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Pharmaceuticals
Volume 14
Issue 6
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Pharmaceuticals, Volume 14, Issue 6 (June 2021) – 106 articles

Cover Story (view full-size image): Site-specific conjugation is one of the promising research areas in antibody–drug conjugate (ADC) development, allowing for controlled conjugation and production of homogeneous ADCs, contrary to highly heterogeneous early-generation ADCs. We report here the characterization of a site-specific DAR2 ADC generated through glycan-based enzymatic remodeling and click chemistry (GlyCLICK™), using innovative native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS) for straightforward identification and quantification of all reaction species. Ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior and the resistance to unfolding of the products along the synthesis. View this paper

Site-specific conjugation is one of the promising research areas in antibody–drug conjugate (ADC) development, allowing for controlled conjugation and production of homogeneous ADCs, contrary to highly heterogeneous early-generation ADCs. We report here the characterization of a site-specific DAR2 ADC generated through glycan-based enzymatic remodeling and click chemistry (GlyCLICK™), using innovative native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS) for straightforward identification and quantification of all reaction species. Ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior and the resistance to unfolding of the products along the synthesis.

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20 pages, 810 KiB  
Review
cfDNA Sequencing: Technological Approaches and Bioinformatic Issues
by Elodie Bohers, Pierre-Julien Viailly and Fabrice Jardin
Pharmaceuticals 2021, 14(6), 596; https://doi.org/10.3390/ph14060596 - 21 Jun 2021
Cited by 38 | Viewed by 11409
Abstract
In the era of precision medicine, it is crucial to identify molecular alterations that will guide the therapeutic management of patients. In this context, circulating tumoral DNA (ctDNA) released by the tumor in body fluids, like blood, and carrying its molecular characteristics is [...] Read more.
In the era of precision medicine, it is crucial to identify molecular alterations that will guide the therapeutic management of patients. In this context, circulating tumoral DNA (ctDNA) released by the tumor in body fluids, like blood, and carrying its molecular characteristics is becoming a powerful biomarker for non-invasive detection and monitoring of cancer. Major recent technological advances, especially in terms of sequencing, have made possible its analysis, the challenge still being its reliable early detection. Different parameters, from the pre-analytical phase to the choice of sequencing technology and bioinformatic tools can influence the sensitivity of ctDNA detection. Full article
(This article belongs to the Special Issue Cell-Free DNA for the Management of Lymphoma)
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<p>Schematic overview of the main steps for blood sample processing and cfDNA extraction. Blood, collected in EDTA or stabilizing tubes, goes through two rounds of centrifugation to obtain plasma samples. CfDNA is isolated from plasma using commercial kits and is quantified and qualified for further analysis.</p> Full article ">
11 pages, 839 KiB  
Review
Whole Body Cryotherapy and Hyperbaric Oxygen Treatment: New Biological Treatment of Depression? A Systematic Review
by Marek Krzystanek, Monika Romańczyk, Stanisław Surma and Agnieszka Koźmin-Burzyńska
Pharmaceuticals 2021, 14(6), 595; https://doi.org/10.3390/ph14060595 - 21 Jun 2021
Cited by 2 | Viewed by 5802
Abstract
Treatment with antidepressants is often insufficiently effective, especially in treatment-resistant depression. In such a situation, it is possible to change the drug, add a second antidepressant, or use pharmacological and non-pharmacological methods of augmenting the effect of pharmacotherapy. New methods that may fall [...] Read more.
Treatment with antidepressants is often insufficiently effective, especially in treatment-resistant depression. In such a situation, it is possible to change the drug, add a second antidepressant, or use pharmacological and non-pharmacological methods of augmenting the effect of pharmacotherapy. New methods that may fall into the scope of multi-module depression treatment as an augmentation of depression treatment are whole body cryotherapy (WBC) and hyperbaric oxygen treatment (HBOT). 545 records were selected and analyzed for these two treatments and finally three clinical trials were selected for analysis. The review also includes data on the possibility of using WBC and HBOT in somatic indications and in organic mental syndromes. Despite the small number of studies on the effectiveness of WBC or HBOT in depression, the current data show that both methods may be effective in the treatment of depression. WBC may be effective in the augmentation of antidepressants, and additionally, it is a method in which a quick antidepressant effect is obtained. HBOT may be effective in endogenous depression, just as it is effective in the treatment of somatic depression symptoms. The results are very preliminary, but if confirmed in subsequent studies, both WBC and HBOT may become new treatment options in treating depression. The authors point to the need and directions for further research into these treatment methods as an augmentation strategy for pharmacological treatment of depression. Full article
(This article belongs to the Special Issue New Drugs and Biologics For Treatment of Central Nervous Dysfunction)
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<p>Flow diagram of studies analysis and selection for review.</p> Full article ">Figure 2
<p>Whole Body Cryotherapy (WBC) and Hyperbaric Treatment (HBOT) could become new methods of antidepressant augmentation, also for treatment resistant depression (TRD).</p> Full article ">
14 pages, 668 KiB  
Review
Zebrafish: An Attractive Model to Study Staphylococcus aureus Infection and Its Use as a Drug Discovery Tool
by Sari Rasheed, Franziska Fries, Rolf Müller and Jennifer Herrmann
Pharmaceuticals 2021, 14(6), 594; https://doi.org/10.3390/ph14060594 - 21 Jun 2021
Cited by 18 | Viewed by 6321
Abstract
Non-mammalian in vivo disease models are particularly popular in early drug discovery. Zebrafish (Danio rerio) is an attractive vertebrate model, the success of which is driven by several advantages, such as the optical transparency of larvae, the small and completely sequenced genome, [...] Read more.
Non-mammalian in vivo disease models are particularly popular in early drug discovery. Zebrafish (Danio rerio) is an attractive vertebrate model, the success of which is driven by several advantages, such as the optical transparency of larvae, the small and completely sequenced genome, the small size of embryos and larvae enabling high-throughput screening, and low costs. In this review, we highlight zebrafish models of Staphyloccoccus aureus infection, which are used in drug discovery and for studying disease pathogenesis and virulence. Further, these infection models are discussed in the context of other relevant zebrafish models for pharmacological and toxicological studies as part of early drug profiling. In addition, we examine key differences to commonly applied models of S. aureus infection based on invertebrate organisms, and we compare their frequency of use in academic research covering the period of January 2011 to January 2021. Full article
(This article belongs to the Special Issue Zebrafish as a Powerful Tool for Drug Discovery 2021)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Percentage of different non-mammalian in vivo models used for <span class="html-italic">Staphylococcus aureus</span> infection (<b>left</b>) and applications of zebrafish models to study <span class="html-italic">S. aureus</span> infection (right) over the last decade. A PubMed database search was performed to identify studies using invertebrates (<span class="html-italic">Galleria mellonella</span>, <span class="html-italic">Caenorhabditis elegans</span>, <span class="html-italic">Drosophila melanogaster</span> and <span class="html-italic">Bombyx mori</span>) and zebrafish embryos/larvae for <span class="html-italic">S. aureus</span> infection models. Studies from January 2011 through January 2021 were included and percentages were calculated based on the total number of publications (<span class="html-italic">n</span> = 282) in this period (see SI). <span class="html-italic">G. mellonella</span> and <span class="html-italic">C. elegans</span> are the most used models for studying different aspects of <span class="html-italic">S. aureus</span> infection and for assessing the in vivo efficacy of new potential antibiotics. Although zebrafish larvae are prominent models for studies of infectious disease and have been used for a broad range of other microorganisms, there are only relatively few literature reports on the use of this model to study <span class="html-italic">S. aureus</span> infection. The fields of applications of zebrafish models of <span class="html-italic">S. aureus</span> infection are depicted on the right. Most studies were performed with the intention of analyzing the pathogenesis and the virulence of different strains. Moreover, zebrafish larvae seem to be a promising platform for studying the in vivo efficacy of new anti-staphylococcal agents and new delivery systems/routes for already known antibiotics.</p> Full article ">Figure 2
<p>Injection sites of zebrafish larvae. Injection into the yolk sac circulation valley, Duct of Cuvier and the caudal vein leads to systemic infection, whereas injection into the yolk body, pericardial cavity, eye, otic vesicle and hindbrain ventricle results in local growth of bacteria. The figure was generated using SketchBook version 8.7.1.</p> Full article ">
12 pages, 2173 KiB  
Communication
The Effects of N-Acetylcysteine on the Rat Mesocorticolimbic Pathway: Role of mGluR5 Receptors and Interaction with Ethanol
by Sandra Fernández-Rodríguez, Claudia Esposito-Zapero, Teodoro Zornoza, Ana Polache, Luis Granero and María José Cano-Cebrián
Pharmaceuticals 2021, 14(6), 593; https://doi.org/10.3390/ph14060593 - 20 Jun 2021
Cited by 3 | Viewed by 2996
Abstract
N-acetylcysteine (NAC) is a prodrug that is marketed as a mucolytic agent and used for the treatment of acetaminophen overdose. Over the last few decades, evidence has been gathered that suggests the potential use of NAC as a new pharmacotherapy for alcohol [...] Read more.
N-acetylcysteine (NAC) is a prodrug that is marketed as a mucolytic agent and used for the treatment of acetaminophen overdose. Over the last few decades, evidence has been gathered that suggests the potential use of NAC as a new pharmacotherapy for alcohol use disorder (AUD), although its mechanism of action is already being debated. In this paper, we set out to assess both the potential involvement of the glutamate metabotropic receptors (mGluR) in the possible dual effect of NAC administered at two different doses and NAC’s effect on ethanol-induced activation. To this aim, 30 or 120 mg/kg of NAC was intraperitoneally administered to rats with the presence or absence of the negative allosteric modulator of mGluR5 (MTEP 0.1 mg/kg). Thereafter, the cFOS IR-cell expression was analyzed. Secondly, we explored the effect of 120 mg/kg of NAC on the neurochemical and behavioral activation induced by intra-VTA ethanol administration (150 nmol). Our results showed that the high NAC dose stimulated cFOS expression in the NAcc, and that this effect was suppressed in the presence of MTEP, thus suggesting the implication of mGluR5. Additionally, high doses could attenuate the ethanol-induced increase in cFOS-expression in the NAcc, probably due to a phenomenon based on the long-term depression of the MSNs. Additional experiments are required to corroborate our hypothesis. Full article
(This article belongs to the Special Issue Repurposing Drug Strategies for CNS Disorders)
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<p>Effect of MTEP administration on cFOS IR-cell expression, induced by low (30 mg/kg) and high doses (120 mg/kg) of NAC. Data are mean ± SD, and represent the number of cells per frame. Asterisks indicate the existence of statistical differences between the marked groups. (Tukey’s test: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 2
<p>Representative images of coronal sections of the NAcc after cFOS staining in experiment 1. Legend: (<b>A</b>) group Veh/Saline; (<b>B</b>) group NAC 30/Saline; (<b>C</b>) group NAC 120/Saline; (<b>D</b>) group Veh/MTEP; (<b>E</b>) group NAC 30/MTEP; (<b>F</b>) Group NAC 120/MTEP.</p> Full article ">Figure 3
<p>Diagram of coronal sections from the brains of rats used in experiment 2, indicating the placement of the tip of the injection cannulae in the posterior VTA. In order to facilitate the inspection of the figure, the placements from animals belonging to the aCSF-treated groups are shown on the right-hand side of the sections, whereas the placements from the EtOH-treated animals are shown on the left-hand side. Numbers indicate the distance from the anterior coordinate to the bregma. Adapted from Paxinos and Watson [<a href="#B26-pharmaceuticals-14-00593" class="html-bibr">26</a>].</p> Full article ">Figure 4
<p>The effects of NAC administration (120 mg/kg), 30 min prior to the intra-VTA administration of EtOH (150 nmol), on the locomotor activity of rats. Data are mean ± SD and represent the distance traveled in 30 min. The Kruskal–Wallis test revealed no statistical differences between the groups.</p> Full article ">Figure 5
<p>Effect of NAC administration (120 mg/kg) on cFOS IR-cell expression in the NAcc, 30 min prior to the intra-VTA administration of EtOH (150 nmol). Data are mean ± SD and represent the number of IR-cells per frame. Asterisks indicate statistical differences between the marked groups. (Tukey’s 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>Representative images of coronal sections of the NAcc after cFOS staining in experiment 2. Legend: (<b>A</b>) group aCSF/Veh; (<b>B</b>) group aCSF/NAC 120; (<b>C</b>) group EtOH/Veh; (<b>D</b>) group EtOH/NAC 120.</p> Full article ">
9 pages, 660 KiB  
Article
Antibiotic Resistance Profile and Biofilm Production of Staphylococcus pseudintermedius Isolated from Dogs in Thailand
by Pavarish Jantorn, Hawaree Heemmamad, Tanawan Soimala, Saowakon Indoung, Jongkon Saising, Julalak Chokpaisarn, Warapond Wanna, Varomyalin Tipmanee and Dennapa Saeloh
Pharmaceuticals 2021, 14(6), 592; https://doi.org/10.3390/ph14060592 - 20 Jun 2021
Cited by 15 | Viewed by 4642
Abstract
Staphylococcus pseudintermedius is a zoonotic pathogen that can cause life-threatening infections in animals and humans. The study of methicillin-resistant S. pseudintermedius (MRSP) and its ability to produce biofilms is important to select the most suitable treatment. The prevalence and characteristics of S. pseudintermedius [...] Read more.
Staphylococcus pseudintermedius is a zoonotic pathogen that can cause life-threatening infections in animals and humans. The study of methicillin-resistant S. pseudintermedius (MRSP) and its ability to produce biofilms is important to select the most suitable treatment. The prevalence and characteristics of S. pseudintermedius isolated from dogs admitted at the Veterinary Teaching Hospital, Prince of Songkla University, Thailand were assessed. Results showed that 28.30% (15/53) of the isolates were MRSP. Amplification of the mecA gene was observed in 93.33% (14/15) MRSP. Methicillin-resistant strains revealed co-resistant patterns against other antibiotics, including chloramphenicol, clindamycin, tetracycline, clarithromycin, ciprofloxacin, and trimethoprim. In this study, all bacterial isolates produced biofilms, while 90.55% of S. pseudintermedius isolates were strong or moderate biofilm producers. Most (45–60%) of the resistant strains were strong biofilm producers, while the correlation between biofilm production and antibiotic resistance was not statistically significant. This is the first study in southern Thailand to investigate the drug-resistant profile of S. pseudintermedius and its ability to form biofilm. The results will contribute to a better understanding of the emergence and prevalence of antimicrobial resistance in S. pseudintermedius. Full article
(This article belongs to the Special Issue Mechanisms of Antibiotic Action and Resistance)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Biofilm formation values (OD570) of <span class="html-italic">S. pseudintermedius</span> isolates (<span class="html-italic">n</span> = 53) obtained by quantitative biofilm production assay. The OD cut-off used to distinguish weak and moderate biofilm producers from strong biofilm producers is 0.09 (dashed line). Categories: non-biofilm producers (OD ≤ 0.09), weak biofilm producers (0.09 &lt; OD ≤ 0.18 (dashed line)), moderate biofilm producers (0.18 &lt; OD ≤ 0.36 (dashed line)), and strong biofilm producers (0.36 &lt; OD). (▲) MRSP; (■) MSSP.</p> Full article ">
18 pages, 590 KiB  
Review
A Comprehensive Review and Perspective on Natural Sources as Dipeptidyl Peptidase-4 Inhibitors for Management of Diabetes
by Sibhghatulla Shaikh, Eun-Ju Lee, Khurshid Ahmad, Syed-Sayeed Ahmad, Jeong-Ho Lim and Inho Choi
Pharmaceuticals 2021, 14(6), 591; https://doi.org/10.3390/ph14060591 - 20 Jun 2021
Cited by 24 | Viewed by 7074
Abstract
Type 2 diabetes mellitus (T2DM) is an increasing global public health problem, and its prevalence is expected to rise in coming decades. Dipeptidyl peptidase-4 (DPP-4) is a therapeutic target for the management of T2DM, and its inhibitors prevent the degradation of glucose-dependent insulinotropic [...] Read more.
Type 2 diabetes mellitus (T2DM) is an increasing global public health problem, and its prevalence is expected to rise in coming decades. Dipeptidyl peptidase-4 (DPP-4) is a therapeutic target for the management of T2DM, and its inhibitors prevent the degradation of glucose-dependent insulinotropic peptide and glucagon-like peptide 1, and thus, maintain their endogenous levels and lower blood glucose levels. Various medicinal plant extracts and isolated bioactive compounds exhibit DPP-4 inhibitory activity. In this review, we discussed different natural sources that have been shown to have anti-diabetic efficacy with a particular emphasis on DPP-4 inhibition. Furthermore, the effect of DPP-4 inhibition on pancreatic beta cell function, skeletal muscle function, and the glucose-lowering mechanisms were also discussed. We believe that scientists looking for novel compounds with therapeutic promise against T2DM will be able to develop antidiabetic drugs using these natural sources. Full article
(This article belongs to the Special Issue Searching for New Therapeutic Targets with Anti-obesity Potential)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Proposed mechanism of DDP-4 inhibition. In response to nutrient intake and/or an enhanced BG level, incretins (GLP-1 and GIP) are released from the gastrointestinal tract. These two incretins enhance insulin synthesis and secretion and inhibit the release of glucagon, and thus, reduce BG levels in healthy individuals. However, in T2DM, DPP-4 rapidly degrades both incretins and renders them inactive. DPP-4 inhibitors act by preventing DPP-4-induced incretin degradation, increasing intact GLP-1 and GIP levels, and improving glucose homeostasis.</p> Full article ">
13 pages, 3959 KiB  
Article
Dextran Sodium Sulphate-Induced Gastrointestinal Injury Further Aggravates the Impact of Galantamine on the Gastric Myoelectric Activity in Experimental Pigs
by Jan Bures, Ilja Tacheci, Jaroslav Kvetina, Vera Radochova, Darina Kohoutova, Martin Valis, Stanislav Rejchrt, Veronika Knoblochova and Jana Zdarova Karasova
Pharmaceuticals 2021, 14(6), 590; https://doi.org/10.3390/ph14060590 - 18 Jun 2021
Cited by 3 | Viewed by 2389
Abstract
Galantamine has been used as a treatment for Alzheimer disease. It has a unique, dual mode of action (inhibitor of acetylcholinesterase and allosteric modulator of nicotinic acetylcholine receptors). Nausea (in about 20%), vomiting (10%) and diarrhoea (5–7%) are the most common side effects. [...] Read more.
Galantamine has been used as a treatment for Alzheimer disease. It has a unique, dual mode of action (inhibitor of acetylcholinesterase and allosteric modulator of nicotinic acetylcholine receptors). Nausea (in about 20%), vomiting (10%) and diarrhoea (5–7%) are the most common side effects. The aim of this study was to assess the effect of galantamine on porcine gastric myoelectric activity without (Group A) and with (Group B) dextran sodium sulphate (DSS)-induced gastrointestinal injury. Galantamine hydrobromide was administrated to twelve pigs as a single intragastric dose (24 mg). Gastric myoelectric activity was investigated by electrogastrography (EGG). Basal (15 min before galantamine administration) and study recordings after galantamine administration (300 min) were evaluated using a running spectral analysis. Results were expressed as dominant frequency of gastric slow waves and power analysis (areas of amplitudes). Altogether, 3780 one-minute EGG recordings were evaluated. In Group A, power was steady from basal values for 180 min, then gradually decreased till 270 min (p = 0.007). In Group B, there was a rapid gradual fall from basal values to those after 120 min (p = 0.007) till 300 min (p ˂ 0.001). In conclusion, galantamine alone revealed an unfavourable effect on porcine myoelectric activity assessed by gastric power. It can be a plausible explanation of galantamine-associated dyspepsia in humans. DSS caused further profound decrease of EGG power. That may indicate that underlying inflammatory, ischaemic or NSAIDs-induced condition of the intestine in humans can have aggravated the effect of galantamine on gastric myoelectric activity. Full article
(This article belongs to the Special Issue New Drugs and Biologics For Treatment of Central Nervous Dysfunction)
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<p>Electrogastrography. Dominant frequency before and after a single intragastric dose of 24 mg galantamine (mean + standard deviation). Explanatory note: Bas: 15-min basal recording before galantamine administration; tn: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 2
<p>Electrogastrography. Dominant frequency before and after a single intragastric dose of 24 mg galantamine in animals with previous 7-day administration of dextran sodium sulphate (mean + standard deviation). Explanatory note: Bas: 15-min basal recording before galantamine administration; t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 3
<p>Electrogastrography. EGG power before and after a single intragastric administration of 24 mg galantamine (mean + standard deviation). Outliers omitted. Axis Y: natural logarithm scale. Explanatory note: Bas: 15-min basal recording before galantamine administration; t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 4
<p>Electrogastrography. EGG power before and after a single intragastric administration of 24 mg galantamine in animals with previous 7-day administration of dextran sodium sulphate (mean + standard deviation). Outliers omitted. Axis Y: natural logarithm scale. Explanatory note: Bas: 15-min basal recording before galantamine administration; t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 5
<p>Wireless capsule enteroscopy. Normal jejunum.</p> Full article ">Figure 6
<p>Wireless capsule enteroscopy. Normal ileum.</p> Full article ">Figure 7
<p>Electrogastrography. Dominant frequency in animals with a shorter small bowel transit time (272 ± 60 min); mean + standard deviation. Explanatory note: t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 8
<p>Electrogastrography. EGG power in animals with a shorter small bowel transit time (272 ± 60 min); mean + standard deviation. Outliers omitted. Axis Y: natural logarithm scale. Explanatory note: t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 9
<p>Electrogastrography. Dominant frequency in animals with a longer small bowel transit time (486 ± 93 min); mean + standard deviation. Explanatory note: t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 10
<p>Electrogastrography. EGG power in animals with a longer small bowel transit time (486 ± 93 min); mean + standard deviation. Outliers omitted. Axis Y: natural logarithm scale. Explanatory note: t: 15-min study recordings after galantamine administration (t1: time interval between 0–15 min ... t20: time interval between 286–300 min).</p> Full article ">Figure 11
<p>Arrangement of electrogastrography in experimental pigs. All recordings were accomplished under general propofol anaesthesia. Monitoring of vital functions was performed throughout the procedures.</p> Full article ">
76 pages, 2419 KiB  
Review
Potentiating Therapeutic Effects of Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer
by Kyu Sic You, Yong Weon Yi, Jeonghee Cho, Jeong-Soo Park and Yeon-Sun Seong
Pharmaceuticals 2021, 14(6), 589; https://doi.org/10.3390/ph14060589 - 18 Jun 2021
Cited by 38 | Viewed by 7714
Abstract
Triple-negative breast cancer (TNBC) is a subset of breast cancer with aggressive characteristics and few therapeutic options. The lack of an appropriate therapeutic target is a challenging issue in treating TNBC. Although a high level expression of epidermal growth factor receptor (EGFR) has [...] Read more.
Triple-negative breast cancer (TNBC) is a subset of breast cancer with aggressive characteristics and few therapeutic options. The lack of an appropriate therapeutic target is a challenging issue in treating TNBC. Although a high level expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis among patients with TNBC, targeted anti-EGFR therapies have demonstrated limited efficacy for TNBC treatment in both clinical and preclinical settings. However, with the advantage of a number of clinically approved EGFR inhibitors (EGFRis), combination strategies have been explored as a promising approach to overcome the intrinsic resistance of TNBC to EGFRis. In this review, we analyzed the literature on the combination of EGFRis with other molecularly targeted therapeutics or conventional chemotherapeutics to understand the current knowledge and to provide potential therapeutic options for TNBC treatment. Full article
(This article belongs to the Special Issue Potential Molecular Targets and Therapeutics in Triple-Negative Breast Cancer)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Milestones of anti-EGFR therapeutics approved globally. Some important milestones of regulatory approval for EGFR inhibitors are presented. See <xref ref-type="table" rid="pharmaceuticals-14-00589-t001">Table 1</xref> and <xref ref-type="table" rid="pharmaceuticals-14-00589-t002">Table 2</xref> for more details. If not specified in parentheses, anti-EGFR therapeutics were approved by the US Food and Drug Administration (US FDA). Abbreviations: BC, breast cancer; CRC, colorectal cancer; HNC, head and neck cancer; NSCLC, non-small cell lung cancer; TC, thyroid cancer; TKIs, tyrosine kinase inhibitors.</p> Full article ">Figure 2
<p>Schematic diagram of the putative EGFR and related signaling pathways in TNBC cells. Abbreviations: ABL1, Abelson murine leukemia viral oncogene homolog 1; ACC1/2, acetyl-CoA carboxylase 1/2; AKT, v-akt oncogene homolog; ALK, anaplastic lymphoma kinase; AMPK, 5′ adenosine monophosphate (AMP)-activated protein kinase; AP-1 activator protein 1; AXL, Anexelekto receptor tyrosine kinase; BCL2, B-cell lymphoma 2; BCL-xL, B-cell lymphoma-extra-large; BRCA1, breast cancer type 1 susceptibility protein; β-TrCP, beta-transducin repeat-containing protein; CDK5, cyclin-dependent-like kinase 5; MYC, cellular myelocytomatosis; DAPK, death-associated protein kinase; DNAPK, DNA-dependent protein kinase; DNMT1, DNA (cytosine-5)-methyltransferase 1; eEF2, eukaryotic elongation factor 2; eEF2K, eukaryotic elongation factor 2 kinase; EGFR, epidermal growth factor receptor; eIF4E, eukaryotic translation initiation factor 4E; ERK, extracellular-signal-regulated kinase; FBW7, F-box and WD repeat domain-containing 7; FGFR, fibroblast growth factor receptor; GSK3β, glycogen synthase kinase-3 beta; HER2, human epidermal growth factor receptor 2; IGF1R, insulin-like growth factor 1 receptor; IκB, nuclear factor of kappa light polypeptide gene enhance in B-cells inhibitor; IKK, IκB kinase; JAK1, Janus kinase 1; JNKs, c-Jun N-terminal kinases; KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homology; LKB1, liver kinase B1; MCL1, myeloid-cell leukemia 1; MEK, MAPK/ERK kinase; MEKK1, mitogen-activated protein kinase kinase kinase 1; MET, mesenchymal–epithelial transition factor; MKK4/7, mitogen-activated protein kinase kinase 4; mTORC1/2, mammalian target of rapamycin complex 1/2; mtp53, mutant p53; NF-κB, nuclear factor of kappa light polypeptide gene enhanced in B-cells; PAK1, p21-activated kinase 1; PDLIM4, PDZ and LI domain 4; PHLPP, PH domain and leucine-rich repeat protein phosphatase; PI3K, phosphoinositide 3-kinase; PDK1, phosphoinositide-dependent kinase-1; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PTEN, phosphatase and tensin homolog; PUMA, p53-upregulated modulator of apoptosis; RAD51, RAD51 (<italic>S. cerevisiae</italic>) homolog; RAF, rapidly accelerated fibrosarcoma kinase; RAS, rat sarcoma; RPS6, ribosomal protein S6; RSK, ribosomal S6 kinase; S6K, S6 kinase; SIRT1, NAD-dependent deacetylase sirtuin-1; SRC, v-src avian sarcoma (Schmidt–Ruppin A2) viral oncogene homolog; STAT3, signal transducer and activator of transcription 3; TAK1, transforming growth factor beta-activated kinase 1; TNFR, tumor necrosis factor receptor; TSC1/2, tuberous sclerosis complex 1/2; ULK1, Unc-51-like autophagy-activating kinase 1; VEGFR, vascular endothelial growth factor receptor; VHL, Von Hippel–Lindau tumor suppressor; XIAP, X-linked inhibitor of apoptosis.</p> Full article ">
14 pages, 4697 KiB  
Article
Protective Effect of Piplartine against LPS-Induced Sepsis through Attenuating the MAPKs/NF-κB Signaling Pathway and NLRP3 Inflammasome Activation
by Chi-Han Huang, Shu-Chi Wang, I-Chen Chen, Yi-Ting Chen, Po-Len Liu, Shih-Hua Fang, Shu-Pin Huang, Hsin-Chih Yeh, Ching-Chih Liu, Po-Yen Lee, Tzu-Chieh Lin, Wei-Chung Cheng, Chia-Cheng Su, Hsin-En Wu, Yuan-Ru Chen and Chia-Yang Li
Pharmaceuticals 2021, 14(6), 588; https://doi.org/10.3390/ph14060588 - 18 Jun 2021
Cited by 25 | Viewed by 4656
Abstract
Piplartine (or Piperlongumine) is a natural alkaloid isolated from Piper longum L., which has been proposed to exhibit various biological properties such as anti-inflammatory effects; however, the effect of piplartine on sepsis has not been examined. This study was performed to examine the [...] Read more.
Piplartine (or Piperlongumine) is a natural alkaloid isolated from Piper longum L., which has been proposed to exhibit various biological properties such as anti-inflammatory effects; however, the effect of piplartine on sepsis has not been examined. This study was performed to examine the anti-inflammatory activities of piplartine in vitro, ex vivo and in vivo using murine J774A.1 macrophage cell line, peritoneal macrophages, bone marrow-derived macrophages and an animal sepsis model. The results demonstrated that piplartine suppresses iNOS and COX-2 expression, reduces PGE2, TNF-α and IL-6 production, decreases the phosphorylation of MAPKs and NF-κB and attenuates NF-κB activity by LPS-activated macrophages. Piplartine also inhibits IL-1β production and suppresses NLRP3 inflammasome activation by LPS/ATP- and LPS/nigericin-activated macrophages. Moreover, piplartine reduces the production of nitric oxide (NO) and TNF-α, IL-6 and IL-1β, decreases LPS-induced tissue damage, attenuates infiltration of inflammatory cells and enhances the survival rate. Collectively, these results demonstrate piplartine exhibits anti-inflammatory activities in LPS-induced inflammation and sepsis and suggest that piplartine might have benefits for sepsis treatment. Full article
(This article belongs to the Special Issue Molecular Mechanism of Inflammasome Activation: Implications in Physiological and Pathological Conditions)
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<p>The effects of piplartine on the cell viability, NO and PGE<sub>2</sub> production, iNOS and COX-2 expression and TNF-α and IL-6 secretion by LPS-activated J774.1 cells. Cells were pretreated with different concentrations (0–10 μM) of piplartine (1 h) and primed with 1 μg/mL LPS (24 h). (<b>a</b>) Cell viability was detected using MTT assay. (<b>b</b>) NO production was measured using Griess reagent assay. (<b>c</b>) The production of PGE<sub>2</sub> was analyzed by ELISA. The expression of COX-2, iNOS and β-Actin (loading control) was determined by Western blotting. Representative images are shown in (<b>d</b>), and the quantified results from three independent experiments are shown in (<b>e</b>,<b>f</b>). The secretions of (<b>g</b>) TNF-α and (<b>h</b>) IL-6 were analyzed using ELISA. Data are presented as means ± SD and the significant differences indicated as * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>The effects of piplartine on both MAPKs and NF-κB signaling pathways in LPS-activated J774.1 cells. (<b>a</b>) Cells were pretreated with different concentrations (5 and 10 μM) of piplartine (1 h) and primed with 1 μg/mL LPS (2 h). Western blotting was performed to examine MAPK-associated protein expression. Representative results from one of three separate Western blotting experiments are shown, and the quantified results from three independent experiments are shown in (<b>b</b>–<b>d</b>). (<b>e</b>,<b>f</b>) Cells were pretreated with different concentrations (5 and 10 μM) of piplartine (1 h) and primed with 1 μg/mL LPS (30 min). Western blotting was executed to examine IκB protein expression in the cytoplasm and NF-κB protein expression between cytoplasm and nucleus. (<b>g</b>) J-blue cells were pretreated with different concentrations (0–10 μM) of piplartine (1 h) and primed with 1 μg/mL LPS (4 h). SEAP activity in the cell culture supernatant was examined. Data are presented as means ± SD and significant differences indicated as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>The effects of piplartine on NLRP3 inflammasome activation in macrophages. J774.1 cells were pretreated with different concentrations (0–10 μM) of piplartine (1 h), primed with 1 μg/mL LPS (5 h), and stimulated with 5 mM ATP (30 min). Inflammasome-associated protein expressions were detected using Western blotting, and β-Actin was used as a loading control. Representative images are shown in (<b>a</b>), and the quantified results from three independent experiments are shown in (<b>b</b>–<b>e</b>). (<b>f</b>,<b>g</b>) Cell culture supernatants were collected, and the levels of IL-1β were examined using ELISA. The colocalization of ASC (red) and caspase-1 (green), and DAPI in LPS/ATP-activated J774.1 cells was examined using confocal microscopy and Imaris software for confocal 3D image reconstruction. Representative images from three independent experiments are shown in (<b>h</b>). (<b>i</b>) The quantification of caspase-1 and ASC of colocalizated signals was analyzed using the threshold of the 2D histogram in panel (<b>h</b>) using Mander’s coefficient. Significant differences are indicated as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>The anti-inflammatory effects of piplartine on murine peritoneal and bone marrow-derived macrophages. (<b>a</b>,<b>f</b>) Cells were pretreated with different concentrations (0–5 μM) of piplartine (1 h) and then treated with 1 μg/mL LPS (24 h). Cell viability was analyzed by MTT assay. (<b>b</b>,<b>g</b>) The production of NO was determined by Griess reagent assay. The levels of (<b>c</b>,<b>h</b>) TNF-α and (<b>d</b>,<b>i</b>) IL-6 were detected using ELISA. (<b>e</b>,<b>j</b>) Cells were pretreated with different concentrations (0–5 μM) of piplartine (1 h), primed with 1 μg/mL LPS (5 h), and stimulated with 5 mM ATP (30 min). Cell culture supernatants were harvested, and levels of IL-1β were examined using ELISA. Significant differences are indicated as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Effects of piplartine on serum NO, TNF-α, IL-6, IL-1β levels, liver and kidney damage markers and mortality in LPS-challenged mice. Mice were intraperitoneally injected with piplartine (10 or 20 mg/kg) or DMSO (control) for 1 h before 50 mg/kg LPS intraperitoneal injection, and the blood samples were collected 4 h after LPS injection. (<b>a</b>) The level of NO in the serum was measured by Griess reagent assay. The levels of (<b>b</b>) TNF-α, (<b>c</b>) IL-6 and (<b>d</b>) IL-1β in the serum were measured by ELISA. (<b>e</b>,<b>f</b>) The levels of CRE and ALT were measured (n = 4). Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test and represented as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. (<b>g</b>) Tissues of lung, liver and kidney were harvested after LPS injection 4 h and stained by H&amp;E-staining kit (200x magnification). The damaged sites and infiltration of inflammatory cells are indicated by black arrows. The figure is representative data from three independent experiments. (<b>h</b>) The survival rate was monitored at different intervals (<span class="html-italic">n</span> = 10). Statistical significance was analyzed using the log-rank test and represented as follows: * <span class="html-italic">p</span> &lt; 0.05 vs. DMSO.</p> Full article ">Figure 6
<p>Piplartine suppresses the production of proinflammatory mediators and cytokines through inhibiting MAPKs/NF-κB signaling pathways and NLRP3 inflammasome activation by LPS-activated macrophages and protects LPS-induced septic shock.</p> Full article ">
19 pages, 4630 KiB  
Review
Small Molecule Inhibitors of Influenza Virus Entry
by Zhaoyu Chen, Qinghua Cui, Michael Caffrey, Lijun Rong and Ruikun Du
Pharmaceuticals 2021, 14(6), 587; https://doi.org/10.3390/ph14060587 - 18 Jun 2021
Cited by 26 | Viewed by 5187
Abstract
Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its [...] Read more.
Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its fusion machinery. In this review, we summarize the advances in HA-targeted development of small molecule inhibitors. Moreover, we discuss the structural basis and mode of action of these inhibitors, and speculate upon future directions toward more potent inhibitors of membrane fusion and potential anti-influenza drugs. Full article
(This article belongs to the Special Issue Recent Strategies in Anti-influenza Therapeutics)
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<p>(<b>a</b>) The structure of a monomer of the HA trimer at neutral pH and the rearranged structure at fusion pH.The subunits HA1 and HA2, are colored blue and multicolored, respectively. The central helix was shown in green, while the short helix was shown in red. Note that the loop between central and short helixes undergoes a “loop-to-helix” transition when induced by lower pH. (<b>b</b>) Phylogenetic tree of influenza HAs. The two groups of IAV are colored brown (group 1) and blue (group 2), while IBV HAs are indicated in black.</p> Full article ">Figure 2
<p>HA inhibitors targeting the receptor binding site. (<b>a</b>–<b>d</b>) The structures of oleanolic acid (<b>a</b>), aureonitol (<b>b</b>), neoechinulin B (<b>c</b>), and N-cyclohexyltautine (<b>d</b>). *, the inhibition has not been validated by bioassays.</p> Full article ">Figure 3
<p>The structures of BMY27709 and its derivatives.</p> Full article ">Figure 4
<p>The structure of JNJ7918 and its chemical modification process. JNJ6715, key modifications were made to improve the HA stem-binding property. JNJ8897, key modifications were made to improve the drug-like property. JNJ4796, key modifications were made to improve the pharmacokinetics property.</p> Full article ">Figure 5
<p>Comparison of CBS1116 with BMY27709, and the structure of optimized compound 16 derived from CBS1116.</p> Full article ">Figure 6
<p>Group 1 specific influenza fusion inhibitors that share similar scaffold with CBS1116.</p> Full article ">Figure 7
<p>Comparison of the structures of GRP-71271 and IY7640 with JNJ4796. * JNJ4796 possesses an additional E-ring.</p> Full article ">Figure 8
<p>Structurally distinct inhibitors specifically targeting group 1 HA mediated fusion process.</p> Full article ">Figure 9
<p>Design of general structure 1 using CL-385319 as starting point and the structure of compound 9d.</p> Full article ">Figure 10
<p>The structures of group 2 specific HA inhibitors targeting the fusion process. (<b>a</b>) TBHQ and its optimized derivative compound 11. (<b>b</b>) The structure of CBS1194. (<b>c</b>) Molecules that facilitate structural rearrangement of group 2 HAs. <sup>#</sup> the compounds did not show antiviral activities in bioassays. (<b>d</b>) The structure of S19. (<b>e</b>)The structure of compound 4c. * the inhibitory effect was defined within H3 subtype.</p> Full article ">Figure 11
<p>The structures of pan-subtype HA fusion inhibitors. (<b>a</b>) The structures of Arbidol and its derivatives. (<b>b</b>) The structure of M090. (<b>c</b>) The structure of camphecene.</p> Full article ">Figure 12
<p>Binding mode of group 1 specific HA inhibitors JNJ4796, CBS1117 and F0045(S). (<b>a</b>)Crystal structures of group 1 HA in complex with molecules JNJ4796 (PDB code: 6cfg), CBS1117 (PDB code: 6vmz) and F0045(S) (PDB code: 6wcr). The fusion peptides (FPs) are shown in red and indicated by arrows. (<b>b</b>) Alignment of bound complexes with JNJ4796 (yellow), CBS1117 (blue) and F0045(S) (brown). In the surface representation of H5 HA, the HA1 and HA2 subunits are colored cyan and pink, respectively. The critical interacting residues were indicated.</p> Full article ">Figure 13
<p>Group 1specific binding of compoundCBS1117. (<b>a</b>) Sequence alignment between group 1 (H1 and H5) and group 2 (H3 and H7) HAs. (<b>b</b>) Structure of H5 HA in complex with CBS1117 (blue). (<b>c</b>) Structure of H3 HA (PDB code: 5t6b). The essential residues involved in CBS1117 binding site are shown in orange. The HA2 position 38 is indicated in red, while other critical variable residues are indicated in brown.</p> Full article ">Figure 14
<p>Binding modes of TBHQ and Arbidol to group 2 HA. (<b>a</b>) The crystal structure of Arbidol/TBHQ in complex with H3 HA is represented with the HA1 shown in gray. The protomers of HA2 trimer is shown in blue, pink, and yellow cartoon separately, while TBHQ and Arbidol are shown as blueand brown sticks, respectively. The N-terminal fusion peptides (FPs) in the trimer has been highlighted in red and indicated by red arrows. The previously predicted binding sites for TBHQ and Arbidol from docking studies are marked by blue and red dashed circles, respectively. (<b>b</b>) The structural basis for HA group-specific inhibition by TBHQ. Comparison of the TBHQ binding site of H3 and corresponding position of H5 clearly shows that the extra turn of the short α-helix in the group 1 HAs precludes TBHQ binding. Potential hydrogen bonds are shown as dotted lines.</p> Full article ">
16 pages, 7736 KiB  
Review
Dipeptidyl Peptidase (DPP)-IV Inhibitors with Antioxidant Potential Isolated from Natural Sources: A Novel Approach for the Management of Diabetes
by Anand-Krishna Singh, Dhananjay Yadav, Neha Sharma and Jun-O Jin
Pharmaceuticals 2021, 14(6), 586; https://doi.org/10.3390/ph14060586 - 18 Jun 2021
Cited by 52 | Viewed by 8851
Abstract
Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia that is predominantly caused by insulin resistance or impaired insulin secretion, along with disturbances in carbohydrate, fat and protein metabolism. Various therapeutic approaches have been used to treat diabetes, including improvement of insulin sensitivity, [...] Read more.
Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia that is predominantly caused by insulin resistance or impaired insulin secretion, along with disturbances in carbohydrate, fat and protein metabolism. Various therapeutic approaches have been used to treat diabetes, including improvement of insulin sensitivity, inhibition of gluconeogenesis, and decreasing glucose absorption from the intestines. Recently, a novel approach has emerged using dipeptidyl peptidase-IV (DPP-IV) inhibitors as a possible agent for the treatment of T2DM without producing any side effects, such as hypoglycemia and exhaustion of pancreatic β-cells. DPP-IV inhibitors improve hyperglycemic conditions by stabilizing the postprandial level of gut hormones such as glucagon-like peptide-1, and glucose-dependent insulinotropic polypeptides, which function as incretins to help upregulate insulin secretion and β-cell mass. In this review, we summarized DPP-IV inhibitors and their mechanism of inhibition, activities of those isolated from various natural sources, and their capacity to overcome oxidative stress in disease conditions. Full article
(This article belongs to the Special Issue Multitarget Drug Discovery and Pharmacology)
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<p>Different approaches for the treatment of hyperglycemia recommended to type 2 diabetes mellitus patients. Abbreviations: thiazolidinedione (TZD), metformin (MET), dipeptidyl peptidase-IV inhibitor (DPP-IV), glucagon-like peptide-1 receptor antagonist (GLP-I RA), sodium-glucose co-transporter-2 inhibitor (SGLT2).</p> Full article ">Figure 2
<p>Diagrammatic representation of the mechanism of DPP-IV and potential role of protecting incretin hormone degradation by DPP-IV enzyme to maintain the homeostatic blood glucose level. Abbreviations: gastrointestinal tract (GI), dipeptidyl peptidase-IV inhibitor (DPP-IV), glucagon-like peptide-1 (GLP-1).</p> Full article ">Figure 3
<p>Role of DPP-IV inhibitors on GLP-1 dependent and independent manner during oxidative stress. Abbreviations: dipeptidyl peptidase-IV (DPP-IV), glucagon-like peptide-1 (GLP-1), Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS).</p> Full article ">
16 pages, 2571 KiB  
Article
HDAC Inhibitor Abrogates LTA−Induced PAI-1 Expression in Pleural Mesothelial Cells and Attenuates Experimental Pleural Fibrosis
by Wei-Lin Chen, Mei-Chuan Chen, Shang-Fu Hsu, Shih-Hsin Hsiao and Chi-Li Chung
Pharmaceuticals 2021, 14(6), 585; https://doi.org/10.3390/ph14060585 - 18 Jun 2021
Cited by 1 | Viewed by 2426
Abstract
Lipoteichoic acid (LTA) stimulates pleural mesothelial cell (PMC) to overproduce plasminogen activator inhibitor-1 (PAI-1), and thus may promote pleural fibrosis in Gram-positive bacteria (GPB) parapneumonic effusion (PPE). Histone deacetylase inhibitor (HDACi) was found to possess anti-fibrotic properties. However, the effects of HDACi on [...] Read more.
Lipoteichoic acid (LTA) stimulates pleural mesothelial cell (PMC) to overproduce plasminogen activator inhibitor-1 (PAI-1), and thus may promote pleural fibrosis in Gram-positive bacteria (GPB) parapneumonic effusion (PPE). Histone deacetylase inhibitor (HDACi) was found to possess anti-fibrotic properties. However, the effects of HDACi on pleural fibrosis remain unclear. The effusion PAI-1 was measured among 64 patients with GPB PPE. Pleural fibrosis was measured as radiographical residual pleural thickening (RPT) and opacity at a 12-month follow-up. The LTA−stimulated human PMCs and intrapleural doxycycline−injected rats were pretreated with or without the pan-HDACi, m-carboxycinnamic acid bis-hydroxamide (CBHA), then PAI-1 and collagen expression and activated signalings in PMCs, and morphologic pleural changes in rats were measured. Effusion PAI-1 levels were significantly higher in GPB PPE patients with RPT > 10 mm (n = 26) than those without (n = 38), and had positive correlation with pleural fibrosis shadowing. CBHA significantly reduced LTA−induced PAI-1 and collagen expression via inhibition of JNK, and decreased PAI-1 promoter activity and mRNA levels in PMCs. Furthermore, in doxycycline−treated rats, CBHA substantially repressed PAI-1 and collagen synthesis in pleural mesothelium and minimized pleural fibrosis. Conclusively, CBHA abrogates LTA−induced PAI-1 and collagen expression in PMCs and attenuates experimental pleural fibrosis. PAI-1 inhibition by HDACi may confer potential therapy for pleural fibrosis. Full article
(This article belongs to the Special Issue Lung Injury and Repair)
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<p>Correlation between pleural fluid PAI-1 level and residual pleural fibrosis among GPB PPE patients: (<b>A</b>) ROC curve for PAI-1 value to predict RPT &gt; 10 mm; (<b>B</b>) Correlation between PAI-1 and residual pleural opacity CXR score. ROC, receiver operating characteristic; AUC, area under the ROC curve; GPB, Gram-positive bacteria; PPE, parapneumonic pleural effusion. Blue dot, GPB PPE with RPT ≤ 10 mm (<span class="html-italic">n</span> = 38); Red dot, GPB PPE with RPT &gt; 10 mm (<span class="html-italic">n</span> = 26).</p> Full article ">Figure 2
<p>CBHA inhibits LTA-induced PAI-1 and collagen expression in human PMCs. (<b>A</b>) Primary pleural mesothelial cells were treated with LTA (0.5–5 μg/mL) for 24 h, and PAI-1 expression was analyzed by western blot and measured by optical densitometry analysis. Data were pooled from four independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting group. (<b>B</b>,<b>C</b>) MeT-5A cells pretreated with various concentrations of CBHA (0.5–2 μM) or vehicle (Dimethyl sulfoxide; DMSO) was stimulated with LTA (1 μg/mL) for 24 h. PAI-1 (<b>B</b>) or collagen-I (<b>C</b>) expression was analyzed by western blot and measured by optical densitometry analysis. Data were pooled from four independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting or vehicle group. <b><sup>#</sup></b> <span class="html-italic">p</span> &lt; 0.05 and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared to the resting group. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 compared to the vehicle (DMSO) group.</p> Full article ">Figure 3
<p>CBHA blocks LTA-induced JNK phosphorylation in human PMCs. (<b>A</b>) MeT-5A cells were treated with vehicle or various signal pathway inhibitors inclusive of parthenolide (Par, 10 μM), LY294002 (LY, 20 μM), PD98059 (PD, 20 μM), SB203580 (SB, 10 μM) and SP600125 (SP, 10 μM), respectively, then stimulated with LTA for 24 h. PAI-1 expression was analyzed by western blot and measured by optical densitometry analysis. Data were pooled from three independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting or vehicle group. (<b>B</b>) MeT-5A cells pretreated with vehicle or SP600125 (SP, 5‒10 μM) were stimulated with LTA for 24 h. Collagen I expression was analyzed by western blot and measured by optical densitometry analysis. Data were pooled from three independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting or vehicle group. (<b>C</b>) MeT-5A cells were treated with LTA for the indicated times, and JNK phosphorylation was evaluated. (<b>D</b>) MeT-5A cells were treated with LTA, with or without CBHA pretreatment (0.5, 1 or 2 μM), and the expression levels of phosphorylated or total JNK were examined in cell lysates by western blot and measured by optical densitometry analysis. Data were pooled from three independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting or vehicle group. (<b>E</b>) MeT-5A cells transfected with control siRNA or JNK3 siRNA were stimulated by LTA for 24 h with or without CBHA pretreatment (1 μM), and the expression of PAI-1 and total JNK were examined by western blot. <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the resting group; * <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 compared with the vehicle (DMSO) group. JNK, c-Jun N-terminal kinases, siRNA, small interfering RNA.</p> Full article ">Figure 4
<p>CBHA inhibits LTA-induced PAI-1 gene transcription in human PMCs. (<b>A</b>) MeT-5A cells were pretreated with the vehicle or the indicated concentrations of CBHA (0.5, 1 or 2 μM), and then stimulated with LTA for 6 h. PAI-1 mRNA level was detected by RT-PCR analysis. Data were pooled from three independent experiments and expressed as average fold change (mean ± SEM) in the expression relative to resting or vehicle group. (<b>B</b>) MeT-5A cells were transfected with both the specific PAI-1 reporter plasmid (p800Luc) and internal plasmid (Renilla), then treated with different concentrations of CBHA, and followed by LTA stimulation for 24 h. The luciferase activity of PAI-1 reporter gene was assessed. Data were pooled from three independent experiments and expressed as average fold change (mean ± SEM) in the activity relative to resting or vehicle group. <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the resting group; * <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 compared with the vehicle (DMSO) group.</p> Full article ">Figure 5
<p>CBHA attenuates doxycycline-induced pleural thickening, PAI-1 expression and collagen deposition in viscera pleurae of rats. Photomicrographs of visceral pleura from haematoxylin and eosin (HE)-stained (<b>A</b>–<b>C</b>), PAI-1 immunostained (<b>D</b>–<b>F</b>) and Masson trichrome-stained (collagen, <b>G</b>–<b>I</b>) paraffin sections of pleura tissue obtained at autopsy 7 days after intrapleural injection with PBS (control), doxycycline (10 mg/Kg) plus PBS, or doxycycline plus CBHA (100 mg/Kg). (<b>J</b>) Histomorphometric quantification of visceral pleural thickness on pleural tissue resected 7 days after intrapleural injection of indicated agents. Ten random measures per section per animal (<span class="html-italic">n</span> = 3 for each group) were obtained. (<b>K</b>) PAI-1 expression in pleural tissue homogenates was analyzed by western blot and measured by optical densitometry analysis. Data were pooled from four experimental rat groups (<span class="html-italic">n</span> = 4 for each group) and expressed as average fold change (mean ± SEM) in the expression relative to PBS or doxycycline plus PBS group. (<b>L</b>) Quantitative percent of collagen-positive area in visceral pleura. Ten random measures per section per animal (<span class="html-italic">n</span> = 3 for each group) were obtained. Scale bar = 20 μm. <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the PBS (control) group; ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 compared with the doxycycline plus PBS group. PBS, phosphate buffered saline.</p> Full article ">Figure 6
<p>Graphic abstract illustrates that CBHA ablates LTA–induced PAI-1 and collagen production in human pleural mesothelial cells via down-regulating the JNK pathway, and attenuates pleural fibrosis (see text for more explications). Single arrow, established pathway. GPB, Gram-positive bacteria; LTA, lipoteichoic acid; CBHA, m-carboxycinnamic acid bis-hydroxamide; JNK, c-Jun N-terminal kinases; Ⓟ, phosphorylated.</p> Full article ">
14 pages, 2970 KiB  
Article
Discovery of Selective Inhibitor Leads by Targeting an Allosteric Site in Insulin-Regulated Aminopeptidase
by Ioannis Temponeras, Lykourgos Chiniadis, Athanasios Papakyriakou and Efstratios Stratikos
Pharmaceuticals 2021, 14(6), 584; https://doi.org/10.3390/ph14060584 - 18 Jun 2021
Cited by 7 | Viewed by 3477
Abstract
Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at [...] Read more.
Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at the C-terminal domain pocket of IRAP. We compiled a library of 2.5 million commercially available compounds from the ZINC database, and performed molecular docking at the target pocket of IRAP and the corresponding pocket of the homologous endoplasmic reticulum aminopeptidase 1 (ERAP1). Of the top compounds that showed high selectivity, 305 were further analyzed by molecular dynamics simulations and free energy calculations, leading to the selection of 33 compounds for in vitro evaluation. Two orthogonal functional assays were employed: one using a small fluorogenic substrate and one following the degradation of oxytocin, a natural peptidic substrate of IRAP. In vitro evaluation suggested that several of the compounds tested can inhibit IRAP, but the inhibition profile was dependent on substrate size, consistent with the allosteric nature of the targeted site. Overall, our results describe several novel leads as IRAP inhibitors and suggest that the C-terminal domain pocket of IRAP is a promising target for developing highly selective IRAP inhibitors. Full article
(This article belongs to the Special Issue Selected Papers from the 6th International Electronic Conference on Medicinal Chemistry (ECMC2020))
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Figure 1
<p>Schematic representations of the internal cavity or ERAP1 (<b>left</b>) and IRAP (<b>right</b>). Figures were constructed based on the coordinates of PDB ID: 6q4r and 4z7i, respectively, retrieved from RCSB Protein Data Bank [<a href="#B17-pharmaceuticals-14-00584" class="html-bibr">17</a>]. Internal cavities are shown in cyan surface representation and proteins in cartoon representations are color-coded by domain. Ligands found in each cavity are shown in sphere representation (C: yellow, N: blue, O: red, H: white). The active sites accommodating an inhibitor for ERAP1 and a peptide analogue for IRAP are indicated. The sites where a bis-tris propane (B3P) and a malic acid (MA) molecule were found in ERAP1 are indicated, as well as the equivalent site of IRAP that was targeted for virtual ligand screening.</p> Full article ">Figure 2
<p>Virtual screening strategy employed for the discovery of lead-like allosteric inhibitors of IRAP, indicating the approximate number of compounds used in each stage. Inset, the distribution of number of compounds (in thousands) as a function of estimated free energy of binding (VINA score). The blue line shows the cumulative distribution and the highlighted region indicates the top 1% (lowest energy) of the compounds selected.</p> Full article ">Figure 3
<p>(<b>A</b>) Crystal structure of IRAP illustrating the position of targeted pocket and color-coded according to its functional domain I–IV. (<b>B</b>) Key residues that comprise the targeted pocket, color-coded according to IRAP domain. (<b>C</b>,<b>D</b>) Representative docked poses of two compounds, <b>5</b> and <b>26</b>, shown in yellow-C sticks. Hydrogen bonding interactions with key residues from domains II and IV of IRAP are indicated with dashed lines and the corresponding distance in Å.</p> Full article ">Figure 4
<p>Representative titrations showing the dose-dependent decrease in hydrolysis of the small substrate Leu-AMC by IRAP upon addition of compounds. The structure of the corresponding compound is indicated in each panel and error bars indicate standard deviation that is only shown if significantly larger than the data point size. Solid lines represent fits to a variable-slope inhibitor vs response model using Graphpad prism™.</p> Full article ">Figure 5
<p>(<b>A</b>) Representative chromatograms of trimming of oxytocin by IRAP as monitored by HPLC. (<b>B</b>) Effect of compound <b>26</b> (inset) on the hydrolysis of 50 μM Leu-AMC by IRAP and ERAP1, and on the hydrolysis of 50 μΜ Arg-AMC by ERAP2. (<b>C</b>) Dose-dependent inhibition of IRAP trimming of oxytocin by compound <b>26.</b> (<b>D</b>) Evaluation of the cellular toxicity of <b>26</b> using the MTT assay.</p> Full article ">
22 pages, 1344 KiB  
Article
Phytosterols and Novel Triterpenes Recovered from Industrial Fermentation Coproducts Exert In Vitro Anti-Inflammatory Activity in Macrophages
by Francisca S. Teixeira, Susana S. M. P. Vidigal, Lígia L. Pimentel, Paula T. Costa, Diana Tavares-Valente, João Azevedo-Silva, Manuela E. Pintado, João C. Fernandes and Luís M. Rodríguez-Alcalá
Pharmaceuticals 2021, 14(6), 583; https://doi.org/10.3390/ph14060583 - 18 Jun 2021
Cited by 12 | Viewed by 3419
Abstract
The unstoppable growth of human population that occurs in parallel with all manufacturing activities leads to a relentless increase in the demand for resources, cultivation land, and energy. In response, currently, there is significant interest in developing strategies to optimize any available resources [...] Read more.
The unstoppable growth of human population that occurs in parallel with all manufacturing activities leads to a relentless increase in the demand for resources, cultivation land, and energy. In response, currently, there is significant interest in developing strategies to optimize any available resources and their biowaste. While solutions initially focused on recovering biomolecules with applications in food, energy, or materials, the feasibility of synthetic biology in this field has been demonstrated in recent years. For instance, it is possible to genetically modify Saccharomyces cerevisiae to produce terpenes for commercial applications (i.e., against malaria or as biodiesel). But the production process, similar to any industrial activity, generates biowastes containing promising biomolecules (from fermentation) that if recovered may have applications in different areas. To test this hypothesis, in the present study, the lipid composition of by-products from the industrial production of β-farnesene by genetically modified Saccharomyces cerevisiae are studied to identify potentially bioactive compounds, their recovery, and finally, their stability and in vitro bioactivity. The assayed biowaste showed the presence of triterpenes, phytosterols, and 1-octacosanol which were recovered through molecular distillation into a single fraction. During the assayed stability test, compositional modifications were observed, mainly for the phytosterols and 1-octacosanol, probably due to oxidative reactions. However, such changes did not affect the in vitro bioactivity in macrophages, where it was found that the obtained fraction decreased the production of TNF-α and IL-6 in lipopolysaccharide (LPS)-induced inflammation. Full article
(This article belongs to the Special Issue Terpenes – Pharmaceutics and Biotechnology)
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<p>Overlay of the Fourier transform infrared spectroscopy (FTIR) spectra of FDR (<b>A</b>), Feed (<b>B</b>), C6 (<b>C</b>), and C7 (<b>D</b>) samples throughout the stability test (T0, T1, T2, and T3).</p> Full article ">Figure 2
<p>Effect of the distilled samples on macrophages. (<b>A</b>,<b>B</b>) Cytotoxicity evaluation of C6 and C7 by measuring the metabolic inhibition (PrestoBlue assay). Dots correspond to T0 samples and squares correspond to T3 samples (<b>C</b>,<b>D</b>) evaluation of TNF-α and IL-6 levels, respectively, in macrophages under an inflammatory stimulus (LPS). Values are expressed as pg of cytokine normalized to the total protein content. Ibuprofen at 1 mM was used as an anti-inflammatory control. One-way ANOVA was used for statistical analysis. ** <span class="html-italic">p</span> ≤ 0.01; **** <span class="html-italic">p</span> ≤ 0.0001.</p> Full article ">Figure 3
<p>Antioxidant values determined by ABTS, DPPH, and ORAC assays for the FDR, Feed, C6, and C7 samples over the accelerated stability storage (T0 vs. T3). A,B,C letters on top of bars for significant differences (<span class="html-italic">p</span> &lt; 0.05) among samples at T0. X,Y,Z for significant differences (<span class="html-italic">p</span> &lt; 0.05) at T3. * significant differences (<span class="html-italic">p</span> &lt; 0.05) along time for a same sample (i.e.. T0 vs. T3).</p> Full article ">
11 pages, 705 KiB  
Article
Electroconvulsive Therapy and Age: Effectiveness, Safety and Tolerability in the Treatment of Major Depression among Patients under and over 65 Years of Age
by Monika Dominiak, Anna Z. Antosik-Wójcińska, Marcin Wojnar and Paweł Mierzejewski
Pharmaceuticals 2021, 14(6), 582; https://doi.org/10.3390/ph14060582 - 18 Jun 2021
Cited by 13 | Viewed by 4586
Abstract
Electroconvulsive therapy (ECT) remains the most effective therapy in treatment-resistant depression. However, the safety of ECT has been consistently questioned, particularly among elderly patients. We assessed the efficacy and safety of ECT in patients before and after 65 years old. The study was [...] Read more.
Electroconvulsive therapy (ECT) remains the most effective therapy in treatment-resistant depression. However, the safety of ECT has been consistently questioned, particularly among elderly patients. We assessed the efficacy and safety of ECT in patients before and after 65 years old. The study was conducted between 2015 and 2018 and included 91 patients (61 under and 29 over 65 years old) with major depression undergoing ECT. The Hamilton Depression Rating Scale was used to evaluate efficacy. Cognitive functions were assessed using: MMSE, RAVLT, Trail Making Test, Stroop Test and Autobiographical Memory Interview-Short Form. ECT was more effective in older patients as compared to younger (p < 0.001). No serious adverse events were observed in either group. Increased blood pressure and arrhythmias were more common in the older compared to the younger group (p = 0.044 and p = 0.047, respectively), while disturbances of consciousness did not differ between groups (p = 0.820). Most of the cognitive functions remained unchanged compared to baseline, whereas the outcomes of MMSE, RAVLT and Stroop tests showed greater improvements in the older compared to the younger group (all p < 0.05). The decline in the retrieval consistency of autobiographical memory was more pronounced in the younger group (p = 0.024). ECT is a highly effective, safe and well-tolerated method of treating depression regardless of age. Full article
(This article belongs to the Special Issue Psychopharmacology of Affective Disorders)
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<p>Cognitive assessment in younger (&lt;65 years old) and older (65 years and above) ECT groups at baseline and endpoint. (<b>A</b>) MMSE—Mini Mental State Examination; (<b>B</b>) RAVLT—Rey Auditory Verbal Learning Test; (<b>C</b>). Stroop. (<b>D</b>) AMI-SF—Autobiographical Memory Interview Short, b—baseline assessment, e—assessment at endpoint. Vertical bars represent Standard Error. * <span class="html-italic">p</span> &lt; 0.05, significant differences between age groups; ** <span class="html-italic">p</span> &lt; 0.01, significant differences between age groups; # <span class="html-italic">p</span> &lt; 0.05, significant differences after ECT treatment between age groups; ## <span class="html-italic">p</span> &lt; 0.01, significant differences after ECT treatment between age groups.</p> Full article ">
26 pages, 1554 KiB  
Review
Confocal Microscopy and Anterior Segment Optical Coherence Tomography Imaging of the Ocular Surface and Bleb Morphology in Medically and Surgically Treated Glaucoma Patients: A Review
by Carmela Carnevale, Ivano Riva, Gloria Roberti, Manuele Michelessi, Lucia Tanga, Alice C. Verticchio Vercellin, Luca Agnifili, Gianluca Manni, Alon Harris, Luciano Quaranta and Francesco Oddone
Pharmaceuticals 2021, 14(6), 581; https://doi.org/10.3390/ph14060581 - 18 Jun 2021
Cited by 7 | Viewed by 3308
Abstract
Glaucoma patients often suffer from ocular surface disease (OSD) caused by the chronic administration of topical anti-glaucoma medications, especially in cases of long-term therapy with preserved or multiple drugs. Additionally, glaucoma surgery may determine ocular surface changes related to the formation and location [...] Read more.
Glaucoma patients often suffer from ocular surface disease (OSD) caused by the chronic administration of topical anti-glaucoma medications, especially in cases of long-term therapy with preserved or multiple drugs. Additionally, glaucoma surgery may determine ocular surface changes related to the formation and location of the filtering bleb, the application of anti-mitotic agents, and the post-operative wound-healing processes within the conjunctiva. Recently, several studies have evaluated the role of advanced diagnostic imaging technologies such as in vivo confocal microscopy (IVCM) and anterior segment-optical coherence tomography (AS-OCT) in detecting microscopic and macroscopic features of glaucoma therapy-related OSD. Their clinical applications are still being explored, with recent particular attention paid to analyzing the effects of new drug formulations and of minimally invasive surgical procedures on the ocular surface status. In this review, we summarize the current knowledge about the main changes of the ocular surface identified at IVCM and AS-OCT in glaucoma patients under medical therapy, or after surgical treatment. Full article
(This article belongs to the Special Issue Ocular Surface Disease and Glaucoma Treatments)
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<p>In vivo confocal microscopy (IVCM) of the ocular surface tissues in multi-treated medically controlled glaucoma. (<b>A</b>–<b>E</b>) Confocal frames taken from a patient controlled with a preserved fixed combination of timolol and dorzolamide and a preservative-free prostaglandin analog (PGA) (three eyedrops per day). (<b>A</b>) Goblet cells (GCs). GCs appear as hyper-reflective elements (black arrowhead) dispersed within the epithelium, and often appear scattered with an evident reduction of their density. (<b>B</b>) Inflammatory infiltrates. Inflammatory infiltrates appear as clusters of small hyper-reflective and mono-nucleate elements (presumably lymphocytes) infiltrating the epithelium of the tarsal or bulbar conjunctiva. (<b>C</b>) Epithelial microcysts. These structures (white asterisk) represent hallmarks of aqueous humor outflow stimulation through the uveo-scleral route, rather than detrimental effects induced by medications. (<b>D</b>) Meibomian glands. These glands appear markedly reduced in their dimension, with hyper-reflectivity of acinar wall and interstice; the black arrow indicates a glandular acinus. (<b>E</b>) CALT. Roundish immune follicles (black asterisk) appear infiltrated by numerous small hyper-reflective mono-nucleate cells (presumably lymphocytes). (<b>F</b>–<b>L</b>) Confocal frames taken from a patient controlled with a preserved fixed combination of timolol and dorzolamide, PGA, and brimonidine (five preserved eyedrops per day). (<b>F</b>,<b>L</b>) Limbal transition epithelium. The transition epithelium of the limbus appears irregular with scattered and highly hyper-reflective inflammatory elements (<b>L</b>), and with evident features of cellular polymegathism (undulated white arrow). (<b>G</b>–<b>I</b>) Cornea. The sub-epithelial layer and the Bowman’s membrane (<b>G</b>,<b>H</b>) present infiltration and activation of numerous dendritic cells (white arrowhead), with alterations of sub-basal nerve plexus morphology (white arrow). The corneal epithelium (<b>I</b>) appears markedly irregular with a higher degree of cellular polymorphism and polymegatysm. Bar represents 50 µm.</p> Full article ">Figure 2
<p>Anterior segment-optical coherence tomography (AS-OCT) of tear meniscus in medically controlled glaucoma. Fluorescein appearance of the tear meniscus and tear film in patients controlled with a preservative-free or preserved PGA mono-therapy (<b>A</b>,<b>B</b>), or with two or more drugs per day (<b>C</b>,<b>D</b>). AS-OCT shows the progressive reduction of the tear meniscus height (arrowhead) with increasing the number of medications, and the cumulative daily dose of preservative, required to control the disease (<b>E</b>–<b>H</b>).</p> Full article ">Figure 3
<p>AS-OCT and IVCM conjunctival bleb features after glaucoma filtration surgery. (<b>A</b>–<b>D</b>) Filtration bleb imaged by AS-OCT. Diffuse (<b>A</b>) or cystic (<b>B</b>) filtration bleb after completely successful trabeculectomy, showing numerous, differently sized hypo-reflective spaces filled with aqueous humor; incapsulated (<b>C</b>) and flat (<b>D</b>) filtration bleb after a failed trabeculectomy without evidence of hypo-reflective intra-bleb wall spaces. (<b>E</b>–<b>N</b>) Bleb-wall imaged by IVCM. In diffuse (<b>E</b>,<b>F</b>) or cystic (<b>G</b>,<b>H</b>) functioning filtration blebs, the bleb-wall epithelium (<b>E</b>,<b>G</b>) shows several microcysts with a loosely arranged stroma (<b>F</b>,<b>H</b>), indicating a good aqueous humor percolation and a minimal hydraulic resistivity through the bleb-wall layers. Opposite features are present in incapsulated (<b>I</b>,<b>L</b>) or flat (<b>M</b>,<b>N</b>) non-functioning filtration blebs: the bleb-wall epithelium (<b>I</b>,<b>M</b>) shows rare microcysts, whereas the stroma (<b>L</b>,<b>N</b>) appears densely arranged. These features indicate an inefficient aqueous humor percolation through the bleb-wall layers. Bar represents 50 µm.</p> Full article ">
12 pages, 2050 KiB  
Article
Capillary Liquid Chromatography for the Determination of Terpenes in Botanical Dietary Supplements
by Henry Daniel Ponce-Rodríguez, Jorge Verdú-Andrés, Pilar Campíns-Falcó and Rosa Herráez-Hernández
Pharmaceuticals 2021, 14(6), 580; https://doi.org/10.3390/ph14060580 - 17 Jun 2021
Cited by 2 | Viewed by 2838
Abstract
Dietary supplements of botanical origin are increasingly consumed due to their content of plant constituents with potential benefits on health and wellness. Among those constituents, terpenes are gaining attention because of their diverse biological activities (anti-inflammatory, antibacterial, geroprotective, and others). While most of [...] Read more.
Dietary supplements of botanical origin are increasingly consumed due to their content of plant constituents with potential benefits on health and wellness. Among those constituents, terpenes are gaining attention because of their diverse biological activities (anti-inflammatory, antibacterial, geroprotective, and others). While most of the existing analytical methods have focused on establishing the terpenic fingerprint of some plants, typically by gas chromatography, methods capable of quantifying representative terpenes in herbal preparations and dietary supplements with combined high sensitivity and precision, simplicity, and high throughput are still necessary. In this study, we have explored the utility of capillary liquid chromatography (CapLC) with diode array detection (DAD) for the determination of different terpenes, namely limonene, linalool, farnesene, α-pinene, and myrcene. An innovative method is proposed that can be applied to quantify the targets at concentration levels as low as 0.006 mg per gram of sample with satisfactory precision, and a total analysis time <30 min per sample. The reliability of the proposed method has been tested by analyzing different dietary supplements of botanical origin, namely three green coffee extract-based products, two fat burnings containing Citrus aurantium (bitter orange), and an herbal preparation containing lime and leaves of orange trees. Full article
(This article belongs to the Special Issue Applications of Liquid Chromatography in Analysis of Pharmaceuticals and Natural Products)
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<p>Chromatograms obtained under the selected conditions at 200 nm and 220 nm for a standard solution of the tested compounds (5 µg mL<sup>−1</sup> each). For other experimental details, see text.</p> Full article ">Figure 2
<p>(<b>a</b>) Chromatograms obtained for a standard solution of linalool (5 µg mL<sup>−1</sup>), for the extract of sample FB-1, and the same extract spiked with linalool (1 µg mL<sup>−1</sup>). (<b>b</b>) normalized spectra of linalool and the peak attributed to linalool in the chromatogram of sample FB-1. (<b>c</b>) Chromatogram of the extract obtained for sample FB-2. For other experimental details, see text.</p> Full article ">Figure 3
<p>Peaks of limonene, myrcene, and farnesene in the chromatogram obtained for sample HP. For other experimental details, see text.</p> Full article ">Figure 4
<p>Analytical features and applicability of the method developed for the analysis of terpenes in botanical dietary supplements by CapLC; strengths in green; weaknesses in red.</p> Full article ">
14 pages, 10778 KiB  
Article
Calcium Chelidonate: Semi-Synthesis, Crystallography, and Osteoinductive Activity In Vitro and In Vivo
by Elena Avdeeva, Ekaterina Porokhova, Igor Khlusov, Tatyana Rybalova, Elvira Shults, Larisa Litvinova, Valeria Shupletsova, Olga Khaziakhmatova, Irina Sukhodolo and Mikhail Belousov
Pharmaceuticals 2021, 14(6), 579; https://doi.org/10.3390/ph14060579 - 17 Jun 2021
Cited by 5 | Viewed by 4880
Abstract
Calcium chelidonate [Ca(ChA)(H2O)3]n was obtained by semi-synthesis using natural chelidonic acid. The structure of the molecular complex was determined by X-ray diffraction analysis. The asymmetric unit of [Ca(ChA)(H2O)3]n includes chelidonic acid coordinated through [...] Read more.
Calcium chelidonate [Ca(ChA)(H2O)3]n was obtained by semi-synthesis using natural chelidonic acid. The structure of the molecular complex was determined by X-ray diffraction analysis. The asymmetric unit of [Ca(ChA)(H2O)3]n includes chelidonic acid coordinated through three oxygen atoms, and three water ligands. The oxygen atoms of acid and oxygen atoms of water from each asymmetric unit are also coordinated to the calcium of another one, forming an infinite linear complex. Calcium geometry is close to the trigonal dodecahedron (D2d). The intra-complex hydrogen bonds additionally stabilize the linear species, which are parallel to the axis. In turn the linear species are packed into the 3D structure through mutual intercomplex hydrogen bonds. The osteogenic activity of the semi-synthetic CaChA was studied in vitro on 21-day hAMMSC culture and in vivo in mice using ectopic (subcutaneous) implantation of CaP-coated Ti plates saturated in vitro with syngeneic bone marrow. The enhanced extracellular matrix ECM mineralization in vitro and ectopic bone tissue formation in situ occurred while a water solution of calcium chelidonate at a dose of 10 mg/kg was used. The test substance promotes human adipose-derived multipotent mesenchymal stromal/stem cells (hAMMSCs), as well as mouse MSCs to differentiate into osteoblasts in vitro and in vivo, respectively. Calcium chelidonate is non-toxic and can stimulate osteoinductive processes. Full article
(This article belongs to the Section Pharmacology)
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<p>The asymmetric unit (<b>a</b>) and the fragment (<b>b</b>) of infinite 8-coordinated calcium complex [Ca(ChA)(H<sub>2</sub>O)<sub>3</sub>]<sub>n</sub> with intracomplex H-bonds (O3–H…O11 and O2–H…O12).</p> Full article ">Figure 2
<p>The in vitro indices (% of control) of human adipose-derived multipotent mesenchymal stromal/stem cells after 21 days of culture in the presence of water solvent (control) or 10 mg/L of CaChA in water solution, Me(Q1–Q3). * Statistical differences (<span class="html-italic">p</span> &lt; 0.05) with the control according to a Mann–Whitney test.</p> Full article ">Figure 3
<p>In vitro osteogenic differentiation of human adipose-derived multipotent mesenchymal stromal/stem cells after 21 days of culture in a standard nutrient medium: (<b>a</b>,<b>c</b>,<b>e</b>)—variants of poor diffuse coloring in control culture (addition of water solvent); (<b>b</b>,<b>d</b>,<b>f</b>)—mineralization nodules after the addition of 10 mg/L of CaChA in water solution. Staining with alizarin red S. Magnification ×200. Scale bars 200 µm.</p> Full article ">Figure 4
<p>Histological sections of tissue lamellae grown on CaP-coated Ti substrates after 45 days of subcutaneous test in mice. Bone tissues with marrow are shown after 35-day peroral administration of water solvent (<b>a</b>–<b>c</b>) or 10 mg/L of CaChA in water solution (<b>d</b>–<b>h</b>). Hematoxylin–eosin staining. Magnification ×200. Scale bars 50 µm.</p> Full article ">
26 pages, 2218 KiB  
Review
Zebrafish as a Screening Model to Study the Single and Joint Effects of Antibiotics
by Roxana Jijie, Gabriela Mihalache, Ioana-Miruna Balmus, Stefan-Adrian Strungaru, Emanuel Stefan Baltag, Alin Ciobica, Mircea Nicoara and Caterina Faggio
Pharmaceuticals 2021, 14(6), 578; https://doi.org/10.3390/ph14060578 - 17 Jun 2021
Cited by 35 | Viewed by 5762
Abstract
The overuse of antibiotics combined with the limitation of wastewater facilities has resulted in drug residue accumulation in the natural environment. Thus, in recent years, the presence of antibiotic residues in the environment has raised concerns over the potential harmful effects on ecosystems [...] Read more.
The overuse of antibiotics combined with the limitation of wastewater facilities has resulted in drug residue accumulation in the natural environment. Thus, in recent years, the presence of antibiotic residues in the environment has raised concerns over the potential harmful effects on ecosystems and human health. The in vivo studies represent an essential step to study the potential impact induced by pharmaceutical exposure. Due to the limitations of traditional vertebrate model systems, zebrafish (Danio rerio) has recently emerged as a promising animal model to study the toxic effects of drugs and their therapeutic efficacy. The present review summarizes the recent advances made on the toxicity of seven representative classes of antibiotics, namely aminoglycosides, β-lactams, macrolides, quinolones, sulfonamides, tetracyclines and polyether antibiotics, in zebrafish, as well as the combined effects of antibiotic mixtures, to date. Despite a significant amount of the literature describing the impact of single antibiotic exposure, little information exists on the effects of antibiotic mixtures using zebrafish as an animal model. Most of the research papers on this topic have focused on antibiotic toxicity in zebrafish across different developmental stages rather than on their efficacy assessment. Full article
(This article belongs to the Special Issue Zebrafish as a Powerful Tool for Drug Discovery 2021)
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<p>Basic behavioral endpoints of the control and DKA exposed groups (6.25, 12.5 and 25 mg/L) obtained from bottom swelling (<b>A</b>) and conditioned place preference (<b>B</b>) tests. The video trials resulting from the behavioral tests were reused in Ethovision XT software (Noldus IT, Wageningen, Netherlands) to generate average heat maps for experiments. Data represent average ± SD (<span class="html-italic">n</span> = 33–36 individuals for bottom swelling test and <span class="html-italic">n</span> = 34–36 individuals for CPP test) evaluated by one way ANOVA (* <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01) [<a href="#B28-pharmaceuticals-14-00578" class="html-bibr">28</a>].</p> Full article ">Figure 1 Cont.
<p>Basic behavioral endpoints of the control and DKA exposed groups (6.25, 12.5 and 25 mg/L) obtained from bottom swelling (<b>A</b>) and conditioned place preference (<b>B</b>) tests. The video trials resulting from the behavioral tests were reused in Ethovision XT software (Noldus IT, Wageningen, Netherlands) to generate average heat maps for experiments. Data represent average ± SD (<span class="html-italic">n</span> = 33–36 individuals for bottom swelling test and <span class="html-italic">n</span> = 34–36 individuals for CPP test) evaluated by one way ANOVA (* <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01) [<a href="#B28-pharmaceuticals-14-00578" class="html-bibr">28</a>].</p> Full article ">Figure 2
<p>Histopathological changes in adult zebrafish eye (<b>A</b>), brain (<b>B</b>), hepatic (<b>C</b>) and spleen (<b>D</b>) tissues after exposure to a series of DKA concentrations. Arrows in Figure 2A indicate photoreceptor cell cysts and intercellular vacuoles; in Figure 2B the “a arrow” shows the decrease in neuron number, “b arrow” shows ventriculomegaly and “c arrow” shows glial cell proliferation and the formation of a glial scar; in Figure 2C the “a arrow” indicates reduced, swollen and vague hepatocytes, “b arrow” shows hepatic parenchyma vacuolar degeneration and “c arrow” denotes blood accumulation and clot formation; in Figure 2D the “d arrow” shows metachromatic granules. Histopathological observations on these organs were performed with hematoxylin and eosin (HE) staining using a standard protocol. The photographs of the tissues were taken when 3 biological replicates showed a high degree of uniformity. [<a href="#B113-pharmaceuticals-14-00578" class="html-bibr">113</a>,<a href="#B115-pharmaceuticals-14-00578" class="html-bibr">115</a>].</p> Full article ">
12 pages, 2530 KiB  
Article
Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells
by Chin-Feng Hsuan, Thung-Lip Lee, Wei-Kung Tseng, Chau-Chung Wu, Chi-Chang Chang, Tsui-Ling Ko, Ya-Ling Chen and Jer-Yiing Houng
Pharmaceuticals 2021, 14(6), 577; https://doi.org/10.3390/ph14060577 - 17 Jun 2021
Cited by 2 | Viewed by 2640
Abstract
The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be [...] Read more.
The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177. Full article
(This article belongs to the Section Natural Products)
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<p>Effects of different extracts from the whole plant, aerial and root of GT on NO production in HUVECs. HUVECs were treated with each extract for 24 h. NO production was determined as the formation of nitrite. Data were obtained from five replicate experiments, and are expressed as mean ± standard deviation. W, whole plant; A, aerial; R, root; E, EA extract; H, hexane extract; Et, ethanol extract; M, methanol extract. A significant difference from the vehicle was indicated as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, (Student’s <span class="html-italic">t</span>-test).</p> Full article ">Figure 2
<p>One-way ANOVA analysis and post-hoc analysis for NO production on various extracts. HUVECs were treated with each extract for 24 h. NO production was determined as the formation of nitrite. Data were obtained from five replicate experiments, and are expressed as mean ± standard deviation. W, whole plant; A, aerial; R, root; E, EA extract; H, hexane extract; Et, ethanol extract; M, methanol extract. A significant difference was indicated as ** <span class="html-italic">p</span> &lt; 0.01 or *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Effect of the R/M GTE on eNOS protein expression in HUVECs. HUVECs was treated with R/M extract for 24 h. Total eNOS protein content was determined by Western blot analysis, using β-actin as an internal control. Data were obtained from four replicate experiments, and are expressed as mean ± standard deviation. A significant difference from the vehicle was indicated as * <span class="html-italic">p</span> &lt; 0.05 or ** <span class="html-italic">p</span> &lt; 0.01 (Student’s <span class="html-italic">t</span>-test).</p> Full article ">Figure 4
<p>Effect of R/M GTE on eNOS mRNA expression in HUVECs. HUVECs were treated with R/M extract for 24 h. Real-time RT–PCR analysis was performed in order to analyze the expression level of mRNA of eNOS. Data were obtained from triplicate experiments, and are expressed as mean ± standard deviation. A significant difference from the vehicle was indicated as * <span class="html-italic">p</span> &lt; 0.05 or ** <span class="html-italic">p</span> &lt; 0.01 (Student’s <span class="html-italic">t</span>-test).</p> Full article ">Figure 5
<p>Effect of the R/M GTE on eNOS activation. HUVECs were treated with R/M extract for 15 min. Total eNOS protein content and phospho-eNOS (P-Ser<sup>1177</sup>-eNOS) were determined by Western blot analysis. Data were obtained from four replicate experiments, and are expressed as mean ± standard deviation. A significant difference from the vehicle was indicated as * <span class="html-italic">p</span> &lt; 0.05 or ** <span class="html-italic">p</span> &lt; 0.01 (Student’s <span class="html-italic">t</span>-test).</p> Full article ">Figure 6
<p>HPLC chromatogram of the W/M GTE (<b>A</b>) and R/M GTE (<b>B</b>). The mobile phase was methanol/0.05% acetic acid (4:6, <span class="html-italic">v</span>/<span class="html-italic">v</span>) with the flow rate of 1.0 mL/min. The detection was performed at 350 nm. Identified components: 1, chlorogenic acid; 2, luteolin-7-glucoside; and 3, luteolin.</p> Full article ">
13 pages, 2616 KiB  
Article
Pharmacokinetics in Zebrafish Embryos (ZFE) Following Immersion and Intrayolk Administration: A Fluorescence-Based Analysis
by Marlly Guarin, Annelii Ny, Noémie De Croze, Jan Maes, Marc Léonard, Pieter Annaert and Peter A. M. de Witte
Pharmaceuticals 2021, 14(6), 576; https://doi.org/10.3390/ph14060576 - 16 Jun 2021
Cited by 10 | Viewed by 3278
Abstract
Zebrafish embryos (ZFE) have increasingly gained in popularity as a model to perform safety screenings of compounds. Although immersion of ZFE is the main route of exposure used, evidence shows that not all small molecules are equally absorbed, possibly resulting in false-negative readouts [...] Read more.
Zebrafish embryos (ZFE) have increasingly gained in popularity as a model to perform safety screenings of compounds. Although immersion of ZFE is the main route of exposure used, evidence shows that not all small molecules are equally absorbed, possibly resulting in false-negative readouts and incorrect conclusions. In this study, we compared the pharmacokinetics of seven fluorescent compounds with known physicochemical properties that were administered to two-cell stage embryos by immersion or by IY microinjection. Absorption and distribution of the dyes were followed at various timepoints up to 120 hpf by spatiotemporal fluorescence imaging. The concentration (10 µM) and dose (2 mg/kg) used were selected as quantities typically applied in preclinical experiments and zebrafish studies. The data show that in the case of a lipophilic compound (log D: 1.73) the immersion procedure resulted in an intrabody exposure which is similar or higher than that seen after the IY microinjection. In contrast, zero to low intrabody exposure was reached after immersion of the embryos with less lipophilic compounds. In the latter case IY microinjection, a technical procedure that can be easily automated, is highly recommended. Full article
(This article belongs to the Special Issue Zebrafish as a Powerful Tool for Drug Discovery 2021)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Representative fluorescent images of embryos exposed to the different fluorescent compounds by immersion (10 µM) for 1 h, before (left panel) and after dechorionation and rinsing (right panel).</p> Full article ">Figure 2
<p>Representative fluorescent images showing the spatiotemporal distribution of the fluorescent dyes in the embryos after immersion (10 µM) and microinjection (2 mg/kg) for several periods of time. (<b>a</b>) animals treated with S-CY3A; (<b>b</b>) S-CY5.5A; (<b>c</b>) S-CY5A; (<b>d</b>) FAMA; (<b>e</b>) TAMRA; (<b>f</b>) R6GA; (<b>g</b>) CY3A. The dash dotted line represents the hatching time and the change of zebrafish medium. 15′ = 0.25 h.</p> Full article ">Figure 2 Cont.
<p>Representative fluorescent images showing the spatiotemporal distribution of the fluorescent dyes in the embryos after immersion (10 µM) and microinjection (2 mg/kg) for several periods of time. (<b>a</b>) animals treated with S-CY3A; (<b>b</b>) S-CY5.5A; (<b>c</b>) S-CY5A; (<b>d</b>) FAMA; (<b>e</b>) TAMRA; (<b>f</b>) R6GA; (<b>g</b>) CY3A. The dash dotted line represents the hatching time and the change of zebrafish medium. 15′ = 0.25 h.</p> Full article ">Figure 3
<p>Semilogarithmic graphs of the fluorescence intensity (RFU) as a function of time, in the case of immersion (IMM) and intrayolk (IY) exposure routes (<b>a</b>) in the whole-body (from 0.25 to 120 h). The dashed line at 72 h indicates the start of the rinsing period. (<b>b</b>) in the yolk (orange line) and non-yolk parts (RoB, Rest of Body) (gray dash line) (from 0.25 to 72 h exposure). Excluded data points are marked as X symbols.</p> Full article ">Figure 4
<p>Relationship between observed RE and Log D for the WB (<b>left</b>) and the RoB (<b>right</b>) of the 7 fluorescent compounds. The dashed line is line of fit.</p> Full article ">
17 pages, 1718 KiB  
Article
Radiopharmaceutical Formulation and Preclinical Testing of 68Ga-Labeled DOTA-MGS5 for the Regulatory Approval of a First Exploratory Clinical Trial
by Anton A. Hörmann, Maximilian Klingler, Christine Rangger, Christian Mair, Clemens Decristoforo, Christian Uprimny, Irene J. Virgolini and Elisabeth von Guggenberg
Pharmaceuticals 2021, 14(6), 575; https://doi.org/10.3390/ph14060575 - 16 Jun 2021
Cited by 11 | Viewed by 3606
Abstract
The new minigastrin analog DOTA-MGS5 is a promising new candidate for targeting cholecystokinin-2 receptor (CCK2R)-expressing tumors. To enable the clinical translation of PET/CT imaging using 68Ga-labeled DOTA-MGS5, different quality and safety aspects need to be considered to comply with the regulatory framework [...] Read more.
The new minigastrin analog DOTA-MGS5 is a promising new candidate for targeting cholecystokinin-2 receptor (CCK2R)-expressing tumors. To enable the clinical translation of PET/CT imaging using 68Ga-labeled DOTA-MGS5, different quality and safety aspects need to be considered to comply with the regulatory framework for clinical trial application. The preparation of the radiopharmaceutical was established using a cassette-based automated synthesis unit. Product specifications, including analytical procedures and acceptance criteria, were adopted from Ph. Eur. monographs for other 68Ga-labeled radiopharmaceuticals. Non-clinical studies included receptor affinity and cell uptake studies using two different CCK2R-expressing cell lines, as well as pharmacokinetic biodistribution studies in BALB/c mice for dosimetry calculations and toxicological studies in Wistar rats. The produced masterbatches fulfilled the defined acceptance criteria. DOTA-MGS5, with confirmed affinity to the CCK2R, showed a high specific cell uptake and no interaction with other receptors in vitro when radiolabeled with gallium-68. Favorable in vivo properties were observed in biodistribution and dosimetry studies. An effective dose of ~0.01 mSv/MBq was estimated for humans utilizing OLINDA/EXM software. A maximum peptide dose of 50 µg was established for the initial clinical dose based on the toxicity study in rats. The standardized production of [68Ga]Ga-DOTA-MGS5 using an automated synthesis module and the performed non-clinical safety studies support a first exploratory clinical trial with this new PET imaging agent. 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>Chemical structure of DOTA-MGS5.</p> Full article ">Figure 2
<p>Scheme of the automated cassette-based production of <sup>68</sup>Ga-DOTA-MGS5.</p> Full article ">Figure 3
<p>Radio-HPLC analysis of <sup>68</sup>Ga-DOTA-MGS5 post preparation (p.p.) and at 1, 2, and 3 h EOS.</p> Full article ">Figure 4
<p>Exemplary binding curves for DOTA-MGS5, Ga-DOTA-MGS5, and pentagastrin.</p> Full article ">Figure 5
<p>Cell uptake of <sup>68</sup>Ga-DOTA-MGS5 in (<b>a</b>) AR42J cells and (<b>b</b>) AR42J cells co-incubated with pentagastrin, as well as (<b>c</b>) A431-CCK2R cells and (<b>d</b>) A431-mock cells for up to 2 h after incubation.</p> Full article ">Figure 6
<p>Cell uptake studies with <sup>68</sup>Ga-DOTA-MGS5 on AR42J cells: (<b>a</b>) Co-incubation with different peptide analogs specific for CCK2R, SSTR, and GRPR; (<b>b</b>) co-incubation with increasing concentrations of the CCK2R antagonist proglumide.</p> Full article ">Figure 7
<p>Biodistribution of <sup>68</sup>Ga-DOTA-MGS5 in BALB/c mice at 5, 20, 60, and 90 min p.i. (<span class="html-italic">n</span> = 12).</p> Full article ">
19 pages, 2275 KiB  
Review
Repurposing Drugs to Treat Heart and Brain Illness
by Maranda S. Cantrell, Alejandro Soto-Avellaneda, Jackson D. Wall, Aaron D. Ajeti, Brad E. Morrison, Lisa R. Warner and Owen M. McDougal
Pharmaceuticals 2021, 14(6), 573; https://doi.org/10.3390/ph14060573 - 16 Jun 2021
Cited by 4 | Viewed by 5424
Abstract
Drug development is a complicated, slow and expensive process with high failure rates. One strategy to mitigate these factors is to recycle existing drugs with viable safety profiles and have gained Food and Drug Administration approval following extensive clinical trials. Cardiovascular and neurodegenerative [...] Read more.
Drug development is a complicated, slow and expensive process with high failure rates. One strategy to mitigate these factors is to recycle existing drugs with viable safety profiles and have gained Food and Drug Administration approval following extensive clinical trials. Cardiovascular and neurodegenerative diseases are difficult to treat, and there exist few effective therapeutics, necessitating the development of new, more efficacious drugs. Recent scientific studies have led to a mechanistic understanding of heart and brain disease progression, which has led researchers to assess myriad drugs for their potential as pharmacological treatments for these ailments. The focus of this review is to survey strategies for the selection of drug repurposing candidates and provide representative case studies where drug repurposing strategies were used to discover therapeutics for cardiovascular and neurodegenerative diseases, with a focus on anti-inflammatory processes where new drug alternatives are needed. Full article
(This article belongs to the Collection Old Pharmaceuticals with New Applications)
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<p>Inhibitory pathway of colchicine. Colchicine inhibits activation of purinergic P2X2/P2X7 receptors and blocks cation uptake and subsequent pro-inflammatory signaling cascades without affecting cell death. Colchicine also inhibits the NALP3 inflammasome pathway, the Rho/ROCK pathway via cytoskeleton rearrangement, and inhibits release of ROS, NO and TNFα. <a href="#pharmaceuticals-14-00573-f001" class="html-fig">Figure 1</a> was created using BioRender.com (adapted from [<a href="#B45-pharmaceuticals-14-00573" class="html-bibr">45</a>]).</p> Full article ">Figure 2
<p>Chemical structure of methotrexate.</p> Full article ">Figure 3
<p>Metabolic pathways of methotrexate. (<b>a</b>) Methotrexate inhibits dihydrofolate reductase (DHFR) and prevents conversion of dihydrobiopterin (BH<sub>2</sub>) to tetrahydrobiopterin (BH<sub>4</sub>), leading to uncoupling of NO synthase. (<b>b</b>) Methotrexate inhibits AICAR transformylase, leading to increased adenosine levels and subsequent anti-inflammatory responses. (<b>c</b>) Methotrexate stimulates lincRNA-p21 expression, leading to increased apoptotic gene expression and subsequent anti-inflammatory responses.</p> Full article ">Figure 4
<p>Autophagy mechanism and associated gene dysfunctions. Autophagy is a cellular mechanism by which metabolites, organelles, proteins and protein aggregates are enveloped by a vesicular membrane to form an autophagosome. This autophagosome is trafficked to a lysosome where fusion occurs, and lysosomal degradative enzymes break down the cargo. Dysfunction in several genes associated with neurodegenerative diseases has been implicated and are known to disrupt autophagy. The schematic image (<a href="#pharmaceuticals-14-00573-f004" class="html-fig">Figure 4</a>) was created using BioRender.com.</p> Full article ">Figure 5
<p>NRF2 signaling and inflammation. Under basal conditions, NRF2 is bound to its repressor, KEAP1 and ultimately degraded by the proteasome following ubiquitination. However, under oxidative stress, free NRF2 translocates to the nucleus and dimerizes with small MAF family proteins. This complex binds to and promotes the expression of genes with an antioxidant response element, such as HO-1. HO-1 directly inhibits pro-inflammatory cytokines while upregulating anti-inflammatory cytokines as well as catalyzing the breakdown of heme into carbon monoxide, free iron, and biliverdin. Carbon monoxide is an inhibitor of the NFκB pathway, resulting in an overall decrease of pro-inflammatory cytokines. The image in <a href="#pharmaceuticals-14-00573-f005" class="html-fig">Figure 5</a> was created using BioRender.com.</p> Full article ">Figure 6
<p>Chemical structure of dimethyl fumarate.</p> Full article ">
32 pages, 8150 KiB  
Review
Cytostatics in Indoor Environment: An Update of Analytical Methods
by M. Francisca Portilha-Cunha, A. Alves and Mónica S. F. Santos
Pharmaceuticals 2021, 14(6), 574; https://doi.org/10.3390/ph14060574 - 15 Jun 2021
Cited by 6 | Viewed by 2531
Abstract
Periodic and adequate environmental monitoring programs are crucial to assess and reduce the occupational exposure of healthcare workers to cytostatics. The analytical methods employed should be rapid, reliable, sensitive, standardized, and include multiple compounds. A critical overview of recent overall procedures for surface [...] Read more.
Periodic and adequate environmental monitoring programs are crucial to assess and reduce the occupational exposure of healthcare workers to cytostatics. The analytical methods employed should be rapid, reliable, sensitive, standardized, and include multiple compounds. A critical overview of recent overall procedures for surface and air contamination with cytostatics in workplace settings is presented, with a focus on sampling, sample preparation, and instrumental considerations. Limitations are also addressed and some recommendations and advice are provided. Since dermal absorption is the main exposure route, surface contamination is the preferred indicator of biological uptake and its methods have significantly improved. In contrast, cytostatics’ inhalation is rare; thus, air contamination has been poorly studied, with little improvement. Still, some elements of the analytical methods have not been extensively explored, namely: the amount of wetting solution, the extraction procedure, surface chemistry and roughness, recovery studies from specific surfaces, and cytostatics stability (in surfaces and during shipping and storage). Furthermore, complete validation data (including precision, accuracy, and instrumental and method detection limits) and estimation of global uncertainty are still lacking in most studies, thus preventing method comparison and proposal of standardized procedures. Full article
(This article belongs to the Special Issue Analytical Techniques in the Pharmaceutical Sciences)
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<p>Schematic representation of the main considerations for the development of an analytical methodology to monitor the contamination of surfaces by cytostatics. (UP)LC–MS/MS—(ultra-performance) liquid chromatography with tandem mass spectrometry; LC–MS—liquid chromatography with mass spectrometry; LC–UV—liquid chromatography with ultraviolet detector; ICP–MS—inductively coupled plasma mass spectrometry; GC–MS—gas chromatography with mass spectrometry.</p> Full article ">
30 pages, 6562 KiB  
Article
Antimicrobial Activity of a Library of Thioxanthones and Their Potential as Efflux Pump Inhibitors
by Fernando Durães, Andreia Palmeira, Bárbara Cruz, Joana Freitas-Silva, Nikoletta Szemerédi, Luís Gales, Paulo Martins da Costa, Fernando Remião, Renata Silva, Madalena Pinto, Gabriella Spengler and Emília Sousa
Pharmaceuticals 2021, 14(6), 572; https://doi.org/10.3390/ph14060572 - 15 Jun 2021
Cited by 13 | Viewed by 4740
Abstract
The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) [...] Read more.
The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 µM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation. Full article
(This article belongs to the Special Issue Identification of Phytochemicals and Derivatives against Infectious Diseases)
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<p>ORTEP view of compounds <b>13</b> and <b>14</b>.</p> Full article ">Figure 2
<p>Relative fluorescence index (RFI) of the thioxanthone derivatives <b>2</b>, <b>3</b>, <b>7</b>, <b>8</b>, and <b>13</b> in <span class="html-italic">S. aureus</span> 272123 (<b>Top</b>) and <span class="html-italic">Salmonella enterica</span> serovar Typhimurium SL1344 (SE03, <b>Bottom</b>). Results are presented as mean ± SD. Statistical comparisons were performed using the <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 vs. control (DMSO 1% <span class="html-italic">v</span>/<span class="html-italic">v</span>)].</p> Full article ">Figure 2 Cont.
<p>Relative fluorescence index (RFI) of the thioxanthone derivatives <b>2</b>, <b>3</b>, <b>7</b>, <b>8</b>, and <b>13</b> in <span class="html-italic">S. aureus</span> 272123 (<b>Top</b>) and <span class="html-italic">Salmonella enterica</span> serovar Typhimurium SL1344 (SE03, <b>Bottom</b>). Results are presented as mean ± SD. Statistical comparisons were performed using the <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 vs. control (DMSO 1% <span class="html-italic">v</span>/<span class="html-italic">v</span>)].</p> Full article ">Figure 3
<p>Molecular visualization of thioxanthones on targets in the BCR of the NorA homology model. (<b>A</b>) General view of compounds <b>2</b> (blue) and <b>3</b> (pink); (<b>B</b>) Interaction between compound <b>2</b> and the BCR; (<b>C</b>) Interaction between compound <b>3</b> and the BCR; (<b>D</b>) in the SBS of AcrB, general view of compounds <b>3</b> (red), <b>7</b> (pink), <b>8</b> (green) and <b>13</b> (blue).</p> Full article ">Figure 4
<p>Cytotoxicity of the thioxanthone derivatives <b>9</b>–<b>17</b> (0–20 μM), in Caco-2 cells, by the neutral red (NR) uptake assay, 24 h after exposure. The results are presented as mean ± SD from four independent experiments, carried out in triplicate. Statistical comparisons were performed using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test [** <span class="html-italic">p</span> &lt;0.01 vs. control (0 μM)].</p> Full article ">Figure 5
<p>Cytotoxicity of the thioxanthone derivatives <b>9</b>–<b>17</b> (0–20 μM), in Caco-2 cells, by the sulforhodamine B binding assay, 24 h after exposure. The results are presented as mean ± SD from four independent experiments, carried out in triplicate. Statistical comparisons were performed using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test [** <span class="html-italic">p</span> &lt;0.01 vs. control (0 μM)].</p> Full article ">Figure 6
<p>Evaluation of the expression levels of P-glycoprotein (P-gp), by flow cytometry, in Caco-2 cells exposed to compounds <b>9</b> and <b>14</b> (20 μM) for 24 h. The results are presented as mean ± SD of three independent experiments (performed in triplicate). Statistical comparisons were made using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test [*** <span class="html-italic">p</span> &lt; 0.001 vs. control (0 μM)].</p> Full article ">Figure 7
<p>P-glycoprotein (P-gp) activity assessed by fluorescence spectroscopy in Caco-2 cells pre-exposed to the thioxanthone derivatives <b>9</b>, <b>10</b>, and <b>12</b>–<b>14</b> (20 μM) for 24 h. The results are presented as mean ± SD of five independent experiments, performed in triplicate. Statistical comparisons were made using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test.</p> Full article ">Figure 8
<p>P-glycoprotein (P-gp) activity assessed by fluorescence spectroscopy in Caco-2 cells pre-exposed to thioxanthone derivatives <b>9</b>, <b>10</b>, and <b>12</b>–<b>14</b> (20 μM) only during the 90 min of the incubation period with the fluorescent substrate (RHO 123, 10 μM). The results are presented as mean ± SD of four independent experiments, performed in triplicate. Statistical comparisons were made using the one-way ANOVA parametric method, followed by Dunnett’s multiple comparisons test [**** <span class="html-italic">p</span> &lt; 0.0001 vs. control (0 μM)].</p> Full article ">
19 pages, 462 KiB  
Review
Benefits of Ginger and Its Constituent 6-Shogaol in Inhibiting Inflammatory Processes
by Iris Bischoff-Kont and Robert Fürst
Pharmaceuticals 2021, 14(6), 571; https://doi.org/10.3390/ph14060571 - 15 Jun 2021
Cited by 90 | Viewed by 11638
Abstract
Ginger (Zingiber officinale Roscoe) is widely used as medicinal plant. According to the Committee on Herbal Medicinal Products (HMPC), dried powdered ginger rhizome can be applied for the prevention of nausea and vomiting in motion sickness (well-established use). Beyond this, a plethora [...] Read more.
Ginger (Zingiber officinale Roscoe) is widely used as medicinal plant. According to the Committee on Herbal Medicinal Products (HMPC), dried powdered ginger rhizome can be applied for the prevention of nausea and vomiting in motion sickness (well-established use). Beyond this, a plethora of pre-clinical studies demonstrated anti-cancer, anti-oxidative, or anti-inflammatory actions. 6-Shogaol is formed from 6-gingerol by dehydration and represents one of the main bioactive principles in dried ginger rhizomes. 6-Shogaol is characterized by a Michael acceptor moiety being reactive with nucleophiles. This review intends to compile important findings on the actions of 6-shogaol as an anti-inflammatory compound: in vivo, 6-shogaol inhibited leukocyte infiltration into inflamed tissue accompanied with reduction of edema swelling. In vitro and in vivo, 6-shogaol reduced inflammatory mediator systems such as COX-2 or iNOS, affected NFκB and MAPK signaling, and increased levels of cytoprotective HO-1. Interestingly, certain in vitro studies provided deeper mechanistic insights demonstrating the involvement of PPAR-γ, JNK/Nrf2, p38/HO-1, and NFκB in the anti-inflammatory actions of the compound. Although these studies provide promising evidence that 6-shogaol can be classified as an anti-inflammatory substance, the exact mechanism of action remains to be elucidated. Moreover, conclusive clinical data for anti-inflammatory actions of 6-shogaol are largely lacking. 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>Chemical structures of 6-shogaol and 6-gingerol.</p> Full article ">
19 pages, 2610 KiB  
Article
Efficient Matrix Cleanup of Soft-Gel-Type Dietary Supplements for Rapid Screening of 92 Illegal Adulterants Using EMR-Lipid dSPE and UHPLC-Q/TOF-MS
by Beom Hee Kim, Wonwoong Lee, You Lee Kim, Ji Hyun Lee and Jongki Hong
Pharmaceuticals 2021, 14(6), 570; https://doi.org/10.3390/ph14060570 - 15 Jun 2021
Cited by 5 | Viewed by 3122
Abstract
An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance [...] Read more.
An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UHPLC-Q/TOF-MS). As representative green chemistry methods, three sample preparation methods (dispersive liquid-liquid microextraction (DLLME), “quick, easy, cheap, effective, rugged, and safe” dispersive solid-phase extraction (QuEChERS-dSPE), and enhanced matrix removal-lipid (EMR-Lipid) dSPE) were evaluated for matrix removal efficiency, recovery rate, and matrix effect. In this study, EMR-Lipid dSPE was shown to effectively remove complicated matrix contents in soft-gels, compared to DLLME and QuEChERS-dSPE. For the rapid screening of a wide range of adulterants, extracted common ion chromatogram (ECIC) and neutral loss scan (NLS) based on specific common MS/MS fragments were applied to randomly collected soft-gel-type dietary supplement samples using UHPLC-Q/TOF-MS. Both ECICs and NLSs enabled rapid and simple screening of multi-class adulterants and could be an alternative to the multiple reaction monitoring (MRM) method. The developed method was validated in terms of limit of detection (LOD), precision, accuracy, recovery, and matrix effects. The range of LODs was 0.1–16 ng/g. The overall precision values were within 0.09–14.65%. The accuracy ranged from 81.6% to 116.6%. The recoveries and matrix effects of 92 illegal adulterants ranged within 16.9–119.4% and 69.8–114.8%, respectively. The established method was successfully applied to screen and identify 92 illegal adulterants in soft-gels. This method can be a promising tool for the high-throughput screening of various adulterants in dietary supplements and could be used as a more environmentally friendly routine analytical method for screening dietary supplements illegally adulterated with multi-class drug substances. Full article
(This article belongs to the Special Issue Analytical Techniques in the Pharmaceutical Sciences)
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<p>Analytical flow for screening of 92 illegal adulterants in soft-gel type dietary supplements.</p> Full article ">Figure 2
<p>Total ion chromatograms of matrix residues after sample pretreatments of soft-gel by (<b>a</b>) QuEChERS-dSPE, (<b>b</b>) EMR-Lipid dSPE, and (<b>c</b>) DLLME methods.</p> Full article ">Figure 3
<p>Comparison of matrix effects and extraction recovery for 92 illegal adulterants according to three pretreatment methods.</p> Full article ">Figure 4
<p>Total ion chromatogram of 92 illegal adulterants under optimized LC-MS conditions in positive ion mode.</p> Full article ">Figure 5
<p>ECICs and NLSs of illegal adulterants after application of EMR-Lipid dSPE pretreatment to soft-gel samples spiked with analytes at 2 μg/g level.</p> Full article ">Figure 6
<p>EIC, ECIC and NLS screening and confirmation of illegal adulterants in soft-gel samples.</p> Full article ">
22 pages, 6486 KiB  
Review
Phytoestrogens for the Management of Endometriosis: Findings and Issues
by Xia Cai, Min Liu, Bing Zhang, Shao-Jie Zhao and Shi-Wen Jiang
Pharmaceuticals 2021, 14(6), 569; https://doi.org/10.3390/ph14060569 - 14 Jun 2021
Cited by 19 | Viewed by 7173
Abstract
Endometriosis, a chronic disease characterized by recurrent pelvic pain and infertility, severely impacts the health and life quality of many women worldwide. Since phytoestrogens are commonly found in a variety of foods, and estrogen is a major pathological factor for the pathogenesis of [...] Read more.
Endometriosis, a chronic disease characterized by recurrent pelvic pain and infertility, severely impacts the health and life quality of many women worldwide. Since phytoestrogens are commonly found in a variety of foods, and estrogen is a major pathological factor for the pathogenesis of endometriosis, their possible involvement cannot be ignored. This review summarizes data on the relationship between phytoestrogen intake and endometriosis risk, and analyzes the findings from in vitro experiments, rodent endometriotic models, and human intervention trials. While favorable results were often obtained from endometrial primary cultures and animal models for resveratrol, isoflavones and puerarin, only resveratrol showed promising results in human intervention trials. Critical issues concerning the current study efforts are discussed: the possible reasons beneath the discrepant observations of estrogenic/anti-estrogenic effects by phytoestrogens; the complicated interplays between phytoestrogens and endogenous estrogens; the shortage of currently used animal models; the necessity to apply reasonable doses of phytoestrogens in experiments. It is expected that the analyses would help to more properly assess the phytoestrogens’ effects on the endometriosis pathogenesis and their potential values for preventive or therapeutic applications. Full article
(This article belongs to the Special Issue Novel Regulators of Female Reproduction)
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<p>The interplay between phytoestrogens and background estrogens. (<b>A</b>). In the absence or the presence of a diminished level of estrogen, e.g., in an estrogen–free experimental system, or inside males, postmenopausal women, or pre-pubertal women, the un-liganded ER molecules are mostly available for phytoestrogen to bind. Despite their relatively low affinity to ER and low transcriptional activation potency, phytoestrogens will bind to ERs, and the consequential effects would be likely estrogenic/agonistic in this situation. (<b>B</b>). In the presence of a high level of estrogens, e.g., addition of estrogens to an in vitro experimental system, or inside the reproductive women, or under some pathological hyperestrogen conditions, phytoestrogens when reaching a sufficient concentration, will effectively compete with estrogens for ER binding. However, phytoestrogen-bound ERs have a lower transcriptional activation potency than estrogen-bound ERs. The final effects will be dependent on the ratio of phytoestrogen-bound ERs verse estrogen-bound ERs. A relatively high phytoestrogen occupancy of ERs would likely leads to an observation of anti-estrogenic/antagonist activities of phytoestrogens. While this can be cited to explain some discrepant results from different laboratories, the simplified model would be modified and further complicated by many factors, including the estrogen/phytoestrogen interactions with non-ER proteins such as SHBG, local estrogen synthesis, tissue-specific expression of ER subtypes, and post-translational modification of ER, etc.</p> Full article ">Figure 2
<p>Molecular pathways and cellular effects leading to the potential therapeutic values of phytoestrogens. Positive findings in support of the application of phytoestrogens for endometriosis management were outlined. Dietary phytoestrogens are absorbed, reach the endometrial epithelial and stromal cells, bind to ER, and participate in gene regulation of a variety of endometriosis-related factors, affecting multiple processes including inflammation, angiogenesis, proliferation, invasion and local estrogen synthesis. These activities would converge to suppress the development, and alleviate the symptoms, of endometriosis. Notes: interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-2 and -9 (MMP-2 and -9), monocarboxylate transporters 1 and 4 (MCT-1 and 4), glucose transporters 1 and 3 (GLUT-1 and GLUT-3), estradiol (E2), proliferating cell nuclear antigen (PCNA), Ki-67 (a proliferative marker), tissue inhibitor of metalloproteinase 1 (TIMP-1), metastasis-associated protein 1 (MTA1), cyclooxygenase-2 (COX-2), estrone (E1).</p> Full article ">
22 pages, 5943 KiB  
Article
Effects of the Hydroethanolic Extract of Lycopodium selago L. on Scopolamine-Induced Memory Deficits in Zebrafish
by Mihai-Vlad Valu, Catalin Ducu, Sorin Moga, Denis Negrea, Lucian Hritcu, Razvan Stefan Boiangiu, Emanuel Vamanu, Tudor Adrian Balseanu, Simone Carradori and Liliana Cristina Soare
Pharmaceuticals 2021, 14(6), 568; https://doi.org/10.3390/ph14060568 - 14 Jun 2021
Cited by 11 | Viewed by 4709
Abstract
This scientific research focused on the production of hydroethanolic extract of the plant species Lycopodium selago L. (L. selago) by the ultrasound-assisted extraction (USAE) and the identification of biocompounds with high antioxidant activity is of interest for possible phytotherapeutic treatment against [...] Read more.
This scientific research focused on the production of hydroethanolic extract of the plant species Lycopodium selago L. (L. selago) by the ultrasound-assisted extraction (USAE) and the identification of biocompounds with high antioxidant activity is of interest for possible phytotherapeutic treatment against Alzheimer’s disease (AD). The extract was phytochemically analyzed to investigate polyphenols, flavonoids, and identify the sesquiterpenoid alkaloid huperzine A (HupA), which is known in the literature for its great relevance in AD. Evaluation and comparison of the antioxidant activity of the extract were performed by four complementary spectrophotometric methods (DPPH, FRAP, ABTS, ORAC). In vitro tests of the extract showed an excellent reciprocal link between the concentration of polyphenols and the measurement of the antioxidant activity of the extract with the sesquiterpenoid HupA. To confirm the antioxidant activity, L. selago hydroethanolic extract was administered in vivo to zebrafish (Danio rerio) with a pattern of scopolamine-induced cognitive impairment. Moreover, this study explored a possible correlation between the expression of oxidative stress markers in the brain tissue with the behavior of the scopolamine zebrafish model. In vivo tests showed that this fern could be used as a nutritional supply and as a phytotherapeutic method to prevent or treat various neurodegenerative diseases that call for high-nutritive-value medications. Full article
(This article belongs to the Special Issue Treatment of Alzheimer Disease)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Chemical structure of the huperzine A (HupA).</p> Full article ">Figure 2
<p><span class="html-italic">Lycopodim selago</span> L. in Cluj County, Romania, 2020 (authors personal collection).</p> Full article ">Figure 3
<p>The HPLC sample chromatogram of HupA. The retention time of sesquiterpene alkaloid HupA peak remained within the range of 14.812–14.813 min (<b>A</b>) as the HupA molecule standard (<b>B</b>), at 14.815 min.</p> Full article ">Figure 4
<p><span class="html-italic">Lycopodium selago</span> hydroethanolic extract (LS: 0.5, 1, and 3 mg/L) improved the locomotion pattern and memory in the Y-maze test. (<b>A</b>) Representative locomotion-tracking pattern of the control, scopolamine (SCOP 100 µM), <span class="html-italic">Lycopodium selago</span> (LS: 0.5, 1, and 3 mg/L) and galantamine (GAL: 1 mg/L) treated groups. (<b>B</b>) Representation of the total time spent in the novel arm in different groups. (<b>C</b>) Representation of the turn angle of zebrafish in the tank in different groups. (<b>D</b>) Representation of the total distance traveled by zebrafish in the tank in different groups. Values are means ± S.E.M. (<span class="html-italic">n</span> = 10). For Tukey’s post-hoc analyses: **** <span class="html-italic">p</span> &lt; 0.0001, *** <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.</p> Full article ">Figure 5
<p><span class="html-italic">Lycopodium selago</span> hydroethanolic extract (LS: 0.5, 1, and 3 mg/L) improved the locomotion pattern and reduced anxiety in the novel tank-diving test (NTT). (<b>A</b>) Representative locomotion-tracking pattern in different groups. (<b>B</b>) Representation of the distance traveled in the top zone in different groups. (<b>C</b>) The time spent by zebrafish in the top/bottom zone of the tank in different groups. (<b>D</b>) Representation of the number of entries in the top zone by zebrafish in the tank in different groups. (<b>E</b>) Representation of the freezing activity by zebrafish in the tank in different groups. (<b>F</b>) Representation of the average velocity of zebrafish in the tank in different groups. (<b>G</b>) Representation of the total distance traveled by zebrafish in the tank in different groups. Values are means ± S.E.M. (<span class="html-italic">n</span> = 10). For Tukey’s post-hoc analyses: **** <span class="html-italic">p</span> &lt; 0.0001, *** <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.</p> Full article ">Figure 6
<p><span class="html-italic">Lycopodium selago</span> hydroethanolic extract (LS: 0.5, 1, and 3 mg/L) improved memory in the novel object recognition test (NOR). (<b>A</b>) Representative locomotion-tracking pattern in different groups. (<b>B</b>) Representation of the percentages of preference in different groups. Values are means ± S.E.M. (<span class="html-italic">n</span> = 10). For Tukey’s post-hoc analyses: **** <span class="html-italic">p</span> &lt; 0.0001, *** <span class="html-italic">p</span> &lt; 0.001, and ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 7
<p><span class="html-italic">Lycopodium selago</span> hydroethanolic extract (LS: 0.5, 1, and 3 mg/L) exhibited an anti-AChE effect and improved the antioxidant status in the zebrafish brain. (<b>A</b>–<b>D</b>) Representation of the enzymes specific activity (CAT, SOD, GPX and GSH) in different groups; (<b>E</b>,<b>F</b>) Representation of the MDA and protein carbonyl levels in different groups; (<b>G</b>) AChE-specific activity. Values are means ± S.E.M. (<span class="html-italic">n</span> = 10). For Tukey’s post-hoc analyses: **** <span class="html-italic">p</span> &lt; 0.0001, *** <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.</p> Full article ">Figure 8
<p>Correlation analyses between behavioral and biochemical parameters (Pearson’s correlation, n = 10): (<b>A</b>) Time in novel arm (% of total time) vs. MDA: r = −0.7693, <span class="html-italic">p</span> &lt; 0.001; (<b>B</b>) Preference vs. MDA: r = −0.7817, <span class="html-italic">p</span> &lt; 0.001; (<b>C</b>) SOD vs. MDA: r = 0.7478, <span class="html-italic">p</span> &lt; 0.01; (<b>D</b>) CAT vs. MDA: r = −0.8081, <span class="html-italic">p</span> &lt; 0.001; (<b>E</b>) GPX vs. MDA: r = −0.7140, <span class="html-italic">p</span> &lt; 0.01; (<b>F</b>) GSH vs. MDA: r = −0.5401, <span class="html-italic">p</span> &lt; 0.05; (<b>G</b>) AChE vs. MDA: r = −0.6455, <span class="html-italic">p</span> &lt; 0.01. Data expressed are time in the novel arm (% of total time), time in the novel arm (s), SOD (U/mg protein), CAT (U/mg protein), GPX (U/mg protein) GSH (µg GSH/µg protein), AChE (nmol/min/mg protein), and MDA (nmol/mg protein).</p> Full article ">
13 pages, 4065 KiB  
Article
Protective Effect of Lusianthridin on Hemin-Induced Low-Density Lipoprotein Oxidation
by Su Wutyi Thant, Noppawan Phumala Morales, Visarut Buranasudja, Boonchoo Sritularak and Rataya Luechapudiporn
Pharmaceuticals 2021, 14(6), 567; https://doi.org/10.3390/ph14060567 - 14 Jun 2021
Cited by 6 | Viewed by 3567
Abstract
Oxidation of low-density lipoprotein (LDL) plays a crucial role in the pathogenesis of atherosclerosis. Hemin (iron (III)-protoporphyrin IX) is a degradation product of hemoglobin that can be found in thalassemia patients. Hemin is a strong oxidant that can cause LDL oxidation and contributes [...] Read more.
Oxidation of low-density lipoprotein (LDL) plays a crucial role in the pathogenesis of atherosclerosis. Hemin (iron (III)-protoporphyrin IX) is a degradation product of hemoglobin that can be found in thalassemia patients. Hemin is a strong oxidant that can cause LDL oxidation and contributes to atherosclerosis in thalassemia patients. Lusianthridin from Dendrobium venustrum is a phenolic compound that possesses antioxidant activity. Hence, lusianthridin could be a promising compound to be used against hemin-induced oxidative stress. The major goal of this study is to evaluate the protective effect of lusianthridin on hemin-induced low-density lipoprotein oxidation (he-oxLDL). Here, various concentrations of lusianthridin (0.25, 0.5, 1, and 2 µM) were preincubated with LDL for 30 min, then 5 µM of hemin was added to initiate the oxidation, and oxidative parameters were measured at various times of incubation (0, 1, 3, 6, 12, 24 h). Lipid peroxidation of LDL was measured by thiobarbituric reactive substance (TBARs) assay and relative electrophoretic mobility (REM). The lipid composition of LDL was analyzed by using reverse-phase HPLC. Foam cell formation with he-oxLDL in RAW 264.7 macrophage cells was detected by Oil Red O staining. The results indicated that lusianthridin could inhibit TBARs formation, decrease REM, decrease oxidized lipid products, as well as preserve the level of cholesteryl arachidonate and cholesteryl linoleate. Moreover, He-oxLDL incubated with lusianthridin for 24 h can reduce the foam cell formation in RAW 264.7 macrophage cells. Taken together, lusianthridin could be a potential agent to be used to prevent atherosclerosis in thalassemia patients. Full article
(This article belongs to the Section Natural Products)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Effect of lusianthridin on TBARs formation in hemin-induced LDL oxidation. LDLs were preincubated with LST or Trolox for 30 min, then hemin was added to induce LDL oxidation. After 24 h treatment, TBARs formations were determined at various time points with TBARs assay. The data are presented as mean ± SEMs (<span class="html-italic">n</span> = 5); * <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. nLDL, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001 <span class="html-italic">vs</span>. he-oxLDL.</p> Full article ">Figure 2
<p>Effect of lusianthridin on relative electrophoretic mobility (REM) in hemin-induced LDL oxidation at various times of incubation. (<b>A</b>) Representative REM in agarose electrophoresis. Lane 1: nLDL, lane 2: he-oxLDL, lane 3: trolox 0.5 µM, lane 4: trolox 1 µM, lane 5: LST 0.25 µM, lane 6: LST 0.5 µM, lane 7: LST 1 µM, lane 8: LST 2 µM; (<b>B</b>) REM data as mean ± SEMs (<span class="html-italic">n</span> = 5), * <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. nLDL; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. he-oxLDL.</p> Full article ">Figure 3
<p>Effects of LST on lipid composition in hemin-induced LDL oxidation for 24 h. (<b>A</b>) Representative chromatogram of lipid composition detected at 210 nm; (<b>B</b>) % free cholesterol FC; (<b>C</b>) % cholesteryl arachidonate, CA; (<b>D</b>) % cholesteryl linoleate, CL; data were presented as mean ± SEMs (<span class="html-italic">n</span> = 4); * <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. nLDL; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. he-oxLDL.</p> Full article ">Figure 4
<p>Effect of lusianthridin on oxidized lipid products detected at 234 nm: (<b>A</b>) at 6 h, (<b>B</b>) at 12 h, and (<b>C</b>) at 24 h in hemin-induced LDL oxidation. Data were presented as mean ± SEMs (<span class="html-italic">n</span> = 4); * <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. nLDL; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. he-oxLDL.</p> Full article ">Figure 5
<p>(<b>A</b>) Representative Oil Red O photographs of RAW 264.7 macrophage cells (40 × magnification) (i) nLDL, (ii) he-oxLDL, (iii) TRO 0.5 µM, (iv) TRO 1 µM, (v) LST 0.25 µM, (vi) LST 0.5 µM, (vii) LST 1 µM, (viii) LST 2 µM. (<b>B</b>) Quantitation of lipid content at 492 nm. Data were presented as mean ± SEMs (<span class="html-italic">n</span> = 4); * <span class="html-italic">p</span> &lt; 0.05 <span class="html-italic">vs</span>. nLDL; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.0001 <span class="html-italic">vs</span>. he-oxLDL. (<b>C</b>) Effect of lusianthridin on RAW 264.7 macrophage cell viability. * <span class="html-italic">p</span> &lt; 0.05 and <sup>***</sup> <span class="html-italic">p</span> &lt; 0.0001 <span class="html-italic">vs</span>. control.</p> Full article ">Figure 6
<p>Structure of lusianthridin.</p> Full article ">
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