Brain metastasis models: What should we aim to achieve better treatments?

Brain metastasis is emerging as a unique entity in oncology based on its particular biology and, consequently, the pharmacological approaches that should be considered. We discuss the current state of modelling this specific progression of cancer and how these experimental models have been used to test multiple pharmacologic strategies over the years. In spite of pre-clinical evidences demonstrating brain metastasis vulnerabilities, many clinical trials have excluded patients with brain metastasis. Fortunately, this trend is getting to an end given the increasing importance of secondary brain tumors in the clinic and a better knowledge of the underlying biology. We discuss emerging trends and unsolved issues that will shape how we will study experimental brain metastasis in the years to come.

Severity of allergic rhinitis impacts sleep and anxiety: results from a large Spanish cohort.

BACKGROUND: Allergic rhinitis (AR) is a highly prevalent disease that generates high social and health care costs and also has a significant effect on quality of life and quality of sleep. It has also been related to some psychological disorders like anxiety or depression. OBJECTIVE: To evaluate anxiety, depression, and quality of sleep and life alteration in a group of patients with perennial AR compared to a group of seasonal AR patients. METHODS: Six-hundred seventy adults (> 18 years) with perennial and seasonal AR were recruited consecutively in 47 centers in Spain. Individuals were grouped in "Perennial" and "Seasonal" according to the seasonality of their symptoms. Anxiety, depression, sleep quality and health related quality of life were evaluated using the Hospital Anxiety and Depression Scale, Medical Outcomes Study Sleep Scale (MOS Sleep Scale) and the Health-related quality of life questionnaire ESPRINT-15, respectively. Both groups of patients were evaluated in and out of the pollen season. RESULTS: AR symptoms are related to worse quality of life and more anxiety and depression symptoms. Indeed, symptom severity also correlates with worse outcomes (quality of life, sleep and depression/anxiety) regardless allergen seasonality. Symptoms severity, compared with seasonality and persistence, is the most important factor related with more anxiety and depression and poor sleep. However, symptoms severity, persistence and seasonality are independently affecting the quality of life in patients with AR. CONCLUSIONS: Although AR symptoms have a great impact on depression and anxiety symptoms, quality of life and quality of sleep in all AR patients, as expected, individuals with more severe AR seem to suffer more intensely their effects.

Increased immune cell infiltration in patient-derived tumor explants treated with Traniplatin: an original Pt(iv) pro-drug based on Cisplatin and Tranilast.

Elevated intra-tumoral immune infiltrate is associated with an improved prognosis in cancer of distinct origins. Traniplatin (TPT) is a novel platinum(iv) pro-drug based on Cisplatin (CDDP) and the marketed drug Tranilast. When compared in vitro to Cisplatin, TPT showed increased cytotoxic activity against colon and lung cancer cells but decreased activity against immune cells. In addition, TPT efficiency was evaluated in tumor explants derived from colorectal cancer samples from patients subjected to intended curative surgery. TPT induced strong intra-tumoral cytotoxic activity yet was associated with an elevated presence of immune cell infiltrate, suggesting a reduced cytotoxic activity against immune cells in colorectal cancer.

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Using peptides to increase transport across the intestinal barrier.

The oral route is the preferred for the administration of drugs; however, it has some serious limitations. One of the main disadvantages is poor permeability across the intestinal barrier. Various approaches are currently being adopted to overcome this issue. In this review, we describe the alternatives that use peptides to enhance intestinal absorption. First, we define the various sources of peptide enhancers followed by the analysis of the absorption mechanism used. We then comment on the possible toxic effects derived from their use as permeation enhancers, as well as potential formulation strategies. Finally, the advantages and drawbacks of peptides as intestinal enhancers are examined.

Update of peptides with antibacterial activity.

For many years a battle has been going on between bacteria and humans, with bacteria trying to survive against the antibiotics used by humans. Bacteria are found to be dominant in this battle since they can develop resistance. In fact, in the last decade multi-, extended- and pan-drug resistant bacteria have been isolated. On the other hand, the number of new antibiotics approved by the FDA has dramatically decreased during the last 20 years. Therefore, there is a desperate need for developing new types of antibacterial agents, where antimicrobial peptides may play an important role. This review provides an update of the recently identified antimicrobial peptides. Three valid approaches for developing a future antibacterial agent, as are the mechanisms of action as well as the in vitro and in vivo assays have been described in depth. In comparison to the antibacterial agents available at present, the targets for most of the antimicrobial peptides are not well known. However several proposals having been introduced for many antimicrobial peptides of different mechanisms of action, there still lies some uncertainty about their utility. Hundreds of antimicrobial peptides have been tested in vitro against all types of bacteria, but in this review we will highlight only those which have been tested against the most important Gram-positive and Gram-negative bacteria. The last step to get a potential antibiotic includes studies with an in vivo model. Therefore only antimicrobial peptides with good activity are tested that have been described in this review.

In vitro activity of several antimicrobial peptides against colistin-susceptible and colistin-resistant Acinetobacter baumannii.

At present, colistin is among the few antibiotics effective against Acinetobacter baumannii clinical isolates. However, in the last few years, colistin-resistant A. baumannii strains have been isolated. Therefore, antibiotics effective against these usually pan-resistant colistin-resistant A. baumannii strains are required. The main objective of this study was to analyse the activity of 15 peptides against colistin-susceptible and colistin-resistant A. baumannii. The MICs were determined by microdilution. Among these 15 antimicrobial peptides (AMPs), melittin, indolicidin and mastoparan showed good activity against both colistin-susceptible and colistin-resistant A. baumannii. Further studies of mastoparan with time-killing curves showed bactericidal activity at MIC ×8 for both colistin-susceptible and colistin-resistant A. baumannii. In conclusion, mastoparan may be a potential alternative for the treatment of colistin-resistant A. baumannii infections.

Serum cholesterol levels and survival after rtPA treatment in acute stroke.

BACKGROUND: According to the reverse epidemiology hypothesis, high cholesterol levels might be protective and associated with greater survival rates under certain conditions. In stroke patients, a clear correlation between lipid levels and mortality after ischaemic and hemorrhagic strokes has been demonstrated. The aim of this study was to analyze the impact of lipid levels on 3-month mortality in patients with ischaemic stroke (IS) homogeneously treated with intravenous rtPA and admitted to a monitored acute stroke unit. METHODS: Retrospective analysis of a prospective cohort of 220 patients with an IS treated with rtPA within the first 4.5 h in a single tertiary hospital from January 2005 to August 2010. RESULTS: Mortality at 3 months was 15.0%. Univariate analysis showed that age, NIHSS at admission, heart failure, and atrial fibrillation were directly related to 3-month mortality; cholesterol, triglycerides, and low density lipoprotein were inversely associated. The death rate by cholesterol level was 5.5% for the highest tertile (>192 mg/dl), 13.7% for the middle (192-155 mg/dl), and 25.7% for the lowest (<155 mg/dl), P = 0.003. Multivariate analysis showed that amongst the lipid determinations, only cholesterol [OR: 0.985 (95% CI: 0.972-0.998), P = 0.021] was inversely associated with 3-month mortality. The 'protective' effect of cholesterol was independent of stroke severity and remained significant in non-lacunar strokes. CONCLUSIONS: Survival of stroke patients receiving current, most effective medical treatment is related to blood cholesterol levels, with an inverse relationship between cholesterol and mortality. The mechanism of this apparently paradoxical situation remains unexplained but merits further research.

Eighteen month study of continuous intraduodenal levodopa infusion in patients with advanced Parkinson's disease: impact on control of fluctuations and quality of life.

Symptom control, daily "on" time, and quality of life (QoL) of nine patients with Advanced Parkinson's Disease was assessed following 18-months treatment with Continuous Intraduodenal Levodopa Infusion (CIDLI). Patients had severe motor fluctuations and dyskinesias and had previously received treatment with oral levodopa and dopamine agonists. There were significant improvements in patients' symptoms on the Unified Parkinson's Disease Rating Scale, and QoL (Parkinson's Disease QoL Questionnaire; Schwab & England Capacity for Daily Living Scale; p < 0.05). Mean (+/-SD) daily "on" time increased from 6.1 +/- 1.9 to 12.0 +/- 3.4 h (p < 0.05). Improved QoL in APD was associated with CIDLI-related improvements in symptom control and increase in daily "on" time.

A proline-rich peptide improves cell transfection of solid lipid nanoparticle-based non-viral vectors.

The aim of this work was to improve the transfection efficacy of solid lipid nanoparticle (SLN)-based non-viral vectors into ARPE-19 cells through the addition of Sweet Arrow Peptide (SAP). First, we prepared SAP-DNA complexes at ratios of at least 50:1, and then incorporated them into the SLNs. All formulations were able to protect DNA, and the peptide favoured the most bioactive form (supercoiled) of open circular DNA turns. In vitro transfection studies of the vectors containing the pCMS-EGFP plasmid in HEK293 and ARPE-19 cell lines revealed that incorporation of SAP led to greater transfection in both cell lines, although via different mechanisms. The presence of SAP in the formulations did not affect the viability of HEK293 or ARPE-19 cells. In HEK293 cells, SAP enabled greater uptake of the vectors, and an SAP to DNA ratio of 50:1 was sufficient for enhancing transfection. In contrast, in ARPE-19 cells, SAP induced a change in the dominant entrance mechanism, from clathrin endocytosis to caveolae/raft-dependent endocytosis, thereby decreasing use of the lysosomal pathway and consequently, reducing vector degradation. The extent to which SAP uses one mechanism or the other largely depends on its concentration in the formulation.

all-D proline-rich cell-penetrating peptides: a preliminary in vivo internalization study.

Proline-rich cell-penetrating peptides, particularly the SAP (sweet arrow peptide), (VRLPPP)(3), have been proposed to be useful intracellular delivery vectors, as a result of their lack of cytotoxicity combined with their capacity to be internalized by cells. A common limitation of the therapeutic use of peptides is metabolic instability. In general, peptides are quickly degraded by proteases upon entry into the bloodstream. The use of all-D-peptide derivatives is emerging as a fruitful strategy to circumvent this degradation problem. In this context, we report on the internalization behaviour, protease-resistance enhancement and self-assembly properties of an all-D version of SAP [(vrlppp)(3)]. The cellular uptake of (vrlppp)(3) was evaluated in an in vivo assay in mice. Both flow cytometry and confocal laser-scanning microscopy experiments showed that a carboxyfluoresceinated version of the molecule, carboxyfluorescein-(vrlppp)(3), is internalized rapidly in white blood cells and kidney cells. Significant fluorescence was also detected in other organs such as the spleen and the liver. Finally, the toxicity of (vrlppp)(3) was examined, and no significant differences in the main biochemical parameters nor in weight were detected compared with controls.

Antibacterial evaluation of a collection of norfloxacin and ciprofloxacin derivatives against multiresistant bacteria.

The objective of this study was to analyse an array of ciprofloxacin and norfloxacin derivatives in order to determine those with good activity against bacteria that already present fluoroquinolone resistance associated with mutations in the gyrA and/or parC genes. Four norfloxacin and 20 ciprofloxacin derivatives were synthesised and tested against quinolone-susceptible and -resistant Escherichia coli, Acinetobacter baumannii, Stenotrophomonas maltophilia and Staphylococcus aureus strains using a microdilution test. Among the derivatives, the 4-methyl-7-piperazine ciprofloxacin derivative showed a minimum inhibitory concentration for 50% of the organisms that was 16- and 8-fold lower than ciprofloxacin for A. baumannii and S. maltophilia, respectively. When the methyl group at position 4 in the piperazine ring was substituted by ethyl, butyl or heptyl groups, activity against A. baumannii steadily decreased. The 7-(4-methyl)-piperazine ciprofloxacin derivative (UB-8902) showed very good activity against these multiresistant microorganisms including A. baumannii and S. maltophilia.

Abbreviated nomenclature for cyclic and branched homo- and hetero-detic peptides.

Amino acid sequences and linear or head-to-tail cyclopeptides can be represented conveniently in one-line text formulae using the three-letter symbols. However, other - but nonetheless important - topologies of peptides are 'side chain-to-head (or tail)', 'backbone-to-backbone', 'side chain-to-side chain' cyclopeptides, 'side chain-to-side chain' connected peptide strands, and branched peptides (like peptide dendrimers). In general, such structures cannot be described using the three-letter symbols in one-line text: a chemical structure editor is required for symbolic representations according to the IUPAC-IUBMB recommendations. The aim of this contribution is to offer an unambiguous and general nomenclature system that enables researchers to represent all cyclic and branched homo- and hetero-detic peptides in a coherent manner in one-line text - as long as their as constituents can be represented in (three)-letter codes. The application of this new nomenclature would overcome the existing difficulties and provide a way to express complex situations in the shortest way in order to highlight more clearly the salient points in a given scientific communication.

Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells.

In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.

AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells: interference with the Akt/NF-kappaB survival pathway.

Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 microM. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and Bisl inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-kappaB activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-kappaB-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancy.

Solid-phase synthesis and characterization of N-methyl-rich peptides.

A library of peptides required for a project investigating the factors relevant for blood-brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.

Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.

Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.

Probing degeneracy in antigen-antibody recognition at the immunodominant site of foot-and-mouth disease virus.

Antigen-antibody binding is regarded as one of the most representative examples of specific molecular recognition in nature. The simplistic view of antigenic recognition in terms of a lock-and-key mechanism is obsolete, as it is evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. This flexibility is the source of complexities such as degeneracy and nonadditivity in antigenic recognition. We have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic GH loop of foot-and-mouth disease virus. Our aim was 2-fold: to explore the extent to which antigenic degeneracy can be extended in this particular case, and to search for potential nonadditive effects in introducing multiple amino acid replacements. Combined analysis of one such multiply substituted peptide by SPR, solution NMR and X-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered.

Synthesis and structure determination of kahalalide F (1,2).

Kahalalide F, the only member of the family of peptides called kahalalides, isolated from the sacoglossan mollusc Elysia rufescens and the green alga Bryopsis sp., with important bioactivity, is in clinical trials for treatment of prostate cancer. An efficient solid-phase synthetic approach is reported. Kahalalide F presents several synthetic difficulties: (i) an ester bond between two beta-branched and sterically hindered amino acids; (ii) a didehydroamino acid; and (iii) a rather hydrophobic sequence with two fragments containing several beta-branched amino acids in a row, one of them terminated with a saturated aliphatic acid. The cornerstones of our strategy were (i) a quasiorthogonal protecting system with allyl, tert-butyl, fluorenyl, and trityl-based groups, (ii) azabenzotriazole coupling reagents, (iii) formation of the didehydroamino acid residue on the solid phase, and (iv) cyclization and final purification in solution. HPLC, high-field NMR, and biological activity studies showed that the correct stereochemistry of the natural product is that proposed by Rinehart et al. whereas the stereochemistry proposed by Scheuer et al. is that of a biologically less active diastereoisomer.

Solid-phase total synthesis of trunkamide A(1).

Marine organisms are a rich source of novel, biologically active compounds. Herein, the solid-phase total synthesis of trunkamide A, currently in preclinical trials, is presented. Trunkamide A contains a thiazoline heterocycle and two residues of Ser and Thr with the hydroxy function modified as reverse prenyl (rPr). Cornerstones of the synthesis are as follows: (i) solid-phase peptide chain elongation using a quasi-orthogonal protecting scheme with tert-butyl and fluorenyl based groups, on a chlorotrityl resin; (ii) concourse of HOAt-based coupling reagents; and (iii) cyclizations in solution. Furthermore, the following synthetic steps are discussed: (i) preparation of the reverse prenyl derivatives of Ser and Thr; (ii) introduction of precursor of thiazoline as a protected amino thionoacid derivative; and (iii) formation of the thiazoline ring with DAST. All these features make this strategy particularly suitable for the large-scale synthesis of trunkamide A and other peptides containing the same motifs.