would enable cost-efficient screening of natural compounds and chemical libraries for molecules that revert the MDRphenotype. Existing fission yeast tools provide genetic, biochemical and cell biological analysis, thereby facilitating identificationof drug targets. Putative inhibitors and modulators of ABC transporters could be used in combination with chemotherapeuticdrugs for the treatment of multidrug resistant tumours.

Antimicrobial Activity of Willowherb (Epilobium angustifolium L.) Leaves and Flowers by Ivan Kosalec, Nevenka Kopjar, Dario Kremer (986-991).
Since the aetiology of benign prostatic hyperplasia (BHP) is still unknown, the use of medicinal herb extractsand products prepared thereof are recommended due to their antimicrobial activity, especially during early stages of BHP.A comparison was performed of the in vitro antimicrobial activity (using broth microdilution assay) of flowers and leavesof willowherb (Epilobium angustifolium L., Onagraceae) from Mt. Velebit (Croatia). The strains (standard ATCC andclinical isolates) of Staphylococcus aureus (including MRSA), Bacillus subtilis, Escherichia coli (including p-fimbriaepositive strain), Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, C. tropicalis, C. dubliniensis and Saccharomycescerevisiae were susceptible with MIC values between 4.6±0.2 and 18.2±0.8 mg/mL. The results of in vitrostudies showed that no differences were found in the antimicrobial activity between the ethanol extracts of leaves andflowers of E. angustifolium. Using the quantitative fluorescent assay with ethidium bromide and acridine orange, the viabilityof C. albicans ATCC 10231 was assessed after in vitro exposure to E. angustifolium leaf and flower ethanol extracts.Apoptosis of C. albicans blastospores dominated over necrosis in all treated samples after short-term exposure with6 to 12 mg/mL of extracts. In addition to the valuable biological activity of E. angustifolium extracts, the data obtainedfrom the in vitro diffusion, the dilution assay and antifungal viability fluorescent assay suggest that leaf and flower ethanolextracts of E. angustifolium L. are a promising complementary herbal therapy of conditions such as BHP.

Hydroxytyrosol Expresses Antifungal Activity In Vitro by Natasa Zoric, Igor Horvat, Nevenka Kopjar, Ante Vucemilovic, Dario Kremer, Sinisa Tomic, Ivan Kosalec (992-998).
Hydroxytyrosol (HT) is a potent antioxidant found in olive oil and leaves. Using several in vitro approaches,we tested antifungal activity of HT. HT showed broad spectrum of antifungal activity against medically important yeastsand dermatophyte strains with MIC values ranging between 97.6 µgml-1 and 6.25 mgml-1. The antimicrobial activity ofHT was also tested using the time-kill methodology. Below the MIC value, HT showed potent damage of cell wall ofCandida albicans ATCC 10231 using fluorescent dye-exclusion method. At the subinhibitory concentration, HT also influenceddimorphic transition of Candida indicating that HT is inhibitor of germ-tube formation as one of the most importantvirulence factor of C. albicans. Furthermore, HT showed disturbances in cell surface hydrophobicity (CSH) of C. albicans.The in vitro results indicate that HT caused a significant cell wall damage and changes in CSH as well as inhibitionof germ-tube formation as virulence factor of C. albicans. The study indicates that HT has a considerable in vitroantifungal activity against medically important yeasts.

Enhanced Antioxidant and Protective Activities on Retinal Ganglion Cells of Carotenoids-Overexpressing Transgenic Carrot by Kee Dong Yoon, Suk-Nam Kang, Ji-Yeong Bae, Haeng-Soon Lee, Sang-Soo Kwak, Insurk Jang, Il-Suk Kim, Cheol Ho Lee, Jung Myung Bae, Shin Woo Lee, Mi-Jeong Ahn (999-1005).
Carotenoids are considered to act as antioxidants and protect humans from serious disorders such as skin degenerationand ageing, cardiovascular disease, certain types of cancer, and age-related diseases of the eye. In this study,these chemopreventive activities of a carotenoids-overexpressing transgenic carrot were evaluated. The results of DPPH,hydroxyl, and superoxide radical scavenging tests demonstrate that the acetone extract obtained from the taproots of thecarrot plants exhibits significant antioxidant activity. A higher activity was detected in the transgenic carrot extract comparedwith the wild-type extract. A chemopreventive activity test for degenerative diseases of the eye revealed that pretreatmentwith the carrot extract reduced cell death in a retinal ganglion cell line, RGC-5 cells exposed to 1-buthionine-(R,S)-sulfoximine and L-glutamic acid.

Investigation of Chemical Compounds, Antioxidant and Antimicrobial Properties of Teucrium arduini L. (Lamiaceae) by Dario Kremer, Iztok Joze Kosir, Ivan Kosalec, Marijana Zovko Koncic, Tanja Potocnik, Andreja Cerenak, Nada Bezic, Sinisa Srecec, Valerija Dunkic (1006-1014).
In this paper chemical composition of the essential oil (analysed by GC and GC-MS), the content of phenoliccompounds (analysed by HPLC), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer),antioxidant and antimicrobial activities of ethanolic extracts were investigated in endemic Teucrium arduini L. in populationof Mt Biokovo (Croatia). The oil was characterized by a high concentration of sesquiterpene hydrocarbons (70.4%) ofwhich β-caryophyllene (35.2%) and germacrene D (18.7%) being the major compounds. Three phenolic compounds(quercetin, ferulic acid and rosmarinic acid) were identified and quantified in ethanolic extract of T. arduini using HPLC.The results also showed that T. arduini is a source of polyphenolic and other antioxidants with radical-scavenging andchelating properties. The ethanol extracts prepared from the leaf of T. arduini showed broad spectrum of antimicrobial activityon Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicansand Aspergillus brasiliensis, which are susceptible on concentration below or equal to 4.00 mg/mL, whilst Microsporumgypseum was resistant at investigated concentrations.

Photodynamic Inactivation of Yeast and Bacteria by Extracts of Alternanthera brasiliana by Nathalia L. Andreazza, Caroline C. de Lourenco, Carlos A. T. Siqueira, Alexandra C.H.F. Sawaya, Tadia F. Lapinski, Adriana Gasparetto, Sonia Khouri, Stella R. Zamuner, Egberto Munin, Marcos Jose Salvador (1015-1022).
This study was undertaken to evaluate the effect of Alternathera brasiliana (Amaranthaceae) extracts as photosensitizingagents in photodynamic antimicrobial therapies (PACT) against Staphylococcus aureus, Staphylococcus epidermidisand Candida dubliniensis. The crude hexane and ethanol extracts were obtained from A. brasiliana whole plantand showed absortion from 650 to 700nm. Also, singlet molecular oxygen (1O2) production (type II photosensitization reaction)was examined, and the results show that 1,3-diphenylisobenzofuran photodegradation was greatly enhanced in thepresence of the A. brasiliana extracts. One plate in each assay was irradiated while the other was not irradiated, the numberof colony-forming units per milliliter (CFU/mL) was obtained, and data analyzed by the Tukey test. The chemicalcomposition of the extracts was determined by chromatographic and spectrometric techniques; steroids, triterpenes, andflavonoids were identified. Laser irradiation alone at 685nm using diode laser, output power of 35mW, and energy of 28J/cm2, or non-irradiated crude extracts in sub-inhibitory concentration did not reduce the number of CFU/mL significantly,whereas irradiated hexane and ethanol extracts, in sub-inhibitory concentrations, inhibited the growth of these microorganisms.The photoactivation of hexane and ethanol extracts of A. brasiliana, in sub-inhibitory concentrations, usingred laser radiation at 685nm had an antimicrobial effect.

Application of Pyrolysis-Gas Chromatography-Mass Spectrometry and Multivariate Analysis to Study Bacteria and Fungi in Biofilms Used for Bioremediation by Dora Melucci, Stefano Fedi, Marcello Locatelli, Clinio Locatelli, Simona Montalbani, Martina Cappelletti (1023-1033).
Biofilms are communities of microorganisms adhering to a surface and embedded in an extracellular polymericmatrix, frequently associated with disease and contamination, and also used for engineering applications such as bioremediation.A mixed biofilm formed by bacteria and fungi may provide an optimal habitat for addressing contaminated areas.To exploit the potential of natural microbial communities consisting of bacteria and fungi, it is essential to understand andcontrol their formation. In this work, a method to discriminate among bacteria of genera Bacillus, Pseudomonas,Rhodococcus with respect to the fungus Pleorotus in a biofilm by means of pyrolysis-gaschromatography-mass spectrometryand multivariate analysis is reported. Methylated fatty acids were chosen as biomarkers of microorganisms in thepyrolysates. In situ thermal hydrolysis and methylation was applied. Pyrograms were used as fingerprints, thus allowingfor the characterization of whole cells analyzed without any sample pretreatment. Normalized pyrographic peak areaswere chosen as variables for chemometric data processing. Principal components analysis was applied as a dataexplorationtool. Satisfactory results were obtained in analyzing a real biofilm. The influence of growth medium on wholebacteria fatty acid cell composition was also explored.

Advances in the Targeting Molecules Modified Chitosan-Based Nanoformulations by Hongliang Du, Xiaoqing Cai, Guangxi Zhai (1034-1052).
Chitosan, a cationic polysaccharide, has prompted the continuous impetus for the development of safe and effectivedrug delivery systems due to its unique properties such as mucoadhesive feature, absorption enhancement and activefunctional groups for chemical modifications. By using chitosan-based nanoformulations, many studies have attemptedto improve the dispersion of loaded hydrophobic drugs in aqueous environment, protect the encapsulated proteinsand genes against enzymatic degradation, and increase their absorption by target tissues. It?s noteworthy that the derivatizationof chitosan-based carriers with a ligand leads to the selective targeting of the nanoformulations to selected cells,thereby facilitating far more sensitive internalization and localization of nanoformulations for diseases? diagnosis andtreatment. As such, this review focuses on some of the most poignant reports of the utility of targeting molecules such ascarbohydrates, antibodies, peptides and some small molecules in chitosan-based nanoformulations for targeted delivery.Additionally, the affinity mechanism of different targeting molecules and the pros and cons of their conjugation strategieswill be illustrated summarily.

Differential Scanning Calorimetry as a Tool to Investigate the Transfer of Anticancer Drugs to Biomembrane Model by Maria Grazia Sarpietro, Maria Lorena Accolla, Christian Celia, Alessandro Grattoni, Francesco Castelli, Massimo Fresta, Mauro Ferrari, Donatella Paolino (1053-1060).
Different anticancer drugs clinically approved by international regulatory organizations present poor watersolubility and low stability after systemic injection. Their administration requires suitable carriers capable of maximizingtherapeutic efficacy. Lipid and polymeric nanotherapeutics, particularly liposomes, are widely used to deliverchemotherapeutics in the clinic. The interaction between chemotherapeutics and biocompatible lipids and polymers canaffect their efficacy and play a pivotal role in chemotherapy. Phospholipids are the main components of liposomes andtheir interactions with therapeutic agents are widely investigated in the pharmaceutical field using differential scanningcalorimetry (DSC). In this work, DSC was exploited to investigate the interaction between hydrophobic chemotherapeutics,i.e. docetaxel, tamoxifen and lapatinib, with lipid vesicles. Lipid carriers are prepared using dimyristoylphosphatidylcholine(DMPC), a phosphatidylcholine derivative, showing the same physicochemical features of the main lipids in thebiological membranes. DMPC was used as a biological membrane model to evaluate interaction, passage, diffusion, andadsorption of chemotherapeutics. These processes were evaluated through the variation of thermotropic parameters of thebiological membrane model. DSC studies were carried out in heating and cooling mode. Results demonstrated a modificationof calorimetric curves and this effect is strictly related to the molar fraction and physicochemical features ofchemotherapeutics. Furthermore, the interaction between chemotherapeutics and biological membranes affects theirinternalization and distribution inside tumors and this process depends on gel-liquid crystal transition of phospholipids.DSC results provide suitable information about this effect and can be used as tool to predict further interaction betweenchemotherapeutics and biological membranes.

Validated RP-HPLC Method for the Simultaneous Analysis of Gemcitabine and LY-364947 in Liposomal Formulations by Shyam S. Bansal, Christian Celia, Silvia Ferrati, Erika Zabre, Mauro Ferrari, Ganesh Palapattu, Alessandro Grattoni (1061-1069).
Combined use of gemcitabine (Gem) and LY-364947 (LY), a TGF-β1 receptor inhibitor, has shown promisefor the treatment of fibrotic pancreatic cancer, by reducing collagen production and improving tumor drug penetration.The preparation and optimization of novel Gem and LY formulations, including co-encapsulation in liposomes, require avalidated method for the simultaneous quantification of both drugs, a method that had yet to be developed. Here we demonstratean RP-HPLC protocol for the simultaneous detection of Gem and LY at 266 and 228 nm with retention times of3.37 and 11.34 mins, respectively. The method, which uses a C18 column and a KH2PO4 (10 mM)-methanol mobile phase,was validated for linearity, precision, accuracy, limits of detection, and robustness. Co-loaded liposomes with both Gemand LY (Gem/LY liposomes) were developed to investigate the protocol applicability to pharmacokinetic analysis andformulation characterization. The method specificity was evaluated in presence of liposomal components in fetal bovineserum (FBS). Finally, the method was demonstrated by quantifying Gem/LY liposomal encapsulation efficiency and concentrationliposomes-spiked FBS.

Niosomes Encapsulating Ibuprofen?Cyclodextrin Complexes: Preparation and Characterization by Carlotta Marianecci, Federica Rinaldi, Sara Esposito, Luisa Di Marzio, Maria Carafa (1070-1078).
A new delivery system based on ibuprofen-β-cyclodextrin (βCd) complexation and its loading into non?ionicsurfactant vesicles (NSVs) was developed to improve ibuprofen therapeutic efficacy in topical formulations. The proposedstrategy exploits the well known solubilizing and stabilizing properties of cyclodextrins together with the high tolerabilityand percutaneous absorption enhancing properties of NSVs. The complexing capacity of Cds in the presence of Ibuprofenin aqueous solution was evaluated by means of phase solubility studies. The technique used to obtain solid ibuprofen-βCdcomplexes was the co-lyophilization method. The influence of the preparation method on the physicochemical propertiesof the final product was evaluated by means of Fourier Transform Infrared Spectroscopy and Differential scanning calorimetrystudies. Ibuprofen-βCd complexes were included in Tween 20/Cholesterol vesicles and characterized in terms ofsize, zeta (ζ)-potential, stability, drug entrapment efficiency and drug release. The best ibuprofen-βCd-NSV system exhibitedin vitro drug permeation properties significantly improved with respect to those of the plain drug suspension.

As a society we are increasingly concerned about our physical appearance. For example, as much as 24% ofpeople in developed countries admittedly exercise to improve their performance. Professional sportsmen and amateursalike are in a constant search for new means that will enable them better sport results in shorter time. Among those means,a prominent place belongs to dietary supplements. However, the producers often advertise products whose use in sports isneither scientifically founded nor safe. This brings on an irrational use of herbal supplements which sometimes leads tounwanted side effects, but is more often of little use. Thus, the aim of this review will be to systematically evaluate someof the herbal supplements that are used as adaptogenic and ergogenic aids in sport. The review will include available dataon Rhodiola rosea, Withania somnifera, Schisandra chinensis, Tribulus terrestris, Vitis vinifera, Citrus aurantium, andothers. Their effects, active ingredients as well as possible adverse effects will be discussed with special focus on clinicalstudies.