27 research outputs found

    Interference with NF-kappaB binding to the MGMT-NFkB1 site using LODN (locked modified ODN).

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    <p>The HEK293T cell line was transiently transfected with either the kB1-MGMT-luc or pNF-kB-Luc reporter gene construct together with NFkappaB/p65. LODN corresponding to the MGMT-kB1 site were added at the indicated concentrations. The CMVβ-galactosidase expression vector (CMVβgal) was included in each transfection to normalize the transfection efficiency. The observed enhancer activity is relative to the corresponding reporter plasmid transfected with NFkappaB/p65. All concentrations of MGMT-kB1 LODN significantly reduced the levels of luciferase expression <i>(p<0.05)</i> compared with cells transfected with control ODN. The control bars indicate the average inhibition of three different concentrations of ODN as indicated for MGMT-kB1 LODN. An asterisk indicates a significant difference of <i>p<0.05</i> and a double asterisk indicates <i>p<0.01</i>.</p

    <i>In-vivo</i> efficacy of the compound drug (kB1-MGMT-LODN and the carrier) with or without temozolomide in A375P human melanoma xenografts.

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    <p>Athymic nude mice were inoculated subcutaneously with 5*10<sup>6</sup> tumor cells (A375P) and randomized into treatment groups. (A) a single dose of IP treatment (indicated by red arrows) was administered as follows: 10% DMSO (control) or TMZ at a dose of 100 mg/kg or 200 mg/Kg or 300 mg/kg. (B) Treatment started on day 5 when the tumors grew to an approximate size of 75 mm<sup>3</sup>. Mice were injected IL (indicated by black arrows) with 25 ug of MGMT-kB1 LODN or with vehicle (5% glucose) on days 5 and 7. On day 6 IP treatments (red arrow) were given with either 100 mg/kg TMZ or with vehicle (10% DMSO). An asterisk indicates a significant difference <i>(p<0.05)</i> between the treated and control group (10% DMSO). Each point represents the average tumor size ± SEM.</p

    The activity induced by the NF-kappaB sites within the MGMT enhancer and their corresponding mutant sites as measured by luciferase fold induction.

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    <p>The HEK293T cell line was transiently transfected with a reporter gene construct either alone or with other plasmids as indicated on the graph. The CMVβ-galactosidase expression vector (CMVβgal) was included in each transfection to normalize the transfection efficiency. The observed enhancer activity is relative to the corresponding reporter plasmid transfected alone. An asterisk indicates a significant difference <i>(p<0.05)</i> compared with the control (reporter plasmid transfected alone).</p

    <i>In-vivo</i> efficacy of long-term repetitive intralesional administration of the compound drug (kB1-MGMT-LODN and the carrier) in A375P human melanoma xenografts.

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    <p>Kaplan Meier survival curve of mice bearing A375P subcutaneous xenograft. IL treatment with either 25 ug of MGMT-kB1-LODN or with control ODN or with vehicle (5% glucose) was started once tumor volume approached 75 mm<sup>3</sup> (on day 6 post inoculation) and treatment was repeated every 4 to 5 days for up to 55 days after tumor induction.</p

    The cytotoxic efficacy of MGMT-kB1-LODN treatment given either in sequence with Temozolomide or as a monotherapy in three tumor cell lines.

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    <p>The effect of combination treatment with MGMT-kB1-decoy LMODNs and TMZ in three tumor cell lines (A) T98G, (B) U87MG and (C) A375P. The cells were transfected with the indicated concentrations of MGMT-kB1-LODN, and 3 hrs later were treated with the indicated doses of TMZ. The percentage of cell survival was evaluated 72 hrs later. (D) The effect of MGMT-kB1-LODN as a monotherapy. Each point represents the average viability percentage ± SEM. (A, B) An asterisk indicates a significant difference of <i>p<0.05</i> and double asterisk indicates <i>p<0.01</i>, between cells treated with MGMT-kB1-LODN and untreated cells. (D) The results are expressed as percentage of cell survival compared with cells treated with control ODN.</p

    Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases

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    <div><p>The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs’ unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a “water-soluble” amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug’s therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.</p></div

    Comparison of passively targeted NSSL and actively targeted peptide-conjugated NSSL.

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    <p><b>(A)</b> Representative fluorescent microscopy images comparing brain accumulation of NSSL and their payload as is (A, A1), β-amyloid NSSL(B,B1), and ApoE NSSL (C,C1) in healthy mice brain showing an increase in the amount of actively targeted NSSL and their payload accumulating, compared to passively targeted NSSL. <b>(B)</b> Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide-conjugated NSSL-MPS in the acute EAE mice model. SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), NSSL-MPS (●), Apo-E NSSL-MPS (▲) or β-amyloid NSSL-MPS (<b>■</b>). * p-value < 0.0001.</p

    Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.

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    <p><sup>a</sup> Significant difference from the control group P<0.0001</p><p><sup>b</sup> Significant difference from the EPC NSSL-TMN treated group P<0.001.</p><p>Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.</p

    Small angle X-ray scattering (SAXS) measurements of NSSL-TMN.

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    <p><b>(A)</b> Radially integrated background-subtracted scattering data (symbols) of DMPC:DPPC NSSL with and without drug, at 4 and 37°C, as indicated in the figure. Note that the curves are shifted in the intensity axis only for clarity of presentation. The solid curves are the corresponding form-factor models of a stack of infinite slabs with a Gaussian electron density profile along the vertical direction. <b>(B)</b> The electron density profiles of the DMPC:DPPC NSSL bilayers (with and without drug at 4 and 37°C) along the normal direction. The density profiles are obtained by fitting the scattering data to the models (see A) with the software X+, choosing a Gaussian electron density profile for the liposome membrane [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130442#pone.0130442.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130442#pone.0130442.ref041" target="_blank">41</a>]. The profile is almost symmetric and very slightly affected by the temperature or the presence of the drug. The arrows point to the profile of the inner and outer PEG layers. <b>(C)</b> The integrated scattering patterns as a function of the magnitude of the scattering vector, q, for EPC liposomes. Note that the curves are shifted in the intensity axis for clarity of presentation. The scattering curves of the EPC NSSL with and without drug, at 4 and 37°C are very similar. These curves are analyzed using the software X+, as in (A). The liposome bilayer is described by a Gaussian electron density profile. <b>(D)</b> The electron density profile in the direction normal to the membrane, calculated using the software X+, is presented for EPC NSSL, with and without drug at 4 and 37°C. The density profile of the membrane is almost unaffected by the temperature or the presence of the drug. Notice that this profile is asymmetric, suggesting that the inner and the outer PEG layers (pointed by an arrow) of the liposome are different.</p

    Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases - Fig 1

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    <p><b>(A)</b> DSC measurements. Samples of SUVs (DMPC:DPPC:Chol:PEG-DSPE, DMPC:DPPC:PEG-DSPE, DMPC:DPPC, DMPC:PEG-DSPE, DPPC:PEG-DSPE, DMPC, DPPC) in saline, and saline in the reference cell, were scanned in the range 10°-80°C, at the heating rate of 1°C/min. <b>(B)</b> Zooming in: Samples of SUVs DMPC:DPPC:Chol:PEG-DSPE, DMPC:DPPC:PEG-DSPE.</p
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