Pharmaceuticals

11 pages, 9689 KiB

Open AccessArticle

APELA Expression in Glioma, and Its Association with Patient Survival and Tumor Grade

by Debolina Ganguly, Chun Cai, Michelle M. Sims, Chuan He Yang, Matthew Thomas, Jinjun Cheng, Ali G. Saad and Lawrence M. Pfeffer

Pharmaceuticals 2019, 12(1), 45; https://doi.org/10.3390/ph12010045 - 26 Mar 2019

Cited by 18 | Viewed by 4733

Abstract

Glioblastoma (GBM) is the most common and deadliest primary adult brain tumor. Invasion, resistance to therapy, and tumor recurrence in GBM can be attributed in part to brain tumor-initiating cells (BTICs). BTICs isolated from various patient-derived xenografts showed high expression of the poorly [...] Read more.

Glioblastoma (GBM) is the most common and deadliest primary adult brain tumor. Invasion, resistance to therapy, and tumor recurrence in GBM can be attributed in part to brain tumor-initiating cells (BTICs). BTICs isolated from various patient-derived xenografts showed high expression of the poorly characterized Apelin early ligand A (APELA) gene. Although originally considered to be a non-coding gene, the APELA gene encodes a protein that binds to the Apelin receptor and promotes the growth of human embryonic stem cells and the formation of the embryonic vasculature. We found that both APELA mRNA and protein are expressed at high levels in a subset of brain tumor patients, and that APELA is also expressed in putative stem cell niche in GBM tumor tissue. Analysis of APELA and the Apelin receptor gene expression in brain tumor datasets showed that high APELA expression was associated with poor patient survival in both glioma and glioblastoma, and APELA expression correlated with glioma grade. In contrast, gene expression of the Apelin receptor or Apelin was not found to be associated with patient survival, or glioma grade. Consequently, APELA may play an important role in glioblastoma tumorigenesis and may be a future therapeutic target. Full article

(This article belongs to the Special Issue Choices of the Journal)

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Figure 1
APELA gene expression in brain tumor-initiating cells (BTICs), and in normal brain and glioma samples. (A) APELA expression was determined by qPCR in RNA extracts from glioblastoma (GBM) patient-derived xenolines (PDXs) grown in bulk cultures or as BTICs, and (B) BTICs isolated from different regions of the BT238 tumor sample (n = 4). (C) RNA was extracted from FFPE blocks of normal brain tissue (5 samples), (LGG) low-grade glioma (18 samples) and (GBM) glioblastoma (24 samples), and APELA expression was measured by qPCR and normalized to ACTIN expression. * p < 0.05. Full article ">Figure 2
Immunohistochemistry (IHC) for APELA protein expression in BTICs and differentiated BTICs. IHC was performed on bulk differentiated and X10 and X16 BTICs. Staining was performed with APELA antisera or pre-immune sera and processed for IHC. Nuclei were counterstained with 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI). Note, the markedly more intense cytoplasmic staining for APELA in BTICs than in bulk GBM cells, and no detectable staining obtained with pre-immune sera. Representative images are shown. Staining with pre-immune sera showed no IHC staining. Full article ">Figure 3
IHC for APELA protein expression in normal brain and glioma samples. IHC was performed on FFPE blocks of normal brain (NB) and GBM tumor tissue using anti-APELA and anti-Nestin Ab and processed for IHC. Representative images are shown. Full article ">Figure 4
APELA is co-expressed with the stem cell marker Nestin at the mRNA level in GBM tumor tissue. Slides were prepared from normal brain (NB) and GBM tumor tissue, subjected to RNA-ISH using the RNAscope technology and images analyzed by confocal microscopy. RNA-ISH was performed with individual gene probes for APELA (green), and Nestin (magenta). Nuclei were DAPI counterstained (blue). Colocalization of APELA and Nestin expression in a cluster of individual cells is evident in the merged image (20× tile-scan magnification) and the 4× magnification of the merged image. Full article ">Figure 5
APELA expression is directly associated with glioma and GBM patient survival. (A,B) Kaplan–Meier curves of the association of APELA, APLNR and APLN expression with patient survival in the (A) Donson (GSE33331) [<a href="#B19-pharmaceuticals-12-00045" class="html-bibr">19</a>] and (B) Vital (GSE43289) [<a href="#B20-pharmaceuticals-12-00045" class="html-bibr">20</a>] microarray dataset analyzed using the GlioVis data portal [<a href="#B18-pharmaceuticals-12-00045" class="html-bibr">18</a>]. Full article ">Figure 6
APELA expression as a function of glioma tumor grade and GBM molecular subtype. APELA, APLNR and APLN expression in the GSE43289 dataset plotted as a function of (A) histological grade in glioma or (B) molecular GBM subtype [<a href="#B20-pharmaceuticals-12-00045" class="html-bibr">20</a>]. Full article ">

11 pages, 3500 KiB

Open AccessArticle

Raman Microspectroscopy as a Tool to Elucidate the Efficacy of Topical Formulations Containing Curcumin

by Ievgeniia Iermak, Ana Paula da Silva, Cristina Kurachi, Vanderlei Salvador Bagnato and Natalia Mayumi Inada

Pharmaceuticals 2019, 12(1), 44; https://doi.org/10.3390/ph12010044 - 23 Mar 2019

Cited by 6 | Viewed by 3694

Abstract

The success of the onychomycosis treatment is directly associated with factors such as the choice of the medication, the administration route, and the pharmaceutical formulation. Photodynamic therapy (PDT) is an emerging and promising technique indicated for onychomycosis treatment. For this application, the main [...] Read more.

The success of the onychomycosis treatment is directly associated with factors such as the choice of the medication, the administration route, and the pharmaceutical formulation. Photodynamic therapy (PDT) is an emerging and promising technique indicated for onychomycosis treatment. For this application, the main challenge is the efficient delivery of the photosensitizer (PS). Curcumin is widely used as a PS, however it is an unstable molecule and it is a challenge to develop a formulation with good penetration into the nail plate, maintaining the stability of curcumin. In this study, the molecular mechanisms underlying the efficacy of two topical formulations containing curcumin used in a clinical trial for onychomycosis treatment were analyzed by Raman microspectroscopy. It is shown that curcumin is present in both formulations in aggregated and non-aggregated states, and in aggregates it is present in different conformations, depending on the interaction with the solvent. This proves to be critical for efficient and uniform PS delivery to the nail and its complete use during the treatment. These analyses are showing how promising Raman microspectroscopy is in understanding the molecular mechanisms of the efficiency of photosensitizers and are helping to improve the development of pharmaceutical formulations. Full article

(This article belongs to the Special Issue Photodynamic Therapy 2019)

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Graphical abstract

14 pages, 1344 KiB

Open AccessArticle

Microencapsulation of Salmonella-Specific Bacteriophage Felix O1 Using Spray-Drying in a pH-Responsive Formulation and Direct Compression Tableting of Powders into a Solid Oral Dosage Form

by Gurinder K. Vinner, Zahra Rezaie-Yazdi, Miika Leppanen, Andrew G.F. Stapley, Mark C. Leaper and Danish J. Malik

Pharmaceuticals 2019, 12(1), 43; https://doi.org/10.3390/ph12010043 - 22 Mar 2019

Cited by 45 | Viewed by 8531

Abstract

The treatment of enteric bacterial infections using oral bacteriophage therapy can be challenging since the harsh acidic stomach environment renders phages inactive during transit through the gastrointestinal tract. Solid oral dosage forms allowing site-specific gastrointestinal delivery of high doses of phages, e.g., using [...] Read more.

The treatment of enteric bacterial infections using oral bacteriophage therapy can be challenging since the harsh acidic stomach environment renders phages inactive during transit through the gastrointestinal tract. Solid oral dosage forms allowing site-specific gastrointestinal delivery of high doses of phages, e.g., using a pH or enzymatic trigger, would be a game changer for the nascent industry trying to demonstrate the efficacy of phages, including engineered phages for gut microbiome modulation in expensive clinical trials. Spray-drying is a scalable, low-cost process for producing pharmaceutical agents in dry powder form. Encapsulation of a model Salmonella-specific phage (Myoviridae phage Felix O1) was carried out using the process of spray-drying, employing a commercially available Eudragit S100^® pH-responsive anionic copolymer composed of methyl methacrylate-co-methacrylic acid formulated with trehalose. Formulation and processing conditions were optimised to improve the survival of phages during spray-drying, and their subsequent protection upon exposure to simulated gastric acidity was demonstrated. Addition of trehalose to the formulation was shown to protect phages from elevated temperatures and desiccation encountered during spray-drying. Direct compression of spray-dried encapsulated phages into tablets was shown to significantly improve phage protection upon exposure to simulated gastric fluid. The results reported here demonstrate the significant potential of spray-dried pH-responsive formulations for oral delivery of bacteriophages targeting gastrointestinal applications. Full article

(This article belongs to the Special Issue Phage Therapy and Phage-Mediated Biological Control)

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Figure 1
Phage Felix O1 spray-dried at varying inlet air drying temperatures in formulations PS04 and PS30 (see text for description) followed by complete release of phages in simulated intestinal fluid (SIF) (titre measured after 3 h of exposure to SIF). The phage titre of feed solution used for spray-drying was 5 × 109 PFU/mL, equating to a theoretical final phage concentration of ~1.7 × 109 PFU/g. * Indicates significant difference in means for samples at a given temperature compared with the spray-dried sample at 100 °C for the same formulation (p < 0.05) using a two-sample t-test. Error bars represent one standard deviation; all measurements were done in triplicate (n = 3). Full article ">Figure 2
Concentration of encapsulated phage Felix O1 released from spray-dried powders after complete dissolution in SIF (~3 h). Samples labelled SIF were exposed to SIF only without acid exposure, whereas samples labelled simulated gastric fluid (SGF)/SIF were first exposed to simulated gastric fluid (pH 2) for 2 h, subsequently centrifuged to remove the acid supernatant, and then SIF (pH 7) was added to the sample to dissolve the polymer. All formulations were spray-dried at 150 °C inlet temperature corresponding to 82 °C outlet temperature. * Indicates significant difference in means (p < 0.05) using a two-sample t-test; n.d. means no difference (p > 0.05). Error bars represent one standard deviation; all measurements were done in triplicate (n = 3). Full article ">Figure 3
Helium ion microscopy (HIM) images of spray-dried PS21 microparticles (inlet drying temperature 150 °C). (a) Spray-dried microparticles were typically <10 µm in size and did not display surface defects such as blow holes. (b) Some particles were spherical, whereas others were flattened spheres, and some had a lens-shaped appearance. (c) A spherical particle about 10 µm in size was cut in half using a neon ion beam and, after 180° rotation, was imaged with a helium ion beam. Two phage virion particles can be seen in the top left-hand corner (red circle). (d) Expanded view of red box area shown in frame (c). Bumps in the inside wall of the sphere were found in microparticles containing phages imaged using a higher magnification (red arrow). (e) Inside wall of a control microparticle not containing phages. Full article ">Figure 4
Encapsulated Felix O1 phages released in SIF from spray-dried powders and corresponding tablets using three different formulations. Samples labelled SIF were exposed to SIF only without acid exposure, whereas samples labelled SGF/SIF were exposed first to simulated gastric fluid (pH 2) for 2 h and were then centrifuged, before the supernatant was withdrawn and SIF was added to the sample. Phage titres were measured after 3 h of exposure to SIF for powders and 5 h of exposure to SIF for tablets to ensure complete dissolution of tablets. * Indicates significant difference in means (p < 0.05) using a two-sample t-test; n.d. means no difference (p > 0.05). Error bars represent one standard deviation; all measurements were done in triplicate (n = 3). Full article ">Figure 5
Storage results for formulations PS04 and PS21 used to spray-dry phage Felix O1. Phage titre was measured after one month and three months of storage at 4 °C and 23 °C. * Indicates a significant difference in means in comparison with the mean value immediately after spray-drying (0 months) using a two-sample t-test, (p < 0.05). Error bars represent one standard deviation; all measurements were done in triplicate (n = 3). Full article ">

14 pages, 1599 KiB

Open AccessArticle

Localization of ^99mTc-GRP Analogs in GRPR-Expressing Tumors: Effects of Peptide Length and Neprilysin Inhibition on Biological Responses

by Aikaterini Kaloudi, Emmanouil Lymperis, Panagiotis Kanellopoulos, Beatrice Waser, Marion de Jong, Eric P. Krenning, Jean Claude Reubi, Berthold A. Nock and Theodosia Maina

Pharmaceuticals 2019, 12(1), 42; https://doi.org/10.3390/ph12010042 - 20 Mar 2019

Cited by 8 | Viewed by 4692

Abstract

The overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR [...] Read more.

The overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR selectivity compared to frog BBN sequences aiming to improve pharmacokinetic profiles. In the present study, we compared two differently truncated human endogenous GRP motifs: GRP(14–27) and GRP(18–27). An acyclic tetraamine was coupled at the N-terminus to allow for stable binding of the SPECT radionuclide ^99mTc. Their biological profiles were compared in PC-3 cells and in mice without or with coinjection of phosphoramidon (PA) to induce transient neprilysin (NEP) inhibition in vivo. The two ^99mTc-N₄-GRP(14/18–27) radioligands displayed similar biological behavior in mice. Coinjection of PA exerted a profound effect on in vivo stability and translated into notably improved radiolabel localization in PC-3 experimental tumors. Hence, this study has shown that promising ^99mTc-radiotracers for SPECT imaging may indeed derive from human GRP sequences. Radiotracer bioavailability was found to be of major significance. It could be improved during in situ NEP inhibition resulting in drastically enhanced uptake in GRPR-expressing lesions. Full article

(This article belongs to the Special Issue Targets, Tracers and Translation – Novel Radiopharmaceuticals Boost Nuclear Medicine)

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Figure 1
Chemical structure of (a) 99mTc-N4-GRP(14–27) (red) and (b) 99mTc-N4-GRP(18–27) (blue). Full article ">Figure 2
Representative radiochromatograms of the radiolabeling reaction mixture of (a) 99mTc-N4-GRP(14–27) (red) and (b) 99mTc-N4-GRP(18–27) (blue), confirming the quantitative formation of high purity radioligands at tR = 14.9 min and tR = 13.3 min, respectively (HPLC system 1). Full article ">Figure 3
(a) [125I-Tyr4]BBN displacement curves from gastrin-releasing peptide receptor (GRPR)-sites on PC-3 cells after 1-h incubation at 22 °C by N4-GRP(14–27) (red solid line—IC50 0.32±0.03 nM), GRP(14–27) (red dashed line—IC50 0.45 ± 0.02 nM), N4-GRP(18–27) (blue solid line—IC50 0.63±0.06 nM) and GRP(18–27) (blue dashed line—IC50 1.66 ± 0.20 nM). (b) GRPR-specific internalization of 99mTc-N4-GRP(14–27) (red solid line) and 99mTc-N4-GRP(18–27) (blue solid line) in PC-3 cells during incubation at 37 °C at 15, 30, 60, and 120 min. Results represent average of cell internalized activity ± sd, n = 3; data is corrected for nonspecific internalization in the presence of 1 µM [Tyr4]BBN. Full article ">Figure 4
Representative radiochromatograms of HPLC analysis of mouse blood samples collected 5 min pi of (a) 99mTc-N4-GRP(14–27) (25.2% intact radiotracer; red dashed line) or (b) 99mTc-N4-GRP(18–27) (31.8% intact radiotracer; blue dashed line) without PA coinjection; the respective radiochromatograms of (c) 99mTc-N4-GRP(14–27) (63.1% intact radiotracer; tR = 29.6 min; red solid line) or (d) 99mTc-N4-GRP(18–27) (68.1% intact radiotracer; tR = 25.1 min; blue solid line) with PA coinjection are also included; the tR of parent radiopeptide was determined by coinjection with the respective radioligand sample in the column (HPLC system 2) and is indicated here by the arrow. Full article ">

31 pages, 9116 KiB

Open AccessReview

BACE-1 and γ-Secretase as Therapeutic Targets for Alzheimer’s Disease

by Miguel A. Maia and Emília Sousa

Pharmaceuticals 2019, 12(1), 41; https://doi.org/10.3390/ph12010041 - 19 Mar 2019

Cited by 96 | Viewed by 10987

Abstract

Alzheimer’s disease (AD) is a growing global health concern with a massive impact on affected individuals and society. Despite the considerable advances achieved in the understanding of AD pathogenesis, researchers have not been successful in fully identifying the mechanisms involved in disease progression. [...] Read more.

Alzheimer’s disease (AD) is a growing global health concern with a massive impact on affected individuals and society. Despite the considerable advances achieved in the understanding of AD pathogenesis, researchers have not been successful in fully identifying the mechanisms involved in disease progression. The amyloid hypothesis, currently the prevalent theory for AD, defends the deposition of β-amyloid protein (Aβ) aggregates as the trigger of a series of events leading to neuronal dysfunction and dementia. Hence, several research and development (R&D) programs have been led by the pharmaceutical industry in an effort to discover effective and safety anti-amyloid agents as disease modifying agents for AD. Among 19 drug candidates identified in the AD pipeline, nine have their mechanism of action centered in the activity of β or γ-secretase proteases, covering almost 50% of the identified agents. These drug candidates must fulfill the general rigid prerequisites for a drug aimed for central nervous system (CNS) penetration and selectivity toward different aspartyl proteases. This review presents the classes of γ-secretase and beta-site APP cleaving enzyme 1 (BACE-1) inhibitors under development, highlighting their structure-activity relationship, among other physical-chemistry aspects important for the successful development of new anti-AD pharmacological agents. Full article

(This article belongs to the Special Issue Therapeutics Agents for Neural Repair)

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15 pages, 2863 KiB

Open AccessArticle

Physical Stability and Viscoelastic Properties of Co-Amorphous Ezetimibe/Simvastatin System

by Justyna Knapik-Kowalczuk, Krzysztof Chmiel, Karolina Jurkiewicz, Natália T. Correia, Wiesław Sawicki and Marian Paluch

Pharmaceuticals 2019, 12(1), 40; https://doi.org/10.3390/ph12010040 - 19 Mar 2019

Cited by 19 | Viewed by 5291

Abstract

The purpose of this paper is to examine the physical stability as well as viscoelastic properties of the binary amorphous ezetimibe–simvastatin system. According to our knowledge, this is the first time that such an amorphous composition is prepared and investigated. The tendency toward [...] Read more.

The purpose of this paper is to examine the physical stability as well as viscoelastic properties of the binary amorphous ezetimibe–simvastatin system. According to our knowledge, this is the first time that such an amorphous composition is prepared and investigated. The tendency toward re-crystallization of the amorphous ezetimibe–simvastatin system, at both standard storage and elevated temperature conditions, have been studied by means of X-ray diffraction (XRD). Our investigations have revealed that simvastatin remarkably improves the physical stability of ezetimibe, despite the fact that it works as a plasticizer. Pure amorphous ezetimibe, when stored at room temperature, begins to re-crystallize after 14 days after amorphization. On the other hand, the ezetimibe-simvastatin binary mixture (at the same storage conditions) is physically stable for at least 1 year. However, the devitrification of the binary amorphous composition was observed at elevated temperature conditions (T = 373 K). Therefore, we used a third compound to hinder the re-crystallization. Finally, both the physical stability as well as viscoelastic properties of the ternary systems containing different concentrations of the latter component have been thoroughly investigated. Full article

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Graphical abstract

14 pages, 996 KiB

Open AccessCommunication

Development of a Microwave-assisted Chemoselective Synthesis of Oxime-linked Sugar Linkers and Trivalent Glycoclusters

by Katherine McReynolds, Dustin Dimas, Grace Floyd and Kara Zeman

Pharmaceuticals 2019, 12(1), 39; https://doi.org/10.3390/ph12010039 - 14 Mar 2019

Cited by 4 | Viewed by 4147

Abstract

A rapid, high-yielding microwave-mediated synthetic procedure was developed and optimized using a model system of monovalent sugar linkers, with the ultimate goal of using this method for the synthesis of multivalent glycoclusters. The reaction occurs between the aldehyde/ketone on the sugars and an [...] Read more.

A rapid, high-yielding microwave-mediated synthetic procedure was developed and optimized using a model system of monovalent sugar linkers, with the ultimate goal of using this method for the synthesis of multivalent glycoclusters. The reaction occurs between the aldehyde/ketone on the sugars and an aminooxy moiety on the linker/trivalent core molecules used in this study, yielding acid-stable oxime linkages in the products and was carried out using equimolar quantities of reactants under mild aqueous conditions. Because the reaction is chemoselective, sugars can be incorporated without the use of protecting groups and the reactions can be completed in as little as 30 min in the microwave. As an added advantage, in the synthesis of the trivalent glycoclusters, the fully substituted trivalent molecules were the major products produced in excellent yields. These results illustrate the potential of this rapid oxime-forming microwave-mediated reaction in the synthesis of larger, more complex glycoconjugates and glycoclusters for use in a wide variety of biomedical applications. Full article

(This article belongs to the Special Issue Carbohydrates 2018)

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14 pages, 3890 KiB

Open AccessReview

Potential of the Other Genetic Information Coded by the Viral RNA Genomes as Antiviral Target

by Alfredo Berzal-Herranz, Cristina Romero-López, Beatriz Berzal-Herranz and Sara Ramos-Lorente

Pharmaceuticals 2019, 12(1), 38; https://doi.org/10.3390/ph12010038 - 13 Mar 2019

Cited by 4 | Viewed by 4179

Abstract

In addition to the protein coding information, viral RNA genomes code functional information in structurally conserved units termed functional RNA domains. These RNA domains play essential roles in the viral cycle (e.g., replication and translation). Understanding the molecular mechanisms behind their function is [...] Read more.

In addition to the protein coding information, viral RNA genomes code functional information in structurally conserved units termed functional RNA domains. These RNA domains play essential roles in the viral cycle (e.g., replication and translation). Understanding the molecular mechanisms behind their function is essential to understanding the viral infective cycle. Further, interfering with the function of the genomic RNA domains offers a potential means of developing antiviral strategies. Aptamers are good candidates for targeting structural RNA domains. Besides its potential as therapeutics, aptamers also provide an excellent tool for investigating the functionality of RNA domains in viral genomes. This review briefly summarizes the work carried out in our laboratory aimed at the structural and functional characterization of the hepatitis C virus (HCV) genomic RNA domains. It also describes the efforts we carried out for the development of antiviral aptamers targeting specific genomic domains of the HCV and the human immunodeficiency virus type-1 (HIV-1). Full article

(This article belongs to the Special Issue Selected Papers from the 4th International Electronic Conference on Medicinal Chemistry)

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Figure 1
The hepatitis C virus (HCV) RNA genome. Upper panel: A schematic representation of the genetic organization of the viral genome. The 5′ and 3′ UTRs flanking the single ORF are depicted by a black line. The viral proteins and their functions are indicated. The translational start and stop codons are marked by arrows. The numbering corresponds to the codon positions in the ORF according to the HCV Con1 isolate, genotype 1b. Lower panel: A structural conservation map of the HCV RNA is represented by gray boxes denoting structurally conserved regions among different viral isolates. The predicted secondary structures of conserved elements in the ORF of the viral RNA genome are shown at the bottom. The color code and labels at the bottom indicate the position where each stem-loop is located. The figure is adapted from Reference [<a href="#B27-pharmaceuticals-12-00038" class="html-bibr">27</a>]. Full article ">Figure 2
A schematic representation of the 5′ and 3′ ends of the HCV genome depicting the highly conserved structural RNA domains: The defined functional RNA–RNA interactions are indicated with broken gray lines. The translational start and stop codons are indicated by arrows. The figure is adapted from Reference [<a href="#B32-pharmaceuticals-12-00038" class="html-bibr">32</a>]. Full article ">Figure 3
The conformational tuning of long-distance functional RNA domains: A summary of the comparative structural analysis of the 5′ and 3′ HCV genomic ends in the presence and absence of each other end. Only nucleotides that show a differential chemical reactivity are indicated with red figures. The solid figures indicate increases in reactivity. The empty figures indicate decreases in reactivity. △ indicates the DMS (dimethyl sulfate) reactivity. ○ indicates the NMIA (N-methyl-isatoic anhydride) reactivity. Full article ">Figure 4
The selection of RNA inhibitors targeting the HCV internal ribosome entry site (IRES): (a) The identification of the aptamer targets within the IRES. The targets determined by the complementary sequence to the consensus motif that define each aptamer’s group are indicated using a colour code on the schematic representation of the secondary structure of the IRES. (b) The inhibition of the IRES activity by a collection of selected aptamers: The IRES-dependent translation was measured in vitro as the Fluc activity. The values are the mean of at least three independent experiments and are referred to the activity in the absence of an RNA inhibitor. Figure adapted from [<a href="#B61-pharmaceuticals-12-00038" class="html-bibr">61</a>]. Full article ">Figure 5
The antiviral activity of selected RNA inhibitors in a human hepatoma cell line. (a) The inhibition of HCV IRES-dependent translation in Huh-7 cells by three RNA inhibitors: The cells were co-transfected with a mix containing the transcripts IRES-Fluc and cap-RLuc mRNAs and an excess of a specific RNA inhibitor. The IRES translation efficiency was measured as Fluc versus RLuc activity and referred to the value obtained in the control reaction without an RNA inhibitor. The activity values are the mean of three independent experiments. (b) The inhibition of viral replication in a Huh-7 cell line-based HCV sub-genomic replication system (Huh-7 NS3-3′): The cells supporting the stable replication of an HCV sub-genomic replicon were transfected with individual RNA inhibitors and incubated for 20 h. The viral replication was measured as the viral RNA amount by qRT-PCR from a total cell RNA extraction. The data are the mean of three independent experiments. Full article ">Figure 6
RNA aptamers selected against the HCV cis-replication element (CRE): (a) Sequence and secondary structure of the 194-nt-long viral RNA fragment used as the target for the selection of anti-HCV aptamers. The highly conserved structural domains of the CRE element (5BSL3.1, 5BSL3.2, and 5BSL3.3) and the 5BSL3.4 that includes the translational stop codon, are indicated. The target sequences complementary to the consensus sequence that define the five groups of selected aptamers are indicated by colored sequences. (b) The inhibition of HCV replication: Huh-7 cells supporting the stable replication of a sub-genomic HCV replicon were transfected with independent aptamers. Viral RNA levels were quantified from the total RNA extracted 18 h post-transfection using qRT-PCR. The bar chart indicates the HCV RNA levels referred to those obtained in the absence of a non-related RNA molecule, RNA80. The values are the mean of at least four independent experiments [<a href="#B67-pharmaceuticals-12-00038" class="html-bibr">67</a>]. The red arrows and boxes indicate the aptamers for which the mechanism of action was further characterized. Full article ">Figure 7
Aptamers selected against the genomic HIV 5′ UTR: A schematic representation of the secondary structure of the 5′ UTR of the viral RNA genome. The well-characterized functional structural elements are depicted. The sequence and secondary structure of the polyA domain is enlarged in the box with blue letters. The octamer consensus sequence of the selected aptamers is indicated in red. Full article ">Figure 8
The in silico-designed RNApt16: (a) The structural analysis of the representative examples of 64-nt-long selected aptamers shows the minimum free energy isoform. The probabilities of every nucleotide to actually hold the structural conformation shown are represented by a colour code (red, highest; dark blue, lowest). The pink, shadowed box indicates the consensus 16-nt-long structural domain. The sequence and secondary structure of the consensus motif and the designed RNApt16 are indicated at the bottom. R, purine; Y, pirimidine; N, any ribonucleotide, (b) HIV-1 inhibition assays: The inhibition of HIV-1 particles production measured as the p24 antigen production by the XIV22, XIV26, and RNApt16 aptamers (compared to a negative control). The data represent the mean of three independent assays. ** The significant differences as compared to the control (p < 0.01). The figure is adapted from Reference [<a href="#B69-pharmaceuticals-12-00038" class="html-bibr">69</a>]. Full article ">

7 pages, 1010 KiB

Open AccessBrief Report

Baricitinib: A 2018 Novel FDA-Approved Small Molecule Inhibiting Janus Kinases

by Annie Mayence and Jean Jacques Vanden Eynde

Pharmaceuticals 2019, 12(1), 37; https://doi.org/10.3390/ph12010037 - 12 Mar 2019

Cited by 40 | Viewed by 9215

Abstract

In 2018, Baricitinib was approved by the Food and Drig Administration (FDA) for the treatment of rheumatoid arthritis. Baricitinib exerts its action by targeting Janus kinases (JAK). In this study, we describe the necessary steps for preparing the drug using two alternative routes. [...] Read more.

In 2018, Baricitinib was approved by the Food and Drig Administration (FDA) for the treatment of rheumatoid arthritis. Baricitinib exerts its action by targeting Janus kinases (JAK). In this study, we describe the necessary steps for preparing the drug using two alternative routes. Full article

(This article belongs to the Special Issue The Story of Successful Drugs and Recent FDA-Approved Molecules)

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19 pages, 10502 KiB

Open AccessArticle

In Silico Study to Identify New Antituberculosis Molecules from Natural Sources by Hierarchical Virtual Screening and Molecular Dynamics Simulations

by Vinícius de S. Pinto, Janay S. C. Araújo, Rai C. Silva, Glauber V. da Costa, Jorddy N. Cruz, Moysés F. De A. Neto, Joaquín M. Campos, Cleydson B. R. Santos, Franco H. A. Leite and Manoelito C. S. Junior

Pharmaceuticals 2019, 12(1), 36; https://doi.org/10.3390/ph12010036 - 12 Mar 2019

Cited by 54 | Viewed by 7812

Abstract

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis, responsible for 1.5 million documented deaths in 2016. The increase in reported cases of M. tuberculosis resistance to the main drugs show the need for the development of new and efficient drugs for [...] Read more.

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis, responsible for 1.5 million documented deaths in 2016. The increase in reported cases of M. tuberculosis resistance to the main drugs show the need for the development of new and efficient drugs for better TB control. Based on these facts, this work aimed to use combined in silico techniques for the discovery of potential inhibitors to β-ketoacyl-ACP synthase (MtKasA). Initially compounds from natural sources present in the ZINC database were selected, then filters were sequentially applied by virtual screening, initially with pharmacophoric modeling, and later the selected compounds (based on QFIT scores) were submitted to the DOCK 6.5 program. After recategorization of the variables (QFIT score and GRID score), compounds ZINC35465970 and ZINC31170017 were selected. These compounds showed great hydrophobic contributions and for each established system 100 ns of molecular dynamics simulations were performed and the binding free energy was calculated. ZINC35465970 demonstrated a greater capacity for the KasA enzyme inhibition, with a ΔG_bind = −30.90 kcal/mol and ZINC31170017 presented a ΔG_bind = −27.49 kcal/mol. These data can be used in other studies that aim at the inhibition of the same biological targets through drugs with a dual action. Full article

(This article belongs to the Special Issue Chemoinformatics and Drug Design)

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23 pages, 1090 KiB

Open AccessReview

Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth

by Paul Hyman

Pharmaceuticals 2019, 12(1), 35; https://doi.org/10.3390/ph12010035 - 11 Mar 2019

Cited by 321 | Viewed by 28013

Abstract

For a bacteriophage to be useful for phage therapy it must be both isolated from the environment and shown to have certain characteristics beyond just killing strains of the target bacterial pathogen. These include desirable characteristics such as a relatively broad host range [...] Read more.

For a bacteriophage to be useful for phage therapy it must be both isolated from the environment and shown to have certain characteristics beyond just killing strains of the target bacterial pathogen. These include desirable characteristics such as a relatively broad host range and a lack of other characteristics such as carrying toxin genes and the ability to form a lysogen. While phages are commonly isolated first and subsequently characterized, it is possible to alter isolation procedures to bias the isolation toward phages with desirable characteristics. Some of these variations are regularly used by some groups while others have only been shown in a few publications. In this review I will describe (1) isolation procedures and variations that are designed to isolate phages with broader host ranges, (2) characterization procedures used to show that a phage may have utility in phage therapy, including some of the limits of such characterization, and (3) results of a survey and discussion with phage researchers in industry and academia on the practice of characterization of phages. Full article

(This article belongs to the Special Issue Phage Therapy and Phage-Mediated Biological Control)

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14 pages, 302 KiB

Open AccessArticle

Chemical Characterization, Antioxidant, Cytotoxic and Microbiological Activities of the Essential Oil of Leaf of Tithonia Diversifolia (Hemsl) A. Gray (Asteraceae)

by Ana Luzia Ferreira Farias, Alex Bruno Lobato Rodrigues, Rosany Lopes Martins, Érica de Menezes Rabelo, Carlos Wagner Ferreira Farias and Sheylla Susan Moreira da Silva de Almeida

Pharmaceuticals 2019, 12(1), 34; https://doi.org/10.3390/ph12010034 - 4 Mar 2019

Cited by 18 | Viewed by 6958

Abstract

The present study aimed to evaluate the chemical composition, antioxidant potential, and the cytotoxic and antimicrobial activity of the essential oil of the plant species Tithonia diversifolia (Hemsl) A. Gray. The essential oil obtained was used to identify the chemical compounds present through [...] Read more.

The present study aimed to evaluate the chemical composition, antioxidant potential, and the cytotoxic and antimicrobial activity of the essential oil of the plant species Tithonia diversifolia (Hemsl) A. Gray. The essential oil obtained was used to identify the chemical compounds present through the techniques of GC-MS and NMR. The antioxidant potential was calculated by the sequestration method of 2,2-diphenyl-1-picrylhydrazyl. For cytotoxic activity, the larval mortality of Artemia salina was evaluated. The main chemical constituents identified are αpinene (9.9%), Limonene (5.40%), (Z)-β-ocimene (4.02%), p-cymen-8-ol (3.0%), Piperitone (11.72%), (E)-nerolidol (3.78%) and Spathulenol (10.8%). In the evaluation of the antimicrobial activity, bacterial strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were used. The results showed that the bacterium E. coli were more susceptible to the presence of the essential oil, presenting minimal inhibitory concentration at the concentrations that were exposed. The essential oil presented antioxidant activity of 54.6% at the concentration of 5 mg·mL⁻¹ and provided a CI₅₀ of 4.30. It was observed that the essential oil of this species was highly toxic against A. salina lavas, as its cytotoxic activity showed an LC₅₀ of 3.11. Thus, it is concluded that T. diversifolia oils are effective in inhibiting bacterial growth and reducing oxidative stress. Full article

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20 pages, 2257 KiB

Open AccessArticle

Characterisation of an Isogenic Model of Cisplatin Resistance in Oesophageal Adenocarcinoma Cells

by Amy M. Buckley, Becky AS. Bibby, Margaret R. Dunne, Susan A. Kennedy, Maria B. Davern, Breandán N. Kennedy, Stephen G. Maher and Jacintha O’Sullivan

Pharmaceuticals 2019, 12(1), 33; https://doi.org/10.3390/ph12010033 - 20 Feb 2019

Cited by 10 | Viewed by 5359

Abstract

Cisplatin (cis-diamminedichloroplatinum) is widely used for the treatment of solid malignancies; however, the development of chemoresistance hinders the success of this chemotherapeutic in the clinic. This study provides novel insights into the molecular and phenotypic changes in an isogenic oesophageal adenocarcinoma (OAC) model [...] Read more.

Cisplatin (cis-diamminedichloroplatinum) is widely used for the treatment of solid malignancies; however, the development of chemoresistance hinders the success of this chemotherapeutic in the clinic. This study provides novel insights into the molecular and phenotypic changes in an isogenic oesophageal adenocarcinoma (OAC) model of acquired cisplatin resistance. Key differences that could be targeted to overcome cisplatin resistance are highlighted. We characterise the differences in treatment sensitivity, gene expression, inflammatory protein secretions, and metabolic rate in an isogenic cell culture model of acquired cisplatin resistance in OAC. Cisplatin-resistant cells (OE33 Cis R) were significantly more sensitive to other cytotoxic modalities, such as 2 Gy radiation (p = 0.0055) and 5-fluorouracil (5-FU) (p = 0.0032) treatment than parental cisplatin-sensitive cells (OE33 Cis P). Gene expression profiling identified differences at the gene level between cisplatin-sensitive and cisplatin-resistant cells, uncovering 692 genes that were significantly altered between OE33 Cis R cells and OE33 Cis P cells. OAC is an inflammatory-driven cancer, and inflammatory secretome profiling identified 18 proteins secreted at significantly altered levels in OE33 Cis R cells compared to OE33 Cis P cells. IL-7 was the only cytokine to be secreted at a significantly higher levels from OE33 Cis R cells compared to OE33 Cis P cells. Additionally, we profiled the metabolic phenotype of OE33 Cis P and OE33 Cis R cells under normoxic and hypoxic conditions. The oxygen consumption rate, as a measure of oxidative phosphorylation, is significantly higher in OE33 Cis R cells under normoxic conditions. In contrast, under hypoxic conditions of 0.5% O₂, the oxygen consumption rate is significantly lower in OE33 Cis R cells than OE33 Cis P cells. This study provides novel insights into the molecular and phenotypic changes in an isogenic OAC model of acquired cisplatin resistance, and highlights therapeutic targets to overcome cisplatin resistance in OAC. Full article

(This article belongs to the Special Issue Anticancer Drugs)

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Oesophageal adenocarcinoma (OAC) cisplatin-sensitive (OE33 Cis P) cells were significantly more sensitive to cisplatin-induced cell death than OAC cisplatin-resistant (OE33 Cis R) cells. The toxicity to a range of increasing concentrations of cisplatin in (A) OE33 Cis P and (B) OE33 Cis R cells following 48 h of treatment was determined using a CCK-8 assay. The 48-h IC50 for (C) OE33 Cis P cells and (D) OE33 Cis R cells was 1.3 µM and 2.8 µM, respectively (n = 3). ** p < 0.01, *** p < 0.001 by an unpaired two-tailed t-test. Full article ">Figure 2
Cisplatin-resistant (OE33 Cis R) oesophageal adenocarcinoma cells are more radiosensitive than cisplatin-sensitive (OE33 Cis P) oesophageal adenocarcinoma (OAC) cells. (A) The sensitivity of cisplatin-sensitive (OE33 Cis P) and cisplatin-resistant (OE33 Cis R) OAC cells to cisplatin was assessed by clonogenic assay (n = 3). (B) There is no difference in the basal cell surviving fraction of cisplatin-sensitive and cisplatin-resistant OAC cells cultured in RPMI media, (n = 3). (C) Surviving fraction of Cis P and Cis R OAC cells following treatment of one 2 Gy fraction of irradiation, (n = 3). (D) The viability of the OE33 Cis R cells was significantly decreased compared to the OE33 Cis P when treated with 12 µM of 5-fluorouracil (5-FU) (n = 4). An unpaired t-test was used to compare between different cell lines, and a paired t-test was used to compare between the same cell line. Data presented as ±SEM * p < 0.05, ** p < 0.01, *** p < 0.0001. Full article ">Figure 3
OE33 Cis R cells have a significantly altered gene expression profile compared to OE33 Cis P cells. Heatmaps were generated from gene expression data after applying a fold change filter ±two. (A) Heatmap showing 42 genes that were significantly upregulated in OE33 Cis R cells with a fold change of greater than two. (B) Heatmap showing 104 genes which were significantly downregulated in OE33 Cis R cells with a fold change of less than minus two. Gene expression values shown as Fragments Per Kilobase of transcript per Million mapped reads (FKPM). Full article ">Figure 4
Inflammatory protein secretions are significantly different in cisplatin-sensitive (OE33 Cis P) versus cisplatin-resistant (OE33 Cis R) OAC cells. The secreted levels of 47 proteins in Cis P and Cis R cells was evaluated by multiplex ELISA; 23 proteins were detected in supernatant of Cis P and Cis R cells; 18 proteins were significantly different between the two cell lines, and interleukin-7 was significantly higher in Cis R cells compared to Cis P cells. Secreted levels of (A) Interleukin-7 (IL-7) (B) C-reactive protein (CRP) (C) Interleukin 12p70 (IL-12p70) (D) Interleukin 10 (IL-10) (E) Tumour necrosis factor α (TNF-α) (F) Macrophage-derived chemokine (MDC) (G) Intracellular adhesion molecule 1 (ICAM-1) (H) Interleukin 6 (IL-6) (I) Interleukin 1β (IL-1β) (J) Interleukin 13 (IL-13) (K) Serum amyloid A (SAA) (L) Thymus and activation regulated chemokine (TARC) (M) Interleukin 4 (IL-4) (N) Interleukin 8 (IL-8) (O) Interleukin 1 receptor antagonist (IL-1RA) (P) Interleukin 1α (IL-1α) (Q) Interleukin 2 (IL-2) (R) Interferon gamma-induced protein 10 (IP-10) in OE33 Cis P and OE33 Cis R cells, all secretions normalised to protein content. (n = 4). Unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Date expressed as ±SEM. Full article ">Figure 5
Cisplatin-resistant (OE33 Cis R) oesophageal adenocarcinoma cells have an altered metabolic phenotype compared to cisplatin-sensitive (OE33 Cis P) cells under normoxic and hypoxic conditions (0.5% O2). (A) The oxygen consumption rate (OCR), which is a measure of oxidative phosphorylation, was evaluated in OE33 Cis P and OE33 Cis R OAC cells using the Seahorse Biosciences XFe24 extracellular flux analyser cultured under normoxic and hypoxic conditions (0.5% O2). OE33 Cis R cells have a significantly higher OCR when compared to OE33 Cis P; cisplatin-sensitive cells, under normoxic conditions (n = 5), unpaired t-test, ** p < 0.01. (B) The extracellular acidification rate (ECAR), which is a measure glycolysis, was evaluated in OE33 Cis P and OE33 Cis R cells using the Seahorse Biosciences XFe24 extracellular flux analyser cultured under normoxic and hypoxic conditions (0.5% O2), (n = 5), unpaired t-test. (C) Difference in the rate of ATP production in OE33 Cis P and OE33 Cis R cells cultured under normoxic and hypoxic conditions (0.5% O2), (n = 5), unpaired t-test. (D) Difference in the rate of proton leak in OE33 Cis P and OE33 Cis R cells cultured under normoxic and hypoxic conditions (0.5% O2), (n = 5), unpaired t-test. (E) Difference in maximal respiration rate in OE33 Cis P and OE33 Cis R cells cultured under normoxic and hypoxic conditions (0.5% O2), (n = 5), unpaired t-test. (F) Difference in non-mitochondrial respiration in OE33 Cis P and OE33 Cis R cells cultured under normoxic and hypoxic conditions (0.5% O2), (n = 5), unpaired t-test. Data presented as ±SEM. Full article ">

15 pages, 1356 KiB

Open AccessArticle

Development and Characterization of Chitosan Microparticles-in-Films for Buccal Delivery of Bioactive Peptides

by Patrícia Batista, Pedro Castro, Ana Raquel Madureira, Bruno Sarmento and Manuela Pintado

Pharmaceuticals 2019, 12(1), 32; https://doi.org/10.3390/ph12010032 - 20 Feb 2019

Cited by 56 | Viewed by 5611

Abstract

Nowadays, bioactive peptides are used for therapeutic applications and the selection of a carrier to deliver them is very important to increase the efficiency, absorption, release, bioavailability and consumer acceptance. The aim of this study was to develop and characterize chitosan-based films loaded [...] Read more.

Nowadays, bioactive peptides are used for therapeutic applications and the selection of a carrier to deliver them is very important to increase the efficiency, absorption, release, bioavailability and consumer acceptance. The aim of this study was to develop and characterize chitosan-based films loaded with chitosan microparticles containing a bioactive peptide (sequence: KGYGGVSLPEW) with antihypertensive properties. Films were prepared by the solvent casting method, while the microparticles were prepared by ionic gelation. The final optimized chitosan microparticles exhibited a mean diameter of 2.5 µm, a polydispersity index of 0.46, a zeta potential of +61 mV and a peptide association efficiency of 76%. Chitosan films were optimized achieving the final formulation of 0.79% (w/v) of chitosan, 6.74% (w/v) of sorbitol and 0.82% (w/v) of citric acid. These thin (±0.100 mm) and transparent films demonstrated good performance in terms of mechanical and biological properties. The oral films developed were flexible, elastic, easy to handle and exhibited rapid disintegration (30 s) and an erosion behavior of 20% when they came into contact with saliva solution. The cell viability (75–99%) was proved by methylthiazolydiphenyl-tetrazolium bromide (MTT) assay with TR146 cells. The chitosan mucoadhesive films loaded with peptide–chitosan microparticles resulted in an innovative approach to perform administration across the buccal mucosa, because these films present a larger surface area, leading to the rapid disintegration and release of the antihypertensive peptide under controlled conditions in the buccal cavity, thus promoting bioavailability. Full article

(This article belongs to the Special Issue Carbohydrates 2018)

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15 pages, 3135 KiB

Open AccessArticle

2-Nitroimidazole-Furanoside Derivatives for Hypoxia Imaging—Investigation of Nucleoside Transporter Interaction, ¹⁸F-Labeling and Preclinical PET Imaging

by Florian C. Maier, Anna Schweifer, Vijaya L. Damaraju, Carol E. Cass, Gregory D. Bowden, Walter Ehrlichmann, Manfred Kneilling, Bernd J. Pichler, Friedrich Hammerschmidt and Gerald Reischl

Pharmaceuticals 2019, 12(1), 31; https://doi.org/10.3390/ph12010031 - 15 Feb 2019

Cited by 5 | Viewed by 4543

Abstract

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives—mimicking nucleosides—have proven their [...] Read more.

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives—mimicking nucleosides—have proven their potential with [¹⁸F]FAZA ([¹⁸F]fluoro-azomycin-α-arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [¹⁸F]fluoro-azomycin-β-deoxyriboside (β-[¹⁸F]FAZDR), with a structure more similar to nucleosides than [¹⁸F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, β-FAZA, α-FAZDR and β-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for β-FAZDR (IC₅₀ 124 ± 33 µM for SLC28A3), but also for FAZA with the non-nucleosidic α-configuration, the interactions were remarkable (290 ± 44 µM {SLC28A1}; 640 ± 10 µM {SLC28A2}). An improved synthesis was developed for β-FAZA. For a PET study in tumor-bearing mice, α-[¹⁸F]FAZDR was synthesized (radiochemical yield: 15.9 ± 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/µmol) and compared to β-[¹⁸F]FAZDR and [¹⁸F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for β-[¹⁸F]FAZDR already at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to [¹⁸F]FMISO (1.37 ± 0.11, n = 5) and α-[¹⁸F]FAZDR (1.93 ± 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5–3.0). Highest clearance from tumor tissue was observed for β-[¹⁸F]FAZDR (56.6 ± 6.8%, 2 h p.i.), followed by α-[¹⁸F]FAZDR (34.2 ± 7.5%) and [¹⁸F]FMISO (11.8 ± 6.5%). In conclusion, both isomers of [¹⁸F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms. Full article

(This article belongs to the Special Issue Targets, Tracers and Translation – Novel Radiopharmaceuticals Boost Nuclear Medicine)

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Figure 1
Chemical structures of the fluorinated 2-nitroimidazole C5-sugars FAZA, β-FAZA and α-, β-FAZDR, synthesized and used in the investigation of their interaction with human nucleoside transporters. Full article ">Figure 2
Exemplary structures and PET images of [18F]FMISO, α-[18F]FAZDR and β-[18F]FAZDR in carcinoma tissue 1 h p.i. (A), calculated tumor-to-muscle ratios (TMR) are shown in B. β-[18F]FAZDR (n = 4) displayed significantly higher TMR in comparison to [18F]FMISO (n = 5) 1 h p.i. (B), while α-[18F]FAZDR-TMR (n = 4) were not significantly different at any time-point. [18F]FMISO displayed significantly higher uptake in carcinomas vs. muscles only at 2 h and 3 h p.i. (C), while α-[18F]FAZDR and β-[18F]FAZDR at all measured time-points (1 h, 2 h and 3 h p.i.). * p < 0.05, ** p < 0.01 (D,E). Full article ">Figure 3
[18F]FMISO clearance was significantly different from carcinoma and muscle tissue (n = 5, A), however lower in comparison to clearance rates from both tissues for α-[18F]FAZDR (n = 4, B) and β-[18F]FAZDR (n = 4, C). Tumor/muscle-clearance ratios were significantly lower for [18F]FMISO in comparison to β-[18F]FAZDR at 2 h p.i. and to both, α-[18F]FAZDR and β-[18F]FAZDR at 3 h p.i. * p < 0.05, ** p < 0.01 (D). Full article ">Figure 4
Exemplary scan of one CT26 colon carcinoma-bearing mouse with [18F]FAZA, α-[18F]FAZDR and β-[18F]FAZDR on three consecutive days, along with structures of used hypoxia tracers. Full article ">Scheme 1
Three-step synthesis of the 5′-fluoro nucleoside analog β-FAZA, starting from 1-(2′-O-acetyl- β-D-arabinofuranosyl)-2-nitroimidazole (β-1; [<a href="#B25-pharmaceuticals-12-00031" class="html-bibr">25</a>]). Reagents for and yields of the individual steps are given in the scheme. Full article ">Scheme 2
Radiosynthesis of α-[18F]FAZDR by nucleophilic substitution from the tosyl precursor α-1 and subsequent hydrolysis with NH4OH. Full article ">

11 pages, 1011 KiB

Open AccessReview

Iron in Lung Pathology

by Vida Zhang, Elizabeta Nemeth and Airie Kim

Pharmaceuticals 2019, 12(1), 30; https://doi.org/10.3390/ph12010030 - 15 Feb 2019

Cited by 35 | Viewed by 6623

Abstract

The lung presents a unique challenge for iron homeostasis. The entire airway is in direct contact with the environment and its iron particulate matter and iron-utilizing microbes. However, the homeostatic and adaptive mechanisms of pulmonary iron regulation are poorly understood. This review provides [...] Read more.

The lung presents a unique challenge for iron homeostasis. The entire airway is in direct contact with the environment and its iron particulate matter and iron-utilizing microbes. However, the homeostatic and adaptive mechanisms of pulmonary iron regulation are poorly understood. This review provides an overview of systemic and local lung iron regulation, as well as the roles of iron in the development of lung infections, airway disease, and lung injury. These mechanisms provide an important foundation for the ongoing development of therapeutic applications. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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18 pages, 347 KiB

Open AccessReview

Inflammation and Depression: A Nervous Plea for Psychiatry to Not Become Immune to Interpretation

by Jan Pieter Konsman

Pharmaceuticals 2019, 12(1), 29; https://doi.org/10.3390/ph12010029 - 14 Feb 2019

Cited by 21 | Viewed by 5602

Abstract

The possibility that inflammation plays a causal role in major depression is an important claim in the emerging field of immunopsychiatry and has generated hope for new treatments. The aims of the present review are first to provide some historical background and to [...] Read more.

The possibility that inflammation plays a causal role in major depression is an important claim in the emerging field of immunopsychiatry and has generated hope for new treatments. The aims of the present review are first to provide some historical background and to consider the evidence in favor of the claim that inflammation is causally involved in major depression. The second part discusses some of the possibilities allowed for by the use of broad ‘umbrella’ concepts, such as inflammation and stress, in terms of proposing new working hypotheses and potential mechanisms. The third part reviews proposed biomarkers of inflammation and depression and the final part addresses how elements discussed in the preceding sections are used in immunopsychiatry. The ‘umbrella’ concepts of inflammation and stress, as well as insufficiently-met criteria based inferences and reverse inferences are being used to some extent in immunopsychiatry. The field is therefore encouraged to specify concepts and constructs, as well as to consider potential alternative interpretations and explanations for findings obtained. The hope is that pointing out some of the potential problems will allow for a clearer picture of immunopsychiatry’s current strengths and limitations and help the field mature. Full article

(This article belongs to the Special Issue Understanding Inflammation-induced Mental Diseases: New Opportunities for Treatment)

17 pages, 2574 KiB

Open AccessArticle

Conformation and Cross-Protection in Group B Streptococcus Serotype III and Streptococcus pneumoniae Serotype 14: A Molecular Modeling Study

by Michelle M. Kuttel and Neil Ravenscroft

Pharmaceuticals 2019, 12(1), 28; https://doi.org/10.3390/ph12010028 - 13 Feb 2019

Cited by 12 | Viewed by 5296

Abstract

Although the branched capsular polysaccharides of Streptococcus agalactiae serotype III (GBSIII PS) and Streptococcus pneumoniae serotype 14 (Pn14 PS) differ only in the addition of a terminal sialic acid on the GBSIII PS side chains, these very similar polysaccharides are immunogenically distinct. Our [...] Read more.

Although the branched capsular polysaccharides of Streptococcus agalactiae serotype III (GBSIII PS) and Streptococcus pneumoniae serotype 14 (Pn14 PS) differ only in the addition of a terminal sialic acid on the GBSIII PS side chains, these very similar polysaccharides are immunogenically distinct. Our simulations of GBSIII PS, Pn14 PS and the unbranched backbone polysaccharide provide a conformational rationale for the different antigenic epitopes identified for these PS. We find that side chains stabilize the proximal

β

dGlc(1→6)

β

dGlcNAc backbone linkage, restricting rotation and creating a well-defined conformational epitope at the branch point. This agrees with the glycotope structure recognized by an anti-GBSIII PS functional monoclonal antibody. We find the same dominant solution conformation for GBSIII and Pn14 PS: aside from the branch point, the backbone is very flexible with a “zig-zag” conformational habit, rather than the helix previously proposed for GBSIII PS. This suggests a common strategy for bacterial evasion of the host immune system: a flexible backbone that is less perceptible to the immune system, combined with conformationally-defined branch points presenting human-mimic epitopes. This work demonstrates how small structural features such as side chains can alter the conformation of a polysaccharide by restricting rotation around backbone linkages. Full article

(This article belongs to the Special Issue Carbohydrates 2018)

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22 pages, 747 KiB

Open AccessReview

Neurodegeneration with Brain Iron Accumulation Disorders: Valuable Models Aimed at Understanding the Pathogenesis of Iron Deposition

by Sonia Levi and Valeria Tiranti

Pharmaceuticals 2019, 12(1), 27; https://doi.org/10.3390/ph12010027 - 9 Feb 2019

Cited by 63 | Viewed by 8534

Abstract

Neurodegeneration with brain iron accumulation (NBIA) is a set of neurodegenerative disorders, which includes very rare monogenetic diseases. They are heterogeneous in regard to the onset and the clinical symptoms, while the have in common a specific brain iron deposition in the region [...] Read more.

Neurodegeneration with brain iron accumulation (NBIA) is a set of neurodegenerative disorders, which includes very rare monogenetic diseases. They are heterogeneous in regard to the onset and the clinical symptoms, while the have in common a specific brain iron deposition in the region of the basal ganglia that can be visualized by radiological and histopathological examinations. Nowadays, 15 genes have been identified as causative for NBIA, of which only two code for iron-proteins, while all the other causative genes codify for proteins not involved in iron management. Thus, how iron participates to the pathogenetic mechanism of most NBIA remains unclear, essentially for the lack of experimental models that fully recapitulate the human phenotype. In this review we reported the recent data on new models of these disorders aimed at highlight the still scarce knowledge of the pathogenesis of iron deposition. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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11 pages, 1162 KiB

Open AccessArticle

Curcumin and (−)- Epigallocatechin-3-Gallate Protect Murine MIN6 Pancreatic Beta-Cells against Iron Toxicity and Erastin-Induced Ferroptosis

by Tugba Kose, Mayra Vera-Aviles, Paul A. Sharp and Gladys O. Latunde-Dada

Pharmaceuticals 2019, 12(1), 26; https://doi.org/10.3390/ph12010026 - 6 Feb 2019

Cited by 95 | Viewed by 10087

Abstract

Ferroptosis is a form of programmed cell death that is characterized by lipid peroxidation and is inducible by iron and the accumulation of reactive oxygen species (ROS). It is triggered by erastin but inhibited by antioxidants such as α-tocopherol, β-carotene, polyphenols, and iron [...] Read more.

Ferroptosis is a form of programmed cell death that is characterized by lipid peroxidation and is inducible by iron and the accumulation of reactive oxygen species (ROS). It is triggered by erastin but inhibited by antioxidants such as α-tocopherol, β-carotene, polyphenols, and iron chelators such as deferoxamine (DFO), nitrilotriacetic acid (NTA), and ethylenediaminetetraacetic acid (EDTA). This study investigated the protective effects of two polyphenols, curcumin and (−)- epigallocatechin-3-gallate (EGCG), against iron loading and erastin-mediated ferroptosis in MIN6 cells. Cells were treated with polyphenols before exposure to iron-induced oxidative stress comprising of 20 μmol/L of 8-hydroxyquinoline (8HQ) and 50 μmol/L of ferric ammonium citrate, (FAC) (8HQ+FAC) or Fenton reaction substrate (FS) (30 μmol/L of FeSO₄ and 0.5 of mmol/L H₂O₂) and 20 μmol/L erastin. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, iron levels were measured by inductively-coupled plasma mass spectrometry (ICP-MS), glutathione and lipid peroxidation were assayed with commercially-available kits. Curcumin and EGCG both significantly protected pancreatic cells against iron-induced oxidative damage. Moreover, both compounds also protected against erastin-induced ferroptosis in pancreatic cells. The polyphenols enhanced cell viability in erastin-treated MIN6 cells in a dose- and time-dependent manner. Furthermore, MIN6 cells exposed to erastin alone showed elevated levels of iron, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) degradation and lipid peroxidation (p < 0.05) compared to cells that were protected by pre-treatment with curcumin or EGCG. Taken together, the data identify curcumin and EGCG as novel ferroptosis inhibitors, which might exert their protective effects by acting as iron chelators and preventing GSH depletion, GPX4 inactivation, and lipid peroxidation in MIN6 cells. The implications of the findings on the effects of iron overload and ferroptosis represent a potential therapeutic strategy against iron-related diseases. Full article

(This article belongs to the Special Issue Plant Phytochemicals on Drug Development)

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Full article ">Figure 1
Protective effects of different polyphenols against iron-mediated toxicity and ferroptosis. MIN6 cells were treated with 20 μM curcumin, quercetin, rutin, EGCG, tannic acid or phytic acid for 24 h and supplemented with (A) Fenton Substrates (FS), (B) 8HQ+FAC for 2 h and (C) MIN6 cells were treated overnight with 20 μM erastin, in the absence or presence of quercetin, rutin, curcumin, tannic acid, phytic acid and EGCG. The percentage of cell viability is relative to control cell samples. Curcumin and EGCG inhibited erastin-induced cell death in a dose-dependent manner. Curcumin and EGCG had a protective effect against ferroptosis at 20 μM in MIN6 for 24 h, with a valuable statistical difference between erastin and cell treated with erastin + 20 μM curcumin or EGCG. All the values are expressed with the mean ± SEM, n = 8. #p < 0.05 control vs. treatment groups, **p < 0.01 and ****p < 0.0001 compared with FS and 8HQ+FAC group only. (C) #p < 0.05 control vs. treatment groups, ****p < 0.0001 vs. erastin only. One-way ANOVA, Tukey post-hoc test. Full article ">Figure 2
Anti-ferroptosis activity of curcumin and EGCG in MIN6 cells. Cells were treated overnight with 20 μM erastin in the absence or presence of curcumin or EGCG. The percentage of cell viability is relative to control cell samples. Curcumin and EGCG inhibited erastin-induced cell death in a dose-dependent manner. Curcumin (A) and EGCG (B) had a protective effect against ferroptosis at 20 μM in MIN6 for 24 h, with valuable statistical difference between erastin and cell treated with erastin + 20 μM curcumin or EGCG. Curcumin (C) and EGCG (D) inhibited erastin-induced cell death in a time-dependent manner in MIN6 with valuable statistical difference between erastin and cell treated with erastin + 20 μM curcumin or EGCG. All the values are expressed with the mean ± SEM, n = 8, #p < 0.05 control vs. treatment groups, *p < 0.05 and ****p < 0.0001 vs. erastin only. One-way ANOVA, Tukey post-hoc test. Full article ">Figure 3
Curcumin and EGCG suppress iron and lipid accumulation in pancreatic cells. (A) Cells were treated overnight with 20 μM erastin in the absence or presence of curcumin or EGCG. Percentage of Fe2+ is relative to control cell samples. Curcumin and EGCG decreased erastin-induced iron accumulation at 20 μM in MIN6 for 24 h, with significant statistical difference between erastin and cell treated with erastin + 20 μM curcumin or EGCG. (B) Percentage of MDA is relative to control cell samples. Curcumin and EGCG decreased erastin-induced lipid peroxidation at 20 μM in MIN6 for 24 h, with valuable statistical difference between erastin and cell treated with erastin + 20 μM curcumin or EGCG. All the values are expressed with the mean ± SEM, n = 3, #p < 0.05 control vs. treatment groups, **p < 0.01 and ****p < 0.0001 vs. erastin only. One-way ANOVA, Tukey post-hoc test. Full article ">Figure 4
Curcumin and EGCG inhibit GSH depletion in pancreatic cells. Cells were treated overnight with 20 μM erastin in the absence or presence of curcumin or EGCG. Percentage GSH level is relative to control cell samples. (A) Curcumin and EGCG decreased erastin-induced GSH level. (B) Western blot analysis showed that curcumin alone significantly suppressed erastin-induced GPX4 level in MIN6 for 24 h. (C) Densitometry of Western blots GPX4 protein bands of GPX4. All the values are expressed with the mean ± SEM, n = 3, # p < 0.05 control vs. treatment groups, *p < 0.05 and **p < 0.01 vs. erastin only. One-way ANOVA, Tukey post-hoc test. Full article ">

8 pages, 1676 KiB

Open AccessArticle

Comparative Study of Two Oxidizing Agents, Chloramine T and Iodo-Gen^®, for the Radiolabeling of β-CIT with Iodine-131: Relevance for Parkinson’s Disease

by Ana Claudia R. Durante, Danielle V. Sobral, Ana Claudia C. Miranda, Érika V. de Almeida, Leonardo L. Fuscaldi, Marycel R. F. F. de Barboza and Luciana Malavolta

Pharmaceuticals 2019, 12(1), 25; https://doi.org/10.3390/ph12010025 - 5 Feb 2019

Cited by 17 | Viewed by 3989

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to alteration of the integrity of dopaminergic transporters (DATs). In recent years, some radiopharmaceuticals have been used in the clinic to [...] Read more.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to alteration of the integrity of dopaminergic transporters (DATs). In recent years, some radiopharmaceuticals have been used in the clinic to evaluate the integrity of DATs. These include tropane derivatives such as radiolabeled β-CIT and FP-CIT with iodine-123 (¹²³I), and TRODAT-1 with metastable technetium-99 (^99mTc). Radiolabeling of β-CIT with radioactive iodine is based on electrophilic radioiodination using oxidizing agents, such as Chloramine T or Iodo-Gen^®. For the first time, the present work performed a comparative study of the radiolabeling of β-CIT with iodine-131 (¹³¹I), using either Chloramine T or Iodo-Gen^® as oxidizing agents, in order to improve the radiolabeling process of β-CIT and to choose the most advantageous oxidizing agent to be used in nuclear medicine. Both radiolabeling methods were similar and resulted in high radiochemical yield (> 95%), with suitable ¹³¹I-β-CIT stability up to 72 h. Although Chloramine T is a strong oxidizing agent, it was as effective as Iodo-Gen^® for β-CIT radiolabeling with ¹³¹I, with the advantage of briefer reaction time and solubility in aqueous medium. Full article

(This article belongs to the Special Issue Targets, Tracers and Translation – Novel Radiopharmaceuticals Boost Nuclear Medicine)

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11 pages, 679 KiB

Open AccessArticle

Camphor, Applied Epidermally to the Back, Causes Snout- and Chest-Grooming in Rats: A Response Mediated by Cutaneous TRP Channels

by Débora T. Ishikawa, Robson Cristiano Lillo Vizin, Cristiane Oliveira de Souza, Daniel Carneiro Carrettiero, Andrej A. Romanovsky and Maria Camila Almeida

Pharmaceuticals 2019, 12(1), 24; https://doi.org/10.3390/ph12010024 - 2 Feb 2019

Cited by 4 | Viewed by 4172

Abstract

Thermoregulatory grooming, a behavioral defense against heat, is known to be driven by skin-temperature signals. Because at least some thermal cutaneous signals that drive heat defenses are likely to be generated by transient receptor potential (TRP) channels, we hypothesized that warmth-sensitive TRPs drive [...] Read more.

Thermoregulatory grooming, a behavioral defense against heat, is known to be driven by skin-temperature signals. Because at least some thermal cutaneous signals that drive heat defenses are likely to be generated by transient receptor potential (TRP) channels, we hypothesized that warmth-sensitive TRPs drive thermoregulatory grooming. Adult male Wistar rats were used. We showed that camphor, a nonselective agonist of several TRP channels, including vanilloid (V) 3, when applied epidermally to the back (500 mg/kg), caused a pronounced self-grooming response, including paw-licking and snout- and chest-“washing”. By the percentage of time spent grooming, the response was similar to the thermoregulatory grooming observed during exposure to ambient warmth (32 °C). Ruthenium red (a non-selective antagonist of TRP channels, including TRPV3), when administered intravenously at a dose of 0.1 mg/kg, attenuated the self-grooming behavior induced by either ambient warmth or epidermal camphor. Furthermore, the intravenous administration of AMG8432 (40 mg/kg), a relatively selective TRPV3 antagonist, also attenuated the self-grooming response to epidermal camphor. We conclude that camphor causes the self-grooming behavior by acting on TRP channels in the skin. We propose that cutaneous warmth signals mediated by TRP channels, possibly including TRPV3, drive thermoregulatory self-grooming in rats. Full article

(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Unlocking the Potential)

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Figure 1
Effects of different Tas on Tb (A), Tsk (B), and grooming (C). An asterisk (*) indicates a significant difference (p < 0.05), as compared to the neutral Ta (26 °C). Full article ">Figure 2
Effect of camphor on Tb (A), Tsk (B), and grooming (C). Camphor, a nonselective transient receptor potential (TRP) agonist, when applied epidermally (e.d.; dose indicated), did not alter Tb or Tsk, but increased the time spent self-grooming, as compared to vehicle (polypropylene glycol, PPG). The experiments were conducted at neutral Ta of 26 °C. An asterisk (*) indicates a significant difference (p < 0.05), as compared to PPG-treated rats. Full article ">Figure 3
Effects of pharmacological blockade of TRP channels on warmth- (A) and camphor- (B,C) induced grooming. Warmth- and camphor-induced self-grooming responses were blocked by pretreatment with the nonselective TRP antagonist ruthenium red (dose indicated). Camphor-induced self-grooming was also blocked by AMG8432, a relatively nonselective TRPV3 antagonist. Both ruthenium red and AMG8432 were injected 30 min prior the exposure to 26 °C or 32 °C, or the camphor or PPG administration. An asterisk (*) indicates a significant difference (p < 0.05) between grooming responses at 32 vs. 26 °C (A), or in saline-pretreated camphor-treated vs. saline-pretreated PPG-treated rats (B). A pound sign (#) indicates a significant difference (p < 0.05) between grooming responses: in ruthenium red- vs. saline-pretreated rats at 32 °C (A); ruthenium red- vs. saline-pretreated camphor-treated rats (B); or in AMG8432-pretreated camphor-treated vs. vehicle-pretreated camphor-treated rats (C). Full article ">

23 pages, 2681 KiB

Open AccessReview

Modulators of Transient Receptor Potential (TRP) Channels as Therapeutic Options in Lung Disease

by Alexander Dietrich

Pharmaceuticals 2019, 12(1), 23; https://doi.org/10.3390/ph12010023 - 1 Feb 2019

Cited by 48 | Viewed by 8108

Abstract

The lungs are essential for gas exchange and serve as the gateways of our body to the external environment. They are easily accessible for drugs from both sides, the airways and the vasculature. Recent literature provides evidence for a role of Transient Receptor [...] Read more.

The lungs are essential for gas exchange and serve as the gateways of our body to the external environment. They are easily accessible for drugs from both sides, the airways and the vasculature. Recent literature provides evidence for a role of Transient Receptor Potential (TRP) channels as chemosensors and essential members of signal transduction cascades in stress-induced cellular responses. This review will focus on TRP channels (TRPA1, TRPC6, TRPV1, and TRPV4), predominantly expressed in non-neuronal lung tissues and their involvement in pathways associated with diseases like asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), lung fibrosis, and edema formation. Recently identified specific modulators of these channels and their potential as new therapeutic options as well as strategies for a causal treatment based on the mechanistic understanding of molecular events will also be evaluated. Full article

(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Unlocking the Potential)

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1 pages, 175 KiB

Open AccessCorrection

Correction: Mateusz, S., et al. Iron Supplementation in Suckling Piglets: An Ostensibly Easy Therapy of Neonatal Iron Deficiency Anemia. Pharmaceuticals 2018, 11, 128

by Mateusz Szudzik, Rafał R. Starzyński, Aneta Jończy, Rafał Mazgaj, Małgorzata Lenartowicz and Paweł Lipiński

Pharmaceuticals 2019, 12(1), 22; https://doi.org/10.3390/ph12010022 - 29 Jan 2019

Cited by 3 | Viewed by 3174

Abstract

The authors wish to make the following corrections to this paper [¹]: the term “liposomal” should be replaced with the term “sucrosomial” in the following places [...]

Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

12 pages, 2080 KiB

Open AccessArticle

On the Absolute Stereochemistry of Tolterodine: A Circular Dichroism Study

by Marcin Górecki, Valerio Zullo, Anna Iuliano and Gennaro Pescitelli

Pharmaceuticals 2019, 12(1), 21; https://doi.org/10.3390/ph12010021 - 26 Jan 2019

Cited by 10 | Viewed by 4647

Abstract

Tolterodine (1) is a potent muscarinic receptor antagonist used in the treatment of overactive urinary bladder (OAB) syndrome. Tolterodine is chiral and it was patented, and is currently marketed, as the l-tartrate salt of the (R)-enantiomer. However, the [...] Read more.

Tolterodine (1) is a potent muscarinic receptor antagonist used in the treatment of overactive urinary bladder (OAB) syndrome. Tolterodine is chiral and it was patented, and is currently marketed, as the l-tartrate salt of the (R)-enantiomer. However, the existing literature does not offer an ultimate proof of a stereoselective mode of action of 1. A second open stereochemical issue concerns the absolute configuration (AC) of 1. Neither the original patents nor subsequent studies have established the AC of 1 in an unambiguous way, although the AC of the l-tartrate salt of 1 was assigned by X-ray diffractometry. Finally, neither electronic nor vibrational circular dichroism (ECD and VCD) spectra of 1 are reported so far. We performed a thorough ECD/VCD study of 1 in different solvents and at variable temperatures. Solvent and temperature dependence highlighted the existence of moderate flexibility which was confirmed by molecular modelling. ECD calculations with time-dependent density functional theory (TDDFT) accurately reproduced the experimental spectra and allowed us to confirm the AC of 1 in an independent way. Full article

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Full article ">Figure 1
Structures of (R)-tolterodine (1), its hydrogen L-tartrate salt 2, and precursor 3. Full article ">Figure 2
Absorption (top) and ECD spectra (bottom) of (R)-tolterodine (1) measured in different solvents. Concentration 1.67 × 10−4 M, cell path length 0.1 cm. Full article ">Figure 3
ECD spectra of (R)-tolterodine (1) measured in MCH and MeOH at variable temperatures. Concentration 1.67 × 10−4 M, cell path length 0.1 cm. Full article ">Figure 4
ECD spectra calculated at CAM-B3LYP/def2-TZVP level for the first 4 low-energy conformers of (R)-1 obtained after ωB97X-D/6-31+G(d) geometry optimizations (shown on the right, with relative energies). Spectra plotted as sum of Gaussians with σ = 0.3 eV, shifted by +13 nm. The molecular diagram emphasizes the formation of the intramolecular hydrogen bond (green). Full article ">Figure 5
Comparison between experimental ECD spectra of (R)-1 measured in MCH at 298 K (left) and 183 K (right) and ECD spectra calculated at CAM-B3LYP/def2-TZVP and B3LYP/def2-TZVP levels as Boltzmann-weighted averages estimated from ωB97X-D/6-31+G(d) energies at the respective temperatures. Spectra plotted as sum of Gaussians with σ = 0.3 eV, shifted by +13 nm, scaling factor 0.31 (CAM-B3LYP); σ = 0.22 eV, shifted by +6 nm, scaling factor 0.6 (B3LYP). Full article ">Figure 6
Experimental (black lines) and calculated (red lines) IR (bottom traces) and VCD (top traces) spectra of (R)-1. Experimental conditions: 0.18 M solution in CCl4, cell path length 100 μm. Calculations run at B3LYP/6-311+G(d,p) level, Boltzmann average over 40 conformers, plotted as sum of Lorentzians with γ = 6 cm−1, scaling factor 0.98. The vertical bands highlight the correspondence between experimental and calculated VCD peaks, while the asterisks (*) indicate experimental IR/VCD peaks with no calculated counterpart. Full article ">Scheme 1
Enantioselective route to (R)-(+)-tolterodine 1. Full article ">

22 pages, 3061 KiB

Open AccessArticle

Identification of Potential Inhibitors from Pyriproxyfen with Insecticidal Activity by Virtual Screening

by Ryan da Silva Ramos, Josivan da Silva Costa, Rai Campos Silva, Glauber Vilhena da Costa, Alex Bruno Lobato Rodrigues, Érica de Menezes Rabelo, Raimundo Nonato Picanço Souto, Carlton Anthony Taft, Carlos Henrique Tomich de Paula da Silva, Joaquín Maria Campos Rosa, Cleydson Breno Rodrigues dos Santos and Williams Jorge da Cruz Macêdo

Pharmaceuticals 2019, 12(1), 20; https://doi.org/10.3390/ph12010020 - 25 Jan 2019

Cited by 41 | Viewed by 7602

Abstract

Aedes aegypti is the main vector of dengue fever transmission, yellow fever, Zika, and chikungunya in tropical and subtropical regions and it is considered to cause health risks to millions of people in the world. In this study, we search to obtain new [...] Read more.

Aedes aegypti is the main vector of dengue fever transmission, yellow fever, Zika, and chikungunya in tropical and subtropical regions and it is considered to cause health risks to millions of people in the world. In this study, we search to obtain new molecules with insecticidal potential against Ae. aegypti via virtual screening. Pyriproxyfen was chosen as a template compound to search molecules in the database Zinc_Natural_Stock (ZNSt) with structural similarity using ROCS (rapid overlay of chemical structures) and EON (electrostatic similarity) software, and in the final search, the top 100 were selected. Subsequently, in silico pharmacokinetic and toxicological properties were determined resulting in a total of 14 molecules, and these were submitted to the PASS online server for the prediction of biological insecticide and acetylcholinesterase activities, and only two selected molecules followed for the molecular docking study to evaluate the binding free energy and interaction mode. After these procedures were performed, toxicity risk assessment such as LD₅₀ values in mg/kg and toxicity class using the PROTOX online server, were undertaken. Molecule ZINC00001624 presented potential for inhibition for the acetylcholinesterase enzyme (insect and human) with a binding affinity value of −10.5 and −10.3 kcal/mol, respectively. The interaction with the juvenile hormone was −11.4 kcal/mol for the molecule ZINC00001021. Molecules ZINC00001021 and ZINC00001624 had excellent predictions in all the steps of the study and may be indicated as the most promising molecules resulting from the virtual screening of new insecticidal agents. Full article

(This article belongs to the Special Issue Design of Enzyme Inhibitors as Potential Drugs)

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20 pages, 2006 KiB

Open AccessArticle

TRPV1 Inhibits the Ventilatory Response to Hypoxia in Adult Rats, but Not the CO₂-Drive to Breathe

by Luis Gustavo A. Patrone, Jaime B. Duarte, Kênia Cardoso Bícego, Alexandre A. Steiner, Andrej A. Romanovsky and Luciane H. Gargaglioni

Pharmaceuticals 2019, 12(1), 19; https://doi.org/10.3390/ph12010019 - 24 Jan 2019

Cited by 3 | Viewed by 3966

Abstract

Receptors of the transient receptor potential (TRP) channels superfamily are expressed in many tissues and have different physiological functions. However, there are few studies investigating the role of these channels in cardiorespiratory control in mammals. We assessed the role of central and peripheral [...] Read more.

Receptors of the transient receptor potential (TRP) channels superfamily are expressed in many tissues and have different physiological functions. However, there are few studies investigating the role of these channels in cardiorespiratory control in mammals. We assessed the role of central and peripheral TRPV1 receptors in the cardiorespiratory responses to hypoxia (10% O₂) and hypercapnia (7% CO₂) by measuring pulmonary ventilation (

{\dot{V}}_{E}

), heart rate (HR), mean arterial pressure (MAP) and body temperature (Tb) of male Wistar rats before and after intraperitoneal (AMG9810 [2.85 µg/kg, 1 mL/kg]) or intracebroventricular (AMG9810 [2.85 µg/kg, 1 µL] or AMG7905 [28.5 μg/kg, 1 µL]) injections of TRPV1 antagonists. Central or peripheral injection of TRPV1 antagonists did not change cardiorespiratory parameters or Tb during room air and hypercapnic conditions. However, the hypoxic ventilatory response was exaggerated by both central and peripheral injection of AMG9810. In addition, the peripheral antagonist blunted the drop in Tb induced by hypoxia. Therefore, the current data provide evidence that TRPV1 channels exert an inhibitory modulation on the hypoxic drive to breathe and stimulate the Tb reduction during hypoxia. Full article

(This article belongs to the Special Issue Transient Receptor Potential (TRP) Channels in Drug Discovery: Unlocking the Potential)

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9 pages, 1234 KiB

Open AccessArticle

[¹⁷⁷Lu]Lu-PSMA-617 Salivary Gland Uptake Characterized by Quantitative In Vitro Autoradiography

by Roswitha Tönnesmann, Philipp T. Meyer, Matthias Eder and Ann-Christin Baranski

Pharmaceuticals 2019, 12(1), 18; https://doi.org/10.3390/ph12010018 - 24 Jan 2019

Cited by 51 | Viewed by 9096

Abstract

Irradiation of salivary glands remains the main dose-limiting side effect of therapeutic PSMA-inhibitors, especially when using alpha emitters. Thus, further advances in radiopharmaceutical design and therapy strategies are needed to reduce salivary gland uptake, thereby allowing the administration of higher doses and potentially [...] Read more.

Irradiation of salivary glands remains the main dose-limiting side effect of therapeutic PSMA-inhibitors, especially when using alpha emitters. Thus, further advances in radiopharmaceutical design and therapy strategies are needed to reduce salivary gland uptake, thereby allowing the administration of higher doses and potentially resulting in improved response rates and better tumor control. As the uptake mechanism remains unknown, this work investigates the salivary gland uptake of [¹⁷⁷Lu]Lu-PSMA-617 by autoradiography studies on pig salivary gland tissue and on PSMA-overexpressing LNCaP cell membrane pellets. Displacement studies were performed with non-labeled PSMA-617 and 2-PMPA, respectively. The uptake of [¹⁷⁷Lu]Lu-PSMA-617 in glandular areas was determined to be partly PSMA-specific, with a high non-specific uptake fraction. The study emphasizes that [¹⁷⁷Lu]Lu-PSMA-617 accumulation in pig salivary glands can be attributed to a combination of both specific and non-specific uptake mechanisms. The observation is of high impact for future design of novel radiopharmaceuticals addressing the dose-limiting salivary gland irradiation of current alpha endoradiotherapy in prostate cancer. Full article

(This article belongs to the Special Issue Targets, Tracers and Translation – Novel Radiopharmaceuticals Boost Nuclear Medicine)

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15 pages, 1793 KiB

Open AccessArticle

L-Ferritin: One Gene, Five Diseases; from Hereditary Hyperferritinemia to Hypoferritinemia—Report of New Cases

by Beatriz Cadenas, Josep Fita-Torró, Mar Bermúdez-Cortés, Inés Hernandez-Rodriguez, José Luis Fuster, María Esther Llinares, Ana María Galera, Julia Lee Romero, Santiago Pérez-Montero, Cristian Tornador and Mayka Sanchez

Pharmaceuticals 2019, 12(1), 17; https://doi.org/10.3390/ph12010017 - 23 Jan 2019

Cited by 22 | Viewed by 9916

Abstract

Ferritin is a multimeric protein composed of light (L-ferritin) and heavy (H-ferritin) subunits that binds and stores iron inside the cell. A variety of mutations have been reported in the L-ferritin subunit gene (FTL gene) that cause the following five diseases: (1) [...] Read more.

Ferritin is a multimeric protein composed of light (L-ferritin) and heavy (H-ferritin) subunits that binds and stores iron inside the cell. A variety of mutations have been reported in the L-ferritin subunit gene (FTL gene) that cause the following five diseases: (1) hereditary hyperferritinemia with cataract syndrome (HHCS), (2) neuroferritinopathy, a subtype of neurodegeneration with brain iron accumulation (NBIA), (3) benign hyperferritinemia, (4) L-ferritin deficiency with autosomal dominant inheritance, and (5) L-ferritin deficiency with autosomal recessive inheritance. Defects in the FTL gene lead to abnormally high levels of serum ferritin (hyperferritinemia) in HHCS and benign hyperferritinemia, while low levels (hypoferritinemia) are present in neuroferritinopathy and in autosomal dominant and recessive L-ferritin deficiency. Iron disturbances as well as neuromuscular and cognitive deficits are present in some, but not all, of these diseases. Here, we identified two novel FTL variants that cause dominant L-ferritin deficiency and HHCS (c.375+2T > A and 36_42delCAACAGT, respectively), and one previously reported variant (Met1Val) that causes dominant L-ferritin deficiency. Globally, genetic changes in the FTL gene are responsible for multiple phenotypes and an accurate diagnosis is useful for appropriate treatment. To help in this goal, we included a diagnostic algorithm for the detection of diseases caused by defects in FTL gene. Full article

(This article belongs to the Special Issue Iron as Therapeutic Targets in Human Diseases)

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Figure 1
Pedigree trees from three studied families affected from dominant L-ferritin deficiency and HHCS. Squares indicate males and circles females. Probands are pointed with an arrow. Filled symbols indicate affected members and asterisks indicate subjects with genetic studies done at BloodGenetics SL. Mutations are named according the HGVS nomenclature. Full article ">Figure 2
Schematic localization of literature reported and new FTL mutations. Mutations described in this work are in bold and new mutations are boxed. The domains of the five alpha helices (A to E) are represented in the protein (NP_000137.2). Mutations are classified as nonsense, frameshift, missense, or splicing. Here we report FTL protein changes using the three-letter amino acid code. Full article ">Figure 3
WT and mutated FTL-IRE fold prediction. Predicted secondary structure of WT and mutated FTL-IRE using Sfold web server [<a href="#B20-pharmaceuticals-12-00017" class="html-bibr">20</a>]. Deletion in hexanucleotide loop (c.-164_158del7) is expected to disturb completely the IRE structure. Nucleotides are numbered from the transcription starting site. Free energy (ΔG) is detailed. Full article ">Figure 4
Algorithm for diagnosis of diseases caused by defects in FTL gene. The following abbreviations were used: F, female; M, male. The mutation nomenclature used follows the HGVS guidelines. Full article ">

15 pages, 596 KiB

Open AccessReview

Can the Efficacy of [¹⁸F]FDG-PET/CT in Clinical Oncology Be Enhanced by Screening Biomolecular Profiles?

by Hazel O’Neill, Vinod Malik, Ciaran Johnston, John V Reynolds and Jacintha O’Sullivan

Pharmaceuticals 2019, 12(1), 16; https://doi.org/10.3390/ph12010016 - 23 Jan 2019

Cited by 13 | Viewed by 9627

Abstract

Positron Emission Tomography (PET) is a functional imaging modality widely used in clinical oncology. Over the years the sensitivity and specificity of PET has improved with the advent of specific radiotracers, increased technical accuracy of PET scanners and incremental experience of Radiologists. However, [...] Read more.

Positron Emission Tomography (PET) is a functional imaging modality widely used in clinical oncology. Over the years the sensitivity and specificity of PET has improved with the advent of specific radiotracers, increased technical accuracy of PET scanners and incremental experience of Radiologists. However, significant limitations exist—most notably false positives and false negatives. Additionally, the accuracy of PET varies between cancer types and in some cancers, is no longer considered a standard imaging modality. This review considers the relative influence of macroscopic tumour features such as size and morphology on 2-Deoxy-2-[¹⁸F]fluoroglucose ([¹⁸F]FDG) uptake by tumours which, though well described in the literature, lacks a comprehensive assessment of biomolecular features which may influence [¹⁸F]FDG uptake. The review aims to discuss the potential influence of individual molecular markers of glucose transport, glycolysis, hypoxia and angiogenesis in addition to the relationships between these key cellular processes and their influence on [¹⁸F]FDG uptake. Finally, the potential role for biomolecular profiling of individual tumours to predict positivity on PET imaging is discussed to enhance accuracy and clinical utility. Full article

(This article belongs to the Special Issue Anticancer Drugs)

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