First-in-human phase I clinical trial of a TLR4-binding DNA aptamer, ApTOLL: Safety and pharmacokinetics in healthy volunteers.

ApTOLL is an aptamer that antagonizes Toll-like receptor 4 and improves functional outcomes in models of ischemic stroke and myocardial infarction. The aim of this study was to characterize the safety and pharmacokinetics of ApTOLL in healthy volunteers. A first-in-human dose-ascending, randomized, placebo-controlled phase I clinical trial to assess safety and pharmacokinetics of ApTOLL (30-min infusion intravenously) was performed in 46 healthy adult male volunteers. The study was divided into two parts: part A included seven single ascending dose levels, and part B had one multiple dose cohort. Safety and pharmacokinetic parameters were evaluated. No serious adverse events or biochemistry alterations were detected at any dose nor at any administration pattern studied. Maximum concentration was detected at the end of the infusion and mean half-life was 9.3 h. Interestingly, exposure increased in the first four levels receiving doses from 0.7 mg to 14 mg (AUC of 2,441.26 h∗ng/mL to 23,371.11 h∗ng/mL) but remained stable thereafter (mean of 23,184.61 h∗ng/mL after 70 mg). Consequently, the multiple dose study did not show any accumulation of ApTOLL. These results show an excellent safety and adequate pharmacokinetic profile that, together with the efficacy demonstrated in nonclinical studies, provide the basis to start clinical trials in patients.

Postharvest sour rot control in lemon fruit by natamycin and an Allium extract.

Citrus sour rot caused by Geotrichum citri-aurantii is one of the most important postharvest diseases in citrus fruit, causing huge economic losses. Traditionally, it has been controlled by the postharvest application of guazatine and propiconazole fungicides, but restrictions in their use make it urgent to find an alternative for sour rot management. Natamycin, a common food preservative, and the organosulfuric compounds extracted from Allium species are safe food additives that control different foodborne pathogens. In the present study, the curative activities of commercial formulations of natamycin (Fruitgard Nat 20) and an Allium extract (PTSO: propyl thiosulfinate oxide; Proallium FRD®), were evaluated for the control of G. citri-aurantii in artificially inoculated lemon fruit. Trials in laboratory and in commercial conditions were carried out to explore the feasibility of including both compounds as part of a safe postharvest sour rot disease control strategy. Under controlled laboratory conditions, sour rot was significatively reduced by 500 mg L-1 of natamycin, 580 mL L-1 of PTSO and 290 mL L-1 of PTSO + 4% of a food coat, applied by immersion. Nevertheless, the maximum dose of PTSO (580 mL L-1) caused phytotoxicity on the fruit rind. In commercial drenching conditions, 290 mL L-1 of PTSO + 4% of a food coat reduced sour rot incidence similar to conventional treatment. In a packing line treatment, spray application of 500 mg L-1 of natamycin with a previous dip in sodium bicarbonate, resulted in nearly 70% reduction of disease incidence compared to conventional salt application. A second commercial experiment revealed that fruit drenching with 290 mL L-1 of PTSO + 4% food coat followed by an in-line cascade application of 500 mg L-1 of natamycin is completely effective for sour rot control after 20 days at 5 °C. Further exposure at room temperature for 7 d showed a 61% reduction in sour rot incidence compared to the control. Results revealed that natamycin and PTSO are promising tools for sour rot control used alone or combined as part of an integrated postharvest strategy.

FPR2 DNA Aptamers for Targeted Therapy of Wound Repair.

Chronic wounds represent a major health problem worldwide. Some of the available therapies based on recombinant proteins usually fail owing to the hostile environment found at the wound bed. Aptamers appear as an attractive alternative to recombinant factors owing in part to their stability, sensitivity, specificity, and low-cost production. In this study, the Cell-Systematic Evolution of Ligands by EXponential Enrichment technology was employed to generate aptamers that specifically recognize and modulate the function of the FPR2, a receptor expressed in a variety of cells involved in wound repair. Three aptamers were obtained that specifically bound to FPR2 stable transfectants generated in HaCaT cells. The targeted aptamers were shown to act as FPR2 agonists in different in vitro functional assays, including wound healing assays, and elicited a similar pattern of response to that obtained with other known FPR2 peptide agonists, such as the human LL37 cathelicidin. We have also obtained in vivo evidence for the prohealing activities of one of these FPR2 aptamers in a skin-humanized mouse model developed by us, previously shown to accurately recreate the main phases of physiological human wound repair process. In conclusion, we provide evidence of the potential therapeutic value of FPR2 aptamers for cutaneous repair.

Anti-HIV Aptamers: Challenges and Prospects.

Human Immunodeficiency Virus (HIV) infection continues to be a significant health burden in many countries around the world. Current HIV treatment through a combination of different antiretroviral drugs (cART) effectively suppresses viral replication, but drug resistance and crossresistance are significant challenges. This has prompted the search for novel targets and agents, such as nucleic acid aptamers. Nucleic acid aptamers are oligonucleotides that attach to the target sites with high affinity and specificity. This review provides a target-by-target account of research into anti-HIV aptamers and summarises the challenges and prospects of this therapeutic strategy, specifically in the unique context of HIV infection.

DNA aptamers targeting Leishmania infantum H3 protein as potential diagnostic tools.

Leishmaniasis is a disease caused by a parasite of the genus Leishmania that affects millions of people worldwide. These parasites are characterized by the presence of a DNA-containing granule, the kinetoplastid, located in the single mitochondrion at the base of the cell's flagellum. Interestingly, these flagellates do not condense chromatin during mitosis, possibly due to the specific molecular features of their histones. Although histones are extremely conserved proteins, kinetoplastid core histone sequences diverge significantly from those of higher eukaryotes. This divergence makes kinetoplastid core histones potential diagnostic and/or therapeutic targets. Aptamers are short single-stranded nucleic acids that are able to recognize target molecules with high affinity and specificity. Their binding capacity is a consequence of the particular three-dimensional structure acquired depending on their sequence. These molecules are currently used for detection, diagnosis and therapeutic purpose. Starting from a previously obtained ssDNA aptamer population against rLiH3 protein we have isolated two individual aptamers, AptLiH3#4 and AptLiH3#10. Next, we have performed ELONA, Western blot and slot blot assays to establish aptamer specificity and affinity for LiH3 histone. In addition, ELONA assays using peptides corresponding to overlapped sequences of LiH3 were made to map the aptamers:LiH3 interaction. Finally, different assays using aptamers were performed in order to evaluate the possibility of using these aptamers as sensing molecule to recognize the endogenous protein LiH3. Our results indicate that both aptamers have high affinity and specificity for the target and are able to detect the endogenous LiH3 histone protein in promastigotes lysates. In silico analysis reveals that these two aptamers have different potential secondary structure among them, however, both of them are able to recognize the same peptide sequences present in the protein. In conclusion, our findings indicate that these aptamers could be used for LiH3 histone detection and, in consequence, as potential biosensing molecules in a diagnostic tool for leishmaniasis.

TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Novel DNA Aptamers Against CCL21 Protein: Characterization and Biomedical Applications for Targeted Drug Delivery to T Cell-Rich Zones.

The chemokine (C-C motif) ligand 21 (CCL21) is a cytokine that attracts CCR7-positive cells to the T cell (paracortical) zone of lymph nodes by directional migration of these cells along the CCL21 gradient. In this article, we sought to mimic this chemotactic mechanism, by identifying a novel aptamer that binds CCL21 with high affinity. In vitro selection of DNA aptamers was performed by the Systematic Evolution of Ligands by Exponential Enrichment. Quantitative polymerase chain reaction (qPCR) and enzyme-linked oligonucleotide assay were used to screen for high-affinity aptamers against human and mouse CCL21 protein, respectively. Three such aptamers were identified. Surface plasmon resonance showed equilibrium dissociation constant (Kd) for these three aptamers in the nano to picomolar range. Cytotoxicity assays showed <10% toxicity in HEK293 and HL-60 cells. Last, in vivo biodistribution was successfully performed and CCL21 chemokine-binding aptamers were quantified within the draining lymph nodes and spleen using qPCR. Fluorescence microscopy revealed that one of the aptamers showed significantly higher presence in the paracortex than the control aptamer. The use of anti-CCL21 aptamers to mimic the chemotaxis mechanism thus represents a promising approach to achieve targeted delivery of drugs to the T cell-rich zones of the lymph node. This may be important for the treatment of HIV infection and the eradication of HIV reservoirs.

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses.

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers' properties as a real tool for viral infection detection and treatment.

Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells.

Elevated expression levels of eukaryotic initiation factor 4E (eIF4E) promote cancer development and progression. MAP kinase interacting kinases (MNKs) modulate the function of eIF4E through the phosphorylation that is necessary for oncogenic transformation. Therefore, pharmacologic MNK inhibitors may provide a nontoxic and effective anticancer strategy. MNK1b is a truncated isoform of MNK1a that is active in the absence of stimuli. Using in vitro selection, high-affinity DNA aptamers to MNK1b were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Four groups of aptamers were identified, and the affinities of one representative for rMNK1b were determined using ELONA and quantitative polymerase chain reaction. Two aptamers, named apMNK2F and apMNK3R, had a lower Kd in the nmol/l range. The secondary structure of the selected aptamers was predicted using mFold, and the QGRS Mapper indicated the presence of potential G-quadruplex structures in both aptamers. The selected aptamers were highly specific against MNK1, showing higher affinity to MNK1b than to MNK1a. Interestingly, both aptamers were able to produce significant translation inhibition and prevent tumor cell proliferation and migration and colony formation in breast cancer cells. These results indicate that MNK1 aptamers have an attractive therapeutic potential.

Tamoxifen therapy in breast cancer: do apolipoprotein E genotype and menopausal state affect plasma lipid changes induced by the drug?

OBJECTIVE: This study examines the lipid profile change produced in response to tamoxifen (TAM) treatment, and its possible relationship with both apolipoprotein E genotype and menopausal state in patients with breast cancer. METHODS: Blood samples were collected from 86 Spanish women with breast cancer before initiating TAM treatment and in the following 6, 12 and 18 months of treatment. Plasma lipid levels (total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol) were determined using an automatic analyzer. Genotypes for apolipoprotein E (ApoE) were identified by PCR-RFLP using the HhaI enzyme. RESULTS: In all patients, significant reductions in total cholesterol and LDL-cholesterol concentrations and a significant increase in triglyceride concentrations were observed after 6, 12, and 18 months of TAM treatment compared to baseline (p<0.01 for each time point). In the subset of APOE4-negative patients, triglyceride concentrations also significantly increased after 6, 12, and 18 months of treatment (p=0.019, p=0.045, p=0.001, respectively), while APOE4-positive patients showed no significant lipid changes at 12 and 18 months. However, after 18 months of TAM treatment the overall triglyceride concentrations had risen by 24.75% in APOE4-negative patients vs 29.9% in APOE4-positive patients. In postmenopausal women, significant reductions in total cholesterol, LDL-cholesterol and LDL/HDL ratios were observed at each time point (p<0.020 for each). CONCLUSIONS: TAM treatment induced similar plasma triglyceride increases in patients with positive or negative APOE genotype. Compared to premenopausal patients, postmenopausal breast cancer patients showed a more beneficial lipid profile change in response to treatment.

Control of HIV-1 RNA load after HAART interruption: relationship with CCR5 co-receptor density and proviral DNA load in HIV-infected patients.

BACKGROUND: CCR5 co-receptor density has been reported to play a role in the level of HIV production. In addition, reports about the relationship between proviral DNA load and plasma HIV load are controversial. OBJECTIVES: To analyse the role of CCR5 co-receptor density and proviral DNA load in the control of plasma HIV-viral load after HAART interruption, comparing patients whose plasma HIV load was persistently below 4log(10) RNA copies/mL, defined as "HIV controllers", with patients who showed a viral load higher than 4log(10) RNA copies/mL, defined as "non-controllers". STUDY DESIGN: Proviral DNA load quantification (N=55) and CCR5 co-receptor density (N=29) were determined in HIV-infected patients on prolonged HAART interruption. RESULTS: Twenty-three percent of our HAART interruption cohort were classified as HIV controllers, while 77% were classified as non-controllers. CCR5 co-receptor density was statistically higher in HIV controllers than in non-controllers, while proviral DNA load was not different between them. CCR5 co-receptor density in activated CD4 cells was independently associated with HIV plasma load after interruption. CONCLUSIONS: The observation of a higher CCR5 co-receptor expression in HIV controllers suggests that HIV infection leads to the selection of CD4 cells with low CCR5 co-receptor density after HAART interruption.

Histone deacetylase 6 regulates human immunodeficiency virus type 1 infection.

Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical alpha-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated alpha-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of alpha-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.

Prevalence and factors involved in discordant responses to highly active antiretroviral treatment in a closely followed cohort of treatment-naïve HIV-infected patients.

The prevalence and the factors involved in discordant responses to highly active antiretroviral therapy were analysed in a closely followed cohort of 51 naïve HIV-infected patients at 48 weeks. A complete treatment response was considered as an increase in CD4 cell count of >or=50 cells/mm3 with a >or=1 log10 decrease in viral load or viral suppression. Virologic response (<50 CD4+ cells/mm3 increase) and immune response (<1 log10 decrease in viral load) were observed in 15.7% and 5.8% of the patients, respectively. We demonstrated that the prevalence of virologic response decreased at week 72 and disappeared after 96 weeks of treatment. This slower response did not correlate with protease inhibitor-based (PI-based) regimens or HCV coinfection. On the other hand, immune response in our cohort could be easily attributable to a simple mechanism, i.e. irregular treatment compliance.

Benzo[f]azino[2,1-a]phthalazinium cations: novel DNA intercalating chromophores with antiproliferative activity.

New azaquinolizinium-type cations have been obtained from isochromane. The synthesis was completed over seven steps and included as the key feature an intramolecular Westphal condensation. This first example of the intramolecular process allowed the preparation of benzo[f]pyrido[2,1-a]phthalazinium and benzo[f]quino[2,1-a]phthalazinium salts, which were evaluated as DNA intercalators, DNA topoisomerase I inhibitors, and antiproliferative compounds. Both cationic systems behave as DNA intercalators and exhibit antiproliferative activity. The pentacyclic benzo[f]quino[2,1-a]phthalazinium cations also have an inhibitory effect on the catalytic activity of DNA topoisomerase I, without trapping of cleavage complexes. Structural characterization using density functional theory indicates that the fused ring systems are slightly nonplanar, and additional molecular modeling studies suggest a preferred orientation for the intercalating chromophores within a typical CpG or TpG intercalation site.

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process.

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.