14 research outputs found
Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function
Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes
Preprint: A drug repurposing screen for whipworms informed by comparative genomics
Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 μM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug repurposing against whipworms. Therefore, we recommend that prior to in vivo studies, future researchers first assess drug absorption by the host, for example, using human intestinal organoids or cell lines, and drug uptake by whipworms using intestinal organoids infected with T. muris
2,4-Diaminothieno[3,2 d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm
The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle
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A drug repurposing screen for whipworms informed by comparative genomics.
Hundreds of millions of people worldwide are infected with the whipworm Trichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested these ex vivo by assessing motility of adult worms of Trichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of ≤50 μM against T. muris ex vivo, and selected nine for testing in vivo. However, the best worm burden reduction seen in mice was just 19%. The high number of ex vivo hits against T. muris shows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug development against whipworms
Properties and activities of resynthesized diaminothienopyrimidines, and other anthelmintics.
<p>RMM: relative molecule mass. HBA: number of hydrogen bond acceptors. HBD: number of hydrogen bond donors. tPSA: topological polar surface area, calculated using DataWarrior [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref020" target="_blank">20</a>]. ROTB: number of rotatable bonds. <sup>a</sup> Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref015" target="_blank">15</a>]. <sup>b</sup> Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref021" target="_blank">21</a>]. <sup>c</sup> Data from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref012" target="_blank">12</a>].</p
Summary of the cytotoxicity in a mouse epithelial cell line of the DATP series.
<p>Mouse CMT-93 rectal epithelial cells were used for this assay. Maximum tested concentration was 100 μM. <i>n =</i> 8, error range (in parentheses) shows 95% confidence interval. EC<sub>50</sub> values in the adult <i>Trichuris</i> paralysis assay are shown for comparison.</p
Reduced worm burden in mice given <i>T</i>. <i>muris</i> eggs that had been treated with diaminothienopyrimidines.
<p><b>(a)</b> Embryonated eggs were soaked in compound for 14 days, washed in water and then used in either <i>in vitro</i> or <i>in vivo</i> hatching assays. <b>(b)</b> Treatment with DATPs reduced the ability of embryonated eggs to hatch in <i>E</i>. <i>coli</i> bacterial suspension after 24 hours. A one-way ANOVA showed a significant difference between treatment groups (F(5,26) = 25.95 p<0.0001) with a post-hoc Dunnett’s compared to DMSO control (**** = p<0.0001) <i>n =</i> 7 (DMSO), <i>n =</i> 5 (DATP compounds) <b>(c)</b> SCID mice were infected with 40 eggs and worm burden assessed at day 15 post infection. The experiment was carried out in two batches, with n = 5 and n = 9 mice respectively in each of the control and treatment groups. Data were normalised for each batch relative to the mean of the DMSO-only control group for that batch. Blue line indicates mean for each treatment group. A two-way ANOVA showed a significant effect of treatment [F(1,24) = 9.569, P = 0.00497] but no effect of batch [F(1,24) = 0.083, P = 0.77618] or interaction [F(1,24) = 0.083 0.77618]. A post-hoc Tukey HSD test showed that the <b>OX02926</b>-treated group was significantly different from the DMSO control group (P = 0.0050).</p
Identification of a diaminothienopyrimidine series from an <i>ex vivo T</i>. <i>muris</i> motility screen.
<p><b>(a)</b> Structure of the hit compound, which was given the identifier <b>OX02926</b>. <b>(b)</b> Hit expansion by testing of structurally-related compounds using library material, assay concentration 100<b>μ</b>M. Significance was determined by a two-sided Mann-Whitney test compared to DMSO-only controls, adjusted for multiple comparisons using the Bonferroni method (for test compounds n = 5, each replicate on different assay plates, each point indicates one assay well). Blue bar indicates mean movement score. <b>(c)</b> Structures and identifiers of additional active compounds from this class. <b>(d)</b> Structures and PubChem CID accession numbers for the two compounds that were not significantly active in this assay.</p
Concentration-response curves for resynthesized DATPs in the <i>T</i>. <i>muris ex vivo</i> adult motility assay.
<p>n = 4 or 5 wells per concentration per compound, each replicate on a different 96-well plate using worms from different mice. Blue line indicates concentration-response curve fitted with the 3-factor log-logistic model using <i>drc</i> [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006487#pntd.0006487.ref016" target="_blank">16</a>]. Figure in parenthesis indicates EC<sub>50</sub> estimate ± standard error from this model. <b>OX03143</b> did not clearly form a sigmoidal curve in the range of concentrations used in this assay so we report the EC50 estimate as > 80μM.</p