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Pharmaceuticals
Volume 14
Issue 12
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Pharmaceuticals, Volume 14, Issue 12 (December 2021) – 134 articles

Cover Story (view full-size image): The limited pH solubility and narrow absorption area of poorly water-soluble drugs can be challenged in developing drug products. This study focuses on combining hot-melt extrusion-based solid dispersion (SD) pellets to improve the bioavailability of itraconazole (ITZ). The bioavailability of ITZ can be enhanced by enhancing solubility and inhibiting precipitation. In this study we evaluate the release behaviors of each ITZ SD pellet and investigate the mechanism of ITZ absorption behaviors in the in vivo gastrointestinal system. View this paper
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13 pages, 566 KiB  
Review
Pharmacological Activity of Garcinia indica (Kokum): An Updated Review
by Sung Ho Lim, Ho Seon Lee, Chang Hoon Lee and Chang-Ik Choi
Pharmaceuticals 2021, 14(12), 1338; https://doi.org/10.3390/ph14121338 - 20 Dec 2021
Cited by 28 | Viewed by 5585
Abstract
Garcinia indica (commonly known as kokum), belonging to the Clusiaceae family (mangosteen family), is a tropical evergreen tree distributed in certain regions of India. It has been used in culinary and industrial applications for a variety of purposes, including acidulant in curries, pickles, [...] Read more.
Garcinia indica (commonly known as kokum), belonging to the Clusiaceae family (mangosteen family), is a tropical evergreen tree distributed in certain regions of India. It has been used in culinary and industrial applications for a variety of purposes, including acidulant in curries, pickles, health drinks, wine, and butter. In particular, G. indica has been used in traditional medicine to treat inflammation, dermatitis, and diarrhea, and to promote digestion. According to several studies, various phytochemicals such as garcinol, hydroxycitric acid (HCA), cyanidin-3-sambubioside, and cyanidin-3-glucoside were isolated from G. indica, and their pharmacological activities were published. This review highlights recent updates on the various pharmacological activities of G. indica. These studies reported that G. indica has antioxidant, anti-obesity, anti-arthritic, anti-inflammatory, antibacterial, hepatoprotective, cardioprotective, antidepressant and anxiolytic effects both in vitro and in vivo. These findings, together with previously published reports of pharmacological activity of various components isolated from G. indica, suggest its potential as a promising therapeutic agent to prevent various diseases. Full article
(This article belongs to the Special Issue Plant and Marine-Derived Natural Product Research in Drug Discovery: Strengths and Perspective)
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<p>Chemical structure of bioactive ingredients isolated from <span class="html-italic">G. indica</span>.</p> Full article ">
20 pages, 4101 KiB  
Article
In Vitro Study of Licorice on IL-1β-Induced Chondrocytes and In Silico Approach for Osteoarthritis
by Akhtar Ali, YoungJoon Park, Jeonghoon Lee, Hyo-Jin An, Jong-Sik Jin, Jong-Hyun Lee, Jaeki Chang, Dong-Keun Kim, Bonhyuk Goo, Yeon Cheol Park, Kang-Hyun Leem, Shin Seong and Wonnam Kim
Pharmaceuticals 2021, 14(12), 1337; https://doi.org/10.3390/ph14121337 - 20 Dec 2021
Cited by 9 | Viewed by 4207
Abstract
Osteoarthritis (OA) is a common degenerative joint disorder that affects joint function, mobility, and pain. The release of proinflammatory cytokines stimulates matrix metalloproteinases (MMPs) and aggrecanase production which further induces articular cartilage degradation. Hypertrophy-like changes in chondrocytes are considered to be an important [...] Read more.
Osteoarthritis (OA) is a common degenerative joint disorder that affects joint function, mobility, and pain. The release of proinflammatory cytokines stimulates matrix metalloproteinases (MMPs) and aggrecanase production which further induces articular cartilage degradation. Hypertrophy-like changes in chondrocytes are considered to be an important feature of OA pathogenesis. A Glycyrrhiza new variety, Wongam (WG), was developed by the Korea Rural Development Administration to enhance the cultivation and quality of Glycyrrhizae Radix et Rhizoma (licorice). This study examined the regulatory effect of WG against hypertrophy-like changes such as RUNX2, Collagen X, VEGFA, MMP-13 induction, and Collagen II reduction induced by IL-1β in SW1353 human chondrocytes. Additionally, in silico methods were performed to identify active compounds in licorice to target chondrocyte hypertrophy-related proteins. WG showed inhibitory effects against IL-1β-induced chondrocyte hypertrophy by regulating both HDAC4 activation via the PTH1R/PKA/PP2A pathway and the SOX9/β-catenin signaling pathway. In silico analysis demonstrated that 21 active compounds from licorice have binding potential with 11 targets related to chondrocyte hypertrophy. Further molecular docking analysis and in vivo studies elicited four compounds. Based on HPLC, isoliquiritigenin and its precursors were identified and quantified. Taken together, WG is a potential therapeutic agent for chondrocyte hypertrophy-like changes in OA. Full article
(This article belongs to the Special Issue Pharmaceuticals and Cosmeceuticals from Plants: Molecular Pharmacology and Toxicology)
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Figure 1
<p>HPLC chromatograms of standard (<b>A</b>) isoliquiritin, liquiritigenin, and isoliquiritigenin. HPLC chromatograms of (<b>B</b>) WG extract.</p> Full article ">Figure 2
<p>Cytotoxicity and effects of WG against IL-1β-induced hypertrophic changes. (<b>A</b>) The cytotoxicity of WG on chondrocytes was determined at different concentrations using MTT assay. (<b>B</b>) Protein expression and quantitative analysis of (<b>C</b>) RUNX2, (<b>D</b>) Collagen X, (<b>E</b>) VEGFA, and (<b>F</b>) Collagen II expression relative to β-actin. (<b>G</b>) MMP-13 production was determined by ELISA. SW1353 cells were pretreated with indicated concentration of WG or DXM for 4 h followed by IL-1β for 24 h. Data shown represent mean ± SEM (<span class="html-italic">n</span> = 3). (### <span class="html-italic">p</span> &lt; 0.001, vs. control; * <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 vs. IL-1β).</p> Full article ">Figure 3
<p>Effect of WG on HDAC4 nuclear translocation. (<b>A</b>) Western blot analysis of cytosolic and nuclear fractions using HDAC4 antibody, (<b>B</b>) quantitative analysis of cytosolic HDAC4 relative β-actin, (<b>C</b>) quantitative analysis of nuclear HDAC4 relative to Lamin A/C. SW1353 cells were pre-treated with indicated concentration of WG or DXM for 4 h in 0.2% BSA media followed by IL-1β for 24 h. (<b>D</b>) Western blot analysis of cytosolic and nuclear fractions using HDAC4 antibody, (<b>E</b>) quantitative analysis of cytosolic HDAC4 relative β-actin, (<b>F</b>) quantitative analysis of nuclear HDAC4 relative to Lamin A/C. (<b>G</b>) Protein expression and quantitative analysis of (<b>H</b>) RUNX2, (<b>I</b>) Collagen X, (<b>J</b>) VEGFA, and (<b>K</b>) Collagen II expression relative to β-actin. SW1353 cells were pre-treated with ODA or H89 in 0.2% BSA media, or cell media was either changed to 0.2% BSA media for 1 h and then treated with WG or DXM for 4 h followed by IL-1β for 24 h. Data shown represent mean ± SEM (<span class="html-italic">n</span> = 3). (# <span class="html-italic">p</span> &lt; 0.05 and ### <span class="html-italic">p</span> &lt; 0.001 vs. control; * <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 vs. IL-1β; † <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 vs. WG).</p> Full article ">Figure 4
<p>Effect of WG on PTH1R. (<b>A</b>) Western blot analysis of PTH1R. (<b>B</b>) Quantitative analysis of PTH1R relative to β-actin. SW1353 cells were pre-treated with indicated concentration of WG or DXM for 4 h in 0.2% BSA media followed by IL-1β for 24 h. (<b>C</b>) Western blot analysis of cytosolic and nuclear fractions using HDAC4 antibody, (<b>D</b>) quantitative analysis of cytosolic HDAC4 relative β-actin, (<b>E</b>) quantitative analysis of nuclear HDAC4 relative to Lamin A/C. SW1353 cells were pre-treated with WG or DXM for 4 h in 0.2% BSA media in the presence or absence of PTH (7–34) followed by IL-1β for 24 h. (# <span class="html-italic">p</span> &lt; 0.05 and ## <span class="html-italic">p</span> &lt; 0.01 vs. control; ** <span class="html-italic">p</span> &lt; 0.01 vs. IL-1β; † <span class="html-italic">p</span> &lt; 0.05 vs. WG).</p> Full article ">Figure 5
<p>WG increased SOX9 expression and attenuated the IL-1β-induced β-catenin signaling pathway. (<b>A</b>) Western blot analysis of SOX9. (<b>B</b>) Quantitative analysis of SOX9 relative to β-actin. (<b>C</b>) Western blot analysis of cytosolic and nuclear and fractions using β-catenin antibody, quantitative analysis of (<b>D</b>) cytosolic β-catenin relative to β-actin and (<b>E</b>) nuclear β-catenin relative to Lamin A/C. SW1353 cells were pre-treated with indicated concentration of WG or DXM for 4 h in 0.2% BSA media followed by IL-1β for 24 h. (<b>F</b>) Western blot analysis of cytosolic and nuclear and fractions using β-catenin antibody, quantitative analysis of (<b>G</b>) cytosolic β-catenin relative to β-actin and (<b>H</b>) nuclear β-catenin relative to Lamin A/C. (<b>I</b>) Western blot analysis of RUNX2, (<b>J</b>) quantitative analysis of RUNX2 relative to β-actin. SW1353 cells were pre-treated with WG or DXM for 4 h in 0.2% BSA media followed by IL-1β for 24 h in the presence or absence of LiCl. Data shown represent mean ± SEM (<span class="html-italic">n</span> = 3). (# <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 vs. control; * <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 vs. IL-1β; † <span class="html-italic">p</span> &lt; 0.05 and ††† <span class="html-italic">p</span> &lt; 0.001 vs. DXM or WG).</p> Full article ">Figure 6
<p>Flow work in silico pharmacology network analysis.</p> Full article ">Figure 7
<p>The 21 active compounds of licorice and its targets in (<b>A</b>) Wnt-signaling pathway and (<b>B</b>) endochondral ossification. (<b>C</b>) Representation of 21 active compounds and their targets (red: targeted and black: non-targeted). (<b>D</b>) The number of targeted genes in Wnt-signaling pathway or endochondral ossification according to 21 active compounds.</p> Full article ">Figure 8
<p>Result of protein docking analysis between 21 active compounds and 11 main proteins. (<b>A</b>) Representation of docking energy. Represented 3D molecular docking between four active compounds which were reported to OA in previous studies with (<b>B</b>) MMP13, (<b>C</b>) SOX9, (<b>D</b>) COL2A1, and (<b>E</b>) COL10A1.</p> Full article ">
23 pages, 4663 KiB  
Article
The Role of TRPA1 Channels in the Central Processing of Odours Contributing to the Behavioural Responses of Mice
by János Konkoly, Viktória Kormos, Balázs Gaszner, Zoltán Sándor, Angéla Kecskés, Ammar Alomari, Alíz Szilágyi, Beatrix Szilágyi, Dóra Zelena and Erika Pintér
Pharmaceuticals 2021, 14(12), 1336; https://doi.org/10.3390/ph14121336 - 20 Dec 2021
Cited by 10 | Viewed by 3773
Abstract
Transient receptor potential ankyrin 1 (TRPA1), a nonselective cation channel, contributes to several (patho)physiological processes. Smell loss is an early sign in several neurodegenerative disorders, such as multiple sclerosis, Parkinson’s and Alzheimer’s diseases; therefore, we focused on its role in olfaction and social [...] Read more.
Transient receptor potential ankyrin 1 (TRPA1), a nonselective cation channel, contributes to several (patho)physiological processes. Smell loss is an early sign in several neurodegenerative disorders, such as multiple sclerosis, Parkinson’s and Alzheimer’s diseases; therefore, we focused on its role in olfaction and social behaviour with the aim to reveal its potential therapeutic use. The presence of Trpa1 mRNA was studied along the olfactory tract of mice by combined RNAscope in situ hybridisation and immunohistochemistry. The aversive effects of fox and cat odour were examined in parallel with stress hormone levels. In vitro calcium imaging was applied to test if these substances can directly activate TRPA1 receptors. The role of TRPA1 in social behaviour was investigated by comparing Trpa1 wild-type and knockout mice (KO). Trpa1 mRNA was detected in the olfactory bulb and piriform cortex, while its expression was weak in the olfactory epithelium. Fox, but not cat odour directly activated TRPA1 channels in TRPA1-overexpressing Chinese Hamster Ovary cell lines. Accordingly, KO animals showed less aversion against fox, but not cat odour. The social interest of KO mice was reduced during social habituation–dishabituation and social interaction, but not during resident–intruder tests. TRPA1 may contribute to odour processing at several points of the olfactory tract and may play an important role in shaping the social behaviour of mice. Thus, TRPA1 may influence the development of certain social disorders, serving as a potential drug target in the future. Full article
(This article belongs to the Section Pharmacology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic representation of the mouse olfactory tract. Red colour shows the investigated brain areas, Reprinted from ref [<a href="#B10-pharmaceuticals-14-01336" class="html-bibr">10</a>]. Abbreviations: OE: olfactory epithelium, OB: olfactory bulb, AON: accessory olfactory nucleus, OT: olfactory tubercle, PC: piriform cortex, EC: entorhinal cortex, AM: amygdala.</p> Full article ">Figure 2
<p>Expression of <span class="html-italic">Trpa1</span> mRNA in the investigated regions of the olfactory system of C57BL/6 mice (<span class="html-italic">n</span> = 4). <span class="html-italic">Trpa1</span> (red) mRNA signal did not colocalise with β-tubulin III (green) immunorective cells in the OE (<b>a</b>). <span class="html-italic">Trpa1</span> (red) mRNA signal colocalised exclusively with NeuN (white) positive neurons in the OB (<b>b</b>). <span class="html-italic">Trpa1</span> (red), Gad1 (white) and Vglut1 (green) mRNA expression in the OB (Bregma 3 mm), (<b>c</b>) and in the PC (<b>d</b>) (Bregma −1.46 mm). <span class="html-italic">Trpa1</span> mRNA signal colocalised both with Gad1 and Vglut1 positive neurons in the OB (<b>c</b>), but it colocalised only with Vglut1 positive neurons in the PC (<b>d</b>). Cell nuclei were counterstained with DAPI (blue) in all areas. Abbreviations: NeuN: neuronal nuclear protein, Gad1: glutamate decarboxylase 1, Vglut1: vesicular glutamate transporter 1, DAPI: 4′,6-diamidino-2-phenylindole. In order to highlight the cell borders, differential interference contrast (DIC) images were merged with the virtual color images. Bars: 10 µm.</p> Full article ">Figure 3
<p>Effect of fox (2-MT) and cat odour (valeric acid) on the calcium response in Chinese Hamster Ovary (CHO) cells expressing human and mouse TRPA1 (hTRPA1 and mTRPA1, respectively) receptors. An increased calcium response was characterised by an enhanced ratio of Fluo-4 AM fluorescence compared to dye-loaded unstimulated cells. 2-MT resulted in a concentration-dependent elevation of the calcium response with an EC<sub>50</sub> value of 5010 μmol/L in human TRPA1-expressing cells and with an EC<sub>50</sub> value of 4419 μmol/L in mouse TRPA1-expressing cells (<b>a</b>). Applying valeric acid, neither human nor mouse TRPA1-expressing cell lines showed an elevated Ca<sup>2+</sup> signal (<b>b</b>). No change of the calcium response was detected in CHO cells not expressing TRPA1 in response to 100, 1000 and 10,000 μmol/L 2-MT or valeric acid. <span class="html-italic">n</span> = 5–6 × 10<sup>4</sup> cells for all types of cell lines, all experiments were performed four times.</p> Full article ">Figure 4
<p>Behavioural differences in <span class="html-italic">Trpa1</span> wild-type (WT) and knockout (KO) mice triggered by the fox odour, 2-MT. Remarkable differences were established in the duration and frequency of sniffing the odour holder (<b>a</b>,<b>b</b>) and freezing behaviour (<b>c</b>,<b>d</b>) between the two groups. The Gantt diagram presents the individual animals (<b>e</b>,<b>f</b>). <span class="html-italic">n</span> = 12 in both (WT and KO) groups, the symbol # shows a significant difference between the two genotypes, in cases with ### <span class="html-italic">p</span> &lt; 0.001, and #### <span class="html-italic">p</span> &lt; 0.0001. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 5
<p>Behavioural differences in <span class="html-italic">Trpa1</span> WT and KO mice triggered by the major component of cat odour, valeric acid. Significant differences were detected in the duration (<b>a</b>) and frequency (<b>b</b>) of sniffing the odour holder between the two groups. However, differences in innate fear (freezing) were not present in this trial (<b>c</b>,<b>d</b>). Individual values are represented on a Gantt diagram (<b>e</b>,<b>f</b>). <span class="html-italic">n</span> = 10 in both (WT and KO) groups, the symbol # shows a significant difference between the two genotypes, in cases with # <span class="html-italic">p</span> &lt; 0.05, and ## <span class="html-italic">p</span> &lt; 0.01. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 6
<p>Serum adrenocorticotropin (ACTH) and corticosterone levels of WT and KO mice after using 2-MT or valeric acid. Applying 2-MT, KO mice showed significantly higher ACTH levels than WTs (<b>a</b>), without significant differences in the corticosterone levels (<b>b</b>). Using valeric acid, no differences in either ACTH (<b>c</b>) or in corticosterone levels (<b>d</b>) were detectable. The symbol # shows a significant difference between the two genotypes, in cases with ## <span class="html-italic">p</span> &lt; 0.01. <span class="html-italic">n</span> = 10 in both (WT and KO) groups. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 7
<p>Results of object (<b>a</b>–<b>c</b>) and social habituation–dishabituation trials (<b>d</b>–<b>g</b>). There was no difference in the duration and frequency of sniffing an object between the genotypes during the object habituation test (<b>a</b>,<b>b</b>). During the social habituation trial, a remarkable decrease in the social behaviour was shown using the first stimulus repeatedly in mice of both genotypes. However, after adding a novel stimulus for mice, the social interactions were again increased (<b>d</b>–<b>f</b>). The social behaviour of KO mice was lower than WT mice during the whole examination period, with significant differences in the 2nd and 5th part of the trial (<b>d</b>). <span class="html-italic">n</span> = 12 in both groups, the symbol # shows a significant difference between KO and WT mice (<span class="html-italic">p</span> &lt; 0.05); * shows a significant difference between the periods compared to the 1st part of the trial in the same group, in cases with * <span class="html-italic">p</span> &lt; 0.05; + shows a significant difference between the 4th and the 5th part of the trial in the same group, in cases with + <span class="html-italic">p</span> &lt; 0.05 and ++ <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 8
<p>Results of the sociability test. During the object habituation phase, the KO animals investigated the objects more times than the WTs (<b>a</b>), but the difference in the duration was not significant (<b>d</b>). During the sociability phase, increased interest was detected towards the social stimulus in both groups without significant genotype differences (<b>b</b>,<b>e</b>). During the social discrimination phase, WT animals demonstrated increased interest towards the novel stimulus mice, but this kind of difference were not detectable in KO mice (<b>c</b>,<b>f</b>). Both genotypes revealed significant social interest as represented by a sociability index higher than 50% (<b>g</b>). However, only WTs showed intact short-term social memory as represented by a discrimination index higher than 0 (<b>h</b>). Schematic representation of the sociability trial (<b>i</b>). <span class="html-italic">n</span> = 5 in WT and <span class="html-italic">n</span> = 7 in KO groups, the symbol # shows a significant difference between KO and WT mice (<span class="html-italic">p</span> &lt; 0.05); * shows a significant difference between the two sides with wired cages, in cases with * <span class="html-italic">p</span> &lt; 0.05; <span>$</span> shows a significant difference in the same group using a single sample t-test, in cases with <span>$</span> <span class="html-italic">p</span> &lt; 0.05, and <span>$</span><span>$</span> <span class="html-italic">p</span> &lt; 0.01. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 9
<p>Results of the social interaction test. No differences were detected in the frequency of social behaviour between the two genotypes (<b>a</b>); however, a decreased duration of sniffing (<b>b</b>) and bout fragmentation (<b>c</b>) were detected in KO mice. <span class="html-italic">n</span> = 12 in both groups, the symbol # shows a significant difference, in cases with # <span class="html-italic">p</span> &lt; 0.05, and ## <span class="html-italic">p</span> &lt; 0.01. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 10
<p>Results of resident–intruder trials. Numbers 1,2,3 represent 10 min trials 1 week apart. No significant differences were found in the frequency of social behaviour between the two genotypes (<b>a</b>). The duration of sniffing decreased significantly in both groups during the subsequent trials, although relevant temporal differences were detectable only in KOs (<b>b</b>). Neither the frequency nor the duration of aggressive interactions were significantly altered by time or by genotype (<b>c</b>,<b>d</b>). <span class="html-italic">n</span> = 12 in both groups, * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 show a significant difference between repeated trials in the same group. Blank triangles represent individual values while dark spots show outliers (characterized by a higher or lower value than mean ± 2 standard deviation (SD)).</p> Full article ">Figure 11
<p>Schematic representation of the process of odour aversion (<b>a</b>) and social behavioural experiments (<b>b</b>), carried out on <span class="html-italic">Trpa1</span> knockout mice and their wild-type siblings.</p> Full article ">
9 pages, 5105 KiB  
Article
Effect of Plasmonic Gold Nanoprisms on Biofilm Formation and Heat Shock Proteins Expression in Human Pathogenic Bacteria
by Rihab Lagha, Fethi Ben Abdallah, Amine Mezni and Othman M. Alzahrani
Pharmaceuticals 2021, 14(12), 1335; https://doi.org/10.3390/ph14121335 - 20 Dec 2021
Cited by 5 | Viewed by 2622
Abstract
Gold nanoparticles have gained interest in biomedical sciences in the areas of nano-diagnostics, bio-labeling, drug delivery, and bacterial infection. In this study, we examined, for the first time, the antibacterial and antibiofilm properties of plasmonic gold nanoprisms against human pathogenic bacteria using MIC [...] Read more.
Gold nanoparticles have gained interest in biomedical sciences in the areas of nano-diagnostics, bio-labeling, drug delivery, and bacterial infection. In this study, we examined, for the first time, the antibacterial and antibiofilm properties of plasmonic gold nanoprisms against human pathogenic bacteria using MIC and crystal violet. In addition, the expression level of GroEL/GroES heat shock proteins was also investigated by western blot. Gold nanoparticles were characterized by TEM and EDX, which showed equilateral triangular prisms with an average edge length of 150 nm. Antibacterial activity testing showed a great effect of AuNPs against pathogenic bacteria with MICs values ranging from 50 μg/mL to 100 μg/mL. Nanoparticles demonstrated strong biofilm inhibition action with a percentage of inhibition ranging from 40.44 to 82.43%. Western blot analysis revealed that GroEL was an AuNPs-inducible protein with an increase of up to 66.04%, but GroES was down-regulated with a reduction of up to 46.81%. Accordingly, plasmonic gold nanoprisms, could be a good candidate for antibiotics substitution in order to treat bacterial infections. Full article
(This article belongs to the Special Issue Recent Advances in Antimicrobial Nanodrugs)
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Figure 1

Figure 1
<p>Characterization of AuNPs. (<b>a</b>) TEM image, (<b>b</b>) particle size distribution, and (<b>c</b>) EDX spectrum of triangular gold nanoprisms. Adapted with permission from [<a href="#B21-pharmaceuticals-14-01335" class="html-bibr">21</a>], Copyright 2014 American Chemical Society.</p> Full article ">Figure 2
<p>GroES heat shock protein expression under AuNPs effect. (<b>a</b>): Western blot analysis; (<b>b</b>): Image-J bands quantification; (A): Control; (B): Treated (AuNPs). S. Typhimurium (1), <span class="html-italic">B. cereus</span> (2), <span class="html-italic">E. coli</span> (3), <span class="html-italic">E. faecalis</span> (4), <span class="html-italic">S. sonnei</span> (5), <span class="html-italic">S. aureus</span> (6), <span class="html-italic">N. gonorrhoeae</span> (7), <span class="html-italic">P. aeruginosa</span> (8), and <span class="html-italic">V. cholerae</span> (9).</p> Full article ">Figure 3
<p>GroEl heat shock protein expression under AuNPs effect. (<b>a</b>): Western blot analysis; (<b>b</b>): Image-J bands quantification; (A): Control; (B): Treated (AuNPs) S. Typhimurium (1), <span class="html-italic">B. cereus</span> (2), <span class="html-italic">E. coli</span> (3), <span class="html-italic">E. faecalis</span> (4), <span class="html-italic">S. sonnei</span> (5), <span class="html-italic">S. aureus</span> (6), <span class="html-italic">N. gonorrhoeae</span> (7), <span class="html-italic">P. aeruginosa</span> (8), and <span class="html-italic">V. cholerae</span> (9).</p> Full article ">
22 pages, 5433 KiB  
Article
Bioactive Compounds in Aegopodium podagraria Leaf Extracts and Their Effects against Fluoride-Modulated Oxidative Stress in the THP-1 Cell Line
by Karolina Jakubczyk, Agnieszka Łukomska, Sylwester Czaplicki, Anna Wajs-Bonikowska, Izabela Gutowska, Norbert Czapla, Małgorzata Tańska and Katarzyna Janda-Milczarek
Pharmaceuticals 2021, 14(12), 1334; https://doi.org/10.3390/ph14121334 - 20 Dec 2021
Cited by 1 | Viewed by 3549
Abstract
Aegopodium podagraria L. (goutweed), a member of the Apiaceae family, is a common perennial plant found all around the world that has been used in folk medicine since antiquity. Goutweed leaves contain polyacetylenes, essential oils, mono- and sesquiterpenes, vitamins, macro- and microelements, and [...] Read more.
Aegopodium podagraria L. (goutweed), a member of the Apiaceae family, is a common perennial plant found all around the world that has been used in folk medicine since antiquity. Goutweed leaves contain polyacetylenes, essential oils, mono- and sesquiterpenes, vitamins, macro- and microelements, and phenolic compounds. In spite of its many health-promoting properties, including antioxidant effects, the plant has not been thoroughly studied. The aim of this study was to investigate the antioxidant properties of different goutweed leaf extracts and their effects on the THP-1 cell line, and also to describe the chemical characteristics of goutweed. Falcarinol and falcarindiol and essential oil were determined by gas chromatography coupled with mass spectrometry. Spectrophotometry was used to measure the total content of polyphenols and antioxidant activity–by DPPH and FRAP methods. Oxidative stress in THP-1 cells was induced via sodium fluoride. Then, goutweed leaf extracts were added to evaluate their influence on antioxidant potential (ABTS) and the activity of antioxidant enzymes. Confocal microscopy was used to visualise the production of cytoplasmic and mitochondrial reactive oxygen species (ROS) and for in vitro imaging of apoptosis. The ethanol extracts have a high total content of polyphenols, polyacetylenes, and essential oil, as well as high antioxidant potential. The main volatiles represented diverse chemical groups, which are both oxygenated derivatives of sesquiterpenes and monoterpenes. We also demonstrated positive effects of the high antioxidant potential and increased activity of antioxidant enzymes on cell cultures under severe fluoride-induced oxidative stress. Extraction at 80 ℃ and the use of ethanol as a solvent increased the antioxidant capacity of the extract. The leaves of Aegopodium podagraria may serve as a valuable source of antioxidants in the daily diet and assist in the prevention and treatment of oxidative stress-mediated conditions, e.g., inflammatory conditions, cardiovascular diseases, neurodegenerative diseases, and even obesity. Full article
(This article belongs to the Special Issue Botanicals: Bioactive Molecules and Therapeutic Properties for Human Health 2021)
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Figure 1
<p>Chemical structures of the polyacetylenes falcarinol [(3<span class="html-italic">R</span>)-heptadeca-1,9(<span class="html-italic">Z</span>)-diene-4,6-diyne-3-ol] and falcarindiol [(3<span class="html-italic">R</span>,8S)-heptadeca-1,9(<span class="html-italic">Z</span>)-diene-4,6-diyne-3,8-diol]. Created with BioRender.com.</p> Full article ">Figure 2
<p>Imaging of apoptosis by confocal microscopy in macrophages cultured with NaF solutions alone or with ethanol extract. THP−1 were cultured with NaF and plant extract for 48 hr as described in Materials and Methods. Cells that are viable are Annexin V-FITC and PI negative; cells that are in early apoptosis are Annexin V-FITC positive and PI negative (green fluorescence); and cells that are in late apoptosis or already dead (necrosis) are both Annexin V-FITC and PI positive (red fluorescence). A dual-pass FITC/rhodamine filter set was applied.</p> Full article ">Figure 2 Cont.
<p>Imaging of apoptosis by confocal microscopy in macrophages cultured with NaF solutions alone or with ethanol extract. THP−1 were cultured with NaF and plant extract for 48 hr as described in Materials and Methods. Cells that are viable are Annexin V-FITC and PI negative; cells that are in early apoptosis are Annexin V-FITC positive and PI negative (green fluorescence); and cells that are in late apoptosis or already dead (necrosis) are both Annexin V-FITC and PI positive (red fluorescence). A dual-pass FITC/rhodamine filter set was applied.</p> Full article ">Figure 2 Cont.
<p>Imaging of apoptosis by confocal microscopy in macrophages cultured with NaF solutions alone or with ethanol extract. THP−1 were cultured with NaF and plant extract for 48 hr as described in Materials and Methods. Cells that are viable are Annexin V-FITC and PI negative; cells that are in early apoptosis are Annexin V-FITC positive and PI negative (green fluorescence); and cells that are in late apoptosis or already dead (necrosis) are both Annexin V-FITC and PI positive (red fluorescence). A dual-pass FITC/rhodamine filter set was applied.</p> Full article ">Figure 3
<p>Imaging of cytoplasmatic superoxides detection by fluorescence microscopy in macrophages cultured with NaF solutions alone or with ethanol extract.</p> Full article ">Figure 3 Cont.
<p>Imaging of cytoplasmatic superoxides detection by fluorescence microscopy in macrophages cultured with NaF solutions alone or with ethanol extract.</p> Full article ">Figure 4
<p>Imaging of mitochondrial superoxides detection by fluorescence microscopy in macrophages cultured with NaF solutions alone or with ethanol extract. THP<sup>−1</sup> were cultured with NaF and extract for 48 h as described in the Materials and Methods. Detection of mitochondrial superoxide synthesis in macrophages was performed using MitoSOX Red indicator (incubation 10 min/37 °C). The reagent is oxidised only by superoxide, and the oxidation product becomes highly fluorescent upon binding to nucleic acids (red fluorescence).</p> Full article ">Figure 5
<p>Methodology used to study <span class="html-italic">Aegopodium podagraria</span> L. extracts. Created with BioRender.com.</p> Full article ">Figure 6
<p>Cell culture with factors incubated with THP-1 line macrophages. The experiment was repeated 5 times.</p> Full article ">
22 pages, 6524 KiB  
Article
An Investigation for Large Volume, Focal Blood-Brain Barrier Disruption with High-Frequency Pulsed Electric Fields
by Melvin F. Lorenzo, Sabrina N. Campelo, Julio P. Arroyo, Kenneth N. Aycock, Jonathan Hinckley, Christopher B. Arena, John H. Rossmeisl, Jr. and Rafael V. Davalos
Pharmaceuticals 2021, 14(12), 1333; https://doi.org/10.3390/ph14121333 - 20 Dec 2021
Cited by 10 | Viewed by 3907
Abstract
The treatment of CNS disorders suffers from the inability to deliver large therapeutic agents to the brain parenchyma due to protection from the blood-brain barrier (BBB). Herein, we investigated high-frequency pulsed electric field (HF-PEF) therapy of various pulse widths and interphase delays for [...] Read more.
The treatment of CNS disorders suffers from the inability to deliver large therapeutic agents to the brain parenchyma due to protection from the blood-brain barrier (BBB). Herein, we investigated high-frequency pulsed electric field (HF-PEF) therapy of various pulse widths and interphase delays for BBB disruption while selectively minimizing cell ablation. Eighteen male Fisher rats underwent craniectomy procedures and two blunt-tipped electrodes were advanced into the brain for pulsing. BBB disruption was verified with contrast T1W MRI and pathologically with Evans blue dye. High-frequency irreversible electroporation cell death of healthy rodent astrocytes was investigated in vitro using a collagen hydrogel tissue mimic. Numerical analysis was conducted to determine the electric fields in which BBB disruption and cell ablation occur. Differences between the BBB disruption and ablation thresholds for each waveform are as follows: 2-2-2 μs (1028 V/cm), 5-2-5 μs (721 V/cm), 10-1-10 μs (547 V/cm), 2-5-2 μs (1043 V/cm), and 5-5-5 μs (751 V/cm). These data suggest that HF-PEFs can be fine-tuned to modulate the extent of cell death while maximizing peri-ablative BBB disruption. Furthermore, numerical modeling elucidated the diffuse field gradients of a single-needle grounding pad configuration to favor large-volume BBB disruption, while the monopolar probe configuration is more amenable to ablation and reversible electroporation effects. Full article
(This article belongs to the Special Issue Drug Delivery Across or Bypassing the Blood–Brain Barrier)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Summary of HF-PEF BBB disruption, resulting in significant diffusion of normally impermeant Gd-EBD; (<b>a</b>) schematic depicting electrode insertion trajectory in rodent brain tissue; (<b>b</b>) coronal view of BBB disruption as seen on a plane traversing the electrode insertion track and as shown by accumulation of Gd-EBD with contrast enhanced T1W MRI and gross tissue sections (sham vs. representative 5-5-5 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s treatment). Without HF-PEFs, no uptake of Gd-EBD is seen; (<b>c</b>) volumetric measurements determined from gross tissue sections, * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01.</p> Full article ">Figure 2
<p>A collagen hydrogel scaffold was leveraged to determine the electric field threshold of cell death for healthy rodent astrocytes: (<b>a</b>) a single-needle grounding ring electrode configuration induces a (<b>b</b>) rotationally symmetric electric field distribution; (<b>c</b>) ablation is quantified as the area of propidium iodide uptake for the 2-2-2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s, 2-5-2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s, 5-2-5 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s, 5-5-5 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s, and 10-1-10 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s groups. The ablation area is mapped to a corresponding electric field and this field is the lethal electric field threshold. In all cases, 600 V was applied.</p> Full article ">Figure 3
<p>Delineation of biological phenomena elicited from PEF therapy for data collected in vivo and in vitro: (<b>a</b>) analysis of the electric field thresholds for cell ablation, reversible electroporation, BBB disruption, and nerve excitation demonstrates high delineation with effects further amplified by modulating the pulse width and interphase delay. (<b>b</b>–<b>d</b>) For the 2-5-2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s waveform, (<b>b</b>) the monopolar probe configuration capitalizes on high electric fields near and adjacent to the electrodes to induce large ablation and reversible electroporation, whereas (<b>d</b>) the single-needle grounding pad configuration capitalizes on a diffuse field gradient to reduce ablation and reversible electroporation effects and maximize BBB disruption. (<b>c</b>) While the bipolar probe has the advantages of a single insertion device, both ablation and BBB disruption areas are smaller and stay confined to the immediate electrode area.</p> Full article ">Figure 4
<p>Representative electric field distributions for the dual monopolar probe, single insertion bipolar probe, and the single-needle distant grounding pad configuration. The electric field distribution is that of a 2-5-2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s waveform with 2 kV applied, simulated to include electroporation effects and coupled Joule heating effects.</p> Full article ">Figure 5
<p>Pulse width and interphase delay modulate the ratio of BBB disruption volume to cell ablation. For the (<b>a</b>) monopolar probe and the (<b>b</b>) single-needle grounding pad configuration, ratios of BBB disruption volume to ablation, largest encapsulated spherical BBB disruption to total BBB disruption volumes, and BBB disruption volume to nerve excitation volume were quantified. Three-dimensional contours across waveforms and configurations were generated to scale to directly compare ablation (magenta) and BBB disruption (blue) volumes. In summary, the monopolar probes are amenable to large ablations (outlined in red box) while the diffuse electric field distribution of the single-needle grounding pad maximizes BBB disruption effects (outlined in red box). Lower pulse widths and higher delays maximize BBB disruption effects, whereas lower pulse widths reduce nerve excitation and ablation.</p> Full article ">Figure 6
<p>A non-dimensional relative efficiency term (Equation (<a href="#FD1-pharmaceuticals-14-01333" class="html-disp-formula">1</a>)) was defined to transcend and normalize BBBd, ablation, and nerve excitation effects across all waveforms and electrode configurations: (<b>a</b>) for a scenario where a neuroparalytic is not administered, all components of Equation (<a href="#FD1-pharmaceuticals-14-01333" class="html-disp-formula">1</a>) are equally weighted at A<sub>1</sub> = A<sub>2</sub> = A<sub>3</sub> = 0.33, resulting in a significant advantage of the 2-5-2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>s waveform for maximizing all desirable effects; (<b>b</b>) for a scenario where a neuroparalytic is administered, all contributions of nerve stimulation in our efficiency terms are nullified (A<sub>1</sub> = A<sub>2</sub> = 0.5, A<sub>3</sub> = 0), further highlighting the advantage of the single-needle grounding pad configuration to maximize BBB disruption and minimize ablation effects.</p> Full article ">
17 pages, 11582 KiB  
Article
Echinocandin Drugs Induce Differential Effects in Cytokinesis Progression and Cell Integrity
by Natalia Yagüe, Laura Gómez-Delgado, M. Ángeles Curto, Vanessa S. D. Carvalho, M. Belén Moreno, Pilar Pérez, Juan Carlos Ribas and Juan Carlos G. Cortés
Pharmaceuticals 2021, 14(12), 1332; https://doi.org/10.3390/ph14121332 - 20 Dec 2021
Cited by 3 | Viewed by 3487
Abstract
Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of [...] Read more.
Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins’ mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs. Full article
(This article belongs to the Special Issue Advances in Antifungal Development: Discovery of New Drugs and Drug Repurposing)
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Figure 1
<p>Morphology of WT and <span class="html-italic">pbr1-8</span> cells after 24 h of growth in the presence of increasing concentrations of anidulafungin (<b>A</b>), caspofungin (<b>B</b>), and micafungin (<b>C</b>) drugs. Early logarithmic-phase cells of the WT and <span class="html-italic">pbr1-8</span> strains growing in YES liquid medium at 28 °C were diluted to a high cell density of 5 × 10<sup>6</sup> in micro-cultures of YES liquid medium containing either DMSO (0.8%, control) or increasing concentrations (1, 2, 4, 10, 20, and 40 µg/mL) of the drugs, grown with shaking for 24 h and imaged by phase-contrast microscopy. The data of this figure are developed in <a href="#pharmaceuticals-14-01332-t002" class="html-table">Table 2</a>. Scale bars, 10 µm.</p> Full article ">Figure 2
<p>Normal cytokinesis (septum synthesis and cell separation) in WT and <span class="html-italic">pbr1-8</span> cells growing in the absence of the three echinocandin drugs. Early logarithmic-phase cells of the indicated strains growing in YES liquid medium at 28 °C were diluted to a cell density of 5 × 10<sup>6</sup> in liquid YES medium containing both calcofluor white (CW, 1.25 µg/mL) and DMSO (0.8%), and then imaged by time-lapse fluorescence microscopy (1 medial z slice, 4 min elapsed time) for 3 h, as described in the Materials and Methods section. The data of this figure are developed in <a href="#pharmaceuticals-14-01332-t003" class="html-table">Table 3</a>, <a href="#pharmaceuticals-14-01332-t004" class="html-table">Table 4</a> and <a href="#pharmaceuticals-14-01332-t005" class="html-table">Table 5</a>. White arrow: first CW-stained septum synthesis. Arrowheads: blue, septum synthesis onset (time 0 for the elapsed time until septum synthesis completion); red, septum completion onset (time 0 for the elapsed time until cell separation onset); orange, cell separation onset. Scale bars, 5 µm.</p> Full article ">Figure 3
<p>Cytokinesis phenotypes in WT and <span class="html-italic">pbr1-8</span> cells growing in the presence of sublethal and lethal concentrations of anidulafungin. The indicated strains were grown and imaged as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a> in the presence of either sublethal ((<b>A</b>), 2 µg/mL) or lethal ((<b>B</b>), 20 µg/mL) concentrations of the drug. The data of this figure are developed in <a href="#pharmaceuticals-14-01332-t003" class="html-table">Table 3</a>, <a href="#pharmaceuticals-14-01332-t004" class="html-table">Table 4</a> and <a href="#pharmaceuticals-14-01332-t005" class="html-table">Table 5</a>. Arrows and arrowheads are as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a>. Scale bars, 5 µm.</p> Full article ">Figure 4
<p>Cytokinesis phenotypes in WT and <span class="html-italic">pbr1-8</span> cells growing in the presence of sublethal and lethal concentrations of caspofungin. The indicated strains were grown and imaged as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a> in the presence of sublethal ((<b>A</b>), 2 µg/mL) or lethal ((<b>B</b>), 20 µg/mL) concentrations of the drug. The data of this figure are developed in <a href="#pharmaceuticals-14-01332-t003" class="html-table">Table 3</a>, <a href="#pharmaceuticals-14-01332-t004" class="html-table">Table 4</a> and <a href="#pharmaceuticals-14-01332-t005" class="html-table">Table 5</a>. Arrows and arrowheads are as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a>. Scale bars, 5 µm.</p> Full article ">Figure 5
<p>Cytokinesis phenotypes in WT and <span class="html-italic">pbr1-8</span> cells growing in the presence of sublethal and lethal concentrations of micafungin. The indicated strains were grown and imaged as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a> in the presence of sublethal ((<b>A</b>), 2 µg/mL) or lethal ((<b>B</b>), 20 µg/mL) concentrations of the drug. The data of this figure are developed in <a href="#pharmaceuticals-14-01332-t003" class="html-table">Table 3</a>, <a href="#pharmaceuticals-14-01332-t004" class="html-table">Table 4</a> and <a href="#pharmaceuticals-14-01332-t005" class="html-table">Table 5</a>. Arrows and arrowheads are as in <a href="#pharmaceuticals-14-01332-f002" class="html-fig">Figure 2</a>. Scale bars, 5 µm.</p> Full article ">
32 pages, 6738 KiB  
Article
New Heterocyclic Combretastatin A-4 Analogs: Synthesis and Biological Activity of Styryl-2(3H)-benzothiazolones
by Gjorgji Atanasov, Rusi I. Rusew, Vladimir M. Gelev, Christo D. Chanev, Rosica Nikolova, Boris L. Shivachev, Ognyan I. Petrov and Margarita D. Apostolova
Pharmaceuticals 2021, 14(12), 1331; https://doi.org/10.3390/ph14121331 - 20 Dec 2021
Cited by 4 | Viewed by 4012
Abstract
Here, we describe the synthesis, characterization, and biological activities of a series of 26 new styryl-2(3H)-benzothiazolone analogs of combretastatin-A4 (CA-4). The cytotoxic activities of these compounds were tested in several cell lines (EA.hy926, A549, BEAS-2B, MDA-MB-231, HT-29, MCF-7, and MCF-10A), and the relations [...] Read more.
Here, we describe the synthesis, characterization, and biological activities of a series of 26 new styryl-2(3H)-benzothiazolone analogs of combretastatin-A4 (CA-4). The cytotoxic activities of these compounds were tested in several cell lines (EA.hy926, A549, BEAS-2B, MDA-MB-231, HT-29, MCF-7, and MCF-10A), and the relations between structure and cytotoxicity are discussed. From the series, compound (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzothiazolone (26Z) exhibits the most potent cytotoxic activity (IC50 0.13 ± 0.01 µM) against EA.hy926 cells. 26Z not only inhibits vasculogenesis but also disrupts pre-existing vasculature. 26Z is a microtubule-modulating agent and inhibits a spectrum of angiogenic events in EA.hy926 cells by interfering with endothelial cell invasion, migration, and proliferation. 26Z also shows anti-proliferative activity in CA-4 resistant cells with the following IC50 values: HT-29 (0.008 ± 0.001 µM), MDA-MB-231 (1.35 ± 0.42 µM), and MCF-7 (2.42 ± 0.48 µM). Cell-cycle phase-specific experiments show that 26Z treatment results in G2/M arrest and mitotic spindle multipolarity, suggesting that drug-induced centrosome amplification could promote cell death. Some 26Z-treated adherent cells undergo aberrant cytokinesis, resulting in aneuploidy that perhaps contributes to drug-induced cell death. These data indicate that spindle multipolarity induction by 26Z has an exciting chemotherapeutic potential that merits further investigation. Full article
(This article belongs to the Topic Compounds with Medicinal Value)
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Figure 1
<p>Structure of natural CA-4 and its synthetic analogs.</p> Full article ">Figure 2
<p>ORTEP view with the atom-numbering scheme of the molecules present in the asymmetric unit of (<b>a</b>) <b>25Z</b>, (<b>b</b>) <b>22Z</b>, and (<b>c</b>) <b>27Z</b>; atomic displacement parameters are at the 50% probability level.</p> Full article ">Figure 3
<p>Overlay based on the common for all compounds (–C–CH=CH–C–) bridge of (<b>a</b>) <b>22Z</b> (grey) and <b>25Z</b> (yellow), (<b>b</b>) <b>22Z</b> (grey) and <b>27Z</b> (purple), and (<b>c</b>) <b>25Z</b> (yellow) and <b>27Z</b> (purple), disclosing the structural flexibility of the molecules.</p> Full article ">Figure 4
<p>Molecular docking of (<b>a</b>) <b>22Z</b>, (<b>b</b>) <b>27Z</b>, (<b>c</b>) <b>26Z</b> and (<b>d</b>) <b>25Z</b> in the colchicine site of α,β-tubulin, along with observed interactions.</p> Full article ">Figure 5
<p>Effect of <b>25Z</b>, <b>26Z</b>, <b>27Z</b>, and CA-4 on colony forming capacity (clonogenic survival) of exponentially growing EA.hy926 cells after treatment for 72 h. (<b>A</b>) EA.hy926 cells were treated with: (1) 0.01 μM DMSO, (2) 25 μM, (3) 10 μM, (4) 2.5 μM, (5) 0.25 μM, and (6) 0.01 μM concentrations of <b>26Z</b>, <b>27Z</b>, <b>25Z</b>, and: (1) 0.01 μM DMSO, (2) 1 nM, (3) 0.5 nM, (4) 0.1 nM, (5) 0.05 nM, and (6) 0.01 nM of CA-4 as a positive control. Cells were allowed to form colonies in fresh medium for 6 days after treatment. (<b>B</b>) Number of colonies formed on day 6 after treatment normalized to control. The presented data were obtained by manual counting and expressed as mean ± SD (<span class="html-italic">n</span> = 3). * <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>Effect of <b>26Z</b> and CA-4 on the cell cycle (<b>A</b>) and EBI competition assay in EA.hy926 cells (<b>B</b>). DNA content was measured in asynchronously growing cells using propidium iodide staining at 4, 6, 8, 24, 48, and 72 h after treatment with 10 nM CA-4 or 300 nM <b>26Z</b>. The results are shown as the percentage of the cells in different cell cycle phases. Cytograms are representative of three independent experiments. In (<b>B</b>), EA.hy926 cells were incubated with or without indicated concentrations of the compounds for 2 h, then 100 μM EBI was added to cells and incubated for another 2 h. Total proteins were lysed and subjected to western blot analysis for β-tubulin and β-actin. VBL: vinblastine; CLC: colchicine; EBI: <span class="html-italic">N</span>,<span class="html-italic">N</span>′-ethylenebis(iodoacetamide).</p> Full article ">Figure 7
<p>Inhibition of cell migration of EA.hy926 cells treated for 24 h with CA-4, <b>25Z</b>, <b>26Z</b>, and <b>27Z</b> in the wound healing assay. Representative images of 3 independent assays. <b>25Z</b>, <b>26Z</b>, and <b>27Z</b>: 50 nM, CA-4: 1 nM, Con: 0.01 µM DMSO.</p> Full article ">Figure 8
<p>Tube-formation assay of EA.hy926 cells cultured on extracellular matrix (Matrigel). (<b>A</b>) Images of formed tubular networks taken after 18 h of incubation and subsequent destruction following 24 h of treatment with equitoxic IC<sub>50</sub> concentrations of CA-4, <b>25Z</b>, <b>26Z</b>, and <b>27Z</b>. (<b>B</b>) Reorganizational analysis of structural network parameters—loop number, tubes length, and branching points. The results are shown as the average ± SD (<span class="html-italic">n</span> = 3). * <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 to 0 h.</p> Full article ">Figure 9
<p>Indirect immunofluorescence staining of α-tubulin (green) and F-actin (red), and simultaneous DAPI chromosome staining (blue) of EA.hy926 cells. (<b>A</b>) Mitosis in control (i: interphase; ii: metaphase; iii: anaphase; iv: metaphase and cytokinesis) and <b>26Z</b> treated cells with IC<sub>50</sub> (130 nM) and IC<sub>80</sub> (300 nM) concentrations for 24 h. Microphotographs were obtained with an Andor Dragonfly 505 Confocal Microscope. (<b>B</b>) Cytoskeleton structure of adherent cells in control conditions and after treatment with <b>26Z</b> at IC<sub>50</sub> (130 nM) and IC<sub>80</sub> (300 nM) for 24 h. Images were acquired with an Axiovert 200 M inverted microscope. Scale bars, 10 μM. Arrowheads—multipolar spindles, arrows—membrane blebbing and apoptotic bodies.</p> Full article ">Figure 10
<p>Ex vivo tubulin polymerization reaction at 37 °C in the presence of DMSO (control) or 20 μM of CA-4, paclitaxel, and <b>26Z</b>. (<b>A</b>) Representative image of western blots from the soluble (S) and polymerized (P) tubulin fractions. (<b>B</b>) Signal intensity analysis showing the stabilizing effect of paclitaxel (PTX) and the inhibitory effect of CA-4 and <b>26Z</b> on tubulin polymerization, illustrated by changes in the supernatant (S) to pellet (P) ratio. M—molecular marker. The results are shown as the average ± SD (<span class="html-italic">n</span> = 3). * <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 to control.</p> Full article ">Figure 11
<p>Fluorescence microscopy of ex vivo polymerized rhodamine-labeled α,β-tubulin. Representative images of formed microtubule fibers in control (DMSO 0.01 μM) and paclitaxel (PTX, 20 μM) samples, and inhibition of the process upon treatment with 20 μM CA-4 and <b>26Z</b>. Aggregates of non-polymerized tubulin are also visible as points with very bright fluorescence (Control, CA-4, and <b>26Z</b>). Scale bar, 10 μm.</p> Full article ">Figure 12
<p>Western blot analysis of soluble and polymerized α,β-tubulin fractions after 6 h of treatment of EA.hy926 cells. (<b>A</b>) Western blot analysis of soluble (S) and polymerized (P) fractions isolated from EA.hy926 cells treated with 0.01 μM DMSO (control) or 1 μM of CA-4, <b>26Z, 27Z</b> and paclitaxel (PTX). (<b>B</b>) Graphic presentation of the soluble vs. polymerized tubulin ratio. (<b>C</b>) Concentration-dependent depolymerization of α,β-tubulin in EA.hy926 cells treated for 6 h with 0.01–10 μM <b>26Z</b>. All results are representative of three independent experiments. M: protein marker. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 compared to control.</p> Full article ">Figure 13
<p>Western blot analysis of cell lysates from EA.hy926 cells treated with <b>26Z</b> and CA-4. Activation of receptor and effector caspases 8, 9, and 3 is shown in representative images following 24 h of treatment with <b>26Z</b> and CA-4.</p> Full article ">Scheme 1
<p>Synthesis of target styryl-benzothiazolones. Reagents and conditions: (<b>a</b>) K<sub>2</sub>CO<sub>3</sub>, 18-crown-6, THF/CH<sub>2</sub>Cl<sub>2</sub> (2:1 <span class="html-italic">v</span>/<span class="html-italic">v</span>), reflux 4–6 h, 57–87%.</p> Full article ">Scheme 2
<p>Synthesis of heterocyclic phosphonium salts <b>11</b>–<b>13</b>, containing a benzothiazolone moiety. Reagents and conditions: (<b>a</b>) (CH<sub>3</sub>)<sub>2</sub>SO<sub>4</sub>, aq. NaOH, r.t., 1 h, 80–89%; (<b>b</b>) <span class="html-italic">N</span>-Bromosuccinimide (NBS), (PhCO)<sub>2</sub>O<sub>2</sub>, CCl<sub>4</sub>, reflux, 3 h, 63–84%; (<b>c</b>) PPh<sub>3</sub>, chlorobenzene, reflux, 85–96%.</p> Full article ">Scheme 3
<p>Synthesis of 4-methyl-2(3<span class="html-italic">H</span>)-benzothiazolone (<b>28</b>). Reagents and conditions: (<b>a</b>) NH<sub>4</sub>SCN, HCl, reflux, 77%; (<b>b</b>) Br<sub>2</sub>, CHCl<sub>3</sub>, 60 °C, 81%; (<b>c</b>) NaNO<sub>2</sub>, HBr, ClCH<sub>2</sub>CH<sub>2</sub>Cl, 5 to 40 °C, 89%; (<b>d</b>) 37% HCl, 2-methoxyethanol, reflux, 85%.</p> Full article ">Scheme 4
<p>Synthesis of 5-methyl-2(3<span class="html-italic">H</span>)-benzothiazolone (<b>29</b>). Reagents and conditions: (<b>a</b>) Na<sub>2</sub>S<sub>x</sub>, CS<sub>2</sub>, water, reflux, 47%; (<b>b</b>) aq. KOH, KMnO<sub>4</sub>; (<b>c</b>) 37% HCl, water, reflux, 88%.</p> Full article ">Scheme 5
<p>Synthesis of 6-methyl-2(3<span class="html-italic">H</span>)-benzothiazolone (<b>30</b>). Reagents and conditions: (<b>a</b>) (CH<sub>3</sub>CO)<sub>2</sub>O, toluene, 87%; (<b>b</b>) Br<sub>2</sub>, AcOH, 76%; (<b>c</b>) 37% HCl, EtOH, 67%; (<b>d</b>) KSC(S)OC<sub>2</sub>H<sub>5</sub>, <span class="html-italic">N</span>-Methyl-2-Pyrrolidone (NMP), 160 °C, 81%; (<b>e</b>) aq. KOH, KMnO<sub>4</sub>; (<b>f</b>) 37% HCl, water, reflux, 91%.</p> Full article ">Scheme 6
<p>Synthesis of 6-methyl-2(3<span class="html-italic">H</span>)-benzothiazolone (<b>30</b>). Reagents and conditions: (<b>a</b>) NH<sub>4</sub>SCN, Br<sub>2</sub>, AcOH, 80%; (<b>b</b>) NaNO<sub>2</sub>, HBr, ClCH<sub>2</sub>CH<sub>2</sub>Cl, 0 °C to r.t, 75%; (<b>c</b>) 37% HCl, 2-methoxyethanol, reflux, 89%.</p> Full article ">Scheme 7
<p>Synthesis of 3-methyl-2(3<span class="html-italic">H</span>)-benzothiazolone-7-carbaldehyde (<b>10</b>). Reagents and conditions: (<b>a</b>) MeOH, Et<sub>3</sub>N, reflux, 79%; (<b>b</b>) 1 eq. <span class="html-italic">n</span>-BuLi, −60 °C, then 1.6 eq. <span class="html-italic">tert</span>-BuLi, −80 to −40 °C, DMF −80 to −20 °C, 60%; (<b>c</b>) 37% HCl, EtOH, reflux, 83%; (<b>d</b>) CH<sub>3</sub>I, K<sub>2</sub>CO<sub>3</sub>, DMF, 35 °C, 84%.</p> Full article ">
17 pages, 1979 KiB  
Article
Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer’s Disease
by Massimiliano Runfola, Michele Perni, Xiaoting Yang, Maria Marchese, Andrea Bacci, Serena Mero, Filippo M. Santorelli, Beatrice Polini, Grazia Chiellini, Daniela Giuliani, Antonietta Vilella, Martina Bodria, Eleonora Daini, Eleonora Vandini, Simon Rudge, Sheraz Gul, Michale O. J. Wakelam, Michele Vendruscolo and Simona Rapposelli
Pharmaceuticals 2021, 14(12), 1330; https://doi.org/10.3390/ph14121330 - 19 Dec 2021
Cited by 7 | Viewed by 3835
Abstract
The identification of effective pharmacological tools for Alzheimer’s disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that [...] Read more.
The identification of effective pharmacological tools for Alzheimer’s disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aβ42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development. Full article
(This article belongs to the Special Issue Therapeutics Agents for Neural Repair)
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Figure 1

Figure 1
<p>SG2 recovers fitness in a <span class="html-italic">C. elegans</span> model of AD. SG2 was administered to the worms following two different treatment regimes, namely early treatment at D0 (<b>A</b>) or late treatment at D4 (<b>B</b>). SG2 dosed at 5, 10 and 20 µM showed significant dose-dependent recovery effect, expressed as body bends/min, on AD worms (<b>A</b>,<b>B</b>). Error bars represent the standard error of the mean (SEM). For statistical tests, One-Way ANOVA was used. <span class="html-italic">p</span> ≤ 0.0001 (****).</p> Full article ">Figure 2
<p>SG2 does not directly inhibit Aβ42 aggregation in in vitro assays. The aggregation of 2 µM Aβ42 was performed in 20 mM sodium phosphate buffer with 0.2 mM EDTA at pH 8. The amyloid-specific dye thioflavin T (ThT) at 20 µM was used as a fluorescence probe to monitor the progression of the aggregation process as depicted in panel (<b>A</b>). SG2 was added to Aβ42 at different concentrations in presence or in absence of preformed fibrils (seeds), as shown in each panel. In the seeded assays, 2% or 50% of preformed fibrils were added to the solution before the start of the aggregation process. Varying the seeds concentration in the assay solution allows to monitor different steps of the Aβ42 aggregation process. Results are presented as normalized fluorescence intensity, which is indicative of the Aβ42 formation, over time. SG2 has a slight effect on primary enucleation (unseeded assay, panel (<b>B</b>)), while does not affect the secondary enucleation (panel (<b>C</b>)) or the elongation step (panel (<b>D</b>)).</p> Full article ">Figure 3
<p>SG2 enhances autophagy in a <span class="html-italic">C. elegans</span> model of AD. (<b>A</b>) GFP structures were counted in 3 different conditions (Fed: 1% DMSO; Starved: 1% DMSO and 6 h of starvation; Treated: 10 µM SG2 in 1% DMSO) of approximately 25–30 worms from two different biological experiments. Bars represent average number of GFP structures normalized to control (Fed) + SEM. For statistical tests, One-Way ANOVA was used <span class="html-italic">p</span> &lt; 0.001 ***. (<b>B</b>) Representative pictures of big muscles cells presenting LGG-1:GFP puncta with white arrows highlighting LGG-1::GFP structures.</p> Full article ">Figure 4
<p>SG2 shows a safe toxicological profile in an in vitro panel. A panel of toxicological assays were performed to test (<b>A</b>) SG2 behaviour over cytotoxicity (U2-OS, HEK293, MCF-7, hTERT), (<b>B</b>) epigenetic modulation (HDAC6, HDAC8, SIRT7), and off-target liability (PDE4C1, Aurora B kinase). All assays were performed at 10 µM in triplicate. Raw data were normalized over positive and negative control and reported as percentage of inhibition. In no assay SG2 showed a percentage of inhibition higher than 40%, where in this profiling a value &lt; 50% is generally considered acceptable.</p> Full article ">Figure 5
<p>SG2 does not show toxicological effects in zebrafish. (<b>A</b>) SG2 at 5 µM was administered to zebrafish embryos at 4 h post fertilization (hpf); the experimental workflow was created with BioRender.com( accessed on 5 May, 2021). All the experiments reported show non-significant values compared to controls, proving a safe impact of SG2 in zebrafish. (<b>B</b>,<b>C</b>) Mortality rate was calculated until 48 hpf (<b>B</b>) (controls <span class="html-italic">N</span> = 545, vehicle <span class="html-italic">N</span> = 281, SG2 <span class="html-italic">N</span> = 224) and no morphological anomaly was observed at 5 days post fertilization (dpf) (<b>C</b>). (<b>D</b>) Cardiotoxicity was evaluated measuring heart rate at 3 dpf. (<b>E</b>,<b>F</b>) Neurological development was evaluated at 30 hpf with burst activity analysis (<b>E</b>) and at 5 dpf with locomotor measurements considering velocity and distance covered (<b>F</b>) (controls <span class="html-italic">N</span> = 366, vehicle <span class="html-italic">N</span> = 212, SG2 <span class="html-italic">N</span> = 246). All experiments were performed at least in triplicate. Error bars represent the SEM. For statistical tests non-parametric one-tailed Mann-Whitney rank sum test was used.</p> Full article ">Figure 6
<p>SG2 improves learning ability in the 5xFAD mouse model of AD. (<b>A</b>) Analysis of MWM learning curve showed no significant differences between experimental groups (repeated measures ANOVA, Time F (3,105) = 21.57, <span class="html-italic">p</span> &lt; 0.0001, Treatment F (3,35) = 0.3141, <span class="html-italic">p</span> = 0.82, Time × Treatment F (9,105) = 1.553, <span class="html-italic">p</span> = 0.14). (<b>B</b>) Learning index analysis demonstrated an improved performance of Tg-SG2 mice compared to Tg-Sal to find the hidden platform. For statistical tests, One-Way ANOVA was used <span class="html-italic">p</span> &lt; 0.02 * (<b>C</b>) The time needed to reach the platform area (<b>C</b>) and the number of entries in the target area (<b>D</b>) were not statistically different between experimental groups. (<b>E</b>) The analysis of search strategy showed an increase in spatially targeted strategies in Tg-SG2 mice compared to Tg-Sal mice (Fisher’s test). Data are shown as mean ± SEM. Experimental groups: Wt-Sal, <span class="html-italic">n</span> = 10; Wt-SG2, <span class="html-italic">n</span> = 10; Tg-Sal, <span class="html-italic">n</span> = 10: Tg-SG2, <span class="html-italic">n</span> = 9.</p> Full article ">
25 pages, 2365 KiB  
Review
Pharmacological Activities of Extracts and Compounds Isolated from Mediterranean Sponge Sources
by Lorenzo Di Cesare Mannelli, Fortunato Palma Esposito, Enrico Sangiovanni, Ester Pagano, Carmen Mannucci, Beatrice Polini, Carla Ghelardini, Mario Dell’Agli, Angelo Antonio Izzo, Gioacchino Calapai, Donatella de Pascale and Paola Nieri
Pharmaceuticals 2021, 14(12), 1329; https://doi.org/10.3390/ph14121329 - 18 Dec 2021
Cited by 6 | Viewed by 4434
Abstract
Marine pharmacology is an exciting and growing discipline that blends blue biotechnology and natural compound pharmacology together. Several sea-derived compounds that are approved on the pharmaceutical market were discovered in sponges, marine organisms that are particularly rich in bioactive metabolites. This paper was [...] Read more.
Marine pharmacology is an exciting and growing discipline that blends blue biotechnology and natural compound pharmacology together. Several sea-derived compounds that are approved on the pharmaceutical market were discovered in sponges, marine organisms that are particularly rich in bioactive metabolites. This paper was specifically aimed at reviewing the pharmacological activities of extracts or purified compounds from marine sponges that were collected in the Mediterranean Sea, one of the most biodiverse marine habitats, filling the gap in the literature about the research of natural products from this geographical area. Findings regarding different Mediterranean sponge species were individuated, reporting consistent evidence of efficacy mainly against cancer, infections, inflammatory, and neurological disorders. The sustainable exploitation of Mediterranean sponges as pharmaceutical sources is strongly encouraged to discover new compounds. Full article
(This article belongs to the Special Issue Chemistry and Biomedical Potential of Marine Natural Products)
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<p>Sponge families that are present in the Mediterranean Sea.</p> Full article ">Figure 2
<p>Mediterranean sponges grouped by geographical localization.</p> Full article ">Figure 3
<p>Graphical representation of sponge activity reports for the therapeutic category. The size of the bubble is proportional to the number of papers individuated.</p> Full article ">Figure 4
<p>Structures of the molecules that have been isolated from Mediterranean sponges.</p> Full article ">Figure 4 Cont.
<p>Structures of the molecules that have been isolated from Mediterranean sponges.</p> Full article ">Figure 4 Cont.
<p>Structures of the molecules that have been isolated from Mediterranean sponges.</p> Full article ">
23 pages, 6490 KiB  
Article
Inhibition Ability of Natural Compounds on Receptor-Binding Domain of SARS-CoV2: An In Silico Approach
by Miroslava Nedyalkova, Mahdi Vasighi, Subrahmanyam Sappati, Anmol Kumar, Sergio Madurga and Vasil Simeonov
Pharmaceuticals 2021, 14(12), 1328; https://doi.org/10.3390/ph14121328 - 18 Dec 2021
Cited by 12 | Viewed by 4215
Abstract
The lack of medication to treat COVID-19 is still an obstacle that needs to be addressed by all possible scientific approaches. It is essential to design newer drugs with varied approaches. A receptor-binding domain (RBD) is a key part of SARS-CoV-2 virus, located [...] Read more.
The lack of medication to treat COVID-19 is still an obstacle that needs to be addressed by all possible scientific approaches. It is essential to design newer drugs with varied approaches. A receptor-binding domain (RBD) is a key part of SARS-CoV-2 virus, located on its surface, that allows it to dock to ACE2 receptors present on human cells, which is followed by admission of virus into cells, and thus infection is triggered. Specific receptor-binding domains on the spike protein play a pivotal role in binding to the receptor. In this regard, the in silico method plays an important role, as it is more rapid and cost effective than the trial and error methods using experimental studies. A combination of virtual screening, molecular docking, molecular simulations and machine learning techniques are applied on a library of natural compounds to identify ligands that show significant binding affinity at the hydrophobic pocket of the RBD. A list of ligands with high binding affinity was obtained using molecular docking and molecular dynamics (MD) simulations for protein–ligand complexes. Machine learning (ML) classification schemes have been applied to obtain features of ligands and important descriptors, which help in identification of better binding ligands. A plethora of descriptors were used for training the self-organizing map algorithm. The model brings out descriptors important for protein–ligand interactions. Full article
(This article belongs to the Special Issue Recent Advances in the Discovery and Development of Biomolecules as Antiviral Drugs)
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<p>Predicted pockets in two different perspectives of views of the representation of the surface of spike fragment with the three pockets as indicated in <a href="#pharmaceuticals-14-01328-t001" class="html-table">Table 1</a>. Pocket 1 in red, Pocket 2 in yellow and pocket 3 in green.</p> Full article ">Figure 2
<p>2-D representation of five ligands that show maximum binding affinity with 6M0J.</p> Full article ">Figure 3
<p>Representation of the surface charge distribution localization for: (<b>a</b>) Amentoflavone; (<b>b</b>) Chrysin; (<b>c</b>) Glycyrrhizin; (<b>d</b>) Myricetin_3′-Rhamnoside; (<b>e</b>) Myricetin_3-(4″-Galloylrhamnoside) with line representation and RBD with surface representation using atom charge scale (red-blue color palette changes from negative (blue) through neutral (white) to positive (red).</p> Full article ">Figure 4
<p>(<b>A</b>) Amentoflavone complexes with RDB. Docking pose for Amentoflavone showing some closed localized amino acids (LEU 335, 368 and CYS 336) of its environment; (<b>B</b>) Hydrogen-acceptor and hydrogen-donor distances for Amentoflavone with LEU 335, 368 and CYS 336.</p> Full article ">Figure 5
<p>Time dependence of the root mean square deviation (RMSD) of the protein from the production run of all seven protein–ligand complexes and compared with protein in water simulation.</p> Full article ">Figure 6
<p>Radius of gyration (Rg) of backbone of the protein from the production run of all seven protein–ligand complexes and compared with protein in water simulation.</p> Full article ">Figure 7
<p>The SASA of the protein in the protein–ligand complex and compared with the protein–water system.</p> Full article ">Figure 8
<p>Hydrogen bonds (HBs) between ligands and protein residues.</p> Full article ">Figure 9
<p>Representative snapshot of protein with (<b>a</b>) ALR, (<b>b</b>) ALS, (<b>c</b>) AMN, (<b>d</b>) APG, (<b>e</b>) GLR, (<b>f</b>) MYG and (<b>g</b>) PSR ligands. Here, solid spheres represent drug molecules, CPK model represents residues of protein, which are within 5 Å of the ligand and cartoon (grey) representation for protein.</p> Full article ">Figure 10
<p>The average number of hydrogen bonds (standard deviation) between the drugs and SARS-CoV-2 spike protein receptor binding domain RBD.</p> Full article ">Figure 11
<p>Minimum distance between amino acids of the protein and selected ligands.</p> Full article ">Figure 12
<p>Correlation between average minimum distance between amino acid residues of the protein and ligand vs. the average HB distance between amino acid residues of the protein and ligand.</p> Full article ">Figure 13
<p>Hierarchical dendrogram for clustering of 83 descriptors.</p> Full article ">Figure 14
<p>Hierarchical dendrogram for clustering of 40 objects.</p> Full article ">Figure 15
<p>Plot of average (standardized) values of each variable for each identified cluster of objects.</p> Full article ">Figure 16
<p>Calinski–Harabasz criterion values at different number of clusters (K).</p> Full article ">Figure 17
<p>The PCA 3D score plot labeled with corresponding docking score values. Member of each cluster found by K-means clustering are depicted with different colors.</p> Full article ">Figure 18
<p>Bar plot of the loading values of 1st PC.</p> Full article ">Figure 19
<p>The Top-Map of SOM. (<b>a</b>) winner neurons for samples are labeled with compound number; (<b>b</b>) winner neurons are labeled with the docking score values.</p> Full article ">Figure 20
<p>The Top-Map of SOM labeled with low (1) high (2) binding score.</p> Full article ">Figure 21
<p>The assignation map of CPANN labeled with low (1) high (2) binding score. The neurons are colored in red or blue based on output layer of the network. The red area is assigned to high docking score compounds and the blue regions.</p> Full article ">Figure 22
<p>The Weight Map of (<b>a</b>) molecular weight (MW) descriptor; (<b>b</b>) maximal electrotopological positive variation (MAXDP) descriptor; (<b>c</b>) 1st component symmetry directional WHIM index weighted by Sanderson electronegativity (G1e) descriptor; (<b>d</b>) modified lead-like score from Congreve (LLS01) descriptor.</p> Full article ">Figure 23
<p>The correlation coefficient between each weight-map (corresponding to each descriptor) with the assignation map.</p> Full article ">
22 pages, 3127 KiB  
Article
Formulation and Characterization of an Effervescent Hydrogen-Generating Tablet
by Moritz Rosch, Kurt Lucas, Jozef Al-Gousous, Ulrich Pöschl and Peter Langguth
Pharmaceuticals 2021, 14(12), 1327; https://doi.org/10.3390/ph14121327 - 18 Dec 2021
Cited by 9 | Viewed by 7004
Abstract
Hydrogen, as a medical gas, is a promising emerging treatment for many diseases related to inflammation and oxidative stress. Molecular hydrogen can be generated through hydrogen ion reduction by a metal, and magnesium-containing effervescent tablets constitute an attractive formulation strategy for oral delivery. [...] Read more.
Hydrogen, as a medical gas, is a promising emerging treatment for many diseases related to inflammation and oxidative stress. Molecular hydrogen can be generated through hydrogen ion reduction by a metal, and magnesium-containing effervescent tablets constitute an attractive formulation strategy for oral delivery. In this regard, saccharide-based excipients represent an important class of potential fillers with high water solubility and sweet taste. In this study, we investigated the effect of different saccharides on the morphological and mechanical properties and the disintegration of hydrogen-generating effervescent tablets prepared by dry granulation. Mannitol was found to be superior to other investigated saccharides and promoted far more rapid hydrogen generation combined with acceptable mechanical properties. In further product optimization involving investigation of lubricant effects, adipic acid was selected for the optimized tablet, due to regulatory considerations. Full article
(This article belongs to the Special Issue Formulation and Evaluation of Tablets of Different Drugs)
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<p>Disintegration time of tablets containing different fillers. Experiments were conducted in a beaker with 200 mL of water as described in the monograph of effervescent tablets in the Ph. Eur. 10.1/0478. Primary and secondary endpoints of disintegration were determined visually. The first measurement was taken when the tablet had partially disintegrated into granular particles (DiG = partial disintegration into granular particles). The second measurement was taken as the endpoint, after the tablet and the particles had disintegrated completely (DT = disintegration).</p> Full article ">Figure 2
<p>Kinetic hydrogen generation measurement (means; <span class="html-italic">n</span> = 3). Kinetics of hydrogen generation were measured using different fillers and lubricants. Means were calculated and plotted against time. SD values can be found in <a href="#pharmaceuticals-14-01327-t001" class="html-table">Table 1</a>.</p> Full article ">Figure 3
<p>Disintegration time vs. porosity. Experiments were conducted in a beaker with 200 mL of water as described in the monograph of effervescent tablets in the Ph. Eur. 10.1/0478. Primary and secondary endpoints of disintegration were determined visually. The first measurement was taken when the tablet had partially disintegrated into granular particles (DiG = partial disintegration into granular particles). The second measurement was taken as the endpoint, after the tablet and the particles had disintegrated completely (DT = disintegration). These values were recorded for different fillers (Mal = maltose; Man = mannitol; Lac = lactose; Dex = dextrates; Man/Adi = mannitol/adipic acid) and were plotted against the porosity of the tablets, which was measured by mercury intrusion.</p> Full article ">Figure 4
<p>Scanning electron microscope pictures of (<b>a</b>) the maltose-based batch (compaction force 40 kN; magnification: 58,480×); (<b>b</b>) the mannitol-based batch (compaction force 40 kN; magnification: 129,440×); (<b>c</b>) the mannitol/adipic acid-based batch (compaction force 25 kN; magnification: 71,350×); (<b>d</b>) the lactose-based batch (compaction force 40 kN; magnification: 53,490×); (<b>e</b>) the dextrates based-batch (compaction force 40 kN; magnification: 50,250×).The analysis confirmed that pores were of the sizes expected based on the mercury porosity measurement.</p> Full article ">Figure 5
<p>Tablet hardness: crushing strength was measured, and the three-point bending test was performed for each formulation (<span class="html-italic">n</span> =10). Tensile strength was calculated from the results.</p> Full article ">Figure 6
<p>Dynamic vapor sorption change in mass analysis. An effervescent granule formulation containing mannitol as filler was investigated. Two cycles of absorption and desorption were performed, 0%-90%-0% P/P<sub>0</sub> in 10% stages. The change in mass was recorded over time. A mass change dm/dt = 0.002% min<sup>−1</sup> or 600 min (whichever occurred first) were selected as criteria for changing the humidity stage.</p> Full article ">Figure 7
<p>Dynamic vapor sorption isotherm analysis. Two sorption and desorption cycles (sorp = sorption; desorp = desorption) of an effervescent granule formulation containing mannitol as a filler from the same DVS measurement as in <a href="#pharmaceuticals-14-01327-f006" class="html-fig">Figure 6</a> are displayed.</p> Full article ">Figure 8
<p>Kinetic hydrogen generation measurement (means; <span class="html-italic">n</span> = 3). Kinetics of hydrogen generation of unpacked tablets of the mannitol/adipic acid-based batch were measured at the starting point t<sub>0</sub>, and after 24 h, 7 days, 14 days, and 8 weeks of storage in a constant climate chamber (25 °C and 60% RH).</p> Full article ">Figure 9
<p>Flow diagram of the manufacturing process.</p> Full article ">
11 pages, 889 KiB  
Article
A Comprehensive Analysis of the Thrombin Binding Aptamer Containing Functionalized Pyrrolo-2’-deoxycytidines
by Weronika Kotkowiak, Zofia Jahnz-Wechmann and Anna Pasternak
Pharmaceuticals 2021, 14(12), 1326; https://doi.org/10.3390/ph14121326 - 18 Dec 2021
Cited by 6 | Viewed by 2426
Abstract
Aptamers constitute an answer for the growing need for targeted therapy development. One of the most well-known representatives of this group of compounds is thrombin binding aptamers (TBA) targeted towards thrombin. The TBA inhibitory activity is determined by its spatial arrangement, which consists [...] Read more.
Aptamers constitute an answer for the growing need for targeted therapy development. One of the most well-known representatives of this group of compounds is thrombin binding aptamers (TBA) targeted towards thrombin. The TBA inhibitory activity is determined by its spatial arrangement, which consists of two G-tetrads linked by two shorter TT loops and one longer TGT loop and folds into a unimolecular, antiparallel G-quadruplex structure. Interesting properties of the aptamer can be further improved via the introduction of a number of chemical modifications. Herein, a comprehensive analysis of the influence of pyrrolo-2’-deoxycytidine (Py-dC) and its derivatives on TBA physicochemical and biological properties has been presented. The studies have shown that the presence of modified residues at the T7 position of the TGT loop has only minor effects on TBA thermodynamic stability without affecting its folding topology. All analyzed oligomers exhibit anticoagulant properties, but only aptamer modified with a decyl derivative of Py-dC was able to inhibit thrombin activity more efficiently than unmodified, parental compounds. Importantly, the same compound also possessed the potential to effectively restrain HeLa cell line growth. Full article
(This article belongs to the Special Issue Potential of the Aptamers to Fill Therapeutic and Diagnostic Gaps)
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<p>Schematic representation of TBA and modified nucleoside residues. Structure of thrombin binding aptamer, TBA (<b>A</b>) and pyrrolo-2’-deoxycytidine (Py-dC) and its derivatives (<b>B</b>).</p> Full article ">Figure 2
<p>Circular dichroism spectra performed at 4 °C (<b>A</b>) and 37 °C (<b>B</b>) and thermal difference spectra (<b>C</b>) of unmodified TBA (black lines) and TBA variants modified with bicyclic Py-dC (ON1, red line), octyl Py-dC (ON2, blue line), decyl Py-dC (ON3, magenta line) and phenyl Py-dC (ON4, green line).</p> Full article ">Figure 3
<p>The antiproliferative activity of G-quadruplexes. The viability of HeLa cells in the presence of TBA variants modified with bicyclic Py-dC (ON1, red bar), octyl Py-dC (ON2, blue bar), decyl Py-dC (ON3, magenta bar) and phenyl Py-dC (ON4, green bar). The presented results are the mean values with ± SEM from two independent experiments.</p> Full article ">
20 pages, 7683 KiB  
Article
The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine
by Francesca Paganelli, Francesca Chiarini, Annalisa Palmieri, Marcella Martinelli, Paola Sena, Jessika Bertacchini, Luca Roncucci, Alessandra Cappellini, Alberto M. Martelli, Massimo Bonucci, Carla Fiorentini and Ivano Hammarberg Ferri
Pharmaceuticals 2021, 14(12), 1325; https://doi.org/10.3390/ph14121325 - 18 Dec 2021
Cited by 3 | Viewed by 3206
Abstract
The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with immunomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement [...] Read more.
The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with immunomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer. Full article
(This article belongs to the Section Natural Products)
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Figure 1

Figure 1
<p>AHCC and ETAS combined treatment inhibits CRC cells growth. (<b>a</b>) MTT analyses were performed to assess cell viability of colon cancer cell lines treated for 48 h with increasing concentrations of AHCC and ETAS as single agents. Results are the mean of at least three independent experiments ± SD; (<b>b</b>) MTT analyses were performed to assess cell viability of colon cancer cell lines treated for 48 h with AHCC and ETAS as single agents or in combination at a fixed ratio (ratio 6:1). Concentrations of compounds used in each single point are reported in the graph. A combination index (CI) was calculated with CompuSyn software and values were plotted as shown in the graph; CI &lt; 0.9 indicates synergism. Results are the mean of at least three independent experiments ± SD; (<b>c</b>) Flow cytometry absolute counts of viable colon cancer cells treated for 48 h with AHCC and ETAS as single agents or in combination (COMB). For HCT-116, HT-29, and SW620 cells, we used 3 mg/mL AHCC + 0.5 mg/mL ETAS. For LOVO cells we employed 7 mg/mL AHCC + 1.16 mg/mL ETAS. “CTRL” indicates non-treated cells used as control cells. The mean ± SD of three independent experiments is plotted. Asterisks indicate statistically significant differences between samples: * <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 2
<p>The combined treatment of AHCC and ETAS has no effects on healthy human colonocytes. (<b>a</b>) Cell viability assays in human primary cell cultures treated with a combination of AHCC and ETAS for 48 h (3 mg/mL for AHCC and 0.5 mg/mL for ETAS). Results are presented as the mean of three independent experiments ± SD conducted on normal mucosal biopsies of eleven patients; (<b>b</b>) Immunofluorescence analysis of Cytokeratin-18 filament bundles well recognizable within the cytoplasm of colonocytes in untreated samples and treated with the combined AHCC and ETAS samples. Magnification 40×, scale bar 10 μm; (<b>c</b>) Cell counts of cells in biopsies from three patients seeded on glass slides and treated for 48 h with a combination of 3 mg/mL for AHCC and 0.5 mg/mL ETAS. The mean ± SD of 3 patients is shown.</p> Full article ">Figure 3
<p>AHCC and ETAS combined treatment induces apoptosis in CRC cells. (<b>a</b>) Apoptosis analysis of colon cancer cell lines treated for 48 h with AHCC and ETAS as single agents or in combination (COMB). For HCT-116 and SW620 cells, we used 3 mg/mL AHCC + 0.5 mg/mL ETAS. For HT-29 and LOVO cells we employed 7 mg/mL AHCC + 1.16 mg/mL ETAS. CTRL indicates untreated cells. The percentage of apoptotic cells is plotted as the mean of three independent experiments ± SD. Asterisks indicate statistically significant differences with respect to untreated cells, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; (<b>b</b>) Western blotting analysis of cleaved caspase 3, cleaved caspase 9 and PARP protein expression in HCT-116 and LOVO cell lines, treated with AHCC and ETAS as single agents or in combination (COMB). For HCT-116 we used 3 mg/mL AHCC + 0.5 mg/mL ETAS; for LOVO we used 7 mg/mL AHCC + 1.16 mg/mL ETAS. GAPDH was used as the loading control; (<b>c</b>) Stress-protein array analysis in HCT-116 and LOVO cells. Each treated sample (COMB; for HCT-116 we used 3 mg/mL AHCC + 0.5 mg/mL ETAS; for LOVO we used 7 mg/mL AHCC + 1.16 mg/mL ETAS) is compared to untreated sample (CTRL). Each protein has a double spot. Histograms indicate densitometric analysis. The following proteins were evaluated: Cytochrome C, BCL-2, p-HSP27 (S78/S82) and p-P53 (S46). Asterisks indicate statistically significant differences with respect to control, * <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 4
<p>AHCC and ETAS combined treatment affects CSC tumorspheres. (<b>a</b>) Representative images show 3D tumorspheres (CSC spheres) suspension cultures derived from HT-29 and SW620 cell lines. Magnification 10×; (<b>b</b>) Quantitative RT-PCR analysis of stemness-associated genes (<span class="html-italic">NANOG</span>, <span class="html-italic">OCT4</span>, <span class="html-italic">MYC</span>, <span class="html-italic">CD326</span>, <span class="html-italic">LGR5</span>, <span class="html-italic">CD24</span>, <span class="html-italic">CD133</span>, <span class="html-italic">ALDH</span> and <span class="html-italic">STAT3</span>) in CSC spheres from HT-29 and SW620 compared to 2D parental cell cultures. Fold change is plotted, <span class="html-italic">p</span> &lt; 0.05; (<b>c</b>) Cell viability in untreated or treated with a combination of AHCC and ETAS (7 mg/mL AHCC + 1.16 mg/mL ETAS) CSC spheres; (<b>d</b>) Quantitative RT-PCR analysis of <span class="html-italic">LGR5</span>, <span class="html-italic">Notch1</span>, <span class="html-italic">CD133</span>, and <span class="html-italic">NANOG</span> genes in untreated or treated with the combination of AHCC and ETAS CSC spheres. Fold change is plotted, * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>AHCC and ETAS combination significantly reduces CRC cell migration. (<b>a</b>) Wound healing and transwell migration assays performed in HCT-116, HT-29, LOVO and SW620 cells treated with increasing concentration of AHCC and ETAS combined treatment (COMB). For combination 1 (COMB (1)), we used 3 mg/mL AHCC + 0.5 mg/mL ETAS. For combination 2 (COMB (2)), we employed 7 mg/mL AHCC + 1.16 mg/mL ETAS. Representative pictures were taken at 0 and 24 h after scratching. For transwell assays, migration was assessed after 24 h of treatment. Magnification 10×. Histograms are plotted as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences with respect to control, * <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; (<b>b</b>) Western blotting analysis of ROCK2, E-cadherin and MMP9 protein expression in HCT-116 and LOVO cell lines, treated with AHCC and ETAS as single agents or in combination (HCT-116 COMB: 3 mg/mL AHCC + 0.5 mg/mL ETAS; LOVO COMB: 7 mg/mL AHCC + 1.16 mg/mL ETAS). GAPDH was used as the loading control. Numbers indicate densitometric analysis plotted as the ratio respect to GAPDH levels.</p> Full article ">Figure 6
<p>AHCC and ETAS combination enhances oxaliplatinum effects in colon cancer cells (<b>a</b>) Flow cytometric absolute counts (with Propidium Iodide) of viable colon cancer cells treated with AHCC and ETAS in combination (AHCC + ETAS), oxliplatinum alone (OXA), or with a combination of AHCC, ETAS and oxaliplatinum (COMB) for 48 h. For HCT-116, HT-29 and SW620 cells, we employed 3 mg/mL AHCC + 0.5 mg/mL ETAS. For LOVO cells we employed 7 mg/mL AHCC + 1.16 mg/mL ETAS. Oxaliplatinum was used at 10 μM for all cell lines. “CTRL” indicates untreated cells. The mean ± SD of three independent experiments is plotted. Asterisks indicate statistically significant differences with respect to different samples. * <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; (<b>b</b>) Flow cytometric analysis of Annexin V-FITC/PI stained CRC cells treated with a combination of AHCC, ETAS and oxaliplatinum (COMB) for 48 h. The percentages of early apoptotic cells (Annexin-V FITC+/PI−; bottom right quadrant) and late apoptotic/necrotic cells (Annexin-V FITC+/PI+; top right quadrant) are shown. “CTRL” indicates untreated cells.</p> Full article ">
13 pages, 19326 KiB  
Article
Preclinical Drug Response Metric Based on Cellular Response Phenotype Provides Better Pharmacogenomic Variables with Phenotype Relevance
by Sanghyun Kim and Sohyun Hwang
Pharmaceuticals 2021, 14(12), 1324; https://doi.org/10.3390/ph14121324 - 17 Dec 2021
Cited by 1 | Viewed by 2727
Abstract
High-throughput screening of drug response in cultured cell lines is essential for studying therapeutic mechanisms and identifying molecular variants associated with sensitivity to drugs. Assessment of drug response is typically performed by constructing a dose-response curve of viability and summarizing it to a [...] Read more.
High-throughput screening of drug response in cultured cell lines is essential for studying therapeutic mechanisms and identifying molecular variants associated with sensitivity to drugs. Assessment of drug response is typically performed by constructing a dose-response curve of viability and summarizing it to a representative, such as IC50. However, this is limited by its dependency on the assay duration and lack of reflections regarding actual cellular response phenotypes. To address these limitations, we consider how each response-phenotype contributes to the overall growth behavior and propose an alternative method of drug response screening that takes into account the cellular response phenotype. In conventional drug response screening methods, the ranking of sensitivity depends on either the metric used to construct the dose-response curve or the representative factor used to summarize the curve. This ambiguity in conventional assessment methods is due to the fact that assessment methods are not consistent with the underlying principles of population dynamics. Instead, the suggested phenotype metrics provide all phenotypic rates of change that shape overall growth behavior at a given dose and better response classification, including the phenotypic mechanism of overall growth inhibition. This alternative high-throughput drug-response screening would improve preclinical pharmacogenomic analysis and the understanding of a therapeutic mechanism of action. Full article
(This article belongs to the Special Issue New Developments in High-Throughput Screening)
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<p>Phenotype dynamics model. (<b>a</b>) A diagrammatic illustration of phenotypic response of the cell upon a drug treatment. Proliferating cells can continue dividing, can enter a state of permanent cell cycle arrest, or can undergo cell death. (<b>b</b>) Differential equations for the population dynamics in each phenotype and (<b>c</b>) their analytical solutions. (<b>d</b>) Typical growth-curve shapes of viable cell depending on the phenotypic rate of change, <math display="inline"><semantics> <mrow> <msub> <mi>k</mi> <mi>p</mi> </msub> <mo>,</mo> <msub> <mi>k</mi> <mi>d</mi> </msub> <mo>,</mo> <mi>s</mi> </mrow> </semantics></math>.</p> Full article ">Figure 2
<p>The dose-response curves of two distinct phenotype responses: (<b>i</b>) senescence-dominant and (<b>ii</b>) negligible-senescence. (<b>a</b>) The rates of change and the corresponding growth curves. The normal growth rate <math display="inline"><semantics> <mrow> <msub> <mi>k</mi> <mn>0</mn> </msub> </mrow> </semantics></math> was assumed as 0.5 (that is, a doubling time = 1.4 days). (<b>b</b>) The dose-response curves of viability, aGR ratio, and also GR ratio.</p> Full article ">Figure 3
<p>Time-dependency of drug response. (<b>a</b>) A diagrammatic illustration of the response classification assay of various cell lines upon a certain drug. (<b>b</b>) Drug responses of two cell lines that have the same GR ratio but different doubling times. (<b>c</b>) Variation of summary factors (IC<sub>50</sub>, EC<sub>50</sub>, E<sub>max</sub>, AUC) along the assay duration. The box plot, along with the ratio of the maximum deviation to the mean, shows uncertainty of summary factors by time-dependency.</p> Full article ">Figure 4
<p>Ambiguity in assessing therapeutic effectiveness by conventional metrics. (<b>a</b>) A diagrammatic illustration of the assay. (<b>b</b>) For each dose-response curve, the summary factors, IC<sub>50</sub>, EC<sub>50</sub>, E<sub>max</sub>, and AUC were extracted. The stars and the circles on dose-response curves correspond to IC<sub>50</sub> and EC<sub>50</sub>, respectively.</p> Full article ">Figure 5
<p>Evaluation of phenotype metric for ovarian cancer cell lines (<b>a</b>) OV1369(R2) and (<b>b</b>) OV1946 treated with olaparib. Fold change of viable cells and the fraction of dead and senescent cells were re-plotted using public raw data (upper). Calculation of the phenotype parameters and classification of drug response are summarized in the table (lower).</p> Full article ">Figure 6
<p>Evaluation of drug response based on (<b>a</b>) the conventional dose-response curve and (<b>b</b>) the phenotype population dynamics at a single dose. Three types of response classification are possible with the phenotype metric.</p> Full article ">
21 pages, 1889 KiB  
Article
2-(Piperidin-4-yl)acetamides as Potent Inhibitors of Soluble Epoxide Hydrolase with Anti-Inflammatory Activity
by Juan Martín-López, Sandra Codony, Clara Bartra, Christophe Morisseau, María Isabel Loza, Coral Sanfeliu, Bruce D. Hammock, José Brea and Santiago Vázquez
Pharmaceuticals 2021, 14(12), 1323; https://doi.org/10.3390/ph14121323 - 17 Dec 2021
Cited by 2 | Viewed by 2923
Abstract
The pharmacological inhibition of soluble epoxide hydrolase (sEH) has been suggested as a potential therapy for the treatment of pain and inflammatory diseases through the stabilization of endogenous epoxyeicosatrienoic acids. Numerous potent sEH inhibitors (sEHI) have been developed, however many contain highly lipophilic [...] Read more.
The pharmacological inhibition of soluble epoxide hydrolase (sEH) has been suggested as a potential therapy for the treatment of pain and inflammatory diseases through the stabilization of endogenous epoxyeicosatrienoic acids. Numerous potent sEH inhibitors (sEHI) have been developed, however many contain highly lipophilic substituents limiting their availability. Recently, a new series of benzohomoadamantane-based ureas endowed with potent inhibitory activity for the human and murine sEH was reported. However, their very low microsomal stability prevented further development. Herein, a new series of benzohomoadamantane-based amides were synthetized, fully characterized, and evaluated as sEHI. Most of these amides were endowed with excellent inhibitory potencies. A selected compound displayed anti-inflammatory effects with higher effectiveness than the reference sEHI, TPPU. Full article
(This article belongs to the Special Issue Design of Enzyme Inhibitors as Potential Drugs 2022)
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<p>Structure of the three sEHI that have entered human clinical trials.</p> Full article ">Figure 2
<p>Structure and some properties of <b>1</b>. The microsomal stability (% remaining at 1 h) of <b>1</b> was 1% in human and in 0.5% in mouse.</p> Full article ">Figure 3
<p>Anti-inflammatory effects of <b>10c</b> in activated BV2 microglial cells. Nitrite levels in the culture media, that indicate nitric oxide generation induced by LPS, were decreased to cell resting levels by co-incubation with <b>10c</b>. However, TPPU was less effective and lead to a partial decrease. Values are mean ±SEM of n = 10–15. Statistics: * <span class="html-italic">p</span> &lt; 0.001 compared to the corresponding control group without LPS; # <span class="html-italic">p</span> &lt; 0.001 compared to the corresponding LPS group without anti-inflammatory agents; <sup><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.001 compared to the corresponding LPS concentration treated with <b>10c</b>.</p> Full article ">Scheme 1
<p>Synthesis of sEHI amides <b>6a</b>–<b>h</b> from polycyclic amines <b>2a</b>–<b>e</b>. (<b>a</b>) EDC·HCl, HOBt, Et<sub>3</sub>N, EtOAc, rt, 24 h (<b>4a</b>–<b>b</b>, <b>4d</b>); (<b>b</b>) HATU, DIPEA, DMF, rt, overnight (<b>4c</b>, <b>4e</b>, <b>6g</b>–<b>h</b>); (<b>c</b>) HCl/Dioxane, rt, 2 h (<b>5a</b>–<b>e</b>); (<b>d</b>) acetyl chloride, Et<sub>3</sub>N, DCM, 0 °C to rt, overnight (<b>6a</b>); (<b>e</b>) propane-2-sulfonyl chloride, Et<sub>3</sub>N, DCM, 0 °C to rt, overnight (<b>6b</b>–<b>f</b>).</p> Full article ">Scheme 2
<p>Structure of known AS2586114 and synthesis of novel sEHI <b>10a–e</b>. (<b>a</b>) HCl/Dioxane 4 M, rt, 2 h; (<b>b</b>) 4-fluoroacetophenone (for <b>9a</b>), 4-fluorobenzonitrile (for <b>9b</b>) or t-butyl 4-fluorobenzoate (for <b>9c</b>), K<sub>2</sub>CO<sub>3</sub>, DMSO, 100 °C, overnight; (<b>c</b>) methyl 3-bromo-4-fluorobenzoate, K<sub>2</sub>CO<sub>3</sub>, DMF, 100 °C, overnight; (<b>d</b>) cyclopropylboronic acid, Pd(Ph<sub>3</sub>)<sub>4</sub>, K<sub>3</sub>PO<sub>4</sub>, dioxane, 100 °C, overnight; (<b>f</b>) <b>9a</b> or <b>9b</b>, HATU, DIPEA, DMF, rt, overnight; (<b>g</b>) <b>9c</b>, HATU, DIPEA, DMF, rt, overnight; then HCl/dioxane 4 M, H<sub>2</sub>O, rt, 2 h. (<b>h</b>) <b>9e</b>, HATU, DIPEA, DMF, rt, overnight; then methanol, KOH, rt, overnight.</p> Full article ">
16 pages, 3733 KiB  
Article
A Humanized Monoclonal Antibody Targeting Extracellular Nicotinamide Phosphoribosyltransferase Prevents Aggressive Prostate Cancer Progression
by Belinda L. Sun, Lin Tang, Xiaoguang Sun, Alexander N. Garcia, Sara M. Camp, Edwin Posadas, Anne E. Cress and Joe G. N. Garcia
Pharmaceuticals 2021, 14(12), 1322; https://doi.org/10.3390/ph14121322 - 17 Dec 2021
Cited by 13 | Viewed by 3317
Abstract
Prostate cancer (PCa) is the major cause of cancer-related death in males; however, effective treatments to prevent aggressive progression remain an unmet need. We have previously demonstrated that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator that promotes PCa invasion. [...] Read more.
Prostate cancer (PCa) is the major cause of cancer-related death in males; however, effective treatments to prevent aggressive progression remain an unmet need. We have previously demonstrated that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator that promotes PCa invasion. In the current study, we further investigate the therapeutic effects of an eNAMPT-neutralizing humanized monoclonal antibody (ALT-100 mAb) in preclinical PCa orthotopic xenograft models. We utilized human aggressive PCa cells (DU145 or PC3) for prostate implantation in SCID mice receiving weekly intraperitoneal injections of either ALT-100 mAb or IgG/PBS (control) for 12 weeks. Prostatic tumors and solid organs were examined for tumor growth, invasion, and metastasis and for biochemical and immunohistochemistry evidence of NFκB activation. ALT-100 mAb treatment significantly improved overall survival of SCID mice implanted with human PCa orthotopic prostate xenografts while inducing tumor necrosis, decreasing PCa proliferation and reducing local invasion and distal metastases. The ALT-100 mAb inhibits NFκB phosphorylation and signaling in PCa cells both in vitro and in vivo. This study demonstrates that eNAMPT neutralization effectively prevents human PCa aggressive progression in preclinical models, indicating its high potential to directly address the unmet need for an effective targeted therapy for patients with aggressive PCa. Full article
(This article belongs to the Special Issue Novel Therapeutic Targets in Cancer)
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<p>The eNAMPT-neutralizing mAb, ALT-100, increases survival of SCID mice with human PCa orthotopic xenografts. (<b>A</b>): DU145<sup>luc</sup> or PC3<sup>luc</sup> cells were implanted into the anterior lobe of prostate (arrow/circle) of SCID mice. (<b>B</b>): PCa tumors grew into larger prostate masses (arrow) at 12 weeks after implantation. (<b>C</b>): Live mice were whole body imaged to monitor tumor growth and location (purple shading indicates PCa tumor in the prostate). (<b>D</b>): Staining showed DU145 xenograft (H&amp;E, 200×, arrow) and strong NAMPT expression (IHC, 200×, brown color). H&amp;E arrow shows DU145 xenograft growing in extracapsular soft tissues of the prostate. NAMPT arrow shows DU145 xenograft with strongly expressed NAMPT staining in brown color. (<b>E</b>): Staining showed PC3 xenograft (H&amp;E, 200×, arrow) and strong NAMPT expression (IHC, 200×, brown color). H&amp;E arrow shows PC3 xenograft invading into capsular tissue of the prostate. NAMPT arrow shows PC3 xenograft with strongly expressed NAMPT staining in brown color. (<b>F</b>): Survival curve of mice with DU145 orthotopic xenograft showed 100% survival in mAb-treated group (mAb) compared to 60% survival in vehicle-treated group (veh) at the study endpoint of 12 weeks (84 days). (<b>G</b>): Survival curve of mice with PC3 orthotopic xenograft showed 60% survival in mAb-treated group compared to 30% survival in vehicle-treated group at the study endpoint of 12 weeks. (<b>H</b>): H&amp;E staining showing dramatic hydronephropathy with dilated renal pelvis (*) in vehicle-treated DU145 PCa-exposed mice, versus H&amp;E staining of kidneys from mice with DU145 xenografts receiving weekly ALT-100 mAb treatment. (<b>I</b>): Subcapsular PC3 metastases grew into a large mass () approximating the size of a kidney, versus unremarkable kidneys of mice with PC3 xenografts receiving weekly ALT-100 mAb treatment. (<b>J</b>): The sizes of prostate DU145 tumors were significantly smaller in ALT-100 mAb-treated group than that in IgG vehicle-treated group, * <span class="html-italic">p</span> &lt; 0.05. (<b>K</b>): The sizes of prostate PC3 tumors were significantly smaller in ALT-100 mAb-treated group than those in IgG vehicle-treated group, * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p>The eNAMPT-neutralizing mAb, ALT-100, inhibits PCa proliferation. (<b>A</b>): IHC for proliferation index Ki67 showed much lower percentage of Ki67-positive DU145 cells (arrows) in mAb-treated compared to vehicle-treated xenografts. (<b>B</b>): IHC for Ki67 showed much lower percentage of positive PC3 cells (arrows) in mAb-treated compared to vehicle-treated xenografts. (<b>C</b>): Statistic analysis on DU145 xenografts showed Ki67 proliferation index was significantly lower in mAb-treated group than in the vehicle treated group (* <span class="html-italic">p</span> &lt; 0.05). (<b>D</b>): Measurement on DU145 xenografts showed significantly smaller tumor size in mAb-treated group than in the vehicle-treated group (* <span class="html-italic">p</span> &lt; 0.05). (<b>E</b>): Statistic analysis on PC3 xenografts showed Ki67 proliferation index was significantly lower in mAb-treated group than in the vehicle-treated group (* <span class="html-italic">p</span> &lt; 0.05). (<b>F</b>): Measurement on PC3 xenografts showed significantly smaller tumor size in ALT-100 mAb-treated group than vehicle-treated group (* <span class="html-italic">p</span> &lt; 0.05). (<b>G</b>): Solid DU145 tumor growth in vehicle-treated xenografts (H&amp;E, 200×), whereas ALT-100 mAb treatment induced extensive tumor necrosis in DU145 xenografts (H&amp;E, 200×). (<b>H</b>): Solid PC3 tumor growth in vehicle-treated xenografts (H&amp;E, 200×), whereas ALT-100 mAb treatment induced tumor necrosis in PC3 xenografts (H&amp;E, 200×).</p> Full article ">Figure 3
<p>The eNAMPT-neutralizing mAb, ALT-100, prevents PCa invasion and metastasis. (<b>A</b>): DU145 xenograft invaded prostate glands (pros) and capsule in vehicle IgG-treated mice (<b>left panel</b>), and lymphovascular invasion (arrows, <b>middle panel</b>) was easily identified in DU145 xenografts in vehicle IgG-treated mice. In mAb-treated mice (<b>right panel</b>), DU145 xenografts appeared to push prostate glands (pro) without infiltration and lymphovascular invasion. (<b>B</b>): Percentage of mice with organ invasion and metastasis in vehicle-treated and mAb-treated DU145 orthotopic xenografts. No distant metastasis was observed in mAb-treated group. (<b>C</b>): PC3 xenograft extensively invaded prostate glands (pros) (<b>left panel</b>) and locally spread and directly invaded into pelvic skeletal muscle (<b>middle panel</b>). In mAb-treated mice (<b>right panel</b>), PC3 grew with a defined border without extensive invasion into the capsule and prostate glands (pros). (<b>D</b>): Percentage of mice with organ invasion and metastasis in vehicle-treated and mAb-treated PC3 orthotopic xenografts. Lower metastasis percentages were observed in mAb-treated group compared to vehicle-treated group. (<b>A</b>,<b>C</b>), H&amp;E 200×.</p> Full article ">Figure 4
<p>The eNAMPT-neutralizing ALT-100 mAb inhibits NFκB phosphorylation in PCa cells and xenografts. DU145 cells were exposed to vehicle (PBS), denatured eNAMPT (den-eNAMPT), anti-eNAMPT mAb (mAb), eNAMPT, or mAb mixed with eNAMPT. Immunoblot for phosphor-NFkB p65 (p-NFkB), total NFkB, and internal control beta-actin showed that mAb-treatment significantly decreased eNAMPT-induced NFκB phosphorylation in DU145 cells (<b>A</b>) and PC3 cells (<b>B</b>). Immunoblot density measurement showed that mAb treatment significantly decreased eNAMPT-induced NFκB phosphorylation level in DU145 cells (<b>C</b>) and PC3 cells (<b>D</b>) (* <span class="html-italic">p</span> &lt; 0.05). IHC for phosphor-NFκB p65 (p-NFκB) showed high levels of NFκB phosphorylation (brown) in vehicle-treated xenografts, whereas the level was markedly decreased in mAb-treated DU145 (<b>E</b>) and PC3 xenografts (<b>F</b>). Measurement of IHC density showed that phosphor-NFκB p65 (p-NFκB) was significantly lower in mAb-treated DU145 (<b>G</b>) and PC3 xenografts (<b>H</b>) compared to vehicle IgG-treated mice (* <span class="html-italic">p</span> &lt; 0.05). 100x. Immunoblots for phosphor-ERK1/2 (p-ERK1/2), total ERK1/2, and internal control beta-actin showed that mAb-treatment significantly decreased eNAMPT-induced ERK1/2 phosphorylation in DU145 (<b>I</b>,<b>K</b>) and PC3 cells (<b>J</b>,<b>L</b>) quantified by immunoblot densitometric measurements (*,** <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
15 pages, 1281 KiB  
Article
Quality Assessment of Investigational Medicinal Products in COVID-19 Clinical Trials: One Year of Activity at the Clinical Trials Office
by Diego Alejandro Dri, Giulia Praticò, Elisa Gaucci, Carlotta Marianecci and Donatella Gramaglia
Pharmaceuticals 2021, 14(12), 1321; https://doi.org/10.3390/ph14121321 - 17 Dec 2021
Cited by 2 | Viewed by 2949
Abstract
One year after the spread of the pandemic, we analyzed the assessment results of the quality documentation submitted to the Clinical Trials Office of the Italian Medicines Agency as part of the request for authorization of clinical trials with a COVID-19 indication. In [...] Read more.
One year after the spread of the pandemic, we analyzed the assessment results of the quality documentation submitted to the Clinical Trials Office of the Italian Medicines Agency as part of the request for authorization of clinical trials with a COVID-19 indication. In this article, we report the classification of the documentation type, an overview of the assessment results, and the related issues focusing on the most frequently detected ones. Relevant data regarding the Investigational Medicinal Products (IMPs) tested in COVID-19 clinical trials and their quality profiles are provided in the perspective of increasing transparency and availability of information. Some criticalities that have been exacerbated by the management of clinical trials during the emergency period are highlighted. Results confirm that IMPs tested in authorized COVID-19 clinical trials are developed in agreement with the same legal requirements for quality, safety, and efficacy as for any other medicinal product in the European Union (EU). The same strong regulatory framework applies, and there is no lowering in the safety profile due to the pandemic; authorized IMPs meet the highest standards of quality. The regulatory network should capitalize on lessons learned from the emergency setting. Some take-home messages are provided that could support the regulatory framework to expand its boundaries by innovating and evolving even though remaining strong and effective. Full article
(This article belongs to the Special Issue COVID-19 in Pharmaceuticals)
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<p>COVID-19 CTs officially submitted with a unique EudraCT number to the CTO on a monthly basis from March 2020 to March 2021.</p> Full article ">Figure 2
<p>Percentage (number) of commercial and non-commercial COVID-19 CTs assessed from March 2020 to March 2021.</p> Full article ">Figure 3
<p>Percentages (number) of the different types of quality documentation for COVID-19 CTs assessed from March 2020 to March 2021.</p> Full article ">Figure 4
<p>Percentage (number) of quality documentation types for commercial and non-commercial COVID-19 CTs assessed from March 2020 to March 2021.</p> Full article ">Figure 5
<p>Number of quality issues combining drug substance (DS) and drug product (DP) classification label in COVID-19 CTs assessed from March 2020 to March 2021.</p> Full article ">
12 pages, 1520 KiB  
Article
Protective Effect of Carotenoid Extract from Orange-Fleshed Sweet Potato on Gastric Ulcer in Mice by Inhibition of NO, IL-6 and PGE2 Production
by Ji-Yeong Bae, Woo-Sung Park, Hye-Jin Kim, Ho-Soo Kim, Kwon-Kyoo Kang, Sang-Soo Kwak and Mi-Jeong Ahn
Pharmaceuticals 2021, 14(12), 1320; https://doi.org/10.3390/ph14121320 - 17 Dec 2021
Cited by 8 | Viewed by 3148
Abstract
Ipomoea batatas (L.) Lam., Convolvulaceae is widely distributed in Asian areas from tropical to warm-temperature regions. Their tubers are known for their antioxidant, anti-bacterial, anti-diabetic, wound healing, anti-inflammatory, and anti-ulcer activities. The preventive and therapeutic effects of orange-fleshed sweet potato on gastric ulcers [...] Read more.
Ipomoea batatas (L.) Lam., Convolvulaceae is widely distributed in Asian areas from tropical to warm-temperature regions. Their tubers are known for their antioxidant, anti-bacterial, anti-diabetic, wound healing, anti-inflammatory, and anti-ulcer activities. The preventive and therapeutic effects of orange-fleshed sweet potato on gastric ulcers have not been investigated. In this study, the carotenoid extract (CE) of orange-fleshed sweet potato was found to protect against gastric ulcers induced by HCl/ethanol in mice. The anti-inflammatory and antioxidant activities of the carotenoid pigment extract were also evaluated as possible evidence of their protective effects. Administration of CE reduced gastric ulcers. Oral administration of CE (100 mg/kg) protected against gastric ulcers by 78.1%, similar to the positive control, sucralfate (77.5%). CE showed potent reducing power and decreased nitric oxide production in a mouse macrophage cell line, RAW 264.7, in a concentration-dependent manner. The production of the inflammatory cytokine interleukin-6 and prostaglandin E2 was also reduced by CE in a dose-dependent manner. The high carotenoid content of orange-fleshed sweet potato could play a role in its protective effect against gastric ulcers. This result suggests the possibility of developing functional products using this nutrient-fortified material. Full article
(This article belongs to the Special Issue Neglected Mushrooms, Food Plants, Nuts: Functional Food Properties and Bioactive Compounds)
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<p>HPLC chromatogram of carotenoid extract (CE) from orange-fleshed sweet potato. <b>1</b>, lutein; <b>2</b>, zeaxanthin; <b>3</b>, ꞵ-cryptoxanthin; <b>4</b>, 13Z-ꞵ-carotene; <b>5</b>, α-carotene; <b>6</b>, all-trans-β-carotene; <b>7</b>, 9Z-ꞵ-carotene.</p> Full article ">Figure 2
<p>The protective effects of carotenoid extract (CE) from orange-fleshed sweet potato on HCl/ethanol-induced gastric ulcer model in mice. (<b>A</b>), normal stomach; (<b>B</b>), glandular stomach treated with vehicle; (<b>C</b>), the stomach of positive control group treated with sucralfate, 100 mg/kg; (<b>D</b>), the stomach of test group treated with CE, 100 mg/kg, <span class="html-italic">p.o.,</span> 1 h before administration of 150 mM HCl/ethanol; (<b>E</b>) Determination of the gastric ulcer area is expressed in the unit of mm<sup>2</sup>. Data are presented as mean ± standard error of the mean (S.E.M). *** <span class="html-italic">p</span> &lt; 0.001, significantly different from gastric ulcer group (<b>B</b>).</p> Full article ">Figure 3
<p>Total antioxidant activity measured by ferric reducing antioxidant power (FRAP) assay for the carotenoid extract (CE). Quercetin is used as a reference compound (concentration range; 1.6–100 µg/mL). The data are presented as mean ± standard deviation.</p> Full article ">Figure 4
<p>Effect of carotenoid extract (CE) on NO production (<b>A</b>); IL-6 production (<b>B</b>); PGE<sub>2</sub> production (<b>C</b>) in LPS-stimulated RAW 264.7 cells. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 vs. LPS-treated control.</p> Full article ">
20 pages, 4865 KiB  
Article
Improved Anticancer Activities of a New Pentafluorothio-Substituted Vorinostat-Type Histone Deacetylase Inhibitor
by Nils Goehringer, Yayi Peng, Bianca Nitzsche, Hannah Biermann, Rohan Pradhan, Rainer Schobert, Marco Herling, Michael Höpfner and Bernhard Biersack
Pharmaceuticals 2021, 14(12), 1319; https://doi.org/10.3390/ph14121319 - 17 Dec 2021
Cited by 7 | Viewed by 2819
Abstract
The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising [...] Read more.
The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate. Full article
(This article belongs to the Special Issue Drug Insight: Histone Deacetylase (HDAC) Inhibitors)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Structures of SAHA (vorinostat) and the new analog SF5-SAHA (ZBG = zinc-binding group).</p> Full article ">Figure 2
<p>Unspecific cytotoxic effects of SF5-SAHA in DU145 prostate cancer and Hep-G2 hepatoblastoma cells. LDH release of cells was measured after 3 and 24 h of incubation with 1, 5 or 10 µM of SF5-SAHA or SAHA. Results show changes in LDH release relative to untreated controls. Data are given as percentage changes relative to basal LDH release of controls. Means ± SEM of n = 3 independent experiments.</p> Full article ">Figure 3
<p>Apoptosis induction by SF5-SAHA. (<b>A</b>) Caspase-3 induction in DU145 or Hep-G2 cells after 24 h treatment with SF5-SAHA and SAHA. Means ± SEM of n = 3 independent experiments. (<b>B</b>) Representative Western blot out of n = 3 experiments, showing changes in the expression of PARP and PARP after cleavage (cl. PARP) in DU145 cells after 24 h treatment with test compounds. (<b>C</b>) From these Western blots gray intensities, of PARP and cleaved PARP specific bands were quantified adjusted to protein loading and normalized to untreated controls. (<b>D</b>) Representative Western blot out of n = 3 experiments, showing changes in the expression of Apaf-1, PARP, and Bcl-2 in Hep-G2 cells after 24 h treatment with SF5-SAHA. Data are given as means ± SEM of n = 3 independent experiments. * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.005, *** <span class="html-italic">p</span> ≤ 0.0005, **** <span class="html-italic">p</span> ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.</p> Full article ">Figure 4
<p>ROS induction (fluorescence microscopy using the ROS-dye CellROX orange) after 6 h, 12 h, and 24 h of incubation with SF5-SAHA and SAHA in DU145 (<b>A</b>) and Hep-G2 cells (<b>B</b>). Orange fluorescence indicates oxidation of the ROS-dye by formed ROS.</p> Full article ">Figure 5
<p>HDAC inhibitory effects of SF5-SAHA in cancer cells. (<b>A</b>) HDAC activity was measured by luminescence display of the acetylation state of histones. HeLa nuclear cell extracts, used as source for histones, where preincubated with substrate and inhibitor. Results are given as relatives to controls, not preincubated with inhibitor, as means ± SEM of n = 3 independent experiment. (<b>B</b>) Representative Western blot out of n = 3 experiments, showing changes in the expression of acetylated histone H3 after 24 h treatment with compounds in DU145 (left) and Hep-G2 (right) cells. (<b>C</b>) From the Western blots gray intensity, mean ± SEM, of acetylated histone H3 specific bands where quantified adjusted to protein loading and normalized to untreated control. * <span class="html-italic">p</span> ≤ 0.05, **** <span class="html-italic">p</span> ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.</p> Full article ">Figure 6
<p>HDAC inhibitory effects of SF5-SAHA in T-cell leukemia/lymphoma cells (Jurkat, Hut78, SMZ1, SupT11). Representative Western blots out of n = 3 experiments, showing changes in the expression of acetylated histone H3 and acetylated α-tubulin after 24 h treatment with SF5-SAHA.</p> Full article ">Figure 7
<p>Inhibition of HDAC1 (top), HDAC2 (middle) and HDAC6 (bottom) by SF5-SAHA and SAHA. HDAC activity was measured by luminescence display of the acetylation state of histones. Human recombinant HDAC1, HDAC2 and HDAC6 enzymes and adjacent fluorgenic HDAC substrates were used to determine subtype specific HDAC activity levels after preincubation with SF5-SAHA and SAHA. Results are given as relatives to controls, not preincubated with inhibitor, as means ± SEM of n = 3 independent experiments. ** <span class="html-italic">p</span> ≤ 0.005, *** <span class="html-italic">p</span> ≤ 0.0005, **** <span class="html-italic">p</span> ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.</p> Full article ">Figure 8
<p>Western blot determined expression of HDAC1, HDAC2, and HDAC6 in DU145 prostate cancer and Hep-G2 liver cancer cells.</p> Full article ">Figure 9
<p>Overlay images of docked SF5-SAHA (green) and SAHA (purple) with HDAC2 in cartoon view (top image) and surface view (bottom image). The interaction of inhibitors with active site amino acids (red) and zinc ion (sphere) is highlighted.</p> Full article ">Figure 10
<p>Inhibitory effects of SF5-SAHA on EGFR signaling pathway protein expression. (<b>A</b>) Representative Western blots of n = 3 independent experiments showing treatment induced changes in the expression of EGFR in DU145 cells after 24 h. β-actin was used as loading control. (<b>B</b>) From these Western blots gray intensity, mean ± SEM, of EGFRs specific bands were quantified and adjusted to protein loading and normalized to untreated control. ** <span class="html-italic">p</span> ≤ 0.005; 2-way ANOVA Dunnett’s post-hoc test.</p> Full article ">Figure 11
<p>Inhibitory effects of SF5-SAHA on DU145 tumor cell migration. Representative scratch assay images of n = 3 independent experiments showing a treatment induced retarded migration of DU145 cells after 24 h. Black lines indicate the initial scratch areas at the corresponding starting points (0 h). Control cells (ctrl) are untreated cells in medium.</p> Full article ">Figure 12
<p>Angiogenesis in fertilized chicken eggs (CAM assay). Representative images of n = 4 independent experiments showing antiangiogenic effects of SF5-SAHA (5 or 10 µM) after 72 h. Control (ctrl) image shows vessels of untreated CAM (PBS control). Red arrows indicate conspicuous branches and vessel diameters.</p> Full article ">Scheme 1
<p>Synthesis of SF5-SAHA.</p> Full article ">
14 pages, 1034 KiB  
Review
Growth Inhibitory Efficacy of Chinese Herbs in a Cellular Model for Triple-Negative Breast Cancer
by Nitin T. Telang, Hareesh B. Nair and George Y. C. Wong
Pharmaceuticals 2021, 14(12), 1318; https://doi.org/10.3390/ph14121318 - 17 Dec 2021
Cited by 6 | Viewed by 3367
Abstract
Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor-α progesterone receptor and human epidermal growth factor receptor-2. Treatment for this breast cancer subtype is restricted to multidrug chemotherapy and survival pathway-based molecularly targeted therapy. The long-term treatment options are associated [...] Read more.
Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor-α progesterone receptor and human epidermal growth factor receptor-2. Treatment for this breast cancer subtype is restricted to multidrug chemotherapy and survival pathway-based molecularly targeted therapy. The long-term treatment options are associated with systemic toxicity, spontaneous and/or acquired tumor resistance and the emergence a of drug-resistant stem cell population. These limitations lead to advanced stage metastatic cancer. Current emphasis is on research directions that identify efficacious, naturally occurring agents representing an unmet need for testable therapeutic alternatives for therapy resistant breast cancer. Chinese herbs are widely used in traditional Chinese medicine in women for estrogen related health issues and also for integrative support for cancer treatment. This review discusses published evidence on a TNBC model for growth inhibitory effects of several mechanistically distinct nontoxic Chinese herbs, most of them nutritional in nature, and identifies susceptible pathways and potential molecular targets for their efficacy. Documented anti-proliferative and pro-apoptotic effects of these herbs are associated with downregulation of RB, RAS, PI3K, and AKT signaling, modulation of Bcl-2/BAX protein expressions and increased caspase activity. This review provides a proof of concept for Chinese herbs as testable alternatives for prevention/therapy of TNBC. Full article
(This article belongs to the Special Issue Potential Molecular Targets and Therapeutics in Triple-Negative Breast Cancer)
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<p>Effect of Chinese herbs on cell cycle progression. Treatment with PC and CO at their respective IC<sub>90</sub> doses increased the ratio, while treatment with DA at IC<sub>90</sub> dose reduced the ratio. The data are presented as G<sub>1</sub>: S + G<sub>2</sub>/M ratio mean ± SD, <span class="html-italic">n</span> = 3 per treatment group, and analyzed by ANOVA with Dunnett’s multiple comparison test (α = 0.05). Contr., control; PC, <span class="html-italic">Psoralea corylifolia</span>; CO, <span class="html-italic">Cornus officinalis</span>. SD, standard deviation; ANOVA, analysis of variance. Data summarized from [<a href="#B14-pharmaceuticals-14-01318" class="html-bibr">14</a>,<a href="#B15-pharmaceuticals-14-01318" class="html-bibr">15</a>,<a href="#B16-pharmaceuticals-14-01318" class="html-bibr">16</a>].</p> Full article ">Figure 2
<p>Effect of Chinese herbs on RB Signaling. Treatment with DA, PC and CO at their respective IC<sub>90</sub> doses decreased the pRB: RB ratio. Data presented as arithmetic means of ASU from duplicate determinations. pRB, phosphorylated RB; RB, retinoblastoma; ASU, arbitrary scanning unit; DA, <span class="html-italic">Dipsacus asperoides</span>; PC, <span class="html-italic">Psoralea corylifolia</span>; CO, <span class="html-italic">Cornus officinalis</span>. Data summarized from [<a href="#B14-pharmaceuticals-14-01318" class="html-bibr">14</a>,<a href="#B15-pharmaceuticals-14-01318" class="html-bibr">15</a>,<a href="#B16-pharmaceuticals-14-01318" class="html-bibr">16</a>].</p> Full article ">Figure 3
<p>Effect of Chinese herbs on CDK4 and CDK 6 expression. Treatment with PC and DA at their respective IC<sub>90</sub> doses inhibited the expressions of CDK4 and CDK6. Data presented as arithmetic means of ASU from duplicate determinations. CDK, cyclin dependent kinase; ASU, arbitrary scanning unit; Contr., Control; Treatment: PC, <span class="html-italic">Psoralea corylifolia</span>; DA, <span class="html-italic">Dipsacus asperoides</span>. Data summarized from [<a href="#B15-pharmaceuticals-14-01318" class="html-bibr">15</a>,<a href="#B16-pharmaceuticals-14-01318" class="html-bibr">16</a>].</p> Full article ">Figure 4
<p>Effect of Chinese herbs on RAS, PI3K, and AKT signaling. Treatment with DA at its IC<sub>90</sub> dose inhibited the phosphorylated: total protein ratio. Data presented as arithmetic means of phosphorylated: total protein ratio of ASU from duplicate determinations. ERK, extracellular receptor kinase; PI3K, phosphatidylinositol 3-kinase, AKT, Protein kinase B; ASU, arbitrary scanning unit. Contr, control; Treatment: DA, <span class="html-italic">Dipsacus asperoides</span>. Data summarized from [<a href="#B16-pharmaceuticals-14-01318" class="html-bibr">16</a>].</p> Full article ">Figure 5
<p>(<b>A</b>): Effect of Chinese herbs on cellular apoptosis. Treatment with DA, CO and PC at their respective IC<sub>90</sub> doses increased % sub G<sub>0</sub> population. The data are presented as % sub G<sub>0</sub> population mean ± SD, <span class="html-italic">n</span> = 3 per treatment group. (<b>B</b>): Effect of Chinese herbs on Caspase 3/7 activity. Treatment with DA, CO and PC at their respective IC<sub>90</sub> doses increased caspase 3/7 activity. The data are presented as RLU mean ± SD, <span class="html-italic">n</span> = 3 per treatment group. The data are analyzed by ANOVA with Dunnett’s multiple comparison test. (α = 0.05). Contr.; control; DA, Dipsacus asperoides; CO, Cornus officinalis; PC, Psoralea corylifolia; RLU, relative luminescent unit; SD, standard deviation; ANOVA, analysis of variance. Data summarized from [<a href="#B14-pharmaceuticals-14-01318" class="html-bibr">14</a>,<a href="#B15-pharmaceuticals-14-01318" class="html-bibr">15</a>,<a href="#B16-pharmaceuticals-14-01318" class="html-bibr">16</a>].</p> Full article ">
15 pages, 3675 KiB  
Article
Azacitidine Omega-3 Self-Assemblies: Synthesis, Characterization, and Potent Applications for Myelodysplastic Syndromes
by Milad Baroud, Elise Lepeltier, Yolla El-Makhour, Nolwenn Lautram, Jerome Bejaud, Sylvain Thepot and Olivier Duval
Pharmaceuticals 2021, 14(12), 1317; https://doi.org/10.3390/ph14121317 - 17 Dec 2021
Cited by 1 | Viewed by 3303
Abstract
5-Azacitidine, a cytidine analogue used as a hypomethylating agent, is one of the main drugs for the treatment of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) in the elderly. However, after administration, it exhibits several limitations, including restricted diffusion and cellular internalization [...] Read more.
5-Azacitidine, a cytidine analogue used as a hypomethylating agent, is one of the main drugs for the treatment of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) in the elderly. However, after administration, it exhibits several limitations, including restricted diffusion and cellular internalization due to its hydrophilicity, and a rapid enzymatic degradation by adenosine deaminase. The aim of this study was to improve the drug cell diffusion and protect it from metabolic degradation via the synthesis of amphiphilic prodrugs and their potential self-assembly. Azacitidine was conjugated to two different omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The carboxylic acid group of the omega-3 fatty acids was effectively conjugated to the amine group of the azacitidine base, yielding two amphiphilic prodrugs. Nanoprecipitation of the obtained prodrugs was performed and self-assemblies were successfully obtained for both prodrugs, with a mean diameter of 190 nm, a polydispersity index below 0.2 and a positive zeta potential. The formation of self-assemblies was confirmed using pyrene as a fluorescent dye, and the critical aggregation concentrations were determined: 400 µM for AzaEPA and 688 µM for AzaDHA. Additionally, the stability of the obtained self-assemblies was studied and after 5 days their final stable arrangement was reached. Additionally, cryo-TEM revealed that the self-assemblies attain a multilamellar vesicle supramolecular structure. Moreover, the obtained self-assemblies presented promising cytotoxicity on a leukemia human cell line, having a low IC50 value, comparable to that of free azacitidine. Full article
(This article belongs to the Special Issue Heterocyclic Compounds and Their Application in Therapy)
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<p>Hydrolysis of azacitidine. In the presence of water, a fast and reversible hydrolysis occurs, producing N-formylribosylguanylurea, followed by a second slow irreversible hydrolysis, producing ribosylguanylurea.</p> Full article ">Figure 2
<p>Formulation of self-assemblies via nanoprecipitation. The conjugates are dissolved in an organic solvent, here acetone, then added drop-wise to a stirring aqueous medium, allowing for the spontaneous formation of self-assemblies. The organic solvent is then evaporated using a rotary evaporator, thus yielding an opalescent aqueous suspension.</p> Full article ">Figure 3
<p>Synthesis of N4-azacitidine DHA (AzaDHA, 1) and N4-azacitidine EPA (AzaEPA, 2).</p> Full article ">Figure 4
<p>Fourier-transform infrared spectroscopy (FTIR) curves and wave numbers of interest between 4000 cm<sup>−1</sup> and 600 cm<sup>−1</sup> of azacitidine (green), EPA (purple), DHA (pink), AzaEPA (black) and AzaDHA (blue).</p> Full article ">Figure 5
<p>Boltzmann-type sigmoid data (red) obtained for the AzaEPA and AzaDHA suspensions in water, showing the CAC value, corresponding to the first sharp decrease point. Graph tangents (blue) are plotted, the tangent’s intersection with the graph (green) determines the CAC.</p> Full article ">Figure 6
<p>Cryo-TEM images of the formed self-assemblies: (<b>A</b>) AzaEPA, (<b>B</b>) AzaDHA.</p> Full article ">Figure 7
<p>Cytotoxicity studies of the self-assemblies compared to the free azacitidine and fatty acids after 24 h of treatment.</p> Full article ">
21 pages, 857 KiB  
Review
Potential of Fatty Acid Amide Hydrolase (FAAH), Monoacylglycerol Lipase (MAGL), and Diacylglycerol Lipase (DAGL) Enzymes as Targets for Obesity Treatment: A Narrative Review
by Justin Matheson, Xin Ming Matthew Zhou, Zoe Bourgault and Bernard Le Foll
Pharmaceuticals 2021, 14(12), 1316; https://doi.org/10.3390/ph14121316 - 17 Dec 2021
Cited by 12 | Viewed by 6051
Abstract
The endocannabinoid system (ECS) plays an integral role in maintaining metabolic homeostasis and may affect hunger, caloric intake, and nutrient absorption. Obesity has been associated with higher levels of the endogenous cannabinoid transmitters (endocannabinoids). Therefore, the ECS is an important target in obesity [...] Read more.
The endocannabinoid system (ECS) plays an integral role in maintaining metabolic homeostasis and may affect hunger, caloric intake, and nutrient absorption. Obesity has been associated with higher levels of the endogenous cannabinoid transmitters (endocannabinoids). Therefore, the ECS is an important target in obesity treatment. Modulating the enzymes that synthesize and degrade endocannabinoids, namely fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and diacylglycerol lipase (DAGL), may be a promising strategy to treat obesity. This review aims to synthesize all studies investigating pharmacological or genetic manipulation of FAAH, MAGL, or DAGL enzymes in association with obesity-related measures. Pharmacological inhibition or genetic deletion of FAAH tended to promote an obesogenic state in animal models, though the relationships between human FAAH polymorphisms and obesity-related outcomes were heterogeneous, which could be due to FAAH having both pro-appetitive and anti-appetitive substrates. Genetic deletion of Mgll and Dagla as well as pharmacological inhibition of DAGL tended to reduce body weight and improve metabolic state in animal studies, though the effects of Mgll manipulation were tissue-dependent. Monitoring changes in body weight in ongoing clinical trials of FAAH inhibitors may clarify whether FAAH inhibition is a potential therapeutic strategy for treatment obesity. More preclinical work is needed to characterize the role of MAGL and DAGL modulation in obesity-related outcomes. Full article
(This article belongs to the Special Issue Searching for New Therapeutic Targets with Anti-obesity Potential)
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<p>Synaptic localization of endocannabinoid synthesis and degradation. 2-AG is synthesized on-demand in the postsynaptic neuron by DAGL. Following synthesis, 2-AG diffuses into the synaptic cleft and activates CB1 receptors at GABA and glutamate terminals. 2-AG signaling can be terminated by MAGL degradation in the presynaptic terminal. AEA can be synthesized through multiple pathways (e.g., through a pathway involving NAPE-PLD), then diffuses into the synapse to activate CB1 receptors. Extracellular AEA undergoes reuptake into the postsynaptic cell and is hydrolyzed by FAAH.</p> Full article ">Figure 2
<p>Flow diagram depicting the process of article identification, screening, and inclusion according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Both preclinical and clinical studies were included in the same article screening process.</p> Full article ">
15 pages, 18784 KiB  
Review
The Therapeutic Potential of Common Herbal and Nano-Based Herbal Formulations against Ovarian Cancer: New Insight into the Current Evidence
by Fatemeh Rezaei-Tazangi, Hossein Roghani-Shahraki, Mahdi Khorsand Ghaffari, Firoozeh Abolhasani Zadeh, Aynaz Boostan, Reza ArefNezhad and Hossein Motedayyen
Pharmaceuticals 2021, 14(12), 1315; https://doi.org/10.3390/ph14121315 - 17 Dec 2021
Cited by 15 | Viewed by 4228
Abstract
Ovarian cancer (OCa) is characterized as one of the common reasons for cancer-associated death in women globally. This gynecological disorder is chiefly named the “silent killer” due to lacking an association between disease manifestations in the early stages and OCa. Because of the [...] Read more.
Ovarian cancer (OCa) is characterized as one of the common reasons for cancer-associated death in women globally. This gynecological disorder is chiefly named the “silent killer” due to lacking an association between disease manifestations in the early stages and OCa. Because of the disease recurrence and resistance to common therapies, discovering an effective therapeutic way against the disease is a challenge. According to documents, some popular herbal formulations, such as curcumin, quercetin, and resveratrol, can serve as an anti-cancer agent through different mechanisms. However, these herbal products may be accompanied by some pharmacological limitations, such as poor bioavailability, instability, and weak water solubility. On the contrary, using nano-based material, e.g., nanoparticles (NPs), micelles, liposomes, can significantly solve these limitations. Therefore, in the present study, we will summarize the anti-cancer aspects of these herbal and-nano-based herbal formulations with a focus on their mechanisms against OCa. Full article
(This article belongs to the Section Natural Products)
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<p>Three main theories regarding the development of ovarian cancer are based on induction of the epithelium of ovarian surface by hormonal receptors, increased induction of pro-inflammatory agents during continuous ovulation, and cancerous cells originating from the fallopian tube. IL-8, Interleukin-8; CCL2/MCP-1, Monocyte chemoattractant protein-1; CCL5/RANTES, CC Chemokine Ligand-5.</p> Full article ">Figure 2
<p>Different endogenous and exogenous factors modify the development and prognosis of ovarian cancer. IL-1β, IL-6, and TNF-α accelerate the growth of cancer cells and affect the prognosis and clinical status of the disease via increasing resistance to chemotherapy and stimulating symptoms. Exogenous factors, such as hypoxia, infection, and chronic inflammation, are the main sources of oxidative stress, namely reactive nitrogen species (RNS) and reactive oxygen species (ROS). They can contribute to ovarian cancer development via genetic instability enhancement, angiogenesis promotion, and abnormality in cell proliferation. One of the most important features of ovarian cancer is genetic changes that mediate the development and progression of tumors. In ovarian cancer, the presence of mutations in <span class="html-italic">PTEN</span>, <span class="html-italic">P53</span>, <span class="html-italic">BRCA1</span><span class="html-italic">,</span> and <span class="html-italic">BRCA2</span> genes, tumor suppressor factors, can lead to ovarian cancer development. IL-1β, Interleukin-1β; IL-6, Interleukin-6; TNF-α, Tumor necrosis factor α; PTEN, Phosphatase and tensin homolog; BRCA1, Breast cancer type 1; BRCA2, Breast cancer type 1.</p> Full article ">Figure 3
<p>Curcumin (CUR) and Quercetin (Que) can exert an anti-cancerous effect on ovarian cancer in many different pathways. CUR triggers AMP-activated protein kinase (AMPK) that leads to stimulation of cell apoptosis and inhibition of cell proliferation. Moreover, CUR can decrease pro-caspase-3, Bcl-XL, and Bcl-2 levels, whereas Bax and p53 levels rise in the treated cells with CUR. These changes lead to ovarian cancer treatment. Furthermore, CUR can exert a significant inhibitory effect on STAT3 and NF-ĸB signaling pathways. Quercetin (Que) can modify many pathways and play a role in ovarian cancer treatment. Que decreases the anti-apoptotic agents, like Bcl-2, Bcl-xL, while it elevates the expression of pro-apoptotic agents, such as Bad and Bid, leading to increased apoptosis and ovarian cancer treatment. In addition, the elevation of cytosolic Ca<sup>2+</sup> levels due to Que consumption can take part in ovarian cancer treatment. Que triggers autophagy by endoplasmic reticulum (ER) stress by the p-STAT3/Bcl-2 axis as well. Bcl-XL, B-cell lymphoma-extra-large; BAX, BCL2-associated X protein; Bcl-2, B-cell lymphoma 2; Bad, BCL2 associated agonist of cell death; Bid, BH3-interacting domain death agonist; STAT, Signal transducer and activator of transcription; NF-ĸB, Nuclear factor-kappaB.</p> Full article ">Figure 4
<p>Resveratrol (Res) can trigger various mechanisms involved in ovarian cancer treatment. Res can stimulate mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) that leads to inhibition of NF-ĸB pathway and subsequently contributes to inflammation suppression and ovarian cancer improvement. Furthermore, the administration of Res can stimulate apoptosome complex formation, caspase activation, and mitochondrial secretion of cytochrome c, which all end in inhibition of growth and stimulation of cell death. In addition, Res suppresses glycolysis in ovarian cancer cells that can be effective in ovarian cancer treatment. Res can act against ovarian cancer through AMPK activation, downregulation of the protein cyclin D1, and inhibition of EMT, STAT3, Notch, and Wnt signaling pathways, leading to ovarian cancer treatment. Moreover, consumption of Res—(Zinc oxide) ZnO nanohybrid can lead to the generation of ROS in ovarian cancer cell lines and exert anti-cancer effects on ovarian cancer. In addition, Res–bovine serum albumin (BSA) NPs induce ovarian cancer cell necrosis and cellular apoptosis, attenuating tumor growth in ovarian cancer. EMT, Epithelial–mesenchymal transition; Cyst C, Cytochrome c; STAT3, Signal transducer, and activator of transcription 3; NF-ĸB, Nuclear factor-kappaB.</p> Full article ">
13 pages, 2504 KiB  
Article
Stable Luminescent Poly(Allylaminehydrochloride)-Templated Copper Nanoclusters for Selectively Turn-Off Sensing of Deferasirox in β-Thalassemia Plasma
by Hung-Ju Lin, Chun-Chi Wang, Hwang-Shang Kou, Cheng-Wei Cheng and Shou-Mei Wu
Pharmaceuticals 2021, 14(12), 1314; https://doi.org/10.3390/ph14121314 - 16 Dec 2021
Cited by 3 | Viewed by 2420
Abstract
Highly stable and facile one-pot copper nanoclusters (Cu NCs) coated with poly(allylamine hydrochloride) (PAH) have been synthesized for selectively sensing deferasirox (DFX) in β-thalassemia plasma. DFX is an important drug used for treating iron overloading in β-thalassemia, but needs to be monitored due [...] Read more.
Highly stable and facile one-pot copper nanoclusters (Cu NCs) coated with poly(allylamine hydrochloride) (PAH) have been synthesized for selectively sensing deferasirox (DFX) in β-thalassemia plasma. DFX is an important drug used for treating iron overloading in β-thalassemia, but needs to be monitored due to certain toxicity. In this study, the PAH-Cu NCs showed highly stable fluorescence with emission wavelengths at 450 nm. The DFX specifically interacted with the copper nanocluster to turn off the fluorescence of the PAH-Cu NCs, and could be selectively quantified through the fluorescence quenching effect. The linear range of DFX in plasma analyzed by PAH-Cu NCs was 1.0–100.0 µg/mL (r = 0.985). The relative standard deviation (RSD) and relative error (RE) were lower than 6.51% and 7.57%, respectively, showing excellent reproducibility of PAH-Cu NCs for sensing DFX in plasma. This method was also successfully applied for an analysis of three clinical plasma samples from β-thalassemia patients taking DFX. The data presented high similarity with that obtained through a capillary electrophoresis method. According to the results, the PAH-Cu NCs could be used as a tool for clinically sensing DFX in human plasma for clinical surveys. Full article
(This article belongs to the Special Issue Synthesis of Metal Nanoparticles and Their Pharmaceutical Applications)
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Graphical abstract
Full article ">Figure 1
<p>The effects of several conditions, including (<b>A</b>) reaction temperature and reaction time; (<b>B</b>) pH values; (<b>C</b>) PAH percentage; (<b>D</b>) LAA concentration, for the synthesis of PAH-Cu NCs.</p> Full article ">Figure 2
<p>The properties of the PAH-Cu NCs of (<b>A</b>) fluorescence emission spectra of PAH-Cu NCs at different excitation wavelengths; (<b>B</b>) excitation (360 nm) and emission (450 nm) wavelength of PAH-Cu NCs; (<b>C</b>) TEM image of PAH-Cu NCs; (<b>D</b>) FT-IR spectra of PAH and PAH-Cu NCs; (<b>E</b>) XPS spectra of PAH-Cu NCs; (<b>F</b>) XPS Cu 2p spectrum of Cu NCs.</p> Full article ">Figure 3
<p>Selectivity evaluation of the PAH-Cu NCs in (<b>A</b>) standard solution and (<b>B</b>) human plasma. All the tested analyte concentrations were 100 μg/mL.</p> Full article ">Figure 4
<p>(<b>A</b>) TEM images of PAH-Cu NCs in the presence of 50 μg/mL DFX; (<b>B</b>) FT-IR spectra of PAH-Cu NCs presented in different concentrations of DFX (5, 10, 30, and 50 μg/mL).</p> Full article ">Figure 5
<p>(<b>A</b>) Fluorescence emission spectra of PAH-Cu NCs with different concentrations of DFX from 1 to 100 μg/mL; (<b>B</b>) the calibration curve of the logarithmic of the DFX concentration versus the fluorescence intensity of PAH-Cu NCs.</p> Full article ">Scheme 1
<p>Schematic illustration of PAH-Cu NCs for sensing DFX in plasma.</p> Full article ">
28 pages, 4744 KiB  
Article
Promising Antiviral Activity of Agrimonia pilosa Phytochemicals against Severe Acute Respiratory Syndrome Coronavirus 2 Supported with In Vivo Mice Study
by Nashwah G. M. Attallah, Aya H. El-Kadem, Walaa A. Negm, Engy Elekhnawy, Thanaa A. El-Masry, Elshaymaa I. Elmongy, Najla Altwaijry, Ashwag S. Alanazi, Gadah Abdulaziz Al-Hamoud and Amany E. Ragab
Pharmaceuticals 2021, 14(12), 1313; https://doi.org/10.3390/ph14121313 - 16 Dec 2021
Cited by 30 | Viewed by 3730
Abstract
The global emergence of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused the entire world’s attention toward searching for a potential remedy for this disease. Thus, we investigated the antiviral activity of Agrimonia pilosa ethanol extract (APEE) [...] Read more.
The global emergence of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused the entire world’s attention toward searching for a potential remedy for this disease. Thus, we investigated the antiviral activity of Agrimonia pilosa ethanol extract (APEE) against SARS-CoV-2 and it exhibited a potent antiviral activity with IC50 of 1.1 ± 0.03 µg/mL. Its mechanism of action was elucidated, and it exhibited a virucidal activity and an inhibition of viral adsorption. Moreover, it presented an immunomodulatory activity as it decreased the upregulation of gene expression of COX-2, iNOS, IL-6, TNF-α, and NF-κB in lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells. A comprehensive analysis of the phytochemical fingerprint of APEE was conducted using LC-ESI-MS/MS technique for the first time. We detected 81 compounds and most of them belong to the flavonoid and coumarin classes. Interestingly, isoflavonoids, procyanidins, and anthocyanins were detected for the first time in A. pilosa. Moreover, the antioxidant activity was evidenced in DPPH (IC50 62.80 µg/mL) and ABTS (201.49 mg Trolox equivalents (TE)/mg) radical scavenging, FRAP (60.84 mg TE/mg), and ORAC (306.54 mg TE/g) assays. Furthermore, the protective effect of APEE was investigated in Lipopolysaccharides (LPS)-induced acute lung injury (ALI) in mice. Lung W/D ratio, serum IL-6, IL-18, IL-1β, HO-1, Caspase-1, caspase-3, TLR-4 expression, TAC, NO, MPO activity, and histopathological examination of lung tissues were assessed. APEE induced a marked downregulation in all inflammation, oxidative stress, apoptosis markers, and TLR-4 expression. In addition, it alleviated all histopathological abnormalities confirming the beneficial effects of APEE in ALI. Therefore, APEE could be a potential source for therapeutic compounds that could be investigated, in future preclinical and clinical trials, in the treatment of patients with COVID-19. Full article
(This article belongs to the Special Issue COVID-19 in Pharmaceuticals)
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Figure 1
<p>The structures and fragmentation pattern in positive ion mode for the identified aglycones.</p> Full article ">Figure 2
<p>A graph showing the cytotoxicity of APEE on Vero-E6 cells using MTT assay to determine CC<sub>50</sub>. The results are expressed as mean ± SD as the experiments were performed in three independent triplicates.</p> Full article ">Figure 3
<p>A curve showing the effect of APEE different concentrations on the viability of NRC-03-nhCoV. The results are expressed as mean ± SD as the experiments were performed in three independent triplicates.</p> Full article ">Figure 4
<p>A graph showing cytotoxicity APEE on PBMCs using MTT to determine IC<sub>50</sub>. The results are expressed as mean ± SD as the experiments were performed in three independent triplicates.</p> Full article ">Figure 5
<p>A chart representing the impact of APEE on the expression of the genes encoding COX-2, iNOS, IL-6, TNF-α, and NF-κB in the LPS-induced PBMCs. The results are expressed as mean ± SD as the experiments were performed in three independent triplicates.</p> Full article ">Figure 6
<p>Impact of APEE pre-treatment on (<b>A</b>) Lung IL-1β level, (<b>B</b>) Serum IL-6 level (<b>C</b>) IL-18 gene expression level, (<b>D</b>) IL-10 gene expression level. Acute lung injury was urged by I.P. injection of LPS (10 mg/kg). APEE 200, 250, and 300 were given I.P. 30 min before LPS injection. Results were expressed as mean ± SD (<span class="html-italic">n</span> = 10/group) as the experiments were performed in three independent triplicates. Significant difference vs. a respective control, b respective LPS group, c respective APEE 200 group, d respective APEE 300 group each at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6 Cont.
<p>Impact of APEE pre-treatment on (<b>A</b>) Lung IL-1β level, (<b>B</b>) Serum IL-6 level (<b>C</b>) IL-18 gene expression level, (<b>D</b>) IL-10 gene expression level. Acute lung injury was urged by I.P. injection of LPS (10 mg/kg). APEE 200, 250, and 300 were given I.P. 30 min before LPS injection. Results were expressed as mean ± SD (<span class="html-italic">n</span> = 10/group) as the experiments were performed in three independent triplicates. Significant difference vs. a respective control, b respective LPS group, c respective APEE 200 group, d respective APEE 300 group each at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>Impact of APEE pre-treatment on (<b>A</b>) HO-1 expression level, (<b>B</b>) Caspase-1 expression level (<b>C</b>) Caspase-3 expression level, (<b>D</b>) Lung Histology score. Acute lung injury was urged by I.P. injection of LPS (10 mg/kg). APEE 200, 250, and 300 were given I.P. 30 min before LPS injection. Results were expressed as mean ± SD (<span class="html-italic">n</span> = 10/group) as the experiments were performed in three independent triplicates. Significant difference vs. a respective control, b respective LPS group, c respective APEE 200 group, d respective APEE 300 group each at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 8
<p>Effect of APEE pre-treatment on the expression of TLR-4 in the lung tissues. The expression levels were measured by western blotting. Acute lung injury was urged by I.P. injection of LPS (10 mg/kg). APEE 200, 250, and 300 were given I.P. 30 min before LPS injection. Results were expressed as mean ± SD (<span class="html-italic">n</span> = 10/group) as the experiments were performed in three independent triplicates. Significant difference vs. a respective control, b respective LPS group, c respective APEE 200 group each at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 9
<p>Histopathological examination of H&amp;E-stained sections of lung tissue indicates the influence of APEE treatment on LPS-induced ALI. (<b>A</b>) A section in lung of the normal control group indicated normal-sized alveoli separated by fibrous septa (blue arrows) and normal-sized bronchiole (black arrow) (H&amp;E X 100). (<b>B</b>) Section in lung of LPS group showed dilated bronchiole (blue arrow) surrounded by marked chronic inflammation and pneumonia (green arrow) and congested vessels (red arrow) (H&amp;E X 200). (<b>C</b>) Section in lung of LPS group showed dilated destructed alveolar walls (emphysema) (red arrow) surrounded by destructed bronchioles (green arrow) and alveolar congestion with fibrosis (blue arrows) (H&amp;E X 100). (<b>D</b>) Section in lung of APEE 200 treated group showed dilated bronchioles (red arrows) surrounded by decreased interstitial inflammation to moderate degree (blue arrows), congested vessels (green arrows) and decreased emphysema (black arrow) (H&amp;E X 100). (<b>E</b>) Section in lung of APEE 250 treated group showed marked remission of inflammation with average-sized of a bronchiole (blue arrow) surrounded by normal-sized alveoli (red arrow) with few congested vessels (black arrow) (H&amp;E X 200). (<b>F</b>) Section in lung of APEE 300 treated group showed focal inflammation (red arrow) surrounded by average-sized of a bronchiole (black arrow) surrounded by normal-sized alveoli (green arrow) with many congested vessels (blue arrows) (H&amp;E X 100).</p> Full article ">Figure 9 Cont.
<p>Histopathological examination of H&amp;E-stained sections of lung tissue indicates the influence of APEE treatment on LPS-induced ALI. (<b>A</b>) A section in lung of the normal control group indicated normal-sized alveoli separated by fibrous septa (blue arrows) and normal-sized bronchiole (black arrow) (H&amp;E X 100). (<b>B</b>) Section in lung of LPS group showed dilated bronchiole (blue arrow) surrounded by marked chronic inflammation and pneumonia (green arrow) and congested vessels (red arrow) (H&amp;E X 200). (<b>C</b>) Section in lung of LPS group showed dilated destructed alveolar walls (emphysema) (red arrow) surrounded by destructed bronchioles (green arrow) and alveolar congestion with fibrosis (blue arrows) (H&amp;E X 100). (<b>D</b>) Section in lung of APEE 200 treated group showed dilated bronchioles (red arrows) surrounded by decreased interstitial inflammation to moderate degree (blue arrows), congested vessels (green arrows) and decreased emphysema (black arrow) (H&amp;E X 100). (<b>E</b>) Section in lung of APEE 250 treated group showed marked remission of inflammation with average-sized of a bronchiole (blue arrow) surrounded by normal-sized alveoli (red arrow) with few congested vessels (black arrow) (H&amp;E X 200). (<b>F</b>) Section in lung of APEE 300 treated group showed focal inflammation (red arrow) surrounded by average-sized of a bronchiole (black arrow) surrounded by normal-sized alveoli (green arrow) with many congested vessels (blue arrows) (H&amp;E X 100).</p> Full article ">
21 pages, 3739 KiB  
Review
Advances in Antifungal Drug Development: An Up-To-Date Mini Review
by Ghada Bouz and Martin Doležal
Pharmaceuticals 2021, 14(12), 1312; https://doi.org/10.3390/ph14121312 - 16 Dec 2021
Cited by 63 | Viewed by 8283
Abstract
The utility of clinically available antifungals is limited by their narrow spectrum of activity, high toxicity, and emerging resistance. Antifungal drug discovery has always been a challenging area, since fungi and their human host are eukaryotes, making it difficult to identify unique targets [...] Read more.
The utility of clinically available antifungals is limited by their narrow spectrum of activity, high toxicity, and emerging resistance. Antifungal drug discovery has always been a challenging area, since fungi and their human host are eukaryotes, making it difficult to identify unique targets for antifungals. Novel antifungals in clinical development include first-in-class agents, new structures for an established target, and formulation modifications to marketed antifungals, in addition to repurposed agents. Membrane interacting peptides and aromatherapy are gaining increased attention in the field. Immunotherapy is another promising treatment option, with antifungal antibodies advancing into clinical trials. Novel targets for antifungal therapy are also being discovered, allowing the design of new promising agents that may overcome the resistance issue. In this mini review, we will summarize the current status of antifungal drug pipelines in clinical stages, and the most recent advancements in preclinical antifungal drug development, with special focus on their chemistry. Full article
(This article belongs to the Special Issue Advances in Antifungal Development: Discovery of New Drugs and Drug Repurposing)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Clinically used antifungals grouped according to their mechanism of action with the most common agents in practice as examples.</p> Full article ">Figure 2
<p>The chemical structures of amphotericin B (an example of a polyene); caspofungin (an example of an echinocandin); flucytosine; fluconazole (an example of an azole); and tavaborole.</p> Full article ">Figure 3
<p>A summative figure showing the classification of new molecules as antifungals in clinical development.</p> Full article ">Figure 4
<p>The chemical structures of (<b>a</b>) T-2307 and (<b>b</b>) pentamidine. The characteristic aryldiamidine moiety is highlighted in grey.</p> Full article ">Figure 5
<p>The chemical structures of (<b>a</b>) 1-[4-butylbenzyl]isoquinoline, (<b>b</b>) manogepix, and (<b>c</b>) fosmanogepix.</p> Full article ">Figure 6
<p>The chemical structures of (<b>a</b>) Nikkomycin Z and (<b>b</b>) Uridine Diphosphate (UDP)-<span class="html-italic">N</span>-acetyl glucosamine.</p> Full article ">Figure 7
<p>The chemical structures of olorofim.</p> Full article ">Figure 8
<p>The chemical structures of (<b>a</b>) VL-2397 and (<b>b</b>) ferrichrome siderophore. To better visualize the structural resemblance, common fragments with ferrochrome siderophore are highlighted in grey.</p> Full article ">Figure 9
<p>The chemical structure of Aureobasidin A.</p> Full article ">Figure 10
<p>Chemical structures of (<b>a</b>) VT-1129, (<b>b</b>) VT-1161, and (<b>c</b>) VT-1598. Differences in chemical structures are highlighted in grey.</p> Full article ">Figure 11
<p>The chemical structures of (<b>a</b>) enfumafungin and (<b>b</b>) ibrexafungerp. The triterpenoid unit is shaded in grey.</p> Full article ">Figure 12
<p>Hydrolysis reaction of the prodrug (<b>a</b>) isavuconazonium by mammalian plasma esterases to yield, (<b>b</b>) isavuconazole, and (<b>c</b>) by-product.</p> Full article ">Figure 13
<p>The chemical structures of (<b>a</b>) albaconazole and (<b>b</b>) iodiconazole.</p> Full article ">Figure 14
<p>The chemical structures of (<b>a</b>) BSG005 and (<b>b</b>) nystatin. The structural modifications are shown in red.</p> Full article ">Figure 15
<p>The chemical structures of (<b>a</b>) anidulafungin and (<b>b</b>) rezafungin. The chemical modification in the structure of rezafungin over anidulafungin is highlighted in grey.</p> Full article ">Figure 16
<p>The chemical structure of the broad-spectrum chelator diethylenetriamine pentaacetate (DTPA).</p> Full article ">Figure 17
<p>The chemical structures of (<b>a</b>) tamoxifen, (<b>b</b>) sertraline, and (<b>c</b>) AR-12.</p> Full article ">Figure 18
<p>The chemical structures of (<b>a</b>) australifungin, (<b>b</b>) galbonolide A, (<b>c</b>) D-threo-PDMP, and (<b>d</b>) BHBM.</p> Full article ">
17 pages, 217175 KiB  
Article
Expanding the Diversity at the C-4 Position of Pyrido[2,3-d]pyrimidin-7(8H)-ones to Achieve Biological Activity against ZAP-70
by Victor Masip, Ángel Lirio, Albert Sánchez-López, Ana B. Cuenca, Raimon Puig de la Bellacasa, Pau Abrisqueta, Jordi Teixidó, José I. Borrell, Albert Gibert and Roger Estrada-Tejedor
Pharmaceuticals 2021, 14(12), 1311; https://doi.org/10.3390/ph14121311 - 15 Dec 2021
Cited by 1 | Viewed by 2810
Abstract
Pyrido[2,3-d]pyrimidin-7(8H)-ones have attracted widespread interest due to their similarity with nitrogenous bases found in DNA and RNA and their potential applicability as tyrosine kinase inhibitors. Such structures, presenting up to five diversity centers, have allowed the synthesis of a [...] Read more.
Pyrido[2,3-d]pyrimidin-7(8H)-ones have attracted widespread interest due to their similarity with nitrogenous bases found in DNA and RNA and their potential applicability as tyrosine kinase inhibitors. Such structures, presenting up to five diversity centers, have allowed the synthesis of a wide range of differently substituted compounds; however, the diversity at the C4 position has mostly been limited to a few substituents. In this paper, a general synthetic methodology for the synthesis of 4-substituted-2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones is described. By using cross-coupling reactions, such as Ullmann, Buchwald–Hartwig, Suzuki–Miyaura, or Sonogashira reactions, catalyzed by Cu or Pd, we were able to describe new potential biologically active compounds. The resulting pyrido[2,3-d]pyrimidin-7(8H)-ones include N-alkyl, N-aryl, O-aryl, S-aryl, aryl, and arylethynyl substituents at C4, which have never been explored in connection with the biological activity of such heterocycles as tyrosine kinase inhibitors, in particular as ZAP-70 inhibitors. Full article
(This article belongs to the Special Issue Heterocyclic Compounds and Their Application in Therapy)
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Figure 1
<p>Pyrido[2,3-<span class="html-italic">d</span>]pyrimidin-7(8<span class="html-italic">H</span>)-ones <b>1</b> and diversity centers of such a scaffold (<b>2</b>).</p> Full article ">Figure 2
<p>Synthetic approaches for pyrido[2,3-<span class="html-italic">d</span>]pyrimidin-7(8<span class="html-italic">H</span>)-ones <b>1</b> from a preformed pyrimidine ring.</p> Full article ">Figure 3
<p>Synthetic approach for pyrido[2,3-<span class="html-italic">d</span>]pyrimidin-7(8<span class="html-italic">H</span>)-ones (<b>10</b>) from a preformed pyridone ring (<b>left</b>). The working hypothesis developed in the present work (<b>right</b>).</p> Full article ">Figure 4
<p>Synthesis of 4-amino-2-(phenylamino)-5,6-dihydropyrido[2,3-<span class="html-italic">d</span>]pyrimidin-7(8<span class="html-italic">H</span>)-one (<b>13</b>). Reaction conditions (a): MW (10 min, 140 °C), dry methanol.</p> Full article ">Figure 5
<p>Synthesis of substrates for C4 decoration. Reaction conditions (a): <span class="html-italic">t</span>-BuONO, H<sub>2</sub>O:DMF (1:5), MW (10 min, 65 °C). (b): BOP, DBU, ACN, 2 days RT. (c): (CF<sub>3</sub>SO<sub>2</sub>)<sub>2</sub>O, dry pyridine, 30 min RT. (d): dry NaI, CH<sub>3</sub>COCl, dry ACN, MW (5 h, 80 °C).</p> Full article ">Figure 6
<p>Synthesis of C4 <span class="html-italic">N</span>-alkyl (18) and <span class="html-italic">N</span>-aryl (19) derivatives from structures 15 and 16. Reaction conditions (a): corresponding amine, ACN, MW (6 h, 140 °C). (b): corresponding amine, ACN, 8–16 h, 100 °C. (c): corresponding aniline, Cs<sub>2</sub>CO<sub>3</sub>, Pd(OAc)<sub>2</sub>, Xphos, dry toluene, O/N, 100 °C.</p> Full article ">Figure 7
<p>Synthesis of C4-substituted <span class="html-italic">O</span>-aryl <b>20</b>, <span class="html-italic">S</span>-aryl <b>21</b>, aryl <b>22</b>, alkylethynyl <b>23</b>, ethynyl <b>24</b>, and arylethinyl <b>25-</b>substituted derivatives from structure <b>17</b>. Reaction conditions (a): corresponding phenol or thiophenol, K<sub>3</sub>PO<sub>4</sub>, CuI, 2-picolinic acid, dry DMSO, O/N, 80 °C. (b): corresponding boronic acid, Cs<sub>2</sub>CO<sub>3</sub>, Pd(PPh<sub>3</sub>)<sub>4</sub>, deoxygenated mixture of 1,4-dioxane/water (10:1), O/N, 90 °C. (c): Sonogashira-type reactions; see the detailed reaction conditions in <a href="#pharmaceuticals-14-01311-f008" class="html-fig">Figure 8</a>.</p> Full article ">Figure 8
<p>(a): Corresponding alkyne (excess reagent), CuI, PdCl<sub>2</sub>(PPh<sub>3</sub>)<sub>2</sub>, Et<sub>3</sub>N, O/N, 65 °C. (b): 1M TBAF/THF, 3 h, RT. (c): Corresponding iodoaryl (excess reagent), CuI, PdCl<sub>2</sub>(PPh<sub>3</sub>)<sub>2</sub>, Et<sub>3</sub>N, 2 days, 65 °C.</p> Full article ">Figure 9
<p>Residual activity percentages at a concentration of 10 µM of the tested compounds (&lt;20% in green, 20–60% yellow, 60–80% red, and &gt;80% white). Results are classified according to the corresponding family of compounds (<b>18</b>, <b>19</b>, <b>20</b>, <b>22</b> and <b>23</b>, <b>24</b>, <b>25</b>).</p> Full article ">Figure 10
<p>Interaction mechanism between one of the compounds with C4 substitution and the pocket of the ZAP-70 protein, predicted by molecular docking. The presence of the C4 substituent clearly allows the molecule to improve the binding on the active site (white arrow).</p> Full article ">
12 pages, 1623 KiB  
Article
Mulberry Leaf Polyphenol Extract and Rutin Induces Autophagy Regulated by p53 in Human Hepatoma HepG2 Cells
by Meng-Hsun Yu, Ming-Chang Tsai, Chi-Chih Wang, Sheng-Wen Wu, Ya-Ju Chang, Cheng-Hsun Wu and Chau-Jong Wang
Pharmaceuticals 2021, 14(12), 1310; https://doi.org/10.3390/ph14121310 - 15 Dec 2021
Cited by 11 | Viewed by 2975
Abstract
The edible leaves of the mulberry (Morus alba L.) plant are used worldwide. They contain abundant polyphenolic compounds with strong anticancer properties. We previously revealed that apoptosis was mediated in p53-negative Hep3B cells, and mulberry leaf polyphenol extract (MLPE) induced autophagy in [...] Read more.
The edible leaves of the mulberry (Morus alba L.) plant are used worldwide. They contain abundant polyphenolic compounds with strong anticancer properties. We previously revealed that apoptosis was mediated in p53-negative Hep3B cells, and mulberry leaf polyphenol extract (MLPE) induced autophagy in p53-transfected Hep3B cells. However, how this autophagy is induced by p53 in human hepatoma HepG2 (p53 wild type) cells remains unclear. In the current study, MLPE induced autophagy, as demonstrated by enhanced acidic vesicular organelle staining, by upregulating beclin-1, increasing LC3-II conversion, and phosphorylating AMPK. In HepG2 cells, these processes were associated with p53. Western blot also revealed phosphatidylinositol-3 kinase (PI3K), p-AKT, and fatty acid synthase (FASN) suppression in MLPE-treated cells. Moreover, treatment with the p53 inhibitor pifithrin-α (PFT-α) inhibited autophagy and increased apoptotic response in MLPE-treated HepG2 cells. PFT-α treatment also reversed MLPE-induced PI3K, p-AKT, and FASN suppression. Thus, co-treatment with MLPE and PFT-α significantly increased caspase-3, caspase-8, and cytochrome c release, indicating that p53 deficiency caused the apoptosis. In addition, rutin, a bioactive polyphenol in MLPE, may affect autophagy in HepG2 cells. This study demonstrates that MLPE is a potential anticancer agent targeting autophagy and apoptosis in cells with p53 status. Moreover, this work provides insight into the mechanism of p53 action in MLPE-induced cytotoxicity in hepatocellular carcinoma. Full article
(This article belongs to the Section Natural Products)
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Figure 1

Figure 1
<p>Composition and characterization of phenolic compounds contained in MLPE. The HPLC chromatogram and absorbance at 280 nm for polyphenols were monitored for MLPE.</p> Full article ">Figure 2
<p>Autophagy-dependent p53 effects of MLPE on HepG2 cells. (<b>A</b>) IC50 of MLPE on the viability in different cell lines. (<b>B</b>) HepG2 cells were treated with 0, 0.25, 0.5 mg/mL of MLPE and solvent for 24 h and subjected to AVO staining. The arrow indicated AVO cells. AVOs values were calculated as the percentage of AVO cells relative to the total number of cells in each random field. Results were statistically analyzed with Student’s t-test. Magnification: 400×. (<b>C</b>) HepG2 cells were pretreated with PFTα for 6 h and treated with 0, 0.5 mg/mL of MLPE and solvent for 24 h and subjected to DAPI/AO staining. (<b>D</b>) Apoptosis effects of MLPE on HepG2 cells. HepG2 cells were treated with 0, 0.25, 0.5, 1, 2 mg/mL of MLPE and solvent (0.05% EtOH) for 24 h and subjected to flow cytometric analysis after PI staining. The figure shows a representative staining profile for 10,000 cells per experiment. Sub G1 was defined as apoptotic cells and represents the average of three independent experiments ±SD, <span class="html-italic">n</span> = 3. *, <span class="html-italic">p</span> &lt; 0.05 compared with E. Magnification: 100×. The figure shows a representative staining profile for 8000 cells per experiment. C, control; E, ethanol.</p> Full article ">Figure 3
<p>Immunoblot analysis of the autophagosome induction of autophagy in HepG2. Cultured cells were treated with 0, 0.125, 0.25, 0.5 mg/mL of MLPE, PFT α for 6 h and treated with various concentrations of MLPE and solvent (0.05% EtOH) for 24 h, and whole-cell extracts were prepared as described in Materials and Methods. Equal amounts of total proteins were loaded in each lane of SDS-polyacrylamide gel (protein concentration is 50 μg/μL). Western hybridization was performed with antibodies against (<b>A</b>,<b>B</b>) PI3K, p-Akt/Akt, Bcl-2, Beclin-1, LC3-I, and LC3-II. (<b>C</b>) Cytochrome C, pro-caspase 3, and pro-caspase 8. (<b>D</b>,<b>E</b>) p-AMPK/AMPK, FASN, and p-P53/P53. Western blot analysis of β-actin was used as an internal control and represents the average of three independent experiments ±SD, <span class="html-italic">n</span> = 3. *, <span class="html-italic">p</span> &lt; 0.05 compared with (<b>E</b>).</p> Full article ">Figure 4
<p>Rutin induced autophagy-dependent p53 in HepG2 cells. (<b>A</b>) HepG2 cells were treated with PFT α for 6 h and treated with 1 mM rutin, 0.1 mM of astragalin for 24 h and subjected to AVO staining. The arrow indicated AVO cells. AVOs values were calculated as the percentage of AVOs cells relative to the total number of cells in each random field. Results were statistically analyzed with Student’s <span class="html-italic">t</span>-test. Magnification: 400×. (<b>B</b>) Cultured cells were treated with 1 mM rutin, 0.1 mM of astragalin, and PFT-α for 24 h, and whole-cell extracts were prepared as described in Materials and Methods. Equal amounts of total proteins were loaded in each lane of SDS-polyacrylamide gel (protein concentration is 50 μg/μL). Western hybridization was performed with antibodies against p-P53/P53, pAkt/Akt, Bcl-2, LC3-I, and LC3-II. (<b>C</b>) PI3K, pAkt/Akt, Bcl-2, Beclin-1, LC3-I, and LC3II. (<b>D</b>) Pro-caspase 3, and pro-caspase 8. (<b>E</b>) pAMPK/AMPK, FASN, and p-P53/P53. Western blot analysis of β-actin was used as an internal control and represents the average of three independent experiments ±SD, <span class="html-italic">n</span> = 3. #, <span class="html-italic">p</span> &lt; 0.05 compared with C. *, <span class="html-italic">p</span> &lt; 0.05 compared PFT α.</p> Full article ">Figure 5
<p>Schematic diagram illustrating the p53-dependent autophagy of HepG2 cell treated with MLPE or rutin.</p> Full article ">
16 pages, 1884 KiB  
Article
Development of an Icariin-Loaded Bilosome-Melittin Formulation with Improved Anticancer Activity against Cancerous Pancreatic Cells
by Nabil A. Alhakamy, Shaimaa M. Badr-Eldin, Waleed S. Alharbi, Mohamed A. Alfaleh, Omar D. Al-hejaili, Hibah M. Aldawsari, Basma G. Eid, Rana Bakhaidar, Filippo Drago, Filippo Caraci and Giuseppe Caruso
Pharmaceuticals 2021, 14(12), 1309; https://doi.org/10.3390/ph14121309 - 15 Dec 2021
Cited by 7 | Viewed by 2826
Abstract
Pancreatic cancer currently represents a severe issue for the entire world. Therefore, much effort has been made to develop an effective treatment against it. Emerging evidence has shown that icariin, a flavonoid glycoside, is an effective anti-pancreatic cancer drug. Melittin, as a natural [...] Read more.
Pancreatic cancer currently represents a severe issue for the entire world. Therefore, much effort has been made to develop an effective treatment against it. Emerging evidence has shown that icariin, a flavonoid glycoside, is an effective anti-pancreatic cancer drug. Melittin, as a natural active biomolecule, has also shown to possess anticancer activities. In the present study, with the aim to increase its effectiveness against cancerous cells, icariin-loaded bilosome-melittin (ICA-BM) was developed. For the selection of an optimized ICA-BM, an experimental design was implemented, which provided an optimized formulation with a particle size equal to 158.4 nm. After estimation of the release pattern, the anti-pancreatic cancer efficacy of this new formulation was evaluated. The MTT assay was employed for the determination of half maximal inhibitory concentration (IC50), providing smaller IC50 for ICA-BM (2.79 ± 0.2 µM) compared to blank-BM and ICA-Raw (free drug) against PNAC1, a human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin. Additionally, cell cycle analysis for ICA-BM demonstrated cell arrest at the S-phase and pre-G1 phase, which indicated a pro-apoptotic behavior of the new developed formulation. The pro-apoptotic and anti-proliferative activity of the optimized ICA-BM against PNAC1 cells was also demonstrated through annexin V staining as well as estimation of caspase-3 and p53 protein levels. It can be concluded that the optimized ICA-BM formulation significantly improved the efficacy of icariin against cancerous pancreatic cells. Full article
(This article belongs to the Section Biopharmaceuticals)
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<p>Preto chart for particle size of ICA-BM formulations, where X1, X2, and X3 represent the concentration of cholesterol:Span 20 ratio, bile salt, and MEL.</p> Full article ">Figure 2
<p>Contours of an estimated response surface for the particle size.</p> Full article ">Figure 3
<p>In vitro release profile of optimized ICA-BM.</p> Full article ">Figure 4
<p>IC<sub>50</sub> of the Blank BM, erlotinib, ICA-Raw, and ICA-BM in the PANC1 cells. Data are the mean of 4 independent experiments ± standard deviation (SD). <sup>ϕϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.001); <sup>θθθ</sup> Significantly different vs. Erlotinib (<span class="html-italic">p</span> &lt; 0.001); <sup>###</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>Effect of Blank BM, ICA-Raw, and ICA-BM on PANC1 cell cycle phases. Data are the mean of 4 independent experiments ± SD. * Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.05); ** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.01); *** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.001); <sup>ϕϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.001); <sup>###</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Effect of Blank BM, ICA-Raw, and ICA-BM on the percentage of apoptotic or necrotic PANC1 cells. Data are the mean of 4 independent experiments ± SD. * Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.05); ** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.01); *** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.001); <sup>ϕϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.001); <sup>##</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.01); <sup>###</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 7
<p>Effect of Blank BM, ICA-Raw, and ICA-BM treatments on caspase-3 enzyme content in PANC1 cells. Data are the mean of 4 independent experiments ± SD. *** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.001); <sup>ϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.01); <sup>ϕϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.001); <sup>###</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 8
<p>Effect of Blank BM, ICA-Raw, and ICA-BM treatments on p53 content in PANC1 cells. Data are the mean of 4 independent experiments ± SD. *** Significantly different vs. Control (<span class="html-italic">p</span> &lt; 0.001); <sup>ϕϕϕ</sup> Significantly different vs. Blank BM (<span class="html-italic">p</span> &lt; 0.001); <sup>###</sup> Significantly different vs. ICA-Raw (<span class="html-italic">p</span> &lt; 0.001).</p> Full article ">
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