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Pharmaceuticals
Volume 14
Issue 7
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Pharmaceuticals, Volume 14, Issue 7 (July 2021) – 105 articles

Cover Story (view full-size image): Cell-Penetrating Peptides (CPPs) and nanobodies could combine into powerful targeting tools, if we optimize their synergy. Nanobodies are low molecular weight antigen-specific binders that show good tissue penetration. However, their fast clearance narrows the time window in which nanobodies distribute into the target tissue. CPPs could increase tissue accumulation of nanobodies, by enhancing their cellular uptake. Several CPPs enhance the uptake of the epidermal growth factor receptor-targeting nanobody 7D12 in monolayer cultures and cell spheroids. The physicochemical properties of each CPP influence those of the whole conjugate. Differences between CPPs suggest that future research should focus on understanding which and how physicochemical properties of CPP conjugates drive optimal biodistribution. View this paper
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19 pages, 2540 KiB  
Review
Recent Advances in the Clinical Translation of Silicon Fluoride Acceptor (SiFA) 18F-Radiopharmaceuticals
by Lexi Gower-Fry, Travis Kronemann, Andreas Dorian, Yinglan Pu, Carolin Jaworski, Carmen Wängler, Peter Bartenstein, Leonie Beyer, Simon Lindner, Klaus Jurkschat, Björn Wängler, Justin J. Bailey and Ralf Schirrmacher
Pharmaceuticals 2021, 14(7), 701; https://doi.org/10.3390/ph14070701 - 20 Jul 2021
Cited by 17 | Viewed by 4803
Abstract
The incorporation of silicon fluoride acceptor (SiFA) moieties into a variety of molecules, such as peptides, proteins and biologically relevant small molecules, has improved the generation of 18F-radiopharmaceuticals for medical imaging. The efficient isotopic exchange radiofluorination process, in combination with the enhanced [...] Read more.
The incorporation of silicon fluoride acceptor (SiFA) moieties into a variety of molecules, such as peptides, proteins and biologically relevant small molecules, has improved the generation of 18F-radiopharmaceuticals for medical imaging. The efficient isotopic exchange radiofluorination process, in combination with the enhanced [18F]SiFA in vivo stability, make it a suitable strategy for fluorine-18 incorporation. This review will highlight the clinical applicability of [18F]SiFA-labeled compounds and discuss the significant radiotracers currently in clinical use. Full article
(This article belongs to the Special Issue Targets, Tracers and Translation, Part 2 - New Horizons in Radiopharmaceutical Development)
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<p>General scheme for the radiofluorination of SiFA compounds. LG = leaving group.</p> Full article ">Figure 2
<p>General scheme showing the radiofluorination of a dialkylfluorosilane SiFA compound.</p> Full article ">Figure 3
<p>General methods for the introduction of SiFA building blocks to targeting vectors. TATE = Tyr<sup>3</sup>-octreotate; RGD = arginine-glycine-aspartate.</p> Full article ">Figure 4
<p>General SiFA building block synthetic scheme used for SiFA-peptide syntheses.</p> Full article ">Figure 5
<p>General structures of [<sup>68</sup>Ga]Ga-DOTA-TATE (Netspot<sup>®</sup>) and [<sup>68</sup>Ga]Ga-DOTA-TOC. TATE = Tyr<sup>3</sup>-octreotate; TOC = Tyr<sup>3</sup>-octreotide.</p> Full article ">Figure 6
<p>General structure of [<sup>18</sup>F]SiTATE. TATE = Tyr<sup>3</sup>-octreotate.</p> Full article ">Figure 7
<p>Comparison of (<b>A</b>) [<sup>68</sup>Ga]Ga-DOTA-TATE and (<b>B</b>) [<sup>18</sup>F]SiTATE in small animal PET/CT imaging with standardized uptake value (SUV) scale [<a href="#B37-pharmaceuticals-14-00701" class="html-bibr">37</a>]. Reprinted with permission from Ref. [<a href="#B37-pharmaceuticals-14-00701" class="html-bibr">37</a>]. Copyright 2015 SNMMI.</p> Full article ">Figure 8
<p>In-patient comparison of PET image quality between (<b>a</b>) [<sup>18</sup>F]SiTATE and (<b>b</b>) [<sup>68</sup>Ga]Ga-DOTA-TOC. Patient was a 39-year old female with Ileum NET. Comparison shows superior image quality with [<sup>18</sup>F]SiTATE, especially for small lesions. Reprinted with permission from Ref. [<a href="#B38-pharmaceuticals-14-00701" class="html-bibr">38</a>]. Copyright 2019 Springer Nature.</p> Full article ">Figure 9
<p>PET/CT comparison between [<sup>18</sup>F]SiTATE and [<sup>68</sup>Ga]Ga-DOTA-TOC in a patient with a falx meningioma with transosseous extension. Images show preferable resolution with [<sup>18</sup>F]SiTATE, therefore yielding a higher quality image. Reprinted with permission from Ref. [<a href="#B34-pharmaceuticals-14-00701" class="html-bibr">34</a>]. Copyright 2021 Wolters Kluwer Health, Inc.</p> Full article ">Figure 10
<p>General structure of [<sup>18</sup>F]rhPSMA-7.</p> Full article ">Figure 11
<p>General structure of [<sup>18</sup>F]SiFA bombesin derivative.</p> Full article ">Figure 12
<p>General structure of bivalent radioligand, [<sup>18</sup>F]BMPPSiF.</p> Full article ">
13 pages, 315 KiB  
Review
Monoclonal Antibodies Targeting CGRP: From Clinical Studies to Real-World Evidence—What Do We Know So Far?
by Theodoros Mavridis, Christina I. Deligianni, Georgios Karagiorgis, Ariadne Daponte, Marianthi Breza and Dimos D. Mitsikostas
Pharmaceuticals 2021, 14(7), 700; https://doi.org/10.3390/ph14070700 - 20 Jul 2021
Cited by 18 | Viewed by 8980
Abstract
Now more than ever is the time of monoclonal antibody use in neurology. In headaches, disease-specific and mechanism-based treatments existed only for symptomatic management of migraines (i.e., triptans), while the standard prophylactic anti-migraine treatments consist of non-specific and repurposed drugs that share limited [...] Read more.
Now more than ever is the time of monoclonal antibody use in neurology. In headaches, disease-specific and mechanism-based treatments existed only for symptomatic management of migraines (i.e., triptans), while the standard prophylactic anti-migraine treatments consist of non-specific and repurposed drugs that share limited safety profiles and high risk for interactions with other medications, resulting in rundown adherence rates. Recent advances in headache science have increased our understanding of the role of calcitonin gene relate peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) pathways in cephalic pain neurotransmission and peripheral or central sensitization, leading to the development of monoclonal antibodies (mAbs) or small molecules targeting these neuropeptides or their receptors. Large scale randomized clinical trials confirmed that inhibition of the CGRP system attenuates migraine, while the PACAP mediated nociception is still under scientific and clinical investigation. In this review, we provide the latest clinical evidence for the use of anti-CGRP in migraine prevention with emphasis on efficacy and safety outcomes from Phase III and real-world studies. Full article
(This article belongs to the Section Biopharmaceuticals)
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16 pages, 4435 KiB  
Article
Development of Machine Learning Models for Accurately Predicting and Ranking the Activity of Lead Molecules to Inhibit PRC2 Dependent Cancer
by Danishuddin, Vikas Kumar, Shraddha Parate, Ashutosh Bahuguna, Gihwan Lee, Myeong Ok Kim and Keun Woo Lee
Pharmaceuticals 2021, 14(7), 699; https://doi.org/10.3390/ph14070699 - 20 Jul 2021
Cited by 4 | Viewed by 4210
Abstract
Disruption of epigenetic processes to eradicate tumor cells is among the most promising interventions for cancer control. EZH2 (Enhancer of zeste homolog 2), a catalytic component of polycomb repressive complex 2 (PRC2), methylates lysine 27 of histone H3 to promote transcriptional silencing and [...] Read more.
Disruption of epigenetic processes to eradicate tumor cells is among the most promising interventions for cancer control. EZH2 (Enhancer of zeste homolog 2), a catalytic component of polycomb repressive complex 2 (PRC2), methylates lysine 27 of histone H3 to promote transcriptional silencing and is an important drug target for controlling cancer via epigenetic processes. In the present study, we have developed various predictive models for modeling the inhibitory activity of EZH2. Binary and multiclass models were built using SVM, random forest and XGBoost methods. Rigorous validation approaches including predictiveness curve, Y-randomization and applicability domain (AD) were employed for evaluation of the developed models. Eighteen descriptors selected from Boruta methods have been used for modeling. For binary classification, random forest and XGBoost achieved an accuracy of 0.80 and 0.82, respectively, on external test set. Contrastingly, for multiclass models, random forest and XGBoost achieved an accuracy of 0.73 and 0.75, respectively. 500 Y-randomization runs demonstrate that the models were robust and the correlations were not by chance. Evaluation metrics from predictiveness curve show that the selected eighteen descriptors predict active compounds with total gain (TG) of 0.79 and 0.59 for XGBoost and random forest, respectively. Validated models were further used for virtual screening and molecular docking in search of potential hits. A total of 221 compounds were commonly predicted as active with above the set probability threshold and also under the AD of training set. Molecular docking revealed that three compounds have reasonable binding energy and favorable interactions with critical residues in the active site of EZH2. In conclusion, we highlighted the potential of rigorously validated models for accurately predicting and ranking the activities of lead molecules against cancer epigenetic targets. The models presented in this study represent the platform for development of EZH2 inhibitors. Full article
(This article belongs to the Section Medicinal Chemistry)
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<p>A framework guiding the development and evaluation of a predictive model for EZH2. The stages are: (1) Data collection, (2) Data cleaning and threshold setting, (3) Development of models, (4) Validation of the model and analysis, (5) Screening of chemical libraries using the validated models.</p> Full article ">Figure 2
<p>ROC curve showing the performance of binary class models. (<b>A</b>) All descriptor set. (<b>B</b>) Selected descriptors set (Boruta method).</p> Full article ">Figure 3
<p>ROC curve showing the performance of multiclass models. (<b>A</b>–<b>C</b>) All descriptors set. (<b>D</b>–<b>F</b>) Selected descriptors (Boruta method).</p> Full article ">Figure 4
<p>Plot showing the chemical space of falsely predicted compounds and their Tc similarities. (<b>A</b>) Distribution of falsely predicted compounds from test set. (<b>B</b>) Density plots of Tc values using MACCS fingerprints. Compared were falsely predicted low and moderate vs. high activity compounds (Orange color), and falsely predicted high activity compounds vs. low and moderate activity compounds of training set (Blue color).</p> Full article ">Figure 5
<p>Box plot showing the frequencies of accuracies from Y-randomization models. (<b>A</b>) Binary class models. (<b>B</b>) Multi-class models. Total 500 Y-randomization runs were performed.</p> Full article ">Figure 6
<p>Applicability domain plot based on principal component analysis (PCA) using eighteen selected descriptors set. (<b>A</b>) Training set. (<b>B</b>) Test set.</p> Full article ">Figure 7
<p>Probabilistic distribution plots. (<b>A</b>) Cumulative gain plot for random forest (green line) and XGBoost (orange line) models (<b>B</b>) Predictiveness curves. Both the plots were plotted using the selected descriptor set.</p> Full article ">Figure 8
<p>Library screening result. (<b>A</b>) Chemical space of selected active hits. (<b>B</b>) Heatmap showing the Tanimoto similarities score (Tc) of selected compounds with high activity compounds from training set. MACCS fingerprints were used to calculate the Tanimoto coefficient similarities.</p> Full article ">Figure 9
<p>Binding analysis of top hits from molecular docking: (<b>A</b>) Reference compound (GSK126), (<b>B</b>) Hit1 (NCI694864), (<b>C</b>) Hit2 (NCI670557) and (<b>D</b>) Hit3 (NCI706726). The active site residues are shown in grey sticks. The protein backbone is shown in light grey wire. Hydrogen bonds were illustrated with green dashed line.</p> Full article ">Figure 10
<p>Two-dimensional schematic representations of protein–ligand interactions between GSK126 (reference compound) and the top three hits: (<b>A</b>) GSK126, (<b>B</b>) Hit1 (NCI694864), (<b>C</b>) Hit2 (NCI670557) and (<b>D</b>) Hit3(NCI706726). The hydrogen bonds, Л-Л, Л-cation, and Л-Sulphur are shown as green, pink and purple dashed lines, respectively.</p> Full article ">
15 pages, 2182 KiB  
Article
Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris
by Tina V. A. Hansen, Richard K. Grencis, Mohamed Issouf, Cédric Neveu and Claude L. Charvet
Pharmaceuticals 2021, 14(7), 698; https://doi.org/10.3390/ph14070698 - 20 Jul 2021
Cited by 5 | Viewed by 3768
Abstract
The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a [...] Read more.
The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species. Full article
(This article belongs to the Special Issue Antiparasitics)
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Graphical abstract
Full article ">Figure 1
<p>Amino-acid alignment of ACR-16-like and ACR-19 subunits from <span class="html-italic">T. muris, T. suis</span> and <span class="html-italic">T. trichiura</span> and the human α7 subunit Predicted signal peptide sequences are shaded in dark green, the Cys-loop, the transmembrane regions (TM1-TM4) and the YxCC motif is shown below the sequences. Conserved amino acids between ACR-16-like and ACR-19 subunit sequences are highlighted in black, conserved amino acids for ACR-16-like and ACR-19 are highlighted with red and green, respectively, nematode specific amino acids are highlighted in grey.</p> Full article ">Figure 2
<p>Effect of anthelmintics and cholinergic agonists on the <span class="html-italic">Tmu</span>-ACR-16-like receptor. A scatter dot plot (mean ± SEM) and representative sample traces show the rank order efficacy in μA of four cholinergic anthelmintics: oxantel (oxa), pyrantel (pyr), morantel (mor), levamisole (lev) and five nicotinic agonists: epibatidine (epi), anabasine (ana), dimethyl phenyl piperazine (DMPP), nicotine (nic) and cytosine (cyt). * <span class="html-italic">p</span> &lt; 0.02. The number of oocytes (<span class="html-italic">n</span>) used for each agonist is given above the traces, the perfusion time of each compound was 10 s as indicated with short bars above the traces.</p> Full article ">Figure 3
<p>Concentration-response relationships. (<b>a</b>) Representative current traces of individual <span class="html-italic">Xenopus</span> oocytes expressing the <span class="html-italic">Tmu</span>-ACR-16-like receptor challenged with increasing concentrations of ACh (blue), oxantel (red) and epibatidine (green). Compounds were applied for 10 s (short bars); (<b>b</b>) Concentration-response curves of ACh, oxantel and epibatidine on the <span class="html-italic">Tmu</span>-ACR-16-like receptor; (<b>c</b>) Concentration-response curves for epibatidine on the ACR-16-like receptor from <span class="html-italic">T. muris</span> (also shown in <b>a</b>) and <span class="html-italic">T. suis</span>; (<b>d</b>) Concentration-response curves for ACh on eggs microinjected either with <span class="html-italic">Tmu-acr-16-like</span> cRNA (also shown in <b>a</b>) or with <span class="html-italic">Tmu-acr-16-like</span> and <span class="html-italic">Tmu-acr-19</span> cRNAs. All current responses are normalized to current responses induced by 300 μM ACh and given as mean ± SEM.</p> Full article ">
20 pages, 4153 KiB  
Article
Albumin Nanoparticle Formulation for Heart-Targeted Drug Delivery: In Vivo Assessment of Congestive Heart Failure
by Nikita Lomis, Ziyab K. Sarfaraz, Aiman Alruwaih, Susan Westfall, Dominique Shum-Tim and Satya Prakash
Pharmaceuticals 2021, 14(7), 697; https://doi.org/10.3390/ph14070697 - 19 Jul 2021
Cited by 7 | Viewed by 3946
Abstract
Congestive heart failure is a fatal cardiovascular disease resulting in tissue necrosis and loss of cardiac contractile function. Inotropic drugs such as milrinone are commonly used to improve the myocardial contractility and heart function. However, milrinone is associated with severe side effects and [...] Read more.
Congestive heart failure is a fatal cardiovascular disease resulting in tissue necrosis and loss of cardiac contractile function. Inotropic drugs such as milrinone are commonly used to improve the myocardial contractility and heart function. However, milrinone is associated with severe side effects and lower circulation time. In this article, a novel protein nanoparticle formulation for heart-targeted delivery of milrinone has been designed and tested. The formulation was prepared using albumin protein conjugated with the targeting ligand, angiotensin II peptide to form nanoparticles following the ethanol desolvation method. The formulation was characterized for size, charge, and morphology and tested in a rat model of congestive heart failure to study pharmacokinetics, biodistribution, and efficacy. The overall cardiac output parameters were evaluated comparing the formulation with the control non-targeted drug, milrinone lactate. This formulation exhibited improved pharmacokinetics with a mean retention time of 123.7 min, half-life of 101.3 min, and clearance rate of 0.24 L/(kg*h). The targeted formulation also significantly improved ejection fraction and fractional shortening parameters thus improving cardiac function. This study demonstrates a new approach in delivering inotropic drugs such as milrinone for superior treatment of congestive heart failure. Full article
(This article belongs to the Special Issue Protein-Based Biomaterial for Pharmaceutical and Biomedical Applications)
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<p>Schematic representation of the surface modification of the HSA molecule for binding with the AT1 peptide through a two-step chemical conjugation reaction using cross-linkers PA-(PEG)<sub>4</sub>-SPA and EDC/Sulfo-NHS.</p> Full article ">Figure 2
<p>1H NMR-based characterization of AT1-HSA, HSA, and AT1 peptide. The AT1 peptide exhibits a spectrum with sharp peaks at δ = 0.5–1.0, δ = 1.1–2.1, δ = 2.8–3.8, δ = 4.0–5.0, and δ = 6.3–7.3. The peaks at δ = 6.8 due to tyrosine and around δ = 3.8–4.0 due to glycine from the AT1 peptide can be seen on the AT1-HSA spectrum around δ = 3.5–4.0 and 6.8 ppm.</p> Full article ">Figure 3
<p>HSA (lower) and AT1-HSA (upper) were analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. The m/z ratio of the AT1-HSA peak was at least 5600 higher than that of the HSA peak, which demonstrated that AT1 was successfully conjugated to the surface of HSA.</p> Full article ">Figure 4
<p>Nanoparticle surface characterization using TEM analysis: (<b>a</b>) Under magnification of 17,800×, AT1-HSA-MRN-NPs of size 190.2 ± 5.7 nm with a zeta potential of −29.5 ± 3.7 mV (Scale = 500 nm); (<b>b</b>) Under magnification of 105,000×, AT1-HSA-MRN-NPs with moderately uniform particle size (Scale = 100 nm); (<b>c</b>) Under 135,000× magnification, the AT1-HSA-MRN-NPs display a dark core surrounded by a bright distinct membrane layer (Scale = 20 nm).</p> Full article ">Figure 5
<p>Pharmacokinetics of AT1-HSA-MRN-NPs (Group II) and MRN-Lactate (Group III) at an initial MRN dose of 50 μg/kg in vivo.</p> Full article ">Figure 6
<p>Tissue distribution of AT1-HSA-MRN-NPs and MRN-Lactate at an initial MRN dose of 50 μg/kg in vivo. The graph shows a representative result of mean ± SD (<span class="html-italic">n</span> = 4). *** <span class="html-italic">p</span> &lt; 0.001 was considered highly significant and ** <span class="html-italic">p</span> &lt; 0.01 was considered significant based on Tukey’s post hoc analysis.</p> Full article ">Figure 7
<p>Percentage LVEF measurements for Groups 1, 2, 3, 4, and 5 at baseline, 48 h post ligation, 60 min, 24 h, 48 h, and 1-week post injections. The graph shows a representative result of mean ± SD (<span class="html-italic">n</span> = 5). *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01 and * <span class="html-italic">p</span> &lt; 0.05 were considered significant based on Tukey’s post hoc analysis.</p> Full article ">Figure 8
<p>Percentage LVFS measurements for Groups 1, 2, 3, 4, and 5 at baseline, 48 h post ligation, 60 min, 24 h, 48 h, and 1-week post injections. The data have been presented as mean ± SD (<span class="html-italic">n</span> = 5). *** <span class="html-italic">p</span> &lt; 0.001 was considered highly significant and ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05 was considered significant based on Tuckey’s post hoc analysis.</p> Full article ">Figure 9
<p>Milrinone levels detected in plasma 24 h post treatment for Groups 2, 3, and 5. The graph shows a representative result of mean ± SD (<span class="html-italic">n</span> = 3). *** <span class="html-italic">p</span> &lt; 0.001 was considered significant based on Tukey’s post hoc analysis.</p> Full article ">Figure 10
<p>Serum analysis performed as a safety test comparing the (<b>a</b>) ALP (U/L); (<b>b</b>) ALT (U/L); (<b>c</b>) AST (U/L) as liver function tests and (<b>d</b>) creatinine (μmol/L); (<b>e</b>) urea (mmol/L) as kidney function tests. The graph shows a representative result of mean ± SD (<span class="html-italic">n</span> = 5). ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05 were considered significant based on Tukey’s post hoc analysis.</p> Full article ">Figure 11
<p>Serum cytokine levels for (<b>a</b>) IL-6 (pg/mL), (<b>b</b>) TNF-α (pg/mL), and (<b>c</b>) IL-10 (pg/mL). Groups 2 and 5 had significantly lower levels of IL-6 and TNF-α and significantly higher levels of IL-10 compared with other groups. The data have been presented as mean ± SD (<span class="html-italic">n</span> = 5). **** <span class="html-italic">p &lt;</span> 0.0001 was considered highly significant and *<span class="html-italic">p &lt;</span> 0.05 was considered significant based on Tukey’s post hoc analysis.</p> Full article ">
21 pages, 3853 KiB  
Article
Development and Characterization of n-Propyl Gallate Encapsulated Solid Lipid Nanoparticles-Loaded Hydrogel for Intranasal Delivery
by Fakhara Sabir, Gábor Katona, Ruba Ismail, Bence Sipos, Rita Ambrus and Ildikó Csóka
Pharmaceuticals 2021, 14(7), 696; https://doi.org/10.3390/ph14070696 - 19 Jul 2021
Cited by 19 | Viewed by 3936
Abstract
The objective of the present study was to develop n-propyl gallate-loaded solid lipid nanoparticles (PG-SLNs) in a hydrogel (HG) formulation using Transcutol-P (TC-P) as a permeation enhancer. Modified solvent injection technique was applied to produce optimized PG-SLNs via the Quality by Design [...] Read more.
The objective of the present study was to develop n-propyl gallate-loaded solid lipid nanoparticles (PG-SLNs) in a hydrogel (HG) formulation using Transcutol-P (TC-P) as a permeation enhancer. Modified solvent injection technique was applied to produce optimized PG-SLNs via the Quality by Design approach and central composite design. The in vitro mucoadhesion, scavenging activity, drug release, permeation studies of PG from PG-SLNs-loaded HG were evaluated under simulated nasal conditions. Compared with in vitro release behavior of PG from SLNs, the drug release from the PG-SLNs-loaded HG showed a lower burst effect and sustained release profile. The cumulative permeation of PG from PG-SLNs-loaded HG with TC-P was 600 μg/cm2 within 60 min, which is 3–60-fold higher than PG-SLNs and native PG, respectively. Raman mapping showed that the distribution of PG-SLNs was more concentrated in HG having lower concentrations of hyaluronic acid. The scavenging assay demonstrated increased antioxidant activity at higher concentrations of HG. Due to enhanced stability and mucoadhesive properties, the developed HG-based SLNs can improve nasal absorption by increasing residence time on nasal mucosa. This study provides in vitro proof of the potential of combining the advantages of SLNs and HG for the intranasal delivery of antioxidants. Full article
(This article belongs to the Special Issue Current Insights on Lipid-Based Nanosystems)
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Full article ">Figure 1
<p>Probability rating of CQA (<b>a</b>) and CPP/CMA (<b>b</b>) elements. The Pareto charts are presented as the calculated severity scores assigned to the elements.</p> Full article ">Figure 2
<p>XRPD diffractogram (<b>a</b>) and FTIR spectra (<b>b</b>) of PG-SLNs and their components.</p> Full article ">Figure 3
<p>Raman chemical mapping of PG-SLNs in HGs with different concentration of HA: 0.5 (<b>a</b>), 1 (<b>b</b>), 2 (<b>c</b>) and 3% <span class="html-italic">w</span>/<span class="html-italic">v</span> (<b>d</b>).</p> Full article ">Figure 4
<p>Swelling study of optimized cross-linked (SLNs-HGCL) and non-cross-linked (SLNs-HGnCL) HG (1% <span class="html-italic">w</span>/<span class="html-italic">v</span> HA). Data are means ± SD (<span class="html-italic">n</span> = 3 independent measurements).</p> Full article ">Figure 5
<p>Viscosity profiles of cross-linked (SLNs-HGCL) and non-cross-linked (SLNs-HGnCL) containing HA in 0.5–1% <span class="html-italic">w</span>/<span class="html-italic">v</span> (<b>a</b>) and 2–3% <span class="html-italic">w</span>/<span class="html-italic">v</span> (<b>b</b>) concentration. Data are means ± SD (<span class="html-italic">n</span> = 3 independent measurements).</p> Full article ">Figure 6
<p>In vitro mucoadhesive studies of SLNs-HGs on agar-mucin gel. Data are means ± SD (<span class="html-italic">n</span> = 5 independent measurements).</p> Full article ">Figure 7
<p>SEM images of optimized lyophilized PG-SLNs (<b>a</b>,<b>b</b>) and 1% <span class="html-italic">w</span>/<span class="html-italic">v</span> SLNs-HGnCL (<b>c</b>) at various resolution.</p> Full article ">Figure 8
<p>In vitro permeation of pure PG dispersion, PG-SLNs, 1% <span class="html-italic">w</span>/<span class="html-italic">v</span> SLNs-HGnCL with and without TC-P (<b>a</b>). In vitro release study of PG dispersion, PG-SLNs, 1% <span class="html-italic">w</span>/<span class="html-italic">v</span> SLNs-HGnCL (<b>b</b>). Data are means ± SD (<span class="html-italic">n</span> = 3 independent measurements). *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 9
<p>Percentage inhibition of the hydrogen-peroxide-scavenging activity of different concentrations of PG-containing SLNs and PG-SLNs-loaded HA-HG in comparison with the initial PG solution. Data are means ± SD (<span class="html-italic">n</span> = 3 independent measurements). Statistical analysis: <span class="html-italic">t</span>-Test.</p> Full article ">
8 pages, 3273 KiB  
Article
Soluble Angiotensin Converting Enzyme 2 (ACE2) Is Upregulated and Soluble Endothelial Nitric Oxide Synthase (eNOS) Is Downregulated in COVID-19-induced Acute Respiratory Distress Syndrome (ARDS)
by Alice G. Vassiliou, Alexandros Zacharis, Chrysi Keskinidou, Edison Jahaj, Maria Pratikaki, Parisis Gallos, Ioanna Dimopoulou, Anastasia Kotanidou and Stylianos E. Orfanos
Pharmaceuticals 2021, 14(7), 695; https://doi.org/10.3390/ph14070695 - 19 Jul 2021
Cited by 32 | Viewed by 4057
Abstract
A damaged endothelium is an underlying condition of the many complications of COVID-19 patients. The increased mortality risk associated with diseases that have underlying endothelial dysfunction, such as acute respiratory distress syndrome (ARDS), suggests that endothelial (e) nitric oxide synthase (NOS)-derived nitric oxide [...] Read more.
A damaged endothelium is an underlying condition of the many complications of COVID-19 patients. The increased mortality risk associated with diseases that have underlying endothelial dysfunction, such as acute respiratory distress syndrome (ARDS), suggests that endothelial (e) nitric oxide synthase (NOS)-derived nitric oxide could be an important defense mechanism. Additionally, intravenous recombinant angiotensin converting enzyme 2 (ACE2) was recently reported as an effective therapy in severe COVID-19, by blocking viral entry, and thus reducing lung injury. Very few studies exist on the prognostic value of endothelium-related protective molecules in severe COVID-19 disease. To this end, serum levels of eNOS, inducible (i) NOS, adrenomedullin (ADM), soluble (s) ACE2 levels, and serum (s) ACE activity were measured on hospital admission in 89 COVID-19 patients, hospitalized either in a ward or ICU, of whom 68 had ARDS, while 21 did not. In our cohort, the COVID-19-ARDS patients had considerably lower eNOS levels compared to the COVID-19 non-ARDS patients. On the other hand, sACE2 was significantly higher in the ARDS patients. iNOS, ADM and sACE activity did not differ. Our results might support the notion of two distinct defense mechanisms in COVID-19-derived ARDS; eNOS-derived nitric oxide could be one of them, while the dramatic rise in sACE2 may also represent an endogenous mechanism involved in severe COVID-19 complications, such as ARDS. These results could provide insight to therapeutical applications in COVID-19. Full article
(This article belongs to the Special Issue Lung Injury and Repair)
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<p>Hospital admission levels of soluble endothelium–related protective molecules in COVID-19 patients. (<b>a</b>) eNOS, (<b>b</b>) iNOS, and (<b>c</b>) ADM were measured in 21 COVID-19 non-ARDS patients and 68 patients with COVID-19-inflicted ARDS on hospital admission (within 24 h). Two–group comparisons were performed with the non-parametric Mann–Whitney test, * <span class="html-italic">p</span> &lt; 0.05. Data are presented as scatter plots, indicating the median value and 25th to 75th centiles. Dashed line, median value of the whole cohort. ADM = adrenomedullin; ARDS = acute respiratory distress syndrome; eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase.</p> Full article ">Figure 2
<p>Hospital admission levels of serum ACE activity and soluble ACE2 in COVID-19 patients. (<b>a</b>) Serum ACE activity, and (<b>b</b>) soluble ACE2 were measured in 21 COVID-19 non-ARDS patients and 68 patients with COVID-19-inflicted ARDS on hospital admission (within 24 h). Two-group comparisons were performed with the non-parametric Mann–Whitney test, **** <span class="html-italic">p</span> &lt; 0.0001. Data are presented as scatter plots, indicating the median value and 25th to 75th centiles. Dashed line, median value of the whole cohort. ARDS = acute respiratory distress syndrome; sACE = serum angiotensin converting enzyme; sACE2 = soluble angiotensin converting enzyme 2.</p> Full article ">
14 pages, 2943 KiB  
Article
The Effect of Cyanine Dye NK-4 on Photoreceptor Degeneration in a Rat Model of Early-Stage Retinitis Pigmentosa
by Shihui Liu, Toshihiko Matsuo, Mary Miyaji and Osamu Hosoya
Pharmaceuticals 2021, 14(7), 694; https://doi.org/10.3390/ph14070694 - 19 Jul 2021
Cited by 5 | Viewed by 4128
Abstract
The present study aimed to evaluate the effects of NK-4 on the apoptosis of photoreceptors in a rat model of retinitis pigmentosa and explore the mechanism underlying anti-apoptosis activity. The Royal College of Surgeons (RCS) rats received an intravitreous injection of NK-4 solution [...] Read more.
The present study aimed to evaluate the effects of NK-4 on the apoptosis of photoreceptors in a rat model of retinitis pigmentosa and explore the mechanism underlying anti-apoptosis activity. The Royal College of Surgeons (RCS) rats received an intravitreous injection of NK-4 solution in the left eye and vehicle control in the right eye. Apoptosis was detected by TUNEL method in frozen sections of the eyes. The retinal tissues of the rats were dissected for RNA-seq analysis. Functional and pathway enrichment analyses of differentially expressed genes (DEGs) were performed by using Metascape and DAVID software. The expression levels of DEGs were confirmed by real-time quantitative PCR (RT-qPCR). The number of apoptotic cells decreased in the outer nuclear layer (ONL) and the thickness of the ONL was significantly thicker in the retina of NK-4-injected eyes, compared with control eyes. Five DEGs were identified by RNA-seq analysis, and Hmox1, Mt1, Atf5, Slc7a11, and Bdh2 were confirmed to be up-regulated by RT-qPCR. Functional and pathway enrichment analysis of the up-regulated genes showed that anti-apoptosis effects of NK-4 in the retina of RCS rats may be related to the pathways of metal ion homeostasis, negative regulation of neuron death, response to toxic substance, and pigment metabolic process. We found a potential mechanism of NK-4, providing a new viewpoint for the development of more therapeutic uses of NK-4 in the future. Full article
(This article belongs to the Special Issue Advances in Ocular Pharmacology)
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<p>NK-4 and experimental design. (<b>A</b>) Chemical structure of NK-4. (<b>B</b>) A series of NK-4 working solutions with different concentrations (0.0001 to 0.1 mg/mL). (<b>C</b>) Experimental schedule. (<b>D</b>) Six retinal sites (a, b, c, d, e, and f) for TUNEL staining. The “a”, “b”, and “c” began at the straight distance of 586, 373, and 160 μm, respectively, superior from the optic nerve head, while “f”, “e”, and “d” began at the straight distance of 586, 373, and 160 μm, respectively, inferior from the optic nerve head.</p> Full article ">Figure 2
<p>TUNEL staining of retinal sections at the site of “f”. TUNEL assay indicated that TUNEL-positive signals (green) were mainly detected in the ONL and were likely to be fewer in retinas treated with NK-4 (0.0001 mg/mL, 0.001 mg/mL, 0.01 mg/mL, and 0.1 mg/mL) than in those with control vehicle. Cell nuclei were counterstained with DAPI (blue). Pair groups: (<b>A</b>) vs. (<b>B</b>), (<b>C</b>) vs. (<b>D</b>), (<b>E</b>) vs. (<b>F</b>), (<b>G</b>) vs. (<b>H</b>). ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer. Scale bar = 25 μm.</p> Full article ">Figure 3
<p>Quantitative analysis of TUNEL-positive cells (mean values of a, b, c, d, e, and f sites) in retinal sections. (<b>A</b>): NK-4 (0.0001 mg/mL)-injected retinas vs. control groups; (<b>B</b>): NK-4 (0.001 mg/mL)-injected retinas vs. control groups; (<b>C</b>): NK-4 (0.01 mg/mL)-injected retinas vs. control groups; (<b>D</b>): NK-4 (0.1 mg/mL)-injected retinas vs. control groups. The number of TUNEL-positive cells was significantly decreased in the ONL of the eyes injected with higher concentrations of NK-4, ranging from 0.001 to 0.1 mg/mL, compared with control vehicle-injected eyes (<span class="html-italic">p</span> &lt; 0.0001, <span class="html-italic">p</span> = 0.001, <span class="html-italic">p</span> &lt; 0.0001, respectively, one-factor ANOVA, post hoc test). <span class="html-italic">**: p</span> &lt; 0.01, <span class="html-italic">***: p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>TUNEL staining of retinal sections at the concentration of 0.1 mg/mL NK-4 (this figure shows the condition of the entire retina in one RCS rat). The thickness of the ONL (white brackets) was thicker in the retina of NK-4-injected eyes, compared with control eyes. Cell nuclei were counterstained with DAPI (blue). Pair groups: (<b>A</b>) vs. (<b>B</b>), (<b>C</b>) vs. (<b>D</b>), (<b>E</b>) vs. (<b>F</b>), (<b>G</b>) vs. (<b>H</b>), (<b>I</b>) vs. (<b>J</b>), (<b>K</b>) vs. (<b>L</b>). ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer. White brackets represent the ONL. Scale bar = 20 μm.</p> Full article ">Figure 5
<p>Quantitative analysis of the thickness of the ONL in retinal sections. (<b>A</b>): NK-4 (0.0001 mg/mL)-injected retinas vs. control groups; (<b>B</b>): NK-4 (0.001 mg/mL)-injected retinas vs. control groups; (<b>C</b>): NK-4 (0.01 mg/mL)-injected retinas vs. control groups; (<b>D</b>): NK-4 (0.1 mg/mL)-injected retinas vs. control groups. The thickness in the ONL: NK-4 (0.1 mg/mL) vs. control vehicle-injected eyes (site a: <span class="html-italic">p</span> = 0.00753, site e: <span class="html-italic">p</span>  =  0.01125, site f: <span class="html-italic">p</span> = 0.00067, Student’s <span class="html-italic">t</span>-test). The thickness in the ONL: NK-4 (0.01 mg/mL) vs. control vehicle-injected eyes (site c: <span class="html-italic">p</span> = 0.00344, site f: <span class="html-italic">p</span> = 0.00055, Student’s <span class="html-italic">t</span>-test). *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.01, ***: <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Pathway enrichment analysis of 5 significant genes up-regulated by NK-4. Heatmap of pathway enrichment analysis in the up-regulated genes. The three enriched terms in the category of biological process (BP) were pigment metabolic process, transition metal ion homeostasis, and negative regulation of neuron death. Nodes are clustered into subnetworks, and the included genes are shown under the respective pathways.</p> Full article ">Figure 7
<p>(<b>A</b>) Analysis of affected functional processes of DEGs. DEGs were submitted for DAVID analysis, and affected functional processes are shown. Significance was accepted at a Benjamini–Hochberg <span class="html-italic">p</span> value of less than 0.01. (<b>B</b>) Quantitative reverse-transcription PCR analysis of <span class="html-italic">Hmox1</span>, <span class="html-italic">Mt1</span>, <span class="html-italic">Slc7a11</span>, <span class="html-italic">Bdh2</span>, and <span class="html-italic">Atf5</span> expression in retinal samples. Since individual rats exhibited differences in expression levels of genes, data represent the mean ± SD obtained for measurements from triplicate wells and are representative of three individual rats. RQ<span class="html-italic">:</span> relative quantification.</p> Full article ">
18 pages, 4787 KiB  
Article
Synthesis and Human Carbonic Anhydrase I, II, IX, and XII Inhibition Studies of Sulphonamides Incorporating Mono-, Bi- and Tricyclic Imide Moieties
by Kalyan K. Sethi, KM Abha Mishra, Saurabh M. Verma, Daniela Vullo, Fabrizio Carta and Claudiu T. Supuran
Pharmaceuticals 2021, 14(7), 693; https://doi.org/10.3390/ph14070693 - 19 Jul 2021
Cited by 5 | Viewed by 3349
Abstract
New derivatives were synthesised by reaction of amino-containing aromatic sulphonamides with mono-, bi-, and tricyclic anhydrides. These sulphonamides were investigated as human carbonic anhydrases (hCAs, EC 4.2.1.1) I, II, IX, and XII inhibitors. hCA I was inhibited with inhibition constants (Kis) ranging from [...] Read more.
New derivatives were synthesised by reaction of amino-containing aromatic sulphonamides with mono-, bi-, and tricyclic anhydrides. These sulphonamides were investigated as human carbonic anhydrases (hCAs, EC 4.2.1.1) I, II, IX, and XII inhibitors. hCA I was inhibited with inhibition constants (Kis) ranging from 49 to >10,000 nM. The physiologically dominant hCA II was significantly inhibited by most of the sulphonamide with the Kis ranging between 2.4 and 4515 nM. hCA IX and hCA XII were inhibited by these sulphonamides in the range of 9.7 to 7766 nM and 14 to 316 nM, respectively. The structure–activity relationships (SAR) are rationalised with the help of molecular docking studies. Full article
(This article belongs to the Section Medicinal Chemistry)
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<p>3D multiple superpositions of four α-hCAs crystal structures (high sequence alignment homology) [<a href="#B23-pharmaceuticals-14-00693" class="html-bibr">23</a>]. The multiple superpositions involved the following crystal structures: 1AZM (hCA I); 1ZFQ (CA II); 3IAI (CA IX); and 1JD0 (CA XII) with the Zn ion shown as red sphere, and its coordinating residues His 94, His 96, and His 119 shown in pink. The protein backbone is shown in green [<a href="#B1-pharmaceuticals-14-00693" class="html-bibr">1</a>,<a href="#B20-pharmaceuticals-14-00693" class="html-bibr">20</a>].</p> Full article ">Figure 2
<p>Resulting designed structural element (HIT) as hCA inhibitor from the outcome of ligand and structure-based drug design (CoMFA, CoMSIA, and pharmacophore modelling) [<a href="#B21-pharmaceuticals-14-00693" class="html-bibr">21</a>].</p> Full article ">Figure 3
<p>The proposed steps of the reaction mechanism.</p> Full article ">Figure 4
<p>Protein ligand interactions in the 3IAI active site. (<b>a</b>) AAZ coordinates to the zinc ion and formed H-bond with THR 199, THR 200, and two water molecules; (<b>b</b>) Compound <b>1</b>, the deprotonate sulphonamide binds to the zinc ion and forms H-bonds with HIS 64 (1.87 Å), and THR 199 (2.156 Å).</p> Full article ">Scheme 1
<p>Synthesis of sulphonamide derivatives 1–13 incorporating phthalic anhydride moiety to different benzene sulphonamides [<a href="#B22-pharmaceuticals-14-00693" class="html-bibr">22</a>,<a href="#B23-pharmaceuticals-14-00693" class="html-bibr">23</a>,<a href="#B24-pharmaceuticals-14-00693" class="html-bibr">24</a>,<a href="#B29-pharmaceuticals-14-00693" class="html-bibr">29</a>]. n = 0 (<b>1</b>, <b>6</b>, <b>9</b>, <b>10</b>, <b>11</b>, <b>12</b>, and <b>13</b>); n = 1 (<b>7</b>); n = 2 (<b>2</b>, <b>8</b>). -(CH<sub>2</sub>)n = <span class="html-italic">para</span> for <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, <b>6</b>, <b>7</b>, <b>8</b>, <b>11</b>, <b>12</b>, and <b>13</b>; -(CH<sub>2</sub>)n = <span class="html-italic">meta</span> for <b>9</b>; -(CH<sub>2</sub>)n = <span class="html-italic">ortho</span> for <b>10</b> in the benzene ring. X = F (<b>5</b>, <b>11</b>); X = Cl (<b>3</b>, <b>12</b>); X = Br (<b>4</b>, <b>13</b>); Z = F (<b>5</b>) in the benzene ring of anhydride. i = glacial acetic acid; ii = stirring with heating.</p> Full article ">
16 pages, 1696 KiB  
Review
Thiophene-Based Compounds with Potential Anti-Inflammatory Activity
by Ryldene Marques Duarte da Cruz, Francisco Jaime Bezerra Mendonça-Junior, Natália Barbosa de Mélo, Luciana Scotti, Rodrigo Santos Aquino de Araújo, Reinaldo Nóbrega de Almeida and Ricardo Olímpio de Moura
Pharmaceuticals 2021, 14(7), 692; https://doi.org/10.3390/ph14070692 - 19 Jul 2021
Cited by 64 | Viewed by 7314
Abstract
Rheumatoid arthritis, arthrosis and gout, among other chronic inflammatory diseases are public health problems and represent major therapeutic challenges. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most prescribed clinical treatments, despite their severe side effects and their exclusive action in improving symptoms, without effectively [...] Read more.
Rheumatoid arthritis, arthrosis and gout, among other chronic inflammatory diseases are public health problems and represent major therapeutic challenges. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most prescribed clinical treatments, despite their severe side effects and their exclusive action in improving symptoms, without effectively promoting the cure. However, recent advances in the fields of pharmacology, medicinal chemistry, and chemoinformatics have provided valuable information and opportunities for development of new anti-inflammatory drug candidates. For drug design and discovery, thiophene derivatives are privileged structures. Thiophene-based compounds, like the commercial drugs Tinoridine and Tiaprofenic acid, are known for their anti-inflammatory properties. The present review provides an update on the role of thiophene-based derivatives in inflammation. Studies on mechanisms of action, interactions with receptors (especially against cyclooxygenase (COX) and lipoxygenase (LOX)), and structure-activity relationships are also presented and discussed. The results demonstrate the importance of thiophene-based compounds as privileged structures for the design and discovery of novel anti-inflammatory agents. The studies reveal important structural characteristics. The presence of carboxylic acids, esters, amines, and amides, as well as methyl and methoxy groups, has been frequently described, and highlights the importance of these groups for anti-inflammatory activity and biological target recognition, especially for inhibition of COX and LOX enzymes. Full article
(This article belongs to the Special Issue Privileged Structures as Leads in Medicinal Chemistry)
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<p>Chemical structures of market anti-inflammatory drugs containing a thiophene moiety (Tinoridine, Tiaprofenic acid, Tenidap and Zileuton.).</p> Full article ">Figure 2
<p>Simplified diagram of COX-1, COX-2 and LOX enzymes activation and beginning of the inflammatory process.</p> Full article ">Figure 3
<p>Chemical structures of thiophene-based compounds active against COX and LOX enzymes.</p> Full article ">Figure 4
<p>Chemical structures of thiophene-based compounds, which modulate gene expression and/or inflammatory cytokines.</p> Full article ">Figure 5
<p>Chemical structures of thiophene-based compounds with anti-inflammatory properties in classic models of inflammation.</p> Full article ">Figure 6
<p>Chemical structures of thiophene-based compounds with anti-inflammatory properties in in silico studies.</p> Full article ">Figure 7
<p>Representative figure of the general mechanisms of action of the thiophene-based compounds presented in this review.</p> Full article ">
15 pages, 370 KiB  
Perspective
Update on Functional Inhibitors of Acid Sphingomyelinase (FIASMAs) in SARS-CoV-2 Infection
by Gwenolé Loas and Pascal Le Corre
Pharmaceuticals 2021, 14(7), 691; https://doi.org/10.3390/ph14070691 - 18 Jul 2021
Cited by 12 | Viewed by 3966
Abstract
The SARS-CoV-2 outbreak is characterized by the need of the search for curative drugs for treatment. In this paper, we present an update of knowledge about the interest of the functional inhibitors of acid sphingomyelinase (FIASMAs) in SARS-CoV-2 infection. Forty-nine FIASMAs have been [...] Read more.
The SARS-CoV-2 outbreak is characterized by the need of the search for curative drugs for treatment. In this paper, we present an update of knowledge about the interest of the functional inhibitors of acid sphingomyelinase (FIASMAs) in SARS-CoV-2 infection. Forty-nine FIASMAs have been suggested in the treatment of SARS-CoV-2 infection using in silico, in vitro or in vivo studies. Further studies using large-sized, randomized and double-blinded controlled clinical trials are needed to evaluate FIASMAs in SARS-CoV-2 infection as off-label therapy. Full article
(This article belongs to the Special Issue COVID-19 in Pharmaceuticals)
34 pages, 2870 KiB  
Review
Dietary Effects of Anthocyanins in Human Health: A Comprehensive Review
by Ana C. Gonçalves, Ana R. Nunes, Amílcar Falcão, Gilberto Alves and Luís R. Silva
Pharmaceuticals 2021, 14(7), 690; https://doi.org/10.3390/ph14070690 - 18 Jul 2021
Cited by 136 | Viewed by 16677
Abstract
In recent years, the consumption of natural-based foods, including beans, fruits, legumes, nuts, oils, vegetables, spices, and whole grains, has been encouraged. This fact is essentially due to their content in bioactive phytochemicals, with the phenolic compounds standing out. Among them, anthocyanins have [...] Read more.
In recent years, the consumption of natural-based foods, including beans, fruits, legumes, nuts, oils, vegetables, spices, and whole grains, has been encouraged. This fact is essentially due to their content in bioactive phytochemicals, with the phenolic compounds standing out. Among them, anthocyanins have been a target of many studies due to the presence of catechol, pyrogallol, and methoxy groups in their chemical structure, which confer notable scavenging, anti-apoptotic, and anti-inflammatory activities, being already recommended as supplementation to mitigate or even attenuate certain disorders, such as diabetes, cancer, and cardiovascular and neurological pathologies. The most well-known anthocyanins are cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside. They are widespread in nature, being present in considerable amounts in red fruits and red vegetables. Overall, the present review intends to discuss the most recent findings on the potential health benefits from the daily intake of anthocyanin-rich foods, as well as their possible pharmacological mechanisms of action. However, before that, some emphasis regarding their chemical structure, dietary sources, and bioavailability was done. Full article
(This article belongs to the Special Issue Plant and Marine-Derived Natural Product Research in Drug Discovery: Strengths and Perspective)
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<p>Biosynthesis pathways of the main anthocyanins found in foods. DAHP: 3-Deoxy-D-arabinoheptulosonate 7-phosphate; CoA: coenzyme A; F3′5′H: Flavonoid 3′, 5′-hydroxylase; FLH: Flavanone 3-hydroxylase; FLS: Flavonol synthase; LAR: Leucoanthocyanidin reductase; ANR: Anthocyanidin reductase; UFGT: UDP glucose flavonoid 3-<span class="html-italic">O</span>-glucosyltransferase; FAOMT: Flavonoid 3’, 5’-methyltransferase (adapted from [<a href="#B25-pharmaceuticals-14-00690" class="html-bibr">25</a>]).</p> Full article ">Figure 2
<p>The basic structure of the flavonoids (<b>A</b>), which include anthocyanidins (e.g., cyanidin and pelargonidin) (<b>B</b>), flavanones (e.g., hesperidin and naringenin) (<b>C</b>), flavanols (e.g., catechin and epicatechin) (<b>D</b>), flavones (e.g., apigenin and luteolin) (<b>E</b>), flavonols (e.g., quercetin and kaempferol) (<b>F</b>), and isoflavones (e.g., daidzein and genistein) (<b>G</b>), differing in the level of oxidation and C ring saturation (adapted from [<a href="#B31-pharmaceuticals-14-00690" class="html-bibr">31</a>]).</p> Full article ">Figure 3
<p>Representation of the main general chemical structure of anthocyanins. Acgal—acetylgalactose; Acglu—acetylglucose; Ara—arabinose; Gal—galactose; Glu—glucose; Rut—rutinoside (adapted from [<a href="#B35-pharmaceuticals-14-00690" class="html-bibr">35</a>,<a href="#B37-pharmaceuticals-14-00690" class="html-bibr">37</a>]).</p> Full article ">Figure 4
<p>Anthocyanin absorption, distribution, metabolism, and excretion. CGB—cytosolic <span class="html-italic">β</span>-glucosidase; SULT—sulfotransferase; UDP-GT—glucuronosyltransferase; COMT—catechol-<span class="html-italic">O</span>-methyl transferase; GLUT—glucose transporters; LPH—lactase-phlorizin hydrolase.</p> Full article ">Figure 5
<p>Health benefits of anthocyanins.</p> Full article ">
15 pages, 4883 KiB  
Article
Small Molecule–Peptide Conjugates as Dimerization Inhibitors of Leishmania infantum Trypanothione Disulfide Reductase
by Alejandro Revuelto, Isabel López-Martín, Héctor de Lucio, Juan Carlos García-Soriano, Nicola Zanda, Sonia de Castro, Federico Gago, Antonio Jiménez-Ruiz, Sonsoles Velázquez and María-José Camarasa
Pharmaceuticals 2021, 14(7), 689; https://doi.org/10.3390/ph14070689 - 17 Jul 2021
Viewed by 3632
Abstract
Trypanothione disulfide reductase (TryR) is an essential homodimeric enzyme of trypanosomatid parasites that has been validated as a drug target to fight human infections. Using peptides and peptidomimetics, we previously obtained proof of concept that disrupting protein–protein interactions at the dimer interface of [...] Read more.
Trypanothione disulfide reductase (TryR) is an essential homodimeric enzyme of trypanosomatid parasites that has been validated as a drug target to fight human infections. Using peptides and peptidomimetics, we previously obtained proof of concept that disrupting protein–protein interactions at the dimer interface of Leishmania infantum TryR (LiTryR) offered an innovative and so far unexploited opportunity for the development of novel antileishmanial agents. Now, we show that linking our previous peptide prototype TRL38 to selected hydrophobic moieties provides a novel series of small-molecule–peptide conjugates that behave as good inhibitors of both LiTryR activity and dimerization. Full article
(This article belongs to the Collection Drug Discovery and Development for Tropical Diseases (TDs))
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<p>(<b>A</b>) An illustration of the <span class="html-italic">Li</span>TryR dimer (PDB entry 6I7N [<a href="#B27-pharmaceuticals-14-00689" class="html-bibr">27</a>]) showing the large channel (yellow) connecting the NADPH entry site to the interfacial cavity [<a href="#B28-pharmaceuticals-14-00689" class="html-bibr">28</a>], as calculated by the CASTp web server [<a href="#B32-pharmaceuticals-14-00689" class="html-bibr">32</a>]; (<b>B</b>) an illustration of a <span class="html-italic">Li</span>TryR monomer showing a similar channel (yellow, 2016 Å<sup>2</sup>, 2550 Å<sup>3</sup>) plus a second one (green, 129 Å<sup>2</sup>, 125 Å<sup>3</sup>) that makes up the putative target site for the dimer-disrupting peptides and SMPCs; (<b>C</b>) theoretical model of the complex between prototype peptide <b>TRL38</b> (C atoms in grey) and a <span class="html-italic">Li</span>TryR monomer (rainbow-colored illustration, with <span class="html-italic">N</span>- and C-termini in blue and red, respectively).</p> Full article ">Figure 2
<p>General structure of the novel small molecule <b>TRL38</b> conjugate.</p> Full article ">Figure 3
<p>Time evolution of the simulated complex between <span class="html-italic">Li</span>TryR monomer (C atoms in green) and SMPC <b>4d</b> (C atoms in cyan). The superimposed snapshots correspond to times 5, 25, and 50 ns (<b>A</b>), 75 and 100 ns (<b>B</b>), and 125 and 150 ns (<b>C</b>) of the unrestrained molecular dynamics trajectory in explicit solvent. The FAD cofactor is displayed as sticks with C atoms colored in pink.</p> Full article ">Scheme 1
<p>Solid-phase synthesis of hydrophobic SMPCs <b>1a</b>–<b>g</b>, <b>2a</b>,<b>d</b>, <b>3a</b>,<b>d</b>, and <b>4a</b>,<b>d</b> by amide coupling. Reagents and conditions: (<b>a</b>) 20% Piperidine/DMF; (<b>b</b>) Fmoc-Ile-OH, HCTU, DIPEA, DMF, MW, 40 °C, 3 x 10 min; (<b>c</b>) DBU/piperidine/DMF; (<b>d</b>) Fmoc-SPACER-OH, HCTU, DIPEA, DMF, rt, 2h; (<b>e</b>) R-CH<sub>2</sub>-COOH, HCTU, DIPEA, DMF, rt, 2 h; (<b>f</b>) TFA, TIPS, and H<sub>2</sub>O.</p> Full article ">Scheme 2
<p>Solid-phase synthesis of hydrophobic SMPCs <b>7</b>–<b>10</b> by reductive amination followed by amine quaternization. Reagents and conditions: (<b>a</b>) DBU, piperidine, DMF; (<b>b</b>) PhCHO (3.6 equivalents); (<b>c</b>) NaBH<sub>3</sub>CN (3.6 equivalents); (<b>d</b>) TFA, TIPS, H<sub>2</sub>O; (<b>e</b>) PhCHO (10 equivalents); (<b>f</b>) NaBH<sub>3</sub>CN (10 equivalents); (<b>g</b>) MeI, 2,6-lutidine.</p> Full article ">
26 pages, 7880 KiB  
Article
Conjugation of Diclofenac with Novel Oleanolic Acid Derivatives Modulate Nrf2 and NF-κB Activity in Hepatic Cancer Cells and Normal Hepatocytes Leading to Enhancement of Its Therapeutic and Chemopreventive Potential
by Maria Narożna, Violetta Krajka-Kuźniak, Barbara Bednarczyk-Cwynar, Małgorzata Kucińska, Robert Kleszcz, Jacek Kujawski, Hanna Piotrowska-Kempisty, Adam Plewiński, Marek Murias and Wanda Baer-Dubowska
Pharmaceuticals 2021, 14(7), 688; https://doi.org/10.3390/ph14070688 - 17 Jul 2021
Cited by 15 | Viewed by 4733
Abstract
Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling [...] Read more.
Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling pathways. This study focused on the synthesis and biological evaluation of four diclofenac (DCL)–OAO derivative conjugates in the context of these pathways’ modification and hepatic cells survival. Treatment with the conjugates 4d, 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide, and 4c, 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester significantly reduced cell viability in comparison to the DCL alone. In THLE-2, immortalized normal hepatocytes treated with these conjugates resulted in the activation of Nrf2 and increased expression in SOD-1 and NQO1, while the opposite effect was observed in the HepG2 hepatoma cells. In both cell lines, reduced activation of the NF-κB and COX-2 expression was observed. In HepG2 cells, conjugates increased ROS production resulting from a reduced antioxidant defense, induced apoptosis, and inhibited cell proliferation. In addition, the OAO morpholide derivative and its DCL hybrid reduced the tumor volume in mice bearing xenografts. In conclusion, our study demonstrated that conjugating diclofenac with the OAO morpholide and a benzyl ester might enhance its anti-cancer activity in HCC. Full article
(This article belongs to the Collection Old Pharmaceuticals with New Applications)
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<p>The chemical structures of the investigated novel oleanolic acid oxime derivatives with diclofenac (DCL–OAO) (<b>4a</b>–<b>4d</b>); (<b>4a</b>) 3-diclofenacoxyiminoolean-12-en-28-oic acid; (<b>4b</b>) 3-diclofenacoxyiminoolean-12-en-28-oic acid methyl ester; (<b>4c</b>) 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester; (<b>4d</b>) 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide.</p> Full article ">Figure 2
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b>) on the viability of THLE-2 (<b>A</b>) and HepG2 (<b>B</b>) cells (after 24 h incubation). Data (mean ± SEM) from three separate experiments are presented. * Significantly different from control—DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s-<span class="html-italic">t</span>-test.</p> Full article ">Figure 3
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on the Nrf2–DNA binding capability, translocation from cytosol to nuclei, and Keap1 protein level in THLE-2 cells (<b>A</b>–<b>C</b>) and HepG2 cells, (<b>D</b>–<b>F</b>) after 24 h incubation. Activated Nrf2 was assessed in terms of the amount of Nrf2 contained in the DNA-binding complexes extracted from the nuclear fraction (<b>A</b>,<b>D</b>). Data (mean ± SEM) from three separate experiments in comparison to control cells set to 100%. Representative immunoblots for the analysis of the cytosolic panels and nuclear (<b>B</b>,<b>E</b>) levels of the Nrf2 protein and cytosolic Keap1 protein (<b>C</b>,<b>F</b>) are shown. The sequence of the bands corresponds to the sequence of the bars in the graph. Lamin and β-actin were used as a loading control. The values were calculated as protein levels in comparison to control cells set to 100%. * Significantly different from control—DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on SOD-1, (<b>A</b>,<b>B</b>) and NQO1, (<b>C</b>,<b>D</b>) mRNA and protein levels in THLE-2 and HepG2 cells after 24 h incubation. The values (mean ± SEM) for the mRNA (<b>B</b>,<b>D</b>) levels were calculated from three separate experiments in comparison to control cells set to 100%. Representative immunoblots of the cytosolic content of SOD-1 and NQO1 in THLE-2 (<b>A</b>,<b>C</b>) and HepG2 cells (<b>B</b>,<b>D</b>) from three separate experiments are shown. The sequence of the bands corresponds to the sequence of the bars in the graph. β-actin was used as a loading control. The values were calculated as protein levels in comparison to control cells set to 100%. * Significantly different from control—DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s-<span class="html-italic">t</span>-test.</p> Full article ">Figure 5
<p>The first poses of the examined ligands ((<b>A</b>) <b>4c</b>, (<b>B</b>) <b>4d</b>, and (<b>C</b>) protonated <b>4d</b>) were determined by the Chimera 1.13.1 package. (<b>D</b>) The superimposition of the docked DCL –OAO derivative conjugates (first poses).</p> Full article ">Figure 6
<p>The effect of DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on the NF-кB p50 and NF-кB p65 binding to DNA and translocation from the cytosol to the nuclei in the THLE-2 cells (<b>A</b>–<b>D</b>) and HepG2 cells (<b>E</b>–<b>H</b>) after 24 h incubation. Activated NF-кB p50 and NF-кB p65 were assessed in terms of the amount of NF-кB p50 (<b>A</b>,<b>E</b>) and NF-кB p65 (<b>C</b>,<b>G</b>) contained in the DNA-binding complexes extracted from the nuclear fraction. The values (mean ± SEM) were calculated from three separate experiments as protein levels in comparison to control cells set to 100%. Representative immunoblots for the analysis of the cytosolic and nuclear levels of proteins NF-кB p50 (<b>B</b>,<b>F</b>) and NF-кB p65 (<b>D</b>,<b>H</b>) are shown. The sequence of the bands corresponds to the sequence of the bars in the graph. Lamin and β-actin were used as a loading control. The values were calculated as protein levels in comparison to control cells set to 100%. * Significantly different from control—DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s-<span class="html-italic">t</span>-test.</p> Full article ">Figure 7
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on the COX-2 protein level (<b>A</b>) in THLE-2 cells, COX-2 mRNA and protein levels in HepG2 (<b>B</b>) and the Nrf2 (<b>C</b>) and NF-κB subunits’ (<b>D</b>,<b>E</b>) transcript levels in HepG2 cells (after 24 h incubation). The values (mean ± SEM) for the mRNA levels in HepG2 cells (<b>B</b>–<b>E</b>) were calculated from three separate experiments in comparison to control cells set to 100%. Representative immunoblots of the cytosolic content of COX-2 in THLE-2 (<b>A</b>) and HepG2 cells (<b>B</b>) from three separate experiments are shown. The sequence of the bands corresponds to the sequence of the bars in the graph. β-actin was used as a loading control. The values were calculated as protein levels in comparison to control cells set to 100%. * Significantly different from control- DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s-<span class="html-italic">t</span>-test.</p> Full article ">Figure 8
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 20 µM concentration) on the cytosolic level of AKT (<b>A</b>), p70 S6 kinase (<b>B</b>), p38 (<b>C</b>), and JNK (<b>D</b>) measured in the cytosolic fractions of the HepG2 cells (after 24 h incubation) and calculated on the MAGPIX<sup>®</sup> System. (<b>E</b>,<b>F</b>) The protein levels of AKT and p-AKT (phosphorylation site Ser 473), respectively assessed by a Western blot, where β-actin was used as a loading control. The results expressing the mean fluorescence intensity (MFI) are presented as the fold of control from three independent measurements. The values (mean ± SEM) were calculated as protein levels in comparison to control cells set to 100%. * Significantly different from control, DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s-<span class="html-italic">t</span>-test.</p> Full article ">Figure 9
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on the cell cycle distribution (<b>A</b>), apoptosis induction (<b>B</b>), and on the proliferation level (<b>C</b>) in HepG2 cells (after 24 h incubation). (<b>A</b>) Graphs represent the percentage of cells in the subG1 (green color before the G1 phase on plots), G1/G0, S, and G2/M phases measured by flow cytometry after propidium iodide staining. Topotecan was used as a positive control. (<b>B</b>) Graphs represent the percentage of cells in the early and late stages of the apoptosis assessed by flow cytometry measurements based on the signal from Annexin V bound to phosphatidylserine externalized in apoptotic cells. A dead cell marker, 7-AAD, was also included. Topotecan was used as a reference for proapoptotic activity. The protein levels of Bax and Caspase-3 is also presented. (<b>C</b>) Graphs representing the percentage of proliferating (Ki67(+)) and nonproliferating (Ki67(−)) cells measured by flow cytometry. Starved cells (cultured in an FBS-free medium) were used as a reference for the anti-proliferative conditions. Representative plots and immunoblots are presented. β-actin was used as a loading control for protein analysis. For all panels, the mean values ± SEM from three independent experiments is shown. Asterisks (*) denote statistically significant changes from control—DMSO treated cells, and panel B denotes statistically significant changes in the percentage of either early or late apoptotic cells, while hashes (#) denote statistically significant changes in the percentage of total apoptotic cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 10
<p>The effect of the DCL and DCL–OAO derivative conjugates (<b>4a</b>–<b>4d</b> in 10 and 20 µM concentrations) on the generation of reactive oxygen species (ROS (+)) (<b>A</b>) and induction of ERK phosphorylation (<b>B</b>) in the HepG2 cells (after 24 h incubation). (<b>A</b>) Graphs representing the relative percentage of cells that are ROS negative and positive (percentage of cells undergoing oxidative stress based on the intracellular detection of superoxide radicals). The kit based on dihydroethidium was used to detect ROS-exhibiting cells (ROS (+)). (<b>B</b>) Graphs representing the percentage of cells expressing ERK 1/2 protein in an active (phosphorylated) state. Phorbol myristate acetate (PMA)-stimulated cells were used as a positive control. The protein levels of ERK 1/2 and p-ERK 1/2 was also shown. Representative plots and immunoblots are presented. β-actin was used as a loading control for protein analysis. The ERK 1/2 antibody used recognizes only its non-phosphorylated form. For all panels, the mean values ± SEM from three independent experiments are shown. Asterisks (*) denote statistically significant changes from control—DMSO-treated cells, <span class="html-italic">p</span> &lt; 0.05. Statistical significance was assessed by Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 11
<p>Anti-cancer efficacy of the DCL, and OAO morpholide derivative and their conjugation in HepG2 tumor xenografts. (<b>A</b>) The body weight of animals during the experiment. (<b>B</b>) The growth curves of the HepG2 tumor xenografts. (<b>C</b>) Kaplan-Meier curves for animals bearing the HepG2 xenografts. (<b>D</b>) Tumor volumes measured ex vivo following the sacrifice of the mice. The volumes of the tumors on day 6, when tumors were measurable in all animals, were set as 1.</p> Full article ">Figure 12
<p>(<b>A</b>) The luminescence images of a representative control mouse, while (<b>B</b>) shows the luminescence images of a representative mouse receiving OAO–morpholide. (<b>C</b>,<b>D</b>) Luminescence measured in regions of interest (ROI) in mice receiving: vehicle and OAO–morpholide. (<b>E</b>,<b>F</b>) Tumor volume changes (expressed as a normalized tumor volume) in representative control and OAO–morpholide-treated mice, respectively, while (<b>G</b>,<b>H)</b> shows corresponding pictures obtained using the Peira Instrument (ultrasound-based) (<b>G</b>) from control mouse, and (<b>H</b>) from a mouse receiving OAO–morpholide.</p> Full article ">
13 pages, 1571 KiB  
Review
Investigating Potential Applications of the Fish Anti-Microbial Peptide Pleurocidin: A Systematic Review
by Katelyn A. M. McMillan and Melanie R. Power Coombs
Pharmaceuticals 2021, 14(7), 687; https://doi.org/10.3390/ph14070687 - 17 Jul 2021
Cited by 9 | Viewed by 3707
Abstract
The anti-microbial peptide (AMP) pleurocidin is found in winter flounder (Pseudopleuronectes americanus), an Atlantic flounder species. There is promising evidence for clinical, aquaculture, and veterinary applications of pleurocidin. This review provides an overview of the current literature available on pleurocidin to [...] Read more.
The anti-microbial peptide (AMP) pleurocidin is found in winter flounder (Pseudopleuronectes americanus), an Atlantic flounder species. There is promising evidence for clinical, aquaculture, and veterinary applications of pleurocidin. This review provides an overview of the current literature available on pleurocidin to guide future research directions. By fully elucidating pleurocidin’s mechanism of action and developing novel treatments against pathogenic microbes, populations of flatfish and humans can be protected. This review consulted publications from PubMed and Environment Complete with search terms such as “pleurocidin”, “winter flounder”, and “antimicrobial”. The fish immune system includes AMPs as a component of the innate immune system. Pleurocidin, one of these AMPs, has been found to be effective against various Gram-positive and Gram-negative bacteria. More investigations are required to determine pleurocidin’s suitability as a treatment against antibiotic-resistant pathogens. There is promising evidence for pleurocidin as a novel anti-cancer therapy. The peptide has been found to display potent anti-cancer effects against human cancer cells. Research efforts focused on pleurocidin may result in novel treatment strategies against antibiotic-resistant bacteria and cancer. More research is required to determine if the peptide is a suitable candidate to be developed into a novel anti-microbial treatment. Some of the microbes susceptible to the peptide are also pathogens of fish, suggesting its suitability as a therapeutic treatment for fish species. Full article
(This article belongs to the Special Issue Antimicrobial Peptides as New Weapons to Fight Antimicrobial Resistance)
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<p>PubMed and Environment Complete diagram of papers included in the review of pleurocidin.</p> Full article ">Figure 2
<p>Helical wheel (<b>a</b>) and net diagrams (<b>b</b>) of pleurocidin [<a href="#B4-pharmaceuticals-14-00687" class="html-bibr">4</a>]. Amino acids are indicated by their single-letter abbreviations and are categorized according to the following colors: red (polar basic), green (polar uncharged), and yellow (nonpolar). Both the wheel and net diagrams demonstrate the division of hydrophobic and hydrophilic components of the peptide. A slightly acidic pH is assumed, as the histidine residues are labelled as polar. The helical wheel diagram was generated using the online program NetWheels [<a href="#B35-pharmaceuticals-14-00687" class="html-bibr">35</a>] and the net diagram was made using Microsoft PowerPoint.</p> Full article ">Figure 3
<p>Role of the AMP pleurocidin in the winter flounder in the defense against pathogens. This peptide is located in the granular cells of winter flounder skin. Upon interacting with a negatively charged membrane, the peptide conforms into an α-helix. Made in © BioRender—<a href="http://biorender.com" target="_blank">biorender.com</a> accessed on 31 May 2021.</p> Full article ">Figure 4
<p>Amino acid sequence of initial pleurocidin peptide NRC-04 [<a href="#B4-pharmaceuticals-14-00687" class="html-bibr">4</a>].</p> Full article ">
15 pages, 2918 KiB  
Article
Antiparasitic Properties of Cardiovascular Agents against Human Intravascular Parasite Schistosoma mansoni
by Raquel Porto, Ana C. Mengarda, Rayssa A. Cajas, Maria C. Salvadori, Fernanda S. Teixeira, Daniel D. R. Arcanjo, Abolghasem Siyadatpanah, Maria de Lourdes Pereira, Polrat Wilairatana and Josué de Moraes
Pharmaceuticals 2021, 14(7), 686; https://doi.org/10.3390/ph14070686 - 16 Jul 2021
Cited by 23 | Viewed by 4059
Abstract
The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Praziquantel is the only drug available to treat schistosomiasis and there is an urgent demand for new anthelmintic agents. Adopting a phenotypic drug [...] Read more.
The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Praziquantel is the only drug available to treat schistosomiasis and there is an urgent demand for new anthelmintic agents. Adopting a phenotypic drug screening strategy, here, we evaluated the antiparasitic properties of 46 commercially available cardiovascular drugs against S. mansoni. From these screenings, we found that amiodarone, telmisartan, propafenone, methyldopa, and doxazosin affected the viability of schistosomes in vitro, with effective concentrations of 50% (EC50) and 90% (EC90) values ranging from 8 to 50 µM. These results were further supported by scanning electron microscopy analysis. Subsequently, the most effective drug (amiodarone) was further tested in a murine model of schistosomiasis for both early and chronic S. mansoni infections using a single oral dose of 400 mg/kg or 100 mg/kg daily for five consecutive days. Amiodarone had a low efficacy in chronic infection, with the worm and egg burden reduction ranging from 10 to 30%. In contrast, amiodarone caused a significant reduction in worm and egg burden in early infection (>50%). Comparatively, treatment with amiodarone is more effective in early infection than praziquantel, demonstrating the potential role of this cardiovascular drug as an antischistosomal agent. Full article
(This article belongs to the Special Issue Antiparasitics)
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<p>Chemical structures of cardiovascular drugs with in vitro schistosomicidal activity.</p> Full article ">Figure 2
<p>Viability of <span class="html-italic">S. mansoni</span> ex vivo following exposure to cardiovascular agents. Adult worms were obtained from mice by perfusion 49 days after infection. Parasites were monitored for up to 72 h and survival was plotted as a percentage over time using the Kaplan–Meier curves. Mean values were derived from a minimum of three experiments, and each experiment was performed with five replicates. Control (dashed line): RPMI 1640 + 0.5% DMSO. PZQ, praziquantel at 2 μM.</p> Full article ">Figure 3
<p>Microscopy observations of <span class="html-italic">S. mansoni</span> male worms after exposure to cardiovascular drugs. Freshly perfused parasites were placed on plates containing various concentrations of cardiovascular agents. (<b>a</b>) Control showing tubercles (Tu) and spines on the surface (arrow); (<b>b</b>) amiodarone 12.5 µM; (<b>c</b>) amiodarone 25 µM; (<b>d</b>) amiodarone 50 µM; (<b>e</b>) telmisartan 12.5 µM; (<b>f</b>) telmisartan 25 µM; (<b>g</b>) telmisartan 50 µM; (<b>h</b>) propafenone 50 µM; (<b>i</b>) propafenone 25 µM. Parasites were monitored for up to 72 h and micrographs of the mid-body region of schistosomes show disintegration (di), roughening (ro), and shrinking (sh). Images were obtained using a JEOL SM-6460LV scanning electron microscope. Scale-bars: 10 μm.</p> Full article ">Figure 4
<p>Effect of amiodarone on the parasite burden of mice harboring either early or chronic <span class="html-italic">S. mansoni</span> infection. Amiodarone (single dose of 400 mg/kg or 100 mg/kg for five consecutive days), praziquantel (PZQ, 400 mg/kg), and vehicle (control) were administered 21 days (early infection) or 49 days (chronic infection) post-infection by oral gavage. On day 63 post-infection, all animals were euthanized, and parasite burdens were determined. Points represent data from individual animals (<span class="html-italic">n</span> = 5 per group). Horizontal bars represent median values. ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 compared with infected untreated control. WBR, worm burden reduction.</p> Full article ">Figure 5
<p>Effect of amiodarone on the egg burden in the tissue of mice harboring either early or chronic <span class="html-italic">S. mansoni</span> infection. Amiodarone (single dose of 400 mg/kg or 100 mg/kg for five consecutive days), praziquantel (PZQ, 400 mg/kg), and vehicle (control) were administered 21 days (early infection) or 49 days (chronic infection) post-infection by oral gavage. On day 63 post-infection, all animals were euthanized, and egg burdens were determined by counting immature eggs in the intestine. Points represent data from individual animals (<span class="html-italic">n</span> = 5 per group). Horizontal bars represent median values. ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 compared with infected untreated control.</p> Full article ">Figure 6
<p>Effect of amiodarone on the egg burden in the tissue of mice harboring either early or chronic <span class="html-italic">S. mansoni</span> infection. Amiodarone (single dose of 400 mg/kg or 100 mg/kg for five consecutive days), praziquantel (PZQ, 400 mg/kg), and vehicle (control) were administered 21 days (early infection) or 49 days (chronic infection) post-infection by oral gavage. On day 63 post-infection, all animals were euthanized, and egg burdens were determined by counting immature eggs in the intestine. Points represent data from individual animals (<span class="html-italic">n</span> = 5 per group). Horizontal bars represent median values. ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 compared with infected untreated control.</p> Full article ">
25 pages, 7881 KiB  
Article
Interaction between DNA, Albumin and Apo-Transferrin and Iridium(III) Complexes with Phosphines Derived from Fluoroquinolones as a Potent Anticancer Drug
by Sandra Amanda Kozieł, Monika Katarzyna Lesiów, Daria Wojtala, Edyta Dyguda-Kazimierowicz, Dariusz Bieńko and Urszula Katarzyna Komarnicka
Pharmaceuticals 2021, 14(7), 685; https://doi.org/10.3390/ph14070685 - 16 Jul 2021
Cited by 18 | Viewed by 3954
Abstract
A group of cytotoxic half-sandwich iridium(III) complexes with aminomethyl(diphenyl)phosphine derived from fluoroquinolone antibiotics exhibit the ability to (i) accumulate in the nucleus, (ii) induce apoptosis, (iii) activate caspase-3/7 activity, (iv) induce the changes in cell cycle leading to G2/M phase arrest, and (v) [...] Read more.
A group of cytotoxic half-sandwich iridium(III) complexes with aminomethyl(diphenyl)phosphine derived from fluoroquinolone antibiotics exhibit the ability to (i) accumulate in the nucleus, (ii) induce apoptosis, (iii) activate caspase-3/7 activity, (iv) induce the changes in cell cycle leading to G2/M phase arrest, and (v) radicals generation. Herein, to elucidate the cytotoxic effects, we investigated the interaction of these complexes with DNA and serum proteins by gel electrophoresis, fluorescence spectroscopy, circular dichroism, and molecular docking studies. DNA binding experiments established that the complexes interact with DNA by moderate intercalation and predominance of minor groove binding without the capability to cause a double-strand cleavage. The molecular docking study confirmed two binding modes: minor groove binding and threading intercalation with the fluoroquinolone part of the molecule involved in pi stacking interactions and the Ir(III)-containing region positioned within the major or minor groove. Fluorescence spectroscopic data (HSA and apo-Tf titration), together with molecular docking, provided evidence that Ir(III) complexes can bind to the proteins in order to be transferred. All the compounds considered herein were found to bind to the tryptophan residues of HSA within site I (subdomain II A). Furthermore, Ir(III) complexes were found to dock within the apo-Tf binding site, including nearby tyrosine residues. Full article
(This article belongs to the Special Issue Privileged Structures as Leads in Medicinal Chemistry)
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<p>Scheme of cytotoxic action mechanism of Ir(III) complexes described by our group in a previous paper (Kozieł, S. et al., Inorganic Chemistry Frontiers, 2020, 7, 3386–3401).</p> Full article ">Figure 2
<p>Stern–Volmer plots of the CT DNA-EB (intercalation), CT DNA-DAPI (binding to a minor groove) and CT DNA-MG (binding to a major groove), system quenched by (<b>A</b>) <b>IrPCp</b>; (<b>B</b>) <b>IrPLm</b>; (<b>C</b>) <b>IrPNr</b>; (<b>D</b>) <b>IrPSf</b>; (I<sub>0</sub> and I—intensity of CT DNA-EB or DAPI or MG in the absence and the presence of increasing concentration [mM] of the compounds; (<b>E</b>) Fluorescence quenching of DAPI-DNA (C = 5 × 10<sup>−5</sup> M) by <b>IrPSf</b> (molar ratios 0.5–10) in 50 mM pH 7.4 phosphate buffer (axis: y—fluorescence intensity; x—wavelength).</p> Full article ">Figure 3
<p>(<b>A</b>) Stern–Volmer plots of the CT DNA–DAPI (binding to a minor groove) system quenched by <b>IrPCp</b>, <b>IrPLm</b>, <b>IrPNr</b>, <b>IrPSf</b>; (<b>B</b>) determined K<sub>DSV</sub> (dynamic quenching constant) values for the studied Ir(III) complexes.</p> Full article ">Figure 4
<p>Circular dichroism spectra of CT DNA with: (<b>A</b>) <b>IrPCp</b>; (<b>B</b>) <b>IrPLm</b>; (<b>C</b>) <b>IrPSf</b>; (<b>D</b>) <b>IrPNr</b>; (<b>E</b>) agarose gel electrophoresis of pBR322 plasmid cleavage by <b>IrPCp</b>, <b>IrPSf</b>, <b>IrPLm</b> and <b>IrPNr</b> in a DMF (each in the 10% DMF) solution, ctrl: plasmid—control. (<b>E1</b>) 1 h of incubation; (<b>E2</b>) 4 h of incubation; (<b>E3</b>) 24 h of incubation.</p> Full article ">Figure 5
<p>Agarose gel electrophoresis of pBR322 plasmid cleavage by: (<b>A</b>) <b>IrPCp</b> and <b>IrPNr</b>. Lanes: 1, plasmid−control; 2, plasmid + 50 μM <b>IrPCp</b>; 3, plasmid + 50 μM <b>IrPCp</b> + 50 μM H<sub>2</sub>O<sub>2</sub>; 4, plasmid + 50 μM <b>IrPCp</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + DMSO; 5, plasmid + 50 μM <b>IrPCp</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.1 M NaN<sub>3</sub>; 6, plasmid + 50 μM <b>IrPCp</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.62 μM SOD; 7, plasmid + 50 μM <b>IrPNr</b>; 8, plasmid + 50 μM <b>IrPNr</b> + 50 μM H<sub>2</sub>O<sub>2</sub>; 9, plasmid + 50 μM <b>IrPNr</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + DMSO; 10, plasmid + 50 μM <b>IrPNr</b> + 50μM H<sub>2</sub>O<sub>2</sub> + 0.1 M NaN<sub>3</sub>; 11, plasmid + 50μM <b>IrPNr</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.62 μM SOD; 12, plasmid + DMF; (<b>B</b>) by <b>IrPLm</b> and <b>IrPSf</b>. Lanes: 1, plasmid−control; 2, plasmid + 50 μM <b>IrPLm</b>; 3, plasmid + 50 μM <b>IrPLm</b> + 50 μM H<sub>2</sub>O<sub>2</sub>; 4, plasmid + 50 μM <b>IrPLm</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + DMSO; 5, plasmid + 50 μM <b>IrPLm</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.1 M NaN<sub>3</sub>; 6, plasmid + 50 μM <b>IrPLm</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.62 μM SOD; 7, plasmid + 50 μM <b>IrPSf</b>; 8, plasmid + 50 μM <b>IrPSf</b> + 50 μM H<sub>2</sub>O<sub>2</sub>; 9, plasmid + 50 μM <b>IrPSf</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + DMSO; 10, plasmid + 50 μM <b>IrPSf</b> + 50μM H<sub>2</sub>O<sub>2</sub> + 0.1 M NaN<sub>3</sub>; 11, plasmid + 50μM <b>IrPSf</b> + 50 μM H<sub>2</sub>O<sub>2</sub> + 0.62 μM SOD; 12, plasmid + DMF.</p> Full article ">Figure 6
<p>Representative structures of the binding modes of DNA-phosphinoiridium(III) complexes: top, minor groove binding; and bottom, threading intercalation.</p> Full article ">Figure 7
<p>(<b>A</b>) Fluorescence quenching of HSA (C = 5 × 10<sup>−5</sup> M) by <b>IrPNr</b>, (molar ratios 0.5, 1, 1.5, 2, 3, 4, 5, 6) in 50 mM pH 7.4 phosphate buffer; (<b>B</b>) Stern–Volmer plots of HSA (C = 10 μM) quenching by <b>PCp</b>, <b>PNr</b>, <b>PSf</b>, <b>PLm</b>, <b>IrPCp</b>, <b>IrPNr</b>, <b>IrPSf</b>, <b>IrPLm</b> (I<sub>0</sub> and I—intensity of HSA in the absence and presence of the increasing amounts of the compounds, respectively).</p> Full article ">Figure 8
<p>Binding mode of HSA-phosphinoiridium(III) complexes. The docked compounds are shown in ball-and-stick representation. Trp214 residue is shown in stick representation in the panels on the left side. In the right side panels are all the HSA residues within 3 Å of the metal complexes. The selected distances are given in Å. Hydrogen atoms are not shown for clarity.</p> Full article ">Figure 9
<p>(<b>A</b>) Fluorescence quenching of apo-Tf (C = 3.6 × 10<sup>−6</sup> M) by IrPCp (molar ratios 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10) in 50 mM pH 7.4 phosphate buffer; (<b>B</b>) Stern–Volmer plots of apo-Tf (C = 3.6 μM) quenching by <b>IrPCp</b>, <b>IrPNr</b>, <b>IrPSf</b>, <b>IrPLm</b>, <b>PCp</b>, <b>PNr</b>, <b>PSf</b>, <b>PLm</b> (I<sub>0</sub> and I—intensity of HSA in the absence and presence of the increasing amounts of the compounds, respectively); (<b>C</b>) Circular dichroism spectra of apo-transferrin with Ir(III) complexes.</p> Full article ">Figure 10
<p>The predicted binding pockets within apo-Tf structure. The N- and C-lobes of apo-Tf are shown in orange and light green colors. The location of #1, #2, #3, and #4 binding sites is specified by the docked <b>IrPCp</b> shown in blue, red, yellow, and dark green colors, respectively.</p> Full article ">Figure 11
<p>Binding mode of apo-Tf-complexes within the #2 binding pocket. The docked compound’s molecules are shown in ball-and-stick representation. All the apo-Tf residues within 3 Å of the metal complexes are presented in stick representation. The selected distances are given in Å. Hydrogen atoms are not shown for clarity.</p> Full article ">Scheme 1
<p>Organometallic iridium(III) complexes used in this study.</p> Full article ">
17 pages, 3345 KiB  
Article
Delaying Effects of Prolactin and Growth Hormone on Aging Processes in Bovine Oocytes Matured In Vitro
by Galina N. Singina, Ekaterina N. Shedova, Alexander V. Lopukhov, Olga S. Mityashova and Irina Y. Lebedeva
Pharmaceuticals 2021, 14(7), 684; https://doi.org/10.3390/ph14070684 - 16 Jul 2021
Cited by 7 | Viewed by 3009
Abstract
Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. [...] Read more.
Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway. Full article
(This article belongs to the Special Issue Novel Regulators of Female Reproduction)
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<p>Effects of prolactin (PRL, 50 ng/mL) and growth hormone (GH, 10 ng/mL) on spontaneous parthenogenetic activation during the aging of bovine cumulus-enclosed oocytes (CEOs) (<b>A</b>) and denuded oocytes (DOs) (<b>B</b>). Data represent means ± SEM of 4–5 replicates using 71–104 oocytes per treatment. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 2
<p>Effects of PRL (50 ng/mL) and GH (10 ng/mL) on spontaneous parthenogenetic activation during the 44 h aging of bovine DOs in the presence and in the absence of PP2 (20 μM), the inhibitor of Src-family tyrosine kinases, and triciribine (TCB, 50 μM), the inhibitor of Akt kinase. Data derived from 4 independent replicates (a total of 79–86 oocytes per each treatment group) are expressed as means ± SEM. Different letters indicate significant differences between means (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Effects of PRL (50 ng/mL) and GH (10 ng/mL) on spontaneous parthenogenetic activation during the 44 h aging of bovine DOs in the presence and in the absence of U0126 (20 μM), the MEK 1/2 inhibitor, and L-NAME (20 μM), the efficient eNOS and nNOS inhibitor (20 μM). Data derived from 4 independent replicates (a total of 71–80 oocytes per each treatment group) are expressed as means ± SEM. Different letters indicate significant differences between means (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>Immunofluorescent detection of PRL receptors in M-II oocytes after the 20 h maturation of bovine CEOs. (<b>A</b>,<b>D</b>) Bright-field images. (<b>B</b>,<b>E</b>) DNA staining with DAPI (blue). (<b>C</b>) Positive staining using the MA1-610 antibody and goat anti-mouse IgG conjugated with Alexa Fluor 488 (green). (<b>F</b>) Negative control performed by omitting the primary antibody. Original magnification: ×400.</p> Full article ">Figure 5
<p>Immunofluorescent detection of GH receptors in M-II oocytes after the 20 h maturation of bovine CEOs. (<b>A</b>,<b>D</b>) Bright-field images. (<b>B</b>,<b>E</b>) DNA staining with DAPI (blue). (<b>C</b>) Positive staining using MAB 263 antibody and goat anti-mouse IgG conjugated with Alexa Fluor 488 (green). (<b>F</b>) Negative control performed by omitting the primary antibody. Original magnification: ×400.</p> Full article ">Figure 6
<p>Representative images of apoptosis in matured or aged bovine CEOs. (<b>A</b>,<b>D</b>) Matured oocytes prior to aging. (<b>B</b>,<b>E</b>) Oocytes aged in the control medium. (<b>C</b>,<b>F</b>) Oocytes aged in the presence of PRL. (<b>A</b>–<b>C</b>) DAPI staining (blue). <span class="html-italic">(</span><b>D</b>–<b>F</b>) TUNEL staining (green). TUNEL-positive chromosomes are indicated by white arrows. Original magnification: ×200.</p> Full article ">Figure 7
<p>Effects of PRL (50 ng/mL) and GH (10 ng/mL) on oocyte apoptosis during the 24 h aging of bovine CEOs and DOs. Data represent means ± SEM of 4 replicates using 75–88 oocytes per treatment. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>Effects of PRL (50 ng/mL) on oocyte apoptosis during the 24 h aging of bovine CEOs in the presence and in the absence of PP2 (20 μM), triciribine (TCB, 50 μM), and calphostin C (CalpC; 1 µM). Data represent means ± SEM of 4 replicates using 52–80 oocytes per treatment. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 9
<p>The developmental capacity of in vitro matured and aged bovine CEOs and DOs. Data represent means ± SEM of 6 replicates using 153–172 oocytes per treatment. Means marked with different letters differ significantly (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 10
<p>Representative images of embryos (day 7) derived from in vitro-matured or aged bovine CEOs. (<b>A</b>,<b>D</b>) Oocytes were fertilized immediately after IVM. (<b>B</b>,<b>E</b>) Oocytes were fertilized following 12 h of aging in the control medium. (<b>C</b>,<b>F</b>) Oocytes were fertilized following 12 h of aging in the medium containing PRL. (<b>A</b>–<b>C</b>) The morphology of IVP blastocysts. Original magnification: ×100. (<b>D</b>–<b>F</b>) The nuclear state of IVP blastocysts, DAPI staining. Original magnification: ×200.</p> Full article ">Figure 11
<p>Effects of PRL (50 ng/mL) and GH (10 ng/mL) during the 12 h aging of bovine CEOs and DOs on the oocyte developmental capacity: (<b>A</b>) cleavage rate, (<b>B</b>) blastocyst rate. Data represent means ± SEM of 6 replicates using 173–184 oocytes per treatment. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 12
<p>The quality of embryos derived from in vitro-matured bovine CEOs aged for 12 h in the absence or in the presence of PRL (50 ng/mL). Data represent means ± SEM for 46 (0 h of aging), 15 (control, 12 h of aging) and 31 (PRL, 12 h of aging) blastocysts on day 7 after IVF. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 13
<p>Effects of PRL (50 ng/mL) during the 12 h aging of bovine CEOs in the presence and in the absence of PP2 (10 μM), triciribine (TCB, 25 μM), and calphostin C (CalpC; 0.5 µM) on the subsequent blastocyst formation. Data represent means ± SEM of 6–7 replicates using 177–212 oocytes per treatment. Means marked with different letters differ significantly (at least <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
17 pages, 3567 KiB  
Article
Efficacy of an Anti-Cellulite Herbal Emgel: A Randomized Clinical Trial
by Ngamrayu Ngamdokmai, Neti Waranuch, Krongkarn Chootip, Katechan Jampachaisri, C. Norman Scholfield and Kornkanok Ingkaninan
Pharmaceuticals 2021, 14(7), 683; https://doi.org/10.3390/ph14070683 - 16 Jul 2021
Cited by 4 | Viewed by 5022
Abstract
Cellulite describes unsightly skin overlying subcutaneous fat around thighs and buttocks of post-pubescent females. A herbal ‘emgel’ containing volatile oils and extracts of A traditional Thai herbal compress was tested in a double-blind, placebo-controlled trial with 18 women aged 20–50 year with severe [...] Read more.
Cellulite describes unsightly skin overlying subcutaneous fat around thighs and buttocks of post-pubescent females. A herbal ‘emgel’ containing volatile oils and extracts of A traditional Thai herbal compress was tested in a double-blind, placebo-controlled trial with 18 women aged 20–50 year with severe cellulite. Appearance of cellulite (primary outcome), thigh circumferences, skin firmness, and cutaneous blood flow (secondary outcomes) were assessed at baseline, 2, 4, 8 and 12 weeks with a 2-week follow-up. Herbal emgel applied onto the thigh skin twice daily reduced cellulite severity scores in every time point. The score was reduced from 13.4 ± 0.3 (baseline) to 12.1 ± 0.3 (week 2) and 9.9 ± 0.6 (week 12). All secondary outcomes improved with both placebo and herbal emgels suggesting that ingredients in the base-formulation might be responsible. Querying of participants, analysis of their diaries, and physical monthly inspections found no adverse events. The herbal emgel safely improved the appearance of cellulite, while the base emgel may play a role for other endpoints. Further studies on the active constituents and their mechanism of action are needed to further explore these factors. Full article
(This article belongs to the Special Issue Promising Anti-inflammatory Medicinal Plants—An Assessment of Bioavailability, Pharmacokinetics, Clinical Efficacy and Overview of New Methodological Approaches)
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<p>Chromatographic analyses of the chemical constituents in the placebo and the herbal emgels. (<b>A</b>) Headspace GC-MS (SIM) total ion chromatograms of 2 mg herbal emgel (black trace), and placebo emgel (red trace, displaced by 2 s), (<b>B</b>) HPLC chromatograms of 20 mg/mL of the herbal emgel (black trace), and the placebo emgel (green trace).</p> Full article ">Figure 2
<p>Participant experience through the trial.</p> Full article ">Figure 3
<p>Representative photographs from a single participant showing thighs in posterior aspect as seen by three evaluators whose estimate is given as cellulite grades. The image analysis used the areas bounded by the yellow boxes and the unitless values were generated by three scans of each image to give means ± SEMs.</p> Full article ">Figure 4
<p>Cellulite changes during the 14 weeks trial. (<b>A</b>) Cellulite grades estimated by three blinded independent evaluators. (<b>B</b>) Cellulite grades expressed as percentages of the grade before treatment (baseline). (<b>C</b>) Normalised cellulite values measured by image analysis. Values are means ± SEMs. <span class="html-italic">p</span>-values <b>in red</b> compare values for herbal gel with corresponding baseline values, those <b>in blue</b> compare values for placebo gel with corresponding baseline values. <span class="html-italic">p</span>-values <b>in black</b> compare herbal and placebo emgel values at the same time point. All statistical testing was performed on data normalised to baseline values for each participant. An absent value indicates <span class="html-italic">p</span> &gt; 0.1. All points are means ± SEM.</p> Full article ">Figure 5
<p>Thigh circumferences 20 (upper) (<b>A</b>) or 10 cm (lower) (<b>B</b>) above the knee treated with either placebo or herbal emgels. The measurements expressed as the corresponding ratio at time 0 (baseline). For the upper thighs, actual baseline values for placebo and herbal emgels were 55.4 ± 1.1 and 55.6 ± 1.1 cm respectively and 47.1 ± 0.9 and 47.6 ± 1.0 cm for the lower thighs. <span class="html-italic">p</span>-value color coded as <a href="#pharmaceuticals-14-00683-f004" class="html-fig">Figure 4</a>.</p> Full article ">Figure 6
<p>Skin firmness recorded from the posterior (<b>A</b>) and anterior (<b>B</b>) aspects of the thighs. All <span class="html-italic">p</span>-values colour coded as <a href="#pharmaceuticals-14-00683-f004" class="html-fig">Figure 4</a>.</p> Full article ">Figure 7
<p>Cutaneous blood flow by laser Doppler flowmetry. All <span class="html-italic">p</span>-values colour coded as <a href="#pharmaceuticals-14-00683-f003" class="html-fig">Figure 3</a>.</p> Full article ">
16 pages, 2513 KiB  
Article
Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy
by Jianling Bi, Garima Dixit, Yuping Zhang, Eric J. Devor, Haley A. Losh, Andreea M. Newtson, Kristen L. Coleman, Donna A. Santillan, Thorsten Maretzky, Kristina W. Thiel and Kimberly K. Leslie
Pharmaceuticals 2021, 14(7), 682; https://doi.org/10.3390/ph14070682 - 16 Jul 2021
Cited by 8 | Viewed by 3631
Abstract
Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was [...] Read more.
Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy. Full article
(This article belongs to the Section Pharmacology)
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<p>Impact of bevacizumab and cediranib on VEGFR signaling endothelial cells and endometrial cancer cells. (<b>A</b>) HUVEC, Hec50, and KLE cells were treated with vehicle control (CT), 1 µM bevacizumab (Bev), 100 ng/mL VEGF-A, or 1 µM cediranib (Ced) for 1 hr, followed by assessment of ERK1/2 phosphorylation at Thr202/Tyr204 or total ERK expression by Western blotting. β-actin: loading control. (<b>B</b>) Cells were transfected with the alkaline phosphatase (AP)-tagged ADAM17 substrate TGF-α, and treated as in (<b>A</b>) for 1 hr. The change in soluble AP-TGF-α was assessed and presented as fold change compared to control (CT). PMA (25 ng/mL) served as a positive control for induction of AP-TGF-α shedding. ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001 versus CT by one-way ANOVA with Tukey’s multiple comparisons test.</p> Full article ">Figure 2
<p>Effect of cediranib and bevacizumab on sensitivity to paclitaxel in patient-derived organoid cultures of primary endometrial tumors. Organoid cultures were treated with 10 nM paclitaxel, 1 µM bevacizumab, 1 µM cediranib, or the combination of paclitaxel with bevacizumab or cediranib for 72 hrs, followed by assessment of cell viability. (<b>A</b>) Data were calculated as the percent (%) cell viability as compared to vehicle control and plotted left-to-right by increasing sensitivity to single-agent paclitaxel. (<b>B</b>) The change in viability with the combination of paclitaxel with either bevacizumab or cediranib was calculated relative to paclitaxel alone. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 versus control; †† <span class="html-italic">p</span> &lt; 0.01, ††† <span class="html-italic">p</span> &lt; 0.001 versus paclitaxel alone; <span>$</span><span>$</span> <span class="html-italic">p</span> &lt; 0.01, <span>$</span><span>$</span><span>$</span> <span class="html-italic">p</span> &lt; 0.001 versus anti-angiogenic agent alone (bevacizumab for bevacizumab + paclitaxel or cediranib for cediranib + paclitaxel treated samples) by ordinary one-way ANOVA with Tukey’s multiple comparisons test.</p> Full article ">Figure 3
<p>Cediranib but not bevacizumab increases sensitivity to paclitaxel in Hec50 endometrial cancer cells. (<b>A</b>) Hec50 (left) or KLE cells (right) were treated with increasing concentrations of paclitaxel in the absence or presence of 1 µM bevacizumab for 72 h; cell viability was determined using WST-1 assay relative to untreated control. (<b>B</b>) Hec50 (left) or KLE cells (right) were treated with increasing concentrations of paclitaxel in the absence or presence of 1 µM cediranib for 72 h; cell viability was determined using WST-1 assay. (<b>C</b>) Hec50 (left) or KLE cells (right) were treated with increasing concentrations of cediranib in the absence or presence of 5 nM paclitaxel for 72 h; cell viability was determined using WST-1 assay. For the combinatorial treatments in (<b>B</b>,<b>C</b>) (green lines), the points on the y-axis at 0 reflect treatment with either cediranib alone (<b>B</b>) or paclitaxel alone (<b>C</b>). For example, single-agent cediranib at 1 µM ((<b>B</b>), Hec50) results in a 40.0% decrease in cell viability as compared to untreated control. Statistical significance was assessed by two-way ANOVA with Sidak’s post-hoc test. n.s.: not significant.</p> Full article ">Figure 4
<p>Comparison of the effect of cediranib and bevacizumab on cell cycle distribution in Hec50 cells treated with paclitaxel. (<b>A</b>) Cell cycle distribution in Hec50 cells treated with DMSO (Control), 14 nM paclitaxel, 1 μM bevacizumab, or a combination of paclitaxel and 1 μM bevacizumab for 24 h. (<b>B</b>) Cell cycle distribution for Hec50 cells treated with vehicle (DMSO), 14 nM paclitaxel, 1 μM cediranib, or a combination of 14 nM paclitaxel and 1 μM cediranib. Insets denote the percentage of cells in the G2/M phase of the cell cycle.</p> Full article ">Figure 5
<p>Molecular effects of anti-angiogenic agents on G2/M cell cycle controllers in endometrial cancer cell models. (<b>A</b>) Hec50 (left) or KLE cells (right) were treated with the indicated agents either alone or in combination with paclitaxel for 24 hrs, followed by assessment of cell cycle controllers by Western blotting. Drug concentrations: 10 μM gefitinib, 1 μM cediranib, 1 μM bevacizumab,14 nM paclitaxel. (<b>B</b>) Expression or phosphorylation of indicated proteins was assessed by Kinex™ KAM-1325 Phosphproteomic Antibody Microarray in Hec50 cells treated with 1 µM bevacizumab or 1 µM cediranib for 24 h. Data were calculated as the percent change from control (%CFC). Phosphosites corresponding to those queried in (<b>A</b>) are indicated with boxes. Pan: antibody total protein expression.</p> Full article ">Figure 6
<p>Bevacizumab regulates more signaling events than cediranib in endometrial cancer cells. Signaling events in response to single-agent bevacizumab or cediranib were analyzed by Kinex™ KAM-1325 Phosphproteomic Antibody Microarray after treatment of Hec50 cells with 1 µM bevacizumab or 1 µM cediranib for 24 h. (<b>A</b>) Depiction of changes in select cell cycle controllers. Data were calculated as the percent change from control (%CFC); “pan” indicates total protein expression. (<b>B</b>) Venn diagram of lead candidates in response to either bevacizumab or cediranib. Overlap indicates signaling events that were shared between the two treatment groups. Up = increased expression/phosphorylation; down = decreased expression/phosphorylation. (<b>C</b>) Table of all lead candidates identified based on the %CFC [<a href="#B28-pharmaceuticals-14-00682" class="html-bibr">28</a>]. Negative %CFC indicates a decrease as compared to control. Full results are provided as <a href="#app1-pharmaceuticals-14-00682" class="html-app">Supplemental Table S2</a>.</p> Full article ">
12 pages, 1700 KiB  
Article
Comprehensive Analysis of Chemotherapeutic Agents That Induce Infectious Neutropenia
by Mashiro Okunaka, Daisuke Kano, Reiko Matsui, Toshikatsu Kawasaki and Yoshihiro Uesawa
Pharmaceuticals 2021, 14(7), 681; https://doi.org/10.3390/ph14070681 - 15 Jul 2021
Cited by 20 | Viewed by 3595
Abstract
Chemotherapy-induced neutropenia (CIN) has been associated with a risk of infections and chemotherapy dose reductions and delays. The chemotherapy regimen remains one of the primary determinants of the risk of neutropenia, with some regimens being more myelotoxic than others. Although a number of [...] Read more.
Chemotherapy-induced neutropenia (CIN) has been associated with a risk of infections and chemotherapy dose reductions and delays. The chemotherapy regimen remains one of the primary determinants of the risk of neutropenia, with some regimens being more myelotoxic than others. Although a number of clinical trials have currently highlighted the risk of CIN with each chemotherapy regimen, only a few ones have comprehensively examined the risk associated with all chemotherapeutic agents. Therefore, this study aimed to investigate the risk factors and characteristics of CIN caused by each neoplastic agent using data from the large voluntary reporting Food and Drug Administration Adverse Event Reporting System database. Initially, univariate analysis showed that an age ≥ 65 years, the female sex, and treatment with chemotherapeutic agents were factors that caused CIN. Then, cluster and component analyses showed that cytotoxic agents (i.e., alkylating agents, antimetabolic agents, antineoplastic antibiotics, platinating agents, and plant-derived alkaloids) were associated with infection following neutropenia. This comprehensive analysis comparing CIN risk suggests that elderly or underweight patients treated with cytotoxic drugs require particularly careful monitoring. Full article
(This article belongs to the Special Issue Cancer Drugs Treatment and Toxicity)
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<p>Flow chart for the construction of the data analysis table. The REAC table was classified into three categories: “suspected medicine”, “concomitant medicine” and “interaction medicine”. We extracted only “primary suspect drug” information relevant to these categories from the REAC table. Duplicate data were then removed from the REAC, DEMO, and DRUG tables. Based on the combined table, only “suspected medicine” information was used to assess the risk of diarrhea. Available information on “suspected medicine”, “concomitant medicine” and “interaction medicine” was used for the time-of-onset analysis. Each table was combined, cleaned up, and then used as the data analysis table.</p> Full article ">Figure 2
<p>Drugs associated with CIN development. The X axis shows the natural lnORs, whereas the Y axis shows the common logarithm of the inverse <span class="html-italic">p</span> value (−log10(<span class="html-italic">p</span> value)) from Fisher’s exact test. The ORs were calculated using cross-tabulation. The dotted line on the Y axis represents <span class="html-italic">p</span> = 0.05. The plot colors indicate Anatomical Therapeutic Chemistry classification, whereas the plot size indicates the common logarithm of the total number of reported adverse events for each drug (−0.25 to 5.75).</p> Full article ">Figure 3
<p>Classification of CIN-related chemotherapeutic agents using hierarchical cluster analysis. The dendrogram shows the relationships between 58 chemotherapeutic agents and CIN. The color map shows the load value of the principal components in red–gray–blue.</p> Full article ">Figure 4
<p>Score plot related with components 1 and 2. The score plot shows the relationships between the drugs and principal components. Each plot indicates a drug. Plot colors indicate log (number of reports).</p> Full article ">Figure 5
<p>Relationships between CIN and drugs using principal component analysis. (<b>a</b>) Score plot; (<b>b</b>) loading vectors. (<b>a</b>) The score plot shows the relationships between the drugs and principal components. Each plot indicates a drug. Plot colors indicate Anatomical Therapeutic Chemistry classification. (<b>b</b>) Loading vectors represent the relationship between side effects and principal components. Each loading vector indicates a side effect.</p> Full article ">Figure 6
<p>Seven information tables included in the FAERS database. The row number shows the number of reports obtained between April 2004 and September 2020.</p> Full article ">
13 pages, 1481 KiB  
Review
Current Status of Baricitinib as a Repurposed Therapy for COVID-19
by Maha Saber-Ayad, Sarah Hammoudeh, Eman Abu-Gharbieh, Rifat Hamoudi, Hamadeh Tarazi, Taleb H. Al-Tel and Qutayba Hamid
Pharmaceuticals 2021, 14(7), 680; https://doi.org/10.3390/ph14070680 - 15 Jul 2021
Cited by 22 | Viewed by 6530
Abstract
The emergence of the COVID-19 pandemic has mandated the instant (re)search for potential drug candidates. In response to the unprecedented situation, it was recognized early that repurposing of available drugs in the market could timely save lives, by skipping the lengthy phases of [...] Read more.
The emergence of the COVID-19 pandemic has mandated the instant (re)search for potential drug candidates. In response to the unprecedented situation, it was recognized early that repurposing of available drugs in the market could timely save lives, by skipping the lengthy phases of preclinical and initial safety studies. BenevolentAI’s large knowledge graph repository of structured medical information suggested baricitinib, a Janus-associated kinase inhibitor, as a potential repurposed medicine with a dual mechanism; hindering SARS-CoV2 entry and combatting the cytokine storm; the leading cause of mortality in COVID-19. However, the recently-published Adaptive COVID-19 Treatment Trial-2 (ACTT-2) positioned baricitinib only in combination with remdesivir for treatment of a specific category of COVID-19 patients, whereas the drug is not recommended to be used alone except in clinical trials. The increased pace of data output in all life sciences fields has changed our understanding of data processing and manipulation. For the purpose of drug design, development, or repurposing, the integration of different disciplines of life sciences is highly recommended to achieve the ultimate benefit of using new technologies to mine BIG data, however, the final say remains to be concluded after the drug is used in clinical practice. This review demonstrates different bioinformatics, chemical, pharmacological, and clinical aspects of baricitinib to highlight the repurposing journey of the drug and evaluates its placement in the current guidelines for COVID-19 treatment. Full article
(This article belongs to the Special Issue COVID-19 in Pharmaceuticals)
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<p>Flow chart of database searching.</p> Full article ">Figure 2
<p>Dual mechanism of action of baracitinib; (<b>A</b>) to inhibit clathrin-mediated endocytosis of the SARS-CoV2, and (<b>B</b>) to inhibit the JAK-mediate release of pro-inflammatory cytokines.</p> Full article ">Figure 3
<p>The effect of baracitinib treatment on ACE2 and TMPRSS2 expression (generated from dataset GSE61552). The dataset compiled the expression profile of systemically barictinib-treated and ruxolitinib-treated C3H/HeJ grafted model of alopecia areata. The analysis revealed a significant reduction of ACE2 and TMPRSS2 expression in the baricitinib-treated samples, compared to untreated control ones. ACE2 = angiotensin-converting enzyme 2, TMPRSS2: transmembrane protease serine 2. ** <span class="html-italic">p</span>-value &lt; 0.01.</p> Full article ">Figure 4
<p>Structures of baricitinib, ruxolitinib, and fedratinib.</p> Full article ">Figure 5
<p>(<b>A</b>) An overlay of ruxolitinib (Purple) on baricitinib (Green); electronic similarity percentage = 82.5%. (<b>B</b>) An overlay of fedratinib (Gray) on ruxolitinib (Purple); electronic similarity percentage = 54.0%. (<b>C</b>) An overlay of fedratinib (Gray) on baricitinib (Green); electronic similarity percentage = 49.1%. Similarity and alignments were measured based on molecular fields descriptors generated by Cresset’s FieldAlign Software (version 1.0.2), [<a href="#B37-pharmaceuticals-14-00680" class="html-bibr">37</a>].</p> Full article ">
17 pages, 12034 KiB  
Article
Suppression of Intracellular Reactive Oxygen Species in Human Corneal Epithelial Cells via the Combination of Quercetin Nanoparticles and Epigallocatechin Gallate and In Situ Thermosensitive Gel Formulation for Ocular Drug Delivery
by Chuda Chittasupho, Taepin Junmahasathien, Jiratchaya Chalermmongkol, Raksakul Wongjirasakul, Phuriwat Leesawat and Siriporn Okonogi
Pharmaceuticals 2021, 14(7), 679; https://doi.org/10.3390/ph14070679 - 15 Jul 2021
Cited by 15 | Viewed by 4078
Abstract
Oxidative stress can cause several severe ophthalmological diseases. In this study, we developed a thermosensitive gel as a delivery system for two antioxidant substances, namely, quercetin and epigallocatechin gallate. The quercetin was loaded in the PLGA nanoparticles using a solvent displacement method. The [...] Read more.
Oxidative stress can cause several severe ophthalmological diseases. In this study, we developed a thermosensitive gel as a delivery system for two antioxidant substances, namely, quercetin and epigallocatechin gallate. The quercetin was loaded in the PLGA nanoparticles using a solvent displacement method. The physical and chemical stability of the quercetin nanoparticles were evaluated, and the degradation kinetics of the quercetin in the nanoparticles was investigated. The in vitro antioxidant and intracellular reactive oxygen species inhibition of the quercetin nanoparticles, combined with the epigallocatechin gallate (EGCG), were determined using a 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and a 2,7-dichlorodihydrofluorescein fluorescent probes, respectively. The thermosensitive gel loaded with the quercetin nanoparticles and EGCG was formulated. We confirmed that quercetin nanoparticles displayed the desired physical characteristics, release kinetics, and stability. The combination of quercetin nanoparticles and EGCG suggested the additive effect of antioxidant activity. We also demonstrated the superior intracellular ROS inhibition activity of the quercetin nanoparticles and EGCG with n-acetyl cysteine. The thermosensitive gel showed an appropriate gelation temperature and time for ocular drug delivery. Our results provide promising prospects for applying the thermosensitive gel loaded with quercetin nanoparticles and EGCG as an efficient drug delivery system for antioxidant activity in human corneal epithelial cells. Full article
(This article belongs to the Section Pharmaceutical Technology)
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<p>The hydrodynamic diameter of the quercetin-loaded PLGA NPs after fresh preparation and storage in deionized water for 2, 4, 8, and 12 weeks at 4 °C and 30 °C, respectively. *, **, and **** indicate <span class="html-italic">p</span> &lt; 0.05, 0.01, and 0.0001 compared with day 0, respectively.</p> Full article ">Figure 2
<p>Polydispersity index of the quercetin-loaded PLGA NPs after fresh preparation and storage in deionized water for 2, 4, 8, and 12 weeks at 4 °C and 30 °C.</p> Full article ">Figure 3
<p>Zeta potential values of the quercetin-loaded PLGA NPs after fresh preparation and storage in deionized water for 2, 4, 8, and 12 weeks at 4 °C and 30 °C. *, **, *** and **** indicate <span class="html-italic">p</span> &lt; 0.05, 0.01, 0.001, and 0.0001 compared with day 0, respectively.</p> Full article ">Figure 4
<p>(<b>A</b>) Hydrodynamic diameter and (<b>B</b>) polydispersity index of the quercetin-loaded PLGA NPs after fresh preparation and storage in phosphate buffer saline for 1, 2, 3, 4, 5, 6, and 7 days at 37 °C. * and ** indicate <span class="html-italic">p</span> &lt; 0.05 and 0.01 compared with day 0, respectively.</p> Full article ">Figure 5
<p>The cumulative release of quercetin from the quercetin-loaded PLGA NPs in phosphate buffer saline, pH 7.4, at 37 °C.</p> Full article ">Figure 6
<p>(<b>A</b>) Percentage remaining of the quercetin in PLGA NPs after storage at 4 °C, 30 °C, and 45 °C for 8 weeks. (<b>B</b>) Second-order kinetics model of the quercetin degradation. (<b>C</b>) Arrhenius plot for the quercetin in PLGA NPs degradation.</p> Full article ">Figure 7
<p>Percentages of the radical scavenging activity of pure quercetin, pure EGCG, quercetin-loaded NPs, quercetin- loaded PLGA NPs combined with EGCG, and blank PLGA NPs, which were determined using the DPPH free radical scavenging method.</p> Full article ">Figure 8
<p>(<b>A</b>) Effect of quercetin-loaded PLGA NPs combined with EGCG on the HCE cell viability after incubation for 24 h. (<b>B</b>) Changes in the morphology and density of the HCE cells after 24 h of exposure to various concentrations of quercetin-loaded PLGA NPs and EGCG.</p> Full article ">Figure 9
<p>Effect of quercetin loaded PLGA NPs combined with EGCG on intracellular ROS generation. (<b>A</b>) Quantification of the hydrogen peroxide levels within the indicated groups. (<b>B</b>) Quantification of the superoxide anion levels within the indicated groups. (<b>C</b>) Fluorescence microscopy of the HCE cells treated with fluorescent probes after cultivation in the presence and absence of simulated-Sun-ray oxidative stress and the treatment with quercetin loaded PLGA NPs and EGCG. *** and **** indicate <span class="html-italic">p</span> &lt; 0.001 and 0.0001 compared with control, respectively.</p> Full article ">Figure 10
<p>Rheology of the in situ gel loaded with quercetin-loaded PLGA NPs and EGCG at 33 °C and room temperature.</p> Full article ">
18 pages, 320 KiB  
Article
Risk of Hospitalization for Adverse Drug Events in Women and Men: A Post Hoc Analysis of an Active Pharmacovigilance Study in Italian Emergency Departments
by Giada Crescioli, Ennio Boscia, Alessandra Bettiol, Silvia Pagani, Giulia Spada, Giuditta Violetta Vighi, Roberto Bonaiuti, Mauro Venegoni, Giuseppe Danilo Vighi, Alfredo Vannacci, Niccolò Lombardi and on behalf of the MEREAFaPS Study Group
Pharmaceuticals 2021, 14(7), 678; https://doi.org/10.3390/ph14070678 - 15 Jul 2021
Cited by 10 | Viewed by 2841
Abstract
This post hoc analysis of an Italian active pharmacovigilance study describes pharmacological differences of ADEs leading to emergency department (ED) visits and hospitalization in women and men. During the study period (January 2007–December 2018), 61,855 reports of ADEs leading to ED visits were [...] Read more.
This post hoc analysis of an Italian active pharmacovigilance study describes pharmacological differences of ADEs leading to emergency department (ED) visits and hospitalization in women and men. During the study period (January 2007–December 2018), 61,855 reports of ADEs leading to ED visits were collected. Overall, 30.6% of ADEs resulted in hospitalization (30% in women and 31% in men). Multivariate logistic regression showed that, among women, drug classes significantly associated with an increased risk of hospitalization were heparins (ROR 1.41, CI 1.13–176), antidepressants (ROR 1.12, CI 1.03–1.23) and antidiabetics (ROR 1.13, CI 1.02–1.24). Among men, only vitamin K antagonists (ROR 1.28, CI 1.09–1.50), opioids (ROR 1.30, CI 1.06–1.60) and digitalis glycosides (ROR 1.32, CI 1.09–1.59) were associated with a higher risk of hospitalization. Overall, older age, multiple suspected drugs and the presence of comorbidities were significantly associated with a higher risk of hospitalization. A significantly reduced risk of hospitalization was observed in both women and men experiencing an adverse event following immunization (ROR 0.36, CI 0.27–0.48 and 0.83, 0.42–0.74, respectively) compared to drugs. Results obtained from this real-world analysis highlight important aspects of drug safety between sexes. Full article
(This article belongs to the Special Issue Adverse Drug Reactions and Gender Differences)
19 pages, 1081 KiB  
Review
Hacking Pancreatic Cancer: Present and Future of Personalized Medicine
by Alessandro Di Federico, Valentina Tateo, Claudia Parisi, Francesca Formica, Riccardo Carloni, Giorgio Frega, Alessandro Rizzo, Dalia Ricci, Mariacristina Di Marco, Andrea Palloni and Giovanni Brandi
Pharmaceuticals 2021, 14(7), 677; https://doi.org/10.3390/ph14070677 - 15 Jul 2021
Cited by 32 | Viewed by 4081
Abstract
Pancreatic cancer (PC) is a recalcitrant disease characterized by high incidence and poor prognosis. The extremely complex genomic landscape of PC has a deep influence on cultivating a tumor microenvironment, resulting in the promotion of tumor growth, drug resistance, and immune escape mechanisms. [...] Read more.
Pancreatic cancer (PC) is a recalcitrant disease characterized by high incidence and poor prognosis. The extremely complex genomic landscape of PC has a deep influence on cultivating a tumor microenvironment, resulting in the promotion of tumor growth, drug resistance, and immune escape mechanisms. Despite outstanding progress in personalized medicine achieved for many types of cancer, chemotherapy still represents the mainstay of treatment for PC. Olaparib was the first agent to demonstrate a significant benefit in a biomarker-selected population, opening the doors for a personalized approach. Despite the failure of a large number of studies testing targeted agents or immunotherapy to demonstrate benefits over standard chemotherapy regimens, some interesting agents, alone or in combination with other drugs, have achieved promising results. A wide spectrum of therapeutic strategies, including immune-checkpoint inhibitors tyrosine kinase inhibitors and agents targeting metabolic pathways or the tumor microenvironment, is currently under investigation. In this review, we aim to provide a comprehensive overview of the current landscape and future directions of personalized medicine for patients affected by PC. Full article
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<p>Main transmembrane receptor and intracellular pathways evaluated as potential therapeutic targets in PC. EGFR: epidermal growth factor receptor; HER-2: human epidermal growth factor receptor 2; NTRK: neurotrophic tyrosine receptor kinase; PTCH1: 12-transmembrane patched protein 1; SMO: 7-transmembrane smoothened protein: RAS: rat sarcoma; RAF: rapidly accelerated fibrosarcoma; MEK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; JAK: Janus kinase; STAT: signal transducer and activator of transcription; PI3K: phosphoinositide-3-kinase; mTOR: mechanistic target of rapamycin; Gli: 5-zinc-finger transcription factor; SSB: single-stranded break; DSB: double-stranded break; PARP: poly ADP-ribose polymerase. Created with BioRender.com (accessed on 14 July 2021).</p> Full article ">Figure 2
<p>Main immune checkpoints acting on cancer cell recognition by effector immune cells. PD-1: programmed death-1; PD-L1: programmed death ligand 1; CTLA-4: cytotoxic T-lymphocyte antigen 4; TCR: T-cell receptor; MHC I: major histocompatibility complex I; + (plus sign): activating signal; − (minus sign): inhibitory signal. Created with BioRender.com (accessed on 14 July 2021).</p> Full article ">
25 pages, 2029 KiB  
Review
Phytomedicines Targeting Cancer Stem Cells: Therapeutic Opportunities and Prospects for Pharmaceutical Development
by Piyush Kumar Gupta, Mrunmayee Saraff, Rekha Gahtori, Nidhi Negi, Surya Kant Tripathi, Jatin Kumar, Sanjay Kumar, Saad Hamad Aldhayan, Sugapriya Dhanasekaran, Mosleh Mohammad Abomughaid, Kamal Dua, Rohit Gundamaraju, Shreesh Ojha, Janne Ruokolainen, Niraj Kumar Jha and Kavindra Kumar Kesari
Pharmaceuticals 2021, 14(7), 676; https://doi.org/10.3390/ph14070676 - 15 Jul 2021
Cited by 15 | Viewed by 6010
Abstract
The presence of small subpopulations of cells within tumor cells are known as cancer stem cells (CSCs). These cells have been the reason for metastasis, resistance with chemotherapy or radiotherapy, and tumor relapse in several types of cancers. CSCs underwent to epithelial–mesenchymal transition [...] Read more.
The presence of small subpopulations of cells within tumor cells are known as cancer stem cells (CSCs). These cells have been the reason for metastasis, resistance with chemotherapy or radiotherapy, and tumor relapse in several types of cancers. CSCs underwent to epithelial–mesenchymal transition (EMT) and resulted in the development of aggressive tumors. CSCs have potential to modulate numerous signaling pathways including Wnt, Hh, and Notch, therefore increasing the stem-like characteristics of cancer cells. The raised expression of drug efflux pump and suppression of apoptosis has shown increased resistance with anti-cancer drugs. Among many agents which were shown to modulate these, the plant-derived bioactive agents appear to modulate these key regulators and were shown to remove CSCs. This review aims to comprehensively scrutinize the preclinical and clinical studies demonstrating the effects of phytocompounds on CSCs isolated from various tumors. Based on the available convincing literature from preclinical studies, with some clinical data, it is apparent that selective targeting of CSCs with plants, plant preparations, and plant-derived bioactive compounds, termed phytochemicals, may be a promising strategy for the treatment of relapsed cancers. Full article
(This article belongs to the Topic Compounds with Medicinal Value)
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<p>CSCs and their overexpressed biomarkers.</p> Full article ">Figure 2
<p>Phytomedicinal compounds targeting the key regulators of anti-cancer drug resistance in CSCs, such as Curcumin/Cinnamic acid: Wnt; Glabridin: SMAD2; Curcumin/Ovatodiolide/Lusianthridin: JAK/STAT; Ovatodiolide/Carnosol/N-butylidenephthalide: EMT; N-butylidenephthalide/Atractylenolide I: PI3K/Akt; Morusin: NF-κB; Berberine: Ras/RAF.</p> Full article ">Figure 3
<p>Phytomedicinal compounds targeting different key regulators of anti-cancer drug resistance in CSCs.</p> Full article ">Figure 3 Cont.
<p>Phytomedicinal compounds targeting different key regulators of anti-cancer drug resistance in CSCs.</p> Full article ">Figure 4
<p>Phytomedicinal compounds targeting Wnt, Sonic Hedgehog, and Notch signaling pathways in CSCs. Abrus agglutinin, Sanguinarine, Diallyl-trisulfide, Koenimbin, Evodiamine, Gomisin M2, Chelerythrine chloride, Sulforaphane, and Ginsenoside-Rb1 inhibit the Wnt/β-catenin signaling pathway. Baicalein, Curcumin, Withaferin A, and Honokiol inhibit the Sonic Hedgehog signaling pathway, and Psoralidin inhibits the Notch signaling pathway.</p> Full article ">
13 pages, 1802 KiB  
Article
An Adenovirus Vector Expressing FMDV RNA Polymerase Combined with a Chimeric VLP Harboring a Neutralizing Epitope as a Prime Boost Strategy to Induce FMDV-Specific Humoral and Cellular Responses
by Giselle Rangel, Verónica Martín, Juan Bárcena, Esther Blanco and Alí Alejo
Pharmaceuticals 2021, 14(7), 675; https://doi.org/10.3390/ph14070675 - 15 Jul 2021
Cited by 5 | Viewed by 3258
Abstract
Foot and mouth disease is a highly contagious disease affecting cattle, sheep, and swine among other cloven-hoofed animals that imposes serious economic burden by its direct effects on farm productivity as well as on commerce of farmed produce. Vaccination using inactivated viral strains [...] Read more.
Foot and mouth disease is a highly contagious disease affecting cattle, sheep, and swine among other cloven-hoofed animals that imposes serious economic burden by its direct effects on farm productivity as well as on commerce of farmed produce. Vaccination using inactivated viral strains of the different serotypes is an effective protective measure, but has several drawbacks including a lack of cross protection and the perils associated with the large-scale growth of infectious virus. We have previously developed chimeric virus-like particles (VLPs) bearing an FMDV epitope which induced strong specific humoral responses in vaccinated pigs but conferred only partial protection against homologous challenge. While this and other FMD vaccines under development mostly rely on the induction of neutralizing responses, it is thought that induction of specific T-cell responses might improve both cross protective efficacy as well as duration of immunity. Therefore, we here describe the development of a recombinant adenovirus expressing the highly conserved nonstructural FMDV 3D protein as well as its capacity to induce specific T-cell responses in a murine model. We further describe the generation of an FMDV serotype C-specific chimeric VLP and analyze the immunogenicity of two different prime-boost strategies combining both elements in mice. This combination can effectively induce both humoral and cellular FMDV-specific responses eliciting high titers of ELISA and neutralizing antibodies anti-FMDV as well as a high frequency of IFNγ-secreting cells. These results provide the basis for further testing of this anti FMD vaccination strategy in cattle or pig, two of the most relevant natural host of this pathogen. Full article
(This article belongs to the Special Issue Current Trends in RNA Virus Vaccines)
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<p>Generation of recombinant adenoviruses expressing the FMDV 3D RNA polymerase. (<b>A</b>), Vero cells were mock infected or infected with the indicated recombinant adenoviruses at an MOI of 1 IFU/cell and fixed at 24 hpi. Upper panel shows phase contrast image of selected fields and lower panel shows GFP expression as analyzed by fluorescence microscopy. (<b>B</b>), Vero cells were mock infected or infected as indicated at an MOI of 10 IFU/cell and harvested at 24 hpi. Samples were analyzed by Western blot using antibodies against FMDV 3D protein or GAPDH as shown. A control lane containing 10 ng of purified recombinant FMDV 3D protein (r3D) was included as a reference and specificity control. The position of MWM (kDa) is indicated on the left.</p> Full article ">Figure 2
<p>The recombinant adenoviruses expressing FMDV 3D protein induce a specific cellular response in vivo. In (<b>A</b>), ELISPOT analysis of IFNγ producing splenocytes in response to stimulation with recombinant protein FMDV 3D from groups of five C57BL/6 mice inoculated subcutaneously with 10<sup>9</sup> IFU of the indicated adenoviruses at days 0 and 14 of the experiment and harvested at day 21. Shown are means + SD for each group of 5 mice and significant differences among groups indicated by ** (<span class="html-italic">p</span> &lt; 0.01) or * (<span class="html-italic">p</span> &lt; 0.05), ns: no significant differences. In (<b>B</b>), groups of five C57BL/6 mice vaccinated as above were challenged via intraperitoneal inoculation at day 23 of the experiment with FMDVC-S8c1 and survival recorded.</p> Full article ">Figure 3
<p>Generation of RHDV VP60-based chimeric VLPs bearing an FMDV_CS8c1 B epitope. In (<b>A</b>), purified recombinant VP60 (1), VP60_OUK bearing the FMDV_OUK isolate epitope (2) or VP60_CS8c1, bearing the FMDV_CS8c1 non-crossreacting epitope (3) were analyzed by Coomassie blue staining (left panel) or Western blot (middle and right panels) with the indicated antibodies. Arrows show position of detected proteins and MWM (kDa) are indicated on the left. In (<b>B</b>), samples from the indicated purified proteins were analyzed by ELISA using the antibodies shown in the legend: 2E7 and 1C9 mAbs recognize RHDV VP60 epitopes, with 1C9 corresponding to a conformational epitope around the insertion site used in this approach; SD6 mAb recognizes the neutralizing epitope found in the G-H loop of protein VP1 from FMDV_CS8c1. Shown are mean + SD of triplicates from one representative assay out of two.</p> Full article ">Figure 4
<p>Specific humoral anti-FMDV responses in VLP/Adenovirus prime boost vaccination regimes. In (<b>A</b>), a schematic view of both regimes used is shown. Groups of ten C57BL/6 mice were vaccinated using sequential adenovirus and VLP combinations. In scheme 1, animals were vaccinated at day 0 and day 21 with 100 µg of purified VP60_CS8c1 VLP and at day 28 with a single subcutaneous inoculation of 10<sup>9</sup> IFU of rhAdV5-3D. In scheme 2, rhAdV5-3D was inoculated at day 0, and VP60_CS8c1 at days 8 and 28. Samples were taken on day 35 in both cases from groups of five animals, sparing the remaining five for the challenge experiment. Corresponding control groups using either rhAd5-lacZ or VP60_OUK VLP or PBS-inoculated animals were included. IgG anti-VP60 antibody titers in sera at day 35 (<b>B</b>) or anti FMDV_CS8c1 B-epitope peptide titers at days 21 and 35 (<b>C</b>) for the indicated groups as determined by ELISA. In (<b>B</b>) mean + SD for each group is shown. In (<b>C</b>), data from individual samples assayed in triplicate is shown and horizontal bars indicate mean titer for each group. In (<b>D</b>), FMDV_CS8c1-specific mean-neutralizing antibody titers were calculated by microneutralization assays on BHK-21 cells for each animal in the group and mean titer is indicated by horizontal bars. Significant differences among groups are indicated by **** (<span class="html-italic">p</span> &lt; 0.0001), ns: no significant differences.</p> Full article ">Figure 5
<p>Cellular response and protection upon FMDV challenge in VLP/Adenovirus prime boost vaccination regimes. (<b>A</b>) ELISPOT analysis of IFNγ-producing splenocytes in response to stimulation with recombinant protein FMDV 3D. Shown are means + SD for each group of 5 mice as indicated and significant differences among groups indicated by ** (<span class="html-italic">p</span> &lt; 0.01), ns: no significant differences. (<b>B</b>) Groups of mice as shown where challenged with FMDV_CS8c1 by intraperitoneal inoculation upon completion of the vaccination schedule and survival over an 8-day period is represented.</p> Full article ">
33 pages, 1080 KiB  
Review
Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)—A Necessity for Future ADC Research and Development
by Manar Hammood, Andrew W. Craig and Jeffrey V. Leyton
Pharmaceuticals 2021, 14(7), 674; https://doi.org/10.3390/ph14070674 - 15 Jul 2021
Cited by 43 | Viewed by 13501
Abstract
Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways (endosomal sorting (recycling, lysosome localization) and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept [...] Read more.
Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways (endosomal sorting (recycling, lysosome localization) and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept of targeted chemotherapy by coupling inhibitory antibodies to cytotoxic payloads. Significant advances in ADC technology and format, and target biology have hastened the FDA approval of nine ADCs (four since 2019). Although the links between aberrant endocytic machinery and cancer are emerging, the impact of dysregulated internalization processes of ADC targets and response rates or resistance have not been well studied. This is despite the reliance on ADC uptake and trafficking to lysosomes for linker cleavage and payload release. In this review, we describe what is known about all the target antigens for the currently approved ADCs. Specifically, internalization efficiency and relevant intracellular sorting activities are described for each receptor under normal processes, and when complexed to an ADC. In addition, we discuss aberrant endocytic processes that have been directly linked to preclinical ADC resistance mechanisms. The implications of endocytosis in regard to therapeutic effectiveness in the clinic are also described. Unexpectedly, information on endocytosis is scarce (absent for two receptors). Moreover, much of what is known about endocytosis is not in the context of receptor-ADC/antibody complexes. This review provides a deeper understanding of the pertinent principles of receptor endocytosis for the currently approved ADCs. Full article
(This article belongs to the Special Issue Recent Insights of Antibody-Drug Conjugate Effectiveness)
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<p>Overview of endocytosis pathways utilized by the target antigens for the currently approved ADCs. Narrow arrows indicate minor utilization by HER2. Dashed arrows indicate that only indirect evidence exists. * Although CD33 utilizes clathrin-mediated endocytosis, it acts independently of AP-2. Green bars for CD30 and BCMA represent disulfide bonds.</p> Full article ">
6 pages, 235 KiB  
Viewpoint
Use of Monoclonal Antibody to Treat COVID-19 in Children and Adolescents: Risk of Abuse of Prescription and Exacerbation of Health Inequalities
by Susanna Esposito, Stefano Zona, Andrea Pession, Lorenzo Iughetti, Giovanni Battista Migliori and Nicola Principi
Pharmaceuticals 2021, 14(7), 673; https://doi.org/10.3390/ph14070673 - 15 Jul 2021
Cited by 4 | Viewed by 3324
Abstract
Monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 in infected patients are a new class of antiviral agents approved as a type of passive immunotherapy. They should be administered to adults and children (≥12 years old, weighing ≥ 40 kg) with SARS-CoV-2 positivity, and who [...] Read more.
Monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 in infected patients are a new class of antiviral agents approved as a type of passive immunotherapy. They should be administered to adults and children (≥12 years old, weighing ≥ 40 kg) with SARS-CoV-2 positivity, and who are suffering from a chronic underlying disease and are at risk of severe COVID-19 and/or hospitalization. The aim of this manuscript is to discuss the benefit-to-risk of mAb therapy to treat COVID-19 in pediatric age, according to current reports. A problem is that the authorization for mAbs use in children was given without studies previously evaluating the efficacy, safety and tolerability of mAbs in pediatric patients. Moreover, although the total number of children with chronic severe underlying disease is not marginal, the risk of severe COVID-19 in pediatric age is significantly reduced than in adults and the role of chronic underlying disease as a risk factor of severe COVID-19 development in pediatric patients is far from being precisely defined. In addition, criteria presently suggested for use of mAbs in children and adolescents are very broad and may cause individual clinicians or institutions to recommend these agents on a case-by-case basis, with an abuse in mAbs prescriptions and an exacerbation of health inequalities while resources are scarce. Several questions need to be addressed before their routine use in clinical practice, including what is their associated benefit-to-risk ratio in children and adolescents, who are the patients that could really have benefit from their use, and if there is any interference of mAb therapy on recommended vaccines. While we wait for answers to these questions from well-conducted research, an effective and safe COVID-19 vaccine for vulnerable pediatric patients remains the best strategy to prevent COVID-19 and represents the priority for public health policies. Full article
(This article belongs to the Section Medicinal Chemistry)
17 pages, 35375 KiB  
Article
Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues
by Qun Zhou, Josephine Kyazike, Ekaterina Boudanova, Michael Drzyzga, Denise Honey, Robert Cost, Lihui Hou, Francis Duffieux, Marie-Priscille Brun, Anna Park and Huawei Qiu
Pharmaceuticals 2021, 14(7), 672; https://doi.org/10.3390/ph14070672 - 14 Jul 2021
Cited by 14 | Viewed by 7170
Abstract
Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free [...] Read more.
Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy. Full article
(This article belongs to the Special Issue Evaluation of the Antitumor Mechanism of Armed Antibodies)
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Figure 1

Figure 1
<p>Different sites in Fc region selected for substitution with cysteine for conjugation. (<b>a</b>) Sites in the CH2 region with N-glycan attached at N297 (in ball and stick) as shown on one of two heavy chains (left in light blue). The sites are in scaled ball and stick in magenta while two C229 residues in yellow from both heavy chains formed an interchain disulfide bond. (<b>b</b>) Sites in the CH3 region as shown on one of two heavy chains (right in green). The sites are in scaled ball and stick in magenta. The Fc structure is from PDB 1E4K.</p> Full article ">Figure 2
<p>SDS-PAGE analysis of PEGylated single cysteine mutants. 27 unpaired single cysteine mutants were PEGylated and applied to 4–12% Bis-Tris NuPAGE under non-reducing and reducing conditions. The gels were stained with Coomassie blue. The wild-type antibody as well as hyperglycosylated mutants, A118N and A118N NNAS (S298N/T299A/Y300S), were also run together as controls for different migrations of the antibody mutants [<a href="#B28-pharmaceuticals-14-00672" class="html-bibr">28</a>]. PageRuler prestained protein ladder (different kDa as shown on left of the gels) was used as protein molecular weight standards (MW Std or Std). The labels related to identities of the different species are shown (on the left side of non-reducing gel and on the right side of gels under reducing condition).</p> Full article ">Figure 3
<p>Different conjugation efficiency and selectivity observed among the unpaired single cysteine mutants. The Coomassie blue stained SDS-PAGE reducing gels were scanned using ProteinSimple. The PAR (<b>a</b>) and percentages of mono-PEGylated (<b>b</b>), un-PEGylated (<b>c</b>), and multi-PEGylated (<b>d</b>) antibody bands were determined with AlphaView software [<a href="#B29-pharmaceuticals-14-00672" class="html-bibr">29</a>]. The red dot lines represent cut offs, and arrows represent top ten single cysteine mutants.</p> Full article ">Figure 4
<p>Plasma stability of PEGylated single cysteine mutants. The PEGylated antibodies were incubated with mouse plasma for 0 and 96 h before being analyzed using western blot with anti-PEG antibody. The percent of mono-PEG at 0 h represents the band area of mono-PEGylated species in the sample at 96 h divided by that at time 0, and then multiplied by 100.</p> Full article ">Figure 5
<p>Comparison of conjugation efficiency (<b>a</b>) and selectivity (<b>b</b>) of single cysteine mutants PEGylated after DTT and TCEP partial reduction. The single unpaired cysteine mutants were partially reduced with either DTT or TCEP to uncap the engineered cysteine residues. After being re-oxidized, the antibody samples were PEGylated and analyzed for conjugation efficiency and selectivity as described in Methods. Selectivity = % mono-PEG% un-PEG—% multi-PEG. The arrows in orange represent the top five mutants identified by conjugation with DTT, while the arrows in blue show the top five mutants identified by conjugation with TCEP.</p> Full article ">Figure 6
<p>SDS-PAGE analysis of PEGylated double cysteine mutants. 38 unpaired double cysteine mutants were PEGylated and applied to 4–12% Bis-Tris NuPAGE under non-reducing and reducing conditions. The gels were stained with Coomassie blue. The wild-type antibody was also PEGylated and run together as controls. The lanes with protein molecular weight standard are labeled as MW Std with different kDa as shown on left of the gels. The initial clone for mutant, A118C + A339C (highlighted in red), showed poor expression and diffuse bands after PEGylation. It was re-sequenced and found to have a sequence mismatch. A second clone for A118C + A339C with the correct sequence was expressed and PEGylated as shown (labelled in black). The labels related to identities of the different species are shown on the right side of top gel under reducing condition.</p> Full article ">Figure 7
<p>Different conjugation efficiency and selectivity observed among the double cysteine mutants. The double cysteine mutants were analyzed using reducing SDS-PAGE and the gels were stained with Coomassie blue and scanned using ProteinSimple. The PAR (<b>a</b>) and percentages of mono- and di-PEGylated (<b>b</b>), un-PEGylated (<b>c</b>), and multi-PEGylated (<b>d</b>) antibody bands were determined with AlphaView software [<a href="#B29-pharmaceuticals-14-00672" class="html-bibr">29</a>]. The red dot lines represent cut offs, and arrows represent top ten double cysteine mutants.</p> Full article ">Figure 8
<p>Heatmap of PEGylated double cysteine mutants. Different colors represent ranges of PAR, mono- and di-PEGylated, multi-PEGylated, and un-PEGylated species. PAR: &gt; 3.4 Green &lt; 4; ≥ 2.5 Yellow &lt; 3.4; &gt; 0 Red &lt; 2.5. Mono- and di-PEGylated: &gt; 80% Green &lt; 100%; ≥ 30% Yellow &lt; 80%; &gt; 5% Red &lt; 30%. Un-PEGylated: ≤ 5% Green &gt; 0%; &gt; 5% Yellow ≤ 10%; &gt; 10% Red &lt; 100%. Multi-PEGylated: &lt; 10% Green &gt; 0%; &gt; 10% Yellow &lt; 20%; &gt; 20% Red &lt; 100%. Selectivity (selectivity = % mono- and di-PEG—% un-PEG—% multi-PEG): ≥ 70% Green &lt; 100%, ≥ 60% yellow &lt; 70%, &gt; 0% red &lt; 60%.</p> Full article ">
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