Pharmaceuticals

16 pages, 4912 KiB

Open AccessArticle

Developing In Situ Chemometric Models with Raman Spectroscopy for Monitoring an API Disproportionation with a Complex Polymorphic Landscape

by Shikhar Mohan, Yi Li, Kevin Chu, Bing Shi, Liliana De La Paz, Prarthana Bakre, Chris Foti, Victor Rucker and Chiajen Lai

Pharmaceuticals 2023, 16(2), 327; https://doi.org/10.3390/ph16020327 - 20 Feb 2023

Cited by 1 | Viewed by 2641

Abstract

An in situ Raman method was developed to characterize the disproportionation of two salts involving a complex polymorphic landscape comprising up to two metastable and one stable freebase forms. Few precedents exist for Raman calibration procedures for solid form quantitation involving more than [...] Read more.

An in situ Raman method was developed to characterize the disproportionation of two salts involving a complex polymorphic landscape comprising up to two metastable and one stable freebase forms. Few precedents exist for Raman calibration procedures for solid form quantitation involving more than two polymorphs, while no literature examples were found for cases with multiple metastable forms. Therefore, a new Raman calibration procedure was proposed by directly using disproportionation experiments to generate multiple calibration samples encompassing a range of polymorph ratios through in-line Raman measurements complemented by off-line reference X-ray diffraction measurements. The developed Raman methods were capable of accurately quantitating each solid form in situ when solid concentration variation was incorporated into the calibration dataset. The kinetic understanding of the thermodynamically driven polymorphic conversions gained from this Raman method guided the selection of the salt best suited for the delivery of the active ingredient in the drug product. This work provided a spectroscopic and mathematical approach for simultaneously quantitating multiple polymorphs from a complex mixture of solids with the objective of real-time monitoring. Full article

(This article belongs to the Special Issue Polymorphs, Salts, and Cocrystals in Drug Delivery)

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29 pages, 3584 KiB

Open AccessArticle

Lyophilized Nasal Inserts of Atomoxetine HCl Solid Lipid Nanoparticles for Brain Targeting as a Treatment of Attention-Deficit/Hyperactivity Disorder (ADHD): A Pharmacokinetics Study on Rats

by Mahmoud H. Teaima, Merhan Taha El-Nadi, Raghda Rabe Hamed, Mohamed A. El-Nabarawi and Rehab Abdelmonem

Pharmaceuticals 2023, 16(2), 326; https://doi.org/10.3390/ph16020326 - 20 Feb 2023

Cited by 9 | Viewed by 3613

Abstract

The study aims to investigate the ability of lyophilized nasal inserts of nanosized atomoxetine HCl solid lipid nanoparticles (ATM-SLNs) to transport atomoxetine (ATM) directly to the brain and overcome the first-pass metabolism. In this case, 16 formulae of (ATM-SLNs) were prepared using hot [...] Read more.

The study aims to investigate the ability of lyophilized nasal inserts of nanosized atomoxetine HCl solid lipid nanoparticles (ATM-SLNs) to transport atomoxetine (ATM) directly to the brain and overcome the first-pass metabolism. In this case, 16 formulae of (ATM-SLNs) were prepared using hot melt emulsification, stirring and ultrasonication method technique. A full factorial design was established with 2⁴ trials by optimization of four variables; lipid type (Compritol 888 ATO or stearic acid) (X1), lipid to drug ratio [(1:2) or (2:1)] (X2), span 60: Pluronic f127 ratio [(1:3) or (3:1)] (X3) and probe sonication time (five or ten minutes) (X4). The prepared SLNs were characterized for entrapment efficiency (EE%), in-vitro drug release after 30 min (Q30min), particle size (PS), zeta potential (ZP) and polydispersity index (PDI). Design Expert^® software was used to select the optimum two formulae. The morphological examination for the optimum two formulae was carried out using a transmission electron microscope (TEM). Furthermore, eight lyophilized nasal inserts were prepared by using a 2³ full factorial design by optimization of three variables: type of (ATM-SLNs) formula (X1), type of polymer (NOVEON AA1 or HPMC K100m) (X2) and concentration of polymer (X3). They were evaluated for nasal inserts’ physicochemical properties. The two optimum inserts were selected by Design Expert^® software. The two optimum insets with the highest desirability values were (S4 and S8). They were subjected to DSC thermal stability study and in-vivo study on rats. They were compared with atomoxetine oral solution, atomoxetine (3 mg/kg, intraperitoneal injection) and the pure atomoxetine solution loaded in lyophilized insert. (ATM-SLNs) showed EE% range of (41.14 mg ± 1.8% to 90.6 mg ± 2.8%), (Q30min%) of (27.11 ± 5.9% to 91.08 ± 0.15%), ZP of (−8.52 ± 0.75 to −28.4 ± 0.212% mV), PS of (320.9 ± 110.81% nm to 936.7 ± 229.6% nm) and PDI of (0.222 ± 0.132% to 0.658 ± 0.03%). Additionally, the two optimum (ATM-SLNs) formulae chosen, i.e., F7 and F9 showed spherical morphology. Nasal inserts had assay of drug content of (82.5 ± 2.5% to 103.94 ± 3.94%), Q15min% of (89.9 ± 6.4% to 100%) and Muco-adhesion strength of (3510.5 ± 140.21 to 9319.5 ± 39.425). DSC results of S4 and S8 showed compatibility of (ATM) with the other excipients. S8 and S4 also showed higher trans-nasal permeation to the brain with brain targeting efficiency of (211.3% and 177.42%, respectively) and drug transport percentages of (52.7% and 43.64%, respectively). To conclude, lyophilized nasal inserts of (ATM-SLNs) enhanced (ATM) trans-nasal drug targeting permeation and brain targeting efficiency. Full article

(This article belongs to the Special Issue Brain Theranostics: Focus on Drug Delivery and Outcomes)

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32 pages, 2247 KiB

Open AccessReview

Biodegradable Electrospun Scaffolds as an Emerging Tool for Skin Wound Regeneration: A Comprehensive Review

by Deepika Sharma, Shriyansh Srivastava, Sachin Kumar, Pramod Kumar Sharma, Rym Hassani, Hamad Ghaleb Dailah, Asaad Khalid and Syam Mohan

Pharmaceuticals 2023, 16(2), 325; https://doi.org/10.3390/ph16020325 - 20 Feb 2023

Cited by 12 | Viewed by 4020

Abstract

Skin is designed to protect various tissues, and because it is the largest and first human bodily organ to sustain damage, it has an incredible ability to regenerate. On account of extreme injuries or extensive surface loss, the normal injury recuperating interaction might [...] Read more.

Skin is designed to protect various tissues, and because it is the largest and first human bodily organ to sustain damage, it has an incredible ability to regenerate. On account of extreme injuries or extensive surface loss, the normal injury recuperating interaction might be inadequate or deficient, bringing about risky and disagreeable circumstances that request the utilization of fixed adjuvants and tissue substitutes. Due to their remarkable biocompatibility, biodegradability, and bioactive abilities, such as antibacterial, immunomodulatory, cell proliferative, and wound mending properties, biodegradable polymers, both synthetic and natural, are experiencing remarkable progress. Furthermore, the ability to convert these polymers into submicrometric filaments has further enhanced their potential (e.g., by means of electrospinning) to impersonate the stringy extracellular grid and permit neo-tissue creation, which is a basic component for delivering a mending milieu. Together with natural biomaterial, synthetic polymers are used to solve stability problems and make scaffolds that can dramatically improve wound healing. Biodegradable polymers, commonly referred to as biopolymers, are increasingly used in other industrial sectors to reduce the environmental impact of material and energy usage as they are fabricated using renewable biological sources. Electrospinning is one of the best ways to fabricate nanofibers and membranes that are very thin and one of the best ways to fabricate continuous nanomaterials with a wide range of biological, chemical, and physical properties. This review paper concludes with a summary of the electrospinning (applied electric field, needle-to-collector distance, and flow rate), solution (solvent, polymer concentration, viscosity, and solution conductivity), and environmental (humidity and temperature) factors that affect the production of nanofibers and the use of bio-based natural and synthetic electrospun scaffolds in wound healing. Full article

(This article belongs to the Special Issue Development of Specific Dosage Form: Wound Dressing)

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13 pages, 2563 KiB

Open AccessArticle

Mucosal Genes Expression in Inflammatory Bowel Disease Patients: New Insights

by Sumaiah J. Alarfaj, Sally Abdallah Mostafa, Walaa A. Negm, Thanaa A. El-Masry, Marwa Kamal, Mohamed Elsaeed and Ahmed Mohamed El Nakib

Pharmaceuticals 2023, 16(2), 324; https://doi.org/10.3390/ph16020324 - 20 Feb 2023

Cited by 6 | Viewed by 2659

Abstract

Individual differences in IBD illness severity, behavior, progression, and therapy response are evident. Since a break in the intestinal epithelial barrier causes IBD to begin, mucosal gene expression in IBD is crucial. Due to its high sensitivity and dynamic nature, molecular analysis of [...] Read more.

Individual differences in IBD illness severity, behavior, progression, and therapy response are evident. Since a break in the intestinal epithelial barrier causes IBD to begin, mucosal gene expression in IBD is crucial. Due to its high sensitivity and dynamic nature, molecular analysis of biomarkers in intestinal biopsies is feasible and provides a reliable means of evaluating localized inflammation. The goal of this investigation was to discover alterations in gene expression in the inflamed mucosa of IBD patients undergoing treatment with 5-amino salicylic acid (5ASA) (N = 39) or anti-TNF drugs (N = 22). The mucosal expression of numerous IBD-related genes was evaluated using qPCR. We discovered that the levels of the proteins Lipocalin-2 (LCN2), Nitric Oxide Synthase 2 (NOS2), Mucin 2 (MUC2), Mucin 5AC (MUC5AC), and Trefoil factor 1 (TFF1), which are overexpressed in untreated IBD patients compared to non-IBD subjects, are decreased by both therapy regimens. On the other hand, anti-TNF medicine helped the levels of ABCB1 and E-cadherin return to normal in IBD patients who were not receiving treatment. Full article

(This article belongs to the Special Issue Drug Treatments for Inflammatory Bowel Diseases)

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12 pages, 808 KiB

Open AccessArticle

Association between WHO First-Step Analgesic Use and Risk of Breast Cancer in Women of Working Age

by Hyun Sook Oh and Hwa Jeong Seo

Pharmaceuticals 2023, 16(2), 323; https://doi.org/10.3390/ph16020323 - 20 Feb 2023

Cited by 3 | Viewed by 2019

Abstract

We assessed the association between breast cancer and analgesic use in women of a specific working-age group. The Korean National Health Insurance Service–National Sample Cohort database (KNHIS–NSC) data were analyzed. We calculated hazard ratios (HRs) with 95% confidence intervals (CIs) for patients’ cancer [...] Read more.

We assessed the association between breast cancer and analgesic use in women of a specific working-age group. The Korean National Health Insurance Service–National Sample Cohort database (KNHIS–NSC) data were analyzed. We calculated hazard ratios (HRs) with 95% confidence intervals (CIs) for patients’ cancer risk based on whether the women participated in economic activity (PEA or not PEA (NPEA) groups) and analgesic use. Additionally, breast cancer incidence variations by age group, and PEA or NPEAs, health behavior, Charlson Comorbidity Index, and analgesic use were evaluated. The PEA group had a higher cancer risk than the NPEA group (HR = 1.542, 95% CI: 1.345–1.768, p < 0.001). Breast cancer risk was high in the PEA, high income, and no history of exercise groups, but significantly reduced in the regular-use-of-analgesics group. Notably, the working age group of 40~49 years, within the PEA group, had the highest HR of breast cancer development (HR = 1.700, 95% CI = 1.361–2.124, p < 0.001); whereas regular analgesic use in those aged 25~39 years decreased breast cancer risk (HR = 0.611, 95% CI = 0.427–0.875, p < 0.05). In conclusion, our results suggest that individuals at a high-risk of comorbidity may benefit from regular use of analgesics, which may prove to be a useful strategy for breast cancer prevention in the Young-aged group. Full article

(This article belongs to the Section Pharmaceutical Technology)

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12 pages, 315 KiB

Open AccessReview

Common Regulators of Lipid Metabolism and Bone Marrow Adiposity in Postmenopausal Women

by Dae-Yong Kim and Seong-Hee Ko

Pharmaceuticals 2023, 16(2), 322; https://doi.org/10.3390/ph16020322 - 20 Feb 2023

Cited by 8 | Viewed by 3193

Abstract

A variety of metabolic disorders are associated with a decrease in estradiol (E2) during natural or surgical menopause. Postmenopausal women are prone to excessive fat accumulation in skeletal muscle and adipose tissue due to the loss of E2 via abnormalities in lipid metabolism [...] Read more.

A variety of metabolic disorders are associated with a decrease in estradiol (E2) during natural or surgical menopause. Postmenopausal women are prone to excessive fat accumulation in skeletal muscle and adipose tissue due to the loss of E2 via abnormalities in lipid metabolism and serum lipid levels. In skeletal muscle and adipose tissue, genes related to energy metabolism and fatty acid oxidation, such as those encoding peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and estrogen-related receptor alpha (ERRα), are downregulated, leading to increased fat synthesis and lipid metabolite accumulation. The same genes regulate lipid metabolism abnormalities in the bone marrow. In this review, abnormalities in lipid metabolism caused by E2 deficiency were investigated, with a focus on genes able to simultaneously regulate not only skeletal muscle and adipose tissue but also bone metabolism (e.g., genes encoding PGC-1α and ERRα). In addition, the mechanisms through which mesenchymal stem cells lead to adipocyte differentiation in the bone marrow as well as metabolic processes related to bone marrow adiposity, bone loss, and osteoporosis were evaluated, focusing on the loss of E2 and lipid metabolic alterations. The work reviewed here suggests that genes underlying lipid metabolism and bone marrow adiposity are candidate therapeutic targets for bone loss and osteoporosis in postmenopausal women. Full article

(This article belongs to the Special Issue Drug Candidates for the Treatment of Metabolic Syndrome)

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20 pages, 3203 KiB

Open AccessArticle

Gamma-Irradiated Non-Capsule Group B Streptococcus Promotes T-Cell Dependent Immunity and Provides a Cross-Protective Reaction

by Yong Zhi, Fengjia Chen, Guangxu Cao and Fang Li

Pharmaceuticals 2023, 16(2), 321; https://doi.org/10.3390/ph16020321 - 20 Feb 2023

Viewed by 3342

Abstract

Group B Streptococcus (GBS) is a Gram-positive bacterium commonly found in the genitourinary tract and is also a leading cause of neonatal sepsis and pneumonia. Despite the current antibiotic prophylaxis (IAP), the disease burdens of late-onset disease in newborns and non-pregnant adult infections [...] Read more.

Group B Streptococcus (GBS) is a Gram-positive bacterium commonly found in the genitourinary tract and is also a leading cause of neonatal sepsis and pneumonia. Despite the current antibiotic prophylaxis (IAP), the disease burdens of late-onset disease in newborns and non-pregnant adult infections are increasing. Recently, inactivation of the pathogens via gamma radiation has been proven to eliminate their replication ability but cause less damage to the antigenicity of the key epitopes. In this study, the non-capsule GBS strain was inactivated via radiation (Rad-GBS) or formalin (Che-GBS), and we further determined its immunogenicity and protective efficacy as vaccines. Notably, Rad-GBS was more immunogenic and gave rise to higher expression of costimulatory molecules in BMDCs in comparison with Che-GBS. Flow cytometric analysis revealed that Rad-GBS induced a stronger CD4⁺ IFN-γ⁺ and CD4⁺IL-17A⁺ population in mice. The protective efficacy was measured through challenge with the highly virulent strain CNCTC 10/84, and the adoptive transfer results further showed that the protective role is reversed by functionally neutralizing antibodies and T cells. Finally, cross-protection against challenges with prevalent serotypes of GBS was induced by Rad-GBS. The higher opsonophagocytic killing activity of sera against multiple serotypes was determined in sera from mice immunized with Rad-GBS. Overall, our results showed that the inactivated whole-cell encapsulated GBS could be an alternative strategy for universal vaccine development against invasive GBS infections. Full article

(This article belongs to the Section Biopharmaceuticals)

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Reduction of the viability of Group B streptococcus treated with gamma-radiation or formalin. Clinical non-capsule GBS isolate NSP14-358 was exposed to various doses of (A) gamma radiation or concentrations of (B) formalin in PBS for 2 h at 37 °C. Bacterial viability was determined by counting CFUs on blood agar plates. The aliquots of bacteria grown in the exponential phase were treated with various concentrations of formaldehyde or doses of gamma radiation; the non-treated was GBS used as control. (C) The levels of protein carbonyl (nmol/mg) in inactivated GBS lysates were used to quantify the damage to protein epitopes according to the commercial kit. (D) The antibody-binding affinities of inactivated GBS were assessed through ELISA using diluted rabbit anti-GBS serum (1/500 dilution). (E) The binding with surface SRRN2N3 epitopes of the antibody by inactivated GBS samples was tested via a dot plot. All data are expressed as the mean ± standard deviation of triplicate samples or three independent experiments, and p-values were determined by performing an unpaired t test. *** Stands for p < 0.001, ** stands for p < 0.01, and * stands for p < 0.05 and NS for not significant. Full article ">Figure 2
Rad-GBS enhances the activation of BM-DCs in comparison to that with Che-GBS. Bone marrow-dendritic cells (BM-DCs) (2 × 106 cells/mL) were isolated and incubated with GM-CSF, followed by stimulation with either Rad-GBS or Che-GBS at 106, 107, and 108 CFU/well for 24 h. The expression of the co-stimulatory makers (A) CD80 and (B) CD86, (C) major histocompatibility complex MHCI, and (D) MHCII was analyzed via flow cytometry. The mean fluorescent intensity (MFI) is representative on the histograms. BM-DCs (2 × 106 cells/mL) were stimulated with Rad-GBS or Che-GBS at MOI for 24 h. Then, the levels of (E) IL-1β, (F) IL-6, (G) TNF-α, and (H) IL-12p70 in the culture supernatant were measured via ELISA. Scatter plots and other data suggest that MFI data are expressed as means ± SEM. All data are expressed as the mean ± standard deviation of triplicate samples or three independent experiments, and p-values were determined by performing an unpaired t test. *** Stands for p < 0.001, ** stands for p < 0.01, * stands for p < 0.05, and NS for not significant. Full article ">Figure 3
Immunoglobulin titers in sera from mice vaccinated with various dosages of Rad-GBS or Che-GBS. (A) Mice (n = 5) were vaccinated with 107 or 108 CFU of Rad-GBS or Che-GBS intramuscularly at two-week intervals, as in the schedule shown, and the mice were immunized with PBS as the control group. Serum levels of GBS-specific immunoglobulin G (IgG). (B) and immunoglobulin M (IgM) (C) were isolated and analyzed 7 days after the last immunization. The antibody levels of subclasses of IgG, such as (D) IgG1 and (E) IgG2a, were further determined. All data are expressed as the mean ± standard deviation of five mouse serum samples and three independent experiments. *** Stands for p < 0.001, ** stands for p < 0.01, * stands for p < 0.05, and NS for not significant. Full article ">Figure 4
Cellular immunogenicity is potently elicited via immunization with Rad-GBS. Mice were intramuscularly immunized with various dosages of Rad-GBS or Che-GBS as shown above, and mice injected with PBS were used as the control group. Seven days after the last vaccination, the spleen cells were isolated, and cell suspensions were re-stimulated with 20 µg/mL GBS whole-cell lysate for 12 h and subsequently used for the flow cytometry analysis. (A) The functional populations of T cells were classified according to the expression of CD4+ and CD8+ of interferon gamma (IFN-γ), interleukin-5 (IL-5), or interleukin-17A(IL-17A) assessed in all mouse groups. Antigen-specific cytokine-producing population in isolated spleen cells. For mouse groups, measurements were made for the population of (B) CD4+IFN-γ+, (C) CD4+ IL5+, (D) CD4+ IL-17A+, and (E) CD8+ IFN-γ+. All data are expressed as the mean ± standard deviation of five splenocyte samples and three independent experiments. ** stands for p < 0.01, * stands for p < 0.05, and NS for not significant. Full article ">Figure 5
Cytokine production by spleen cells of vaccinated mice. Single-cell suspensions of spleen cells were isolated from mice above and were treated with 20 µg/mL GBS whole-cell lysate for 24 h. Supernatants were collected for the determination of cytokine levels, including (A) IFN-γ, (B) IL-5, and (C) IL-17A in the supernatant. All data are expressed as the mean ± standard deviation of five cell culture supernatant samples and three independent experiments, and p-values were determined by performing an unpaired t test. *** Stands for p < 0.001, ** stands for p < 0.01, and NS for not significant. Full article ">Figure 6
Evaluation of Rad-GBS-induced protection and increased survival in response to CNCTC 10/84 challenge. Mice (n = 5) were intramuscularly vaccinated with 108 CFU of Rad-GBS or Che-GBS as described above. (A) All mice were intravenously challenged with 5 × 108 CFU of CNCTC 10/84 GBS, and survival in each group was recorded daily for one week. To investigate the protection origins from immune responses, adoptive transfers of vaccinated mouse serum or CD4+or CD8+ T cells with GBS challenge was used. (B) Serum (300 μL), (C) Splenic CD4+ T cells, or (D) CD8+ T cells from alum or vaccinated mice was inoculated intraperitoneally into naïve C57BL/6 mice (n = 5). At 24 h following transfer, mice were also challenged intravenously with CNCTC 10/84 GBS. Mouse survival was monitored for 7 days. The survival of mice was determined using Kaplan–Meier survival analysis, and the significance of the difference was analyzed using a log-rank test. ** stands for p < 0.01 and * stands for p < 0.05 and NS for not significant. Full article ">Figure 7
Cross-reactive protection highly induces functional antibodies against multiple serotypes via Rad-GBS vaccination. Mice (n = 5) were vaccinated intramuscularly with 107 CFU of Rad-GBS supplemented with alum (20 µg), and the cohort was administrated PBS as a control. Mice were challenged intravenously with a lethal dose of GBS strains (A) A909 (Ia), (B) COH1 (III), (C) NCTC 1/32 (IV), and (D) 2603VR (V) at 7 days after the last immunization. Survival of the mice was observed and recorded for one week. (E) Opsonic killing activity of the sera isolated from Rad-GBS- or Che-GBS-immunized mice against multiple serotypes of GBS strains (A909, COH1, NCTC1/32, and 2603V/R) was examined. The differentiated HL-60 cells (4 × 106 cells) and 10µL of baby rabbit complement were added into the mixed suspension of the diluted sera (1:500) isolated from the immunized mice and bacteria, while the negative control group contained only differentiated HL-60 cells and heat-inactivated baby rabbit complement. All data were expressed as the means ± standard deviation per group. The survival of mice was determined using Kaplan–Meier survival analysis, and the significance of the difference was analyzed using a log-rank test. *** Stands for p < 0.001, ** stands for p < 0.01, * stands for p < 0.05, and NS for not significant. Full article ">

20 pages, 1518 KiB

Open AccessReview

Treatment of Fabry Disease: Established and Emerging Therapies

by Muhammad Umer and Dinesh K. Kalra

Pharmaceuticals 2023, 16(2), 320; https://doi.org/10.3390/ph16020320 - 20 Feb 2023

Cited by 18 | Viewed by 6776

Abstract

Fabry disease (FD) is a rare, X-linked inherited disorder of glycosphingolipid metabolism. It leads to the progressive accumulation of globotriaosylceramide within lysosomes due to a deficiency of α-galactosidase A enzyme. It involves multiple organs, predominantly the renal, cardiac, and cerebrovascular systems. Early diagnosis [...] Read more.

Fabry disease (FD) is a rare, X-linked inherited disorder of glycosphingolipid metabolism. It leads to the progressive accumulation of globotriaosylceramide within lysosomes due to a deficiency of α-galactosidase A enzyme. It involves multiple organs, predominantly the renal, cardiac, and cerebrovascular systems. Early diagnosis and treatment are critical to prevent progression to irreversible tissue damage and organ failure, and to halt life-threatening complications that can significantly reduce life expectancy. This review will focus on the established and emerging treatment options for FD. Full article

(This article belongs to the Section Pharmacology)

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25 pages, 7076 KiB

Open AccessArticle

Profiling of Secondary Metabolites of Optimized Ripe Ajwa Date Pulp (Phoenix dactylifera L.) Using Response Surface Methodology and Artificial Neural Network

by Fanar Alshammari, Md Badrul Alam, Marufa Naznin, Ahsan Javed, Sunghwan Kim and Sang-Han Lee

Pharmaceuticals 2023, 16(2), 319; https://doi.org/10.3390/ph16020319 - 20 Feb 2023

Cited by 6 | Viewed by 3130

Abstract

The date palm (Phoenix dactylifera L.) is a popular edible fruit consumed all over the world and thought to cure several chronic diseases and afflictions. The profiling of the secondary metabolites of optimized ripe Ajwa date pulp (RADP) extracts is scarce. The [...] Read more.

The date palm (Phoenix dactylifera L.) is a popular edible fruit consumed all over the world and thought to cure several chronic diseases and afflictions. The profiling of the secondary metabolites of optimized ripe Ajwa date pulp (RADP) extracts is scarce. The aim of this study was to optimize the heat extraction (HE) of ripe Ajwa date pulp using response surface methodology (RSM) and artificial neural network (ANN) modeling to increase its polyphenolic content and antioxidant activity. A central composite design was used to optimize HE to achieve the maximum polyphenolic compounds and antioxidant activity of target responses as a function of ethanol concentration, extraction time, and extraction temperature. From RSM estimates, 75.00% ethanol and 3.7 h (extraction time), and 67 °C (extraction temperature) were the optimum conditions for generating total phenolic content (4.49 ± 1.02 mgGAE/g), total flavonoid content (3.31 ± 0.65 mgCAE/g), 2,2-diphenyl-1-picrylhydrazyl (11.10 ± 0.78 % of inhibition), and cupric-reducing antioxidant capacity (1.43 µM ascorbic acid equivalent). The good performance of the ANN was validated using statistical metrics. Seventy-one secondary metabolites, including thirteen new bioactive chemicals (hebitol II, 1,2-di-(syringoyl)-hexoside, naringin dihydrochalcone, erythron-guaiacylglycerol-β-syringaresinol ether hexoside, erythron-1-(4′-O-hexoside-3,5-dimethoxyphenyl)-2-syrngaresinoxyl-propane-1,3-diol, 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid, linustatin and 1-deoxynojirimycin galactoside), were detected using high-resolution mass spectroscopy. The results revealed a significant concentration of phytoconstituents, making it an excellent contender for the pharmaceutical and food industries. Full article

(This article belongs to the Special Issue Exploring Natural Metabolites to Identify Novel Potential Therapeutic Agents)

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20 pages, 7463 KiB

Open AccessArticle

Protective Potential of Saussurea costus (Falc.) Lipsch. Roots against Cyclophosphamide-Induced Pulmonary Injury in Rats and Its In Vitro Antiviral Effect

by Nashwah G. M. Attallah, Amal Kabbash, Walaa A. Negm, Engy Elekhnawy, Reem Binsuwaidan, Omnia Momtaz Al-Fakhrany, Moataz A. Shaldam, Ehssan Moglad, Marwa Tarek, Nehal Samir and Heba M. Fawzy

Pharmaceuticals 2023, 16(2), 318; https://doi.org/10.3390/ph16020318 - 18 Feb 2023

Cited by 7 | Viewed by 2781

Abstract

Diseases and infections of the respiratory tract are common global causes of morbidity and mortality. Our study attempts to elucidate a novel remedy for respiratory ailments, in addition to identifying and quantifying the metabolites of Saussurea costus root extract (SCRE) using HPLC. Then, [...] Read more.

Diseases and infections of the respiratory tract are common global causes of morbidity and mortality. Our study attempts to elucidate a novel remedy for respiratory ailments, in addition to identifying and quantifying the metabolites of Saussurea costus root extract (SCRE) using HPLC. Then, in vitro antiviral and in vivo lung protective effects were elucidated. The in vitro antiviral potential of SCRE was analyzed via plaque assay against the low pathogenic human coronavirus (HCoV-229E) and human influenza virus (H1N1). The value of the half maximal inhibitory concentrations (IC₅₀) of SCRE against HCoV-229E and H1N1 influenza virus were 23.21 ± 1.1 and 47.6 ± 2.3 µg/mL, respectively. SCRE showed a histological improvement, namely a decrease in inducible nitric oxide synthase (iNOS) and caspase-3 immunoexpression in in vivo cyclophosphamide (CP)-induced acute lung injury (ALI). Moreover, there was a considerable decline in microRNA-let-7a gene expression and a significant rise in heme oxygenase-1 (HO-1) gene expression, with a marked decrease in the malondialdehyde (MDA) level. Molecular docking studies revealed that the major constituents of SCRE have a good affinity for caspase-3, HO-1, and iNOS proteins. In conclusion, a traditional plant SCRE could be a promising source of novel therapeutic agents for treating and protecting respiratory tract diseases. More future investigations should be carried out to reveal its efficacy clinically. Full article

(This article belongs to the Special Issue Discovery, Metabolism and Potential Bio-Activities of Natural Products in Traditional Medicine)

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19 pages, 3755 KiB

Open AccessEditor’s ChoiceReview

Computational Chemistry for the Identification of Lead Compounds for Radiotracer Development

by Chia-Ju Hsieh, Sam Giannakoulias, E. James Petersson and Robert H. Mach

Pharmaceuticals 2023, 16(2), 317; https://doi.org/10.3390/ph16020317 - 18 Feb 2023

Cited by 12 | Viewed by 6466

Abstract

The use of computer-aided drug design (CADD) for the identification of lead compounds in radiotracer development is steadily increasing. Traditional CADD methods, such as structure-based and ligand-based virtual screening and optimization, have been successfully utilized in many drug discovery programs and are highlighted [...] Read more.

The use of computer-aided drug design (CADD) for the identification of lead compounds in radiotracer development is steadily increasing. Traditional CADD methods, such as structure-based and ligand-based virtual screening and optimization, have been successfully utilized in many drug discovery programs and are highlighted throughout this review. First, we discuss the use of virtual screening for hit identification at the beginning of drug discovery programs. This is followed by an analysis of how the hits derived from virtual screening can be filtered and culled to highly probable candidates to test in in vitro assays. We then illustrate how CADD can be used to optimize the potency of experimentally validated hit compounds from virtual screening for use in positron emission tomography (PET). Finally, we conclude with a survey of the newest techniques in CADD employing machine learning (ML). Full article

(This article belongs to the Special Issue Feature Reviews in Radiopharmaceutical Sciences: Highlights of Two-Decade Development)

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Workflow from VS to lead compounds identification for radiotracer development. “A, B, and C” in the last two steps of the workflow are represented as the fragment “A”, “B”, and “C” for structure–activity relationship studies. Full article ">Figure 2
Illustration of the key interactions between amino acid residues in the binding site and the crystallographic ligand. (A) µ-opioid receptor and a morphinan antagonist (PDB ID: 4DKL) [<a href="#B77-pharmaceuticals-16-00317" class="html-bibr">77</a>], (B) dopamine D2 receptor and risperidone (PDB ID: 6CM4) [<a href="#B78-pharmaceuticals-16-00317" class="html-bibr">78</a>], and (C) histamine H1 receptor and doxepin (PDB ID: 3RZE) [<a href="#B79-pharmaceuticals-16-00317" class="html-bibr">79</a>]. Full article ">Figure 3
A summary workflow from Ferrie et al. that identified lead compounds from structural-based VS. Full article ">Figure 4
A summary workflow from Kim et al. that identified lead compounds from ligand-based VS. Full article ">Figure 5
(a) BOILED-Egg plot of the testing radiotracer dataset, including 211 BBB-penetrated and 31 not BBB-penetrated radioligands from the literature. (b) Pie charts of true positive (TP), false negative (FN), true negative (TN), and false positive (FP) rates for BOILED-Egg plot, CNS-MPO, CNS PET MPO, and DeePred-BBB. The total number of not BBB-penetrated compounds for DeePred-BBB is 30 due to the conversion failure of one of the compounds from the program. Full article ">Figure 6
Three putative alpha-synuclein binding sites, Sites 2, 3/13, and 9, identified from the blind docking studies. Site 2 and Site 9 were confirmed via in vitro photo-cross-linking and mass spectrometry studies. [3H]tg-190b and IL-4-42 are the radioligand and photoaffinity probes for Site 2. [3H]BF-2846 and CLX1 are the radioligand and photoaffinity probes for Site 9. Site 2 and Site 9 probes were used to test in silico hits from the Exemplar screen and Site 9 optimization based on MDS. Full article ">

18 pages, 4673 KiB

Open AccessArticle

Therapeutic Effect of Costunolide in Autoimmune Hepatitis: Network Pharmacology and Experimental Validation

by Zheng Huang, Shangshu Nie, Shuhui Wang, Han Wang, Jin Gong, Wei Yan, Dean Tian and Mei Liu

Pharmaceuticals 2023, 16(2), 316; https://doi.org/10.3390/ph16020316 - 17 Feb 2023

Cited by 8 | Viewed by 2789

Abstract

Novel treatments for autoimmune hepatitis (AIH) are highly demanded due to the limitations of existing therapeutic agents. Costunolide is a promising candidate due to its anti-inflammatory and hepatoprotective function, but its effect in AIH remains obscure. In this study, we integrated network pharmacology [...] Read more.

Novel treatments for autoimmune hepatitis (AIH) are highly demanded due to the limitations of existing therapeutic agents. Costunolide is a promising candidate due to its anti-inflammatory and hepatoprotective function, but its effect in AIH remains obscure. In this study, we integrated network pharmacology and experimental validation to reveal the effect and mechanism of costunolide in AIH. A total of 73 common targets of costunolide and AIH were obtained from databases. Pathway enrichment analysis indicated that PI3K-AKT pathway was the core pathway of costunolide in AIH. Protein–protein interaction network analysis and molecular docking revealed that SRC and IGF1R might play critical roles. In two murine AIH models, costunolide significantly attenuated liver injury, inflammation, and fibrosis reflected by the liver gross appearance, serum transaminases, necrosis area, spleen index, immune cell infiltration, and collagen deposition. Western blot and immunohistochemistry confirmed that phosphorylated AKT, SRC, and IGF1R were upregulated in AIH models, and costunolide administration could inhibit the phosphorylation of these proteins. In summary, costunolide significantly ameliorates murine AIH. The therapeutic effect might work by suppressing the activation of PI3K-AKT pathway and inhibiting the phosphorylation of SRC and IGF1R. Our research reveals the potent therapeutic effect of costunolide in AIH and the potential role of SRC and IGF1R in AIH for the first time, which may further contribute to the novel drug development for AIH and other autoimmune diseases. Full article

(This article belongs to the Special Issue Plant-Derived Anti-inflammatory Agents: Molecular Pharmacology and Further Studies)

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26 pages, 9226 KiB

Open AccessReview

PSMA-Targeted Nanotheranostics for Imaging and Radiotherapy of Prostate Cancer

by Niranjan Meher, Henry F. VanBrocklin, David M. Wilson and Robert R. Flavell

Pharmaceuticals 2023, 16(2), 315; https://doi.org/10.3390/ph16020315 - 17 Feb 2023

Cited by 17 | Viewed by 4471

Abstract

Targeted nanotheranostic systems offer significant benefits due to the integration of diagnostic and therapeutic functionality, promoting personalized medicine. In recent years, prostate-specific membrane antigen (PSMA) has emerged as an ideal theranostic target, fueling multiple new drug approvals and changing the standard of care [...] Read more.

Targeted nanotheranostic systems offer significant benefits due to the integration of diagnostic and therapeutic functionality, promoting personalized medicine. In recent years, prostate-specific membrane antigen (PSMA) has emerged as an ideal theranostic target, fueling multiple new drug approvals and changing the standard of care in prostate cancer (PCa). PSMA-targeted nanosystems such as self-assembled nanoparticles (NPs), liposomal structures, water-soluble polymers, dendrimers, and other macromolecules are under development for PCa theranostics due to their multifunctional sensing and therapeutic capabilities. Herein, we discuss the significance and up-to-date development of “PSMA-targeted nanocarrier systems for radioligand imaging and therapy of PCa”. The review also highlights critical parameters for designing nanostructured radiopharmaceuticals for PCa, including radionuclides and their chelators, PSMA-targeting ligands, and the EPR effect. Finally, prospects and potential for clinical translation is discussed. Full article

(This article belongs to the Special Issue Application of Radiolabeled Agents: Imaging, Therapy, Multimodal Approaches and Beyond)

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12 pages, 1307 KiB

Open AccessArticle

Cyclotron-Based Production of ⁶⁷Cu for Radionuclide Theranostics via the ⁷⁰Zn(p,α)⁶⁷Cu Reaction

by Santiago Andrés Brühlmann, Martin Walther, Martin Kreller, Falco Reissig, Hans-Jürgen Pietzsch, Torsten Kniess and Klaus Kopka

Pharmaceuticals 2023, 16(2), 314; https://doi.org/10.3390/ph16020314 - 17 Feb 2023

Cited by 8 | Viewed by 4368

Abstract

Theranostic matched pairs of radionuclides have aroused interest during the last couple of years, and in that sense, copper is one element that has a lot to offer, and although ⁶¹Cu and ⁶⁴Cu are slowly being established as diagnostic radionuclides for [...] Read more.

Theranostic matched pairs of radionuclides have aroused interest during the last couple of years, and in that sense, copper is one element that has a lot to offer, and although ⁶¹Cu and ⁶⁴Cu are slowly being established as diagnostic radionuclides for PET, the availability of the therapeutic counterpart ⁶⁷Cu plays a key role for further radiopharmaceutical development in the future. Until now, the ⁶⁷Cu shortage has not been solved; however, different production routes are being explored. This project aims at the production of no-carrier-added ⁶⁷Cu with high radionuclidic purity with a medical 30MeV compact cyclotron via the ⁷⁰Zn(p,α)⁶⁷Cu reaction. With this purpose, proton irradiation of electrodeposited ⁷⁰Zn targets was performed followed by two-step radiochemical separation based on solid-phase extraction. Activities of up to 600MBq ⁶⁷Cu at end of bombardment, with radionuclidic purities over 99.5% and apparent molar activities of up to 80MBq/nmol, were quantified. Full article

(This article belongs to the Special Issue New Trends in Applications and Production of Metal Radionuclides for Nuclear Medicine)

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14 pages, 1280 KiB

Open AccessReview

Novel Theranostic Approaches Targeting CCR4-Receptor, Current Status and Translational Prospectives: A Systematic Review

by Joana Gorica, Maria Silvia De Feo, Ferdinando Corica, Marko Magdi Abdou Sidrak, Miriam Conte, Luca Filippi, Orazio Schillaci, Giuseppe De Vincentis and Viviana Frantellizzi

Pharmaceuticals 2023, 16(2), 313; https://doi.org/10.3390/ph16020313 - 17 Feb 2023

Cited by 4 | Viewed by 2441

Abstract

Background: With the high mortality rate of malignant tumors, there is a need to find novel theranostic approaches to provide an early diagnosis and targeted therapy. The chemokine receptor 4 (CCR4) is highly expressed in various tumors and plays an important role in [...] Read more.

Background: With the high mortality rate of malignant tumors, there is a need to find novel theranostic approaches to provide an early diagnosis and targeted therapy. The chemokine receptor 4 (CCR4) is highly expressed in various tumors and plays an important role in tumor pathogenesis. This systematic review aims to provide a complete overview on clinical and preclinical applications of the CCR4 receptor as a target for theranostics, using a systematic approach to classify and assemble published studies performed on humans and animals, sorted by field of application and specific tumor. Methods: A systematic literature search of articles suiting the inclusion criteria was conducted on Pubmed, Scopus, Central, and Web of Science databases, including papers published from January 2006 to November 2022. Eligible studies had to be performed on humans and/or in vivo/in vitro studying CCR4 expression in tumors. The methodological quality was assessed through the Critical Appraisal Skills Programme (CASP) assessing only the studies performed on humans. Results: A total of 17 articles were screened. The articles were assessed for eligibility with the exclusion of 4 articles. Ultimately, 13 articles were selected for the qualitative analysis, and six articles were selected for the critical appraisal skills program. Conclusions: The development of new radionuclides and radiopharmaceuticals targeting CCR4 show promising results in the theranostics of CCR4 sensible tumors. Although to widen its use in clinical practice, further translation of preclinical to clinical data is needed. Full article

(This article belongs to the Special Issue Targeted Radionuclide Therapy (TRNT) in Modern Cancer Management)

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17 pages, 11868 KiB

Open AccessReview

Protein Interactome of Amyloid-β as a Therapeutic Target

by Vladimir F. Lazarev, Elizaveta A. Dutysheva, Igor E. Kanunikov, Irina V. Guzhova and Boris A. Margulis

Pharmaceuticals 2023, 16(2), 312; https://doi.org/10.3390/ph16020312 - 16 Feb 2023

Cited by 6 | Viewed by 3909

Abstract

The amyloid concept of Alzheimer’s disease (AD) assumes the β-amyloid peptide (Aβ) as the main pathogenic factor, which injures neural and other brain cells, causing their malfunction and death. Although Aβ has been documented to exert its cytotoxic effect in a solitary manner, [...] Read more.

The amyloid concept of Alzheimer’s disease (AD) assumes the β-amyloid peptide (Aβ) as the main pathogenic factor, which injures neural and other brain cells, causing their malfunction and death. Although Aβ has been documented to exert its cytotoxic effect in a solitary manner, there is much evidence to claim that its toxicity can be modulated by other proteins. The list of such Aβ co-factors or interactors includes tau, APOE, transthyretin, and others. These molecules interact with the peptide and affect the ability of Aβ to form oligomers or aggregates, modulating its toxicity. Thus, the list of potential substances able to reduce the harmful effects of the peptide should include ones that can prevent the pathogenic interactions by specifically binding Aβ and/or its partners. In the present review, we discuss the data on Aβ-based complexes in AD pathogenesis and on the compounds directly targeting Aβ or the destructors of its complexes with other polypeptides. Full article

(This article belongs to the Special Issue New Perspective in Alzheimer's Disease Treatment)

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26 pages, 2297 KiB

Open AccessReview

Intranasal Lipid Nanoparticles Containing Bioactive Compounds Obtained from Marine Sources to Manage Neurodegenerative Diseases

by Joana Torres, Inês Costa, Andreia F. Peixoto, Renata Silva, José Manuel Sousa Lobo and Ana Catarina Silva

Pharmaceuticals 2023, 16(2), 311; https://doi.org/10.3390/ph16020311 - 16 Feb 2023

Cited by 4 | Viewed by 3306

Abstract

Marine sources contain several bioactive compounds with high therapeutic potential, such as remarkable antioxidant activity that can reduce oxidative stress related to the pathogenesis of neurodegenerative diseases. Indeed, there has been a growing interest in these natural sources, especially those resulting from the [...] Read more.

Marine sources contain several bioactive compounds with high therapeutic potential, such as remarkable antioxidant activity that can reduce oxidative stress related to the pathogenesis of neurodegenerative diseases. Indeed, there has been a growing interest in these natural sources, especially those resulting from the processing of marine organisms (i.e., marine bio-waste), to obtain natural antioxidants as an alternative to synthetic antioxidants in a sustainable approach to promote circularity by recovering and creating value from these bio-wastes. However, despite their expected potential to prevent, delay, or treat neurodegenerative diseases, antioxidant compounds may have difficulty reaching the brain due to the need to cross the blood–brain barrier (BBB). In this regard, alternative delivery systems administered by different routes have been proposed, including intranasal administration of lipid nanoparticles, such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), which have shown promising results. Intranasal administration shows several advantages, including the fact that molecules do not need to cross the BBB to reach the central nervous system (CNS), as they can be transported directly from the nasal cavity to the brain (i.e., nose-to-brain transport). The benefits of using SLN and NLC for intranasal delivery of natural bioactive compounds for the treatment of neurodegenerative diseases have shown relevant outcomes through in vitro and in vivo studies. Noteworthy, for bioactive compounds obtained from marine bio-waste, few studies have been reported, showing the open potential of this research area. This review updates the state of the art of using SLN and NLC to transport bioactive compounds from different sources, in particular, those obtained from marine bio-waste, and their potential application in the treatment of neurodegenerative diseases. Full article

(This article belongs to the Special Issue Current Insights on Lipid-Based Nanosystems 2023)

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Common pathophysiological mechanisms underlying the most prevalent and debilitating neurodegenerative diseases. Neurodegenerative diseases are a group of debilitant conditions that result from the progressive damage inflicted to the neuronal cells and nervous system, with abnormal deposition of proteins, and with the progressive loss of synapses and neurons. Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis are examples of complex neurodegenerative diseases sharing several common pathophysiological mechanisms, such as: (1) iron overload, (2) mitochondrial dysfunction and oxidative stress, (3) neuroinflammation, and (4) protein misfolding. Iron has essential functions in the brain and, therefore, needs to cross the blood–brain barrier (BBB) to reach this organ. The most elucidating hypothesis of the passage of iron through the luminal membrane of the capillary endothelium mainly occurs through the transferrin/transferrin receptor (Tf/TfR) pathway. This process starts with the binding of the complex ferric iron (Fe3+)-Tf to the extracellular portion of transferrin receptor (TfR), followed by the endocytosis of the complex, formation of endosome, and acidification of the microenvironment within endosome. Next, occurs the dissociation of iron from Tf and the reduction of ferric iron (Fe3+) to Fe2+ by the ferrireductase six-transmembrane epithelial antigen of prostate 3 (STEAP3). Fe2+ accumulates in cytoplasm, forming the labile iron pool (LIP), and the excess of intracellular iron is then stored in ferritin. Ferritinophagy is defined as the autophagic degradation of ferritin, a process mediated by nuclear receptor coactivator 4 (NCOA4). Ferritin, in combination with NCOA4, is transported to the lysosomes for degradation, being then the iron released for cellular physiological activities. However, when this metal is in excess, it participates in Fenton reaction leading to a cycle between the two redox states and prompting the generation of •OH, promoting lipid peroxidation and ferroptosis, a new type of regulated cell death. Ferroptosis is also characterized by an inhibition of System Xc-, with the consequent decrease in glutathione peroxidase 4 (GPX4) activity and promotion of lipid peroxidation, leading to neuronal damage. Mitochondria are essential organelles for eukaryotic life, producing most of the energy or adenosine triphosphate (ATP) required by the cell, being responsible for cellular respiration and oxidative phosphorylation. Changes in the correct functioning or in structures involved in this process lead to a decrease in ATP production, to the accumulation of reactive oxygen species (ROS), and to the release of apoptosis-inducing factors, leading to oxidative stress and cell death. Neuroinflammation is another pathological mechanism present in neurodegenerative diseases, and results from the presence of chronically activated glial cells (astrocytes and microglia) in the brain, which release cytokines and chemokines that are toxic to neurons. Finally, protein misfolding and aggregation of specific proteins into toxic products is a common feature of neurodegenerative diseases. Depending on the type of protein involved and the pathology in question, its aggregation promotes different consequences. For example, in Alzheimer’s disease, amyloid beta peptide (Aβ), originating from the fragmentation of amyloid precursor protein (APP), accumulates in the brain in the form of senile plaques. In Parkinson’s disease, α-synuclein (α-syn) is often found accumulated and aggregated and has several harmful effects. GR: Glutathione reductase; GSH: Reduced glutathione; GSSG: Glutathione disulfide. Full article ">Figure 2
Possible transport routes of bioactive compounds or drugs to the brain after intranasal administration. Direct route: passage through the olfactory or trigeminal nerves, avoiding crossing the blood–brain barrier (BBB). Indirect route: absorption through the respiratory mucosa into the systemic circulation and across the BBB (adapted from Nguyen et al. [<a href="#B15-pharmaceuticals-16-00311" class="html-bibr">15</a>,<a href="#B169-pharmaceuticals-16-00311" class="html-bibr">169</a>]). Full article ">

23 pages, 4355 KiB

Open AccessArticle

3D-Printing of Capsule Devices as Compartmentalization Tools for Supported Reagents in the Search of Antiproliferative Isatins

by Camilla Malatini, Carlos Carbajales, Mariángel Luna, Osvaldo Beltrán, Manuel Amorín, Christian F. Masaguer, José M. Blanco, Silvia Barbosa, Pablo Taboada and Alberto Coelho

Pharmaceuticals 2023, 16(2), 310; https://doi.org/10.3390/ph16020310 - 16 Feb 2023

Cited by 3 | Viewed by 2718

Abstract

The application of high throughput synthesis methodologies in the generation of active pharmaceutical ingredients (APIs) currently requires the use of automated and easily scalable systems, easy dispensing of supported reagents in solution phase organic synthesis (SPOS), and elimination of purification and extraction steps. [...] Read more.

The application of high throughput synthesis methodologies in the generation of active pharmaceutical ingredients (APIs) currently requires the use of automated and easily scalable systems, easy dispensing of supported reagents in solution phase organic synthesis (SPOS), and elimination of purification and extraction steps. The recyclability and recoverability of supported reagents and/or catalysts in a rapid and individualized manner is a challenge in the pharmaceutical industry. This objective can be achieved through a suitable compartmentalization of these pulverulent reagents in suitable devices for it. This work deals with the use of customized polypropylene permeable-capsule devices manufactured by 3D printing, using the fused deposition modeling (FDM) technique, adaptable to any type of flask or reactor. The capsules fabricated in this work were easily loaded “in one step” with polymeric reagents for use as scavengers of isocyanides in the work-up process of Ugi multicomponent reactions or as compartmentalized and reusable catalysts in copper-catalyzed cycloadditions (CuAAC) or Heck palladium catalyzed cross-coupling reactions (PCCCRs). The reaction products are different series of diversely substituted isatins, which were tested in cancerous cervical HeLa and murine 3T3 Balb fibroblast cells, obtaining potent antiproliferative activity. This work demonstrates the applicability of 3D printing in chemical processes to obtain anticancer APIs. Full article

(This article belongs to the Special Issue 3D Printing of Drug Formulations)

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Graphical abstract
Full article ">Figure 1
(a) EDX and FESEM of IRA-120-Cu(0) catalyst for CuAAC. (b) IRA-120-Pd(0) for Heck reactions (see graphics in detail in <a href="#app1-pharmaceuticals-16-00310" class="html-app">supplementary material Figure S2</a>). Full article ">Figure 2
(a) The 3D design of a unicompartmental capsule (see also <a href="#app1-pharmaceuticals-16-00310" class="html-app">Figure S1</a>); (b) bicompartmental capsule; (c) polypropylene mesh (bar scale 1 mm); (d) 3D printer and components; (e,f) 3D printing of a unicompartmental capsule; (g) bicompartmental capsule; (h,i) parietal view of the 3D-printing process of Capsule@IRA-120-Cu(0) and Capsule@IRA-120-Pd(0); (j) view of the porous area (mesh) of Capsule@IRA-120-Pd(0). (k) View of Capsule@Amberlyst-15, in an isocyanide scavenging assay, in MeOH. Full article ">Figure 3
(a) ORTEP diagram of compound 17, obtained by single crystal X-ray diffraction, showing the molecular conformation and numbering. (b) Crystal packing of 17. Full article ">Figure 4
(a) Parietal view of the compartments during the 3D-printing process of capsule@Amberlyst-15 and the magnetic stirrer. (b) Bicompartimental 3D-printed Capsule@Amberlyst-15 finished, before scavenging. (c) Reaction mixture during treatment with Capsule@Amberlyst-15 polypropylene capsule. (d) Washing of the capsule after scavenging. (e) Capsule after TFA/DCM treatment, 1 h. Full article ">Figure 5
(a) Heck reaction mixture using Capsule@IRA-120-Pd(0). (b) Capsule@ IRA-120-Cu(0) during CuAAC. (c) Different shapes and sizes of 3D-printed capsule prototypes. Full article ">Figure 6
(a) Cell viability of Series 1 (compounds 11–22) in Balb and (b) HeLa cells. (c) Cell viability of Series 2 and 3 (compounds 25–33) in Balb and (d) HeLa cells. Full article ">Chart 1
Chemical structures of the isatin compounds synthesized (series 1). Compounds 1: (X = H), 2 (X = I), 3 (X = Br), 4 (X = NO2), 5 (X = Cl, X7 = Cl). Full article ">Scheme 1
(a) Synthesis of IRA-120-Pd(0) for Heck reactions and (b) IRA-120-Cu(0) catalyst for CuAAC. (c) Chemical scavenging of isocyanides by Amberlyst-15 and recycling process in TFA. (d) Structure of the PP monomer. Full article ">Scheme 2
General conditions for Ugi: isatin derivative 1–5 (200 mg, 1 equiv), InCl3 (10 mol%), 150 mg of MS 3 Å and methanol (1 mL), n-butylamine (1.5 equiv), isocyanide (1.5 equiv), and carboxylic acid (1.5 equiv), rt. Full article ">Scheme 3
(a) K2CO3 anh., propargyl bromide, MeCN, 50 °C. (b) Capsule@IRA-120-Cu(0), alkyne, R-N3, MeCN, 10 h. (c) K2CO3 anh., 4-iodo-bencyl bromide, MeCN, 50 °C, 12 h. (d) K2CO3 anh., benzyl bromide, MeCN, 70 °C. (e) Capsule@IRA-120-Pd(0), methyl acrylate (exc), 90 °C, sodium acetatate, DMF/H2O, 24 h. (f) Capsule@IRA-120-Pd(0), methyl acrylate, sodium acetatate, 70 °C, DMF/H2O, 24 h. (g) Capsule@IRA-120-Pd(0), methyl acrylate (exc), sodium acetatate, 90 °C, DMF/H2O, 12 h. Full article ">

11 pages, 2497 KiB

Open AccessArticle

Coupling of NIR Spectroscopy and Chemometrics for the Quantification of Dexamethasone in Pharmaceutical Formulations

by Alessandra Biancolillo, Claudia Scappaticci, Martina Foschi, Claudia Rossini and Federico Marini

Pharmaceuticals 2023, 16(2), 309; https://doi.org/10.3390/ph16020309 - 16 Feb 2023

Cited by 4 | Viewed by 2644

Abstract

Counterfeit or substandard drugs are pharmaceutical formulations in which the active pharmaceutical ingredients (APIs) have been replaced or ingredients do not comply with the drug leaflet. With the outbreak of the COVID-19 pandemic, fraud associated with the preparation of substandard or counterfeit drugs [...] Read more.

Counterfeit or substandard drugs are pharmaceutical formulations in which the active pharmaceutical ingredients (APIs) have been replaced or ingredients do not comply with the drug leaflet. With the outbreak of the COVID-19 pandemic, fraud associated with the preparation of substandard or counterfeit drugs is expected to grow, undermining health systems already weakened by the state of emergency. Analytical chemistry plays a key role in tackling this problem, and in implementing strategies that permit the recognition of uncompliant drugs. In light of this, the present work represents a feasibility study for the development of a NIR-based tool for the quantification of dexamethasone in mixtures of excipients (starch and lactose). Two different regression strategies were tested. The first, based on the coupling of NIR spectra and Partial Least Squares (PLS) provided good results (root mean square error in prediction (RMSEP) of 720 mg/kg), but the most accurate was the second, a strategy exploiting sequential preprocessing through orthogonalization (SPORT), which led (on the external set of mixtures) to an R²_pred of 0.9044, and an RMSEP of 450 mg/kg. Eventually, Variable Importance in Projection (VIP) was applied to interpret the obtained results and determine which spectral regions contribute most to the SPORT model. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: New Techniques for Characterization of Drugs and Drug Products)

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13 pages, 2157 KiB

Open AccessCommunication

Characterization of Potential Intoxications with Medicines in a Regional Setting

by Tânia Nascimento, Teresa Santos, Fátima Rato and Ana Luísa De Sousa-Coelho

Pharmaceuticals 2023, 16(2), 308; https://doi.org/10.3390/ph16020308 - 16 Feb 2023

Viewed by 2214

Abstract

The Portuguese Poison Information Center (from Portuguese—CIAV) is a call center that offers medical assistance in case of possible intoxication with any kind of product, including medicines. This center´s main goal is to inform and guide the general public and health professionals. This [...] Read more.

The Portuguese Poison Information Center (from Portuguese—CIAV) is a call center that offers medical assistance in case of possible intoxication with any kind of product, including medicines. This center´s main goal is to inform and guide the general public and health professionals. This work aimed to analyze and compare data corresponding to the telephone calls from the Algarve region (South of Portugal), received by CIAV during 2019 and 2020, regarding potential intoxications with medicines. To this end, data provided by CIAV on possible cases of medication intoxication in the Algarve region were collected, including the number of calls received, the place of origin of the call, the age group and sex of the intoxicated individual, the route of exposure to the drug, the circumstances of contact with the substance, the existence of symptoms, and the drug or drugs involved in the potential intoxication. The results showed that the number of cases slightly decreased in 2020 (n = 1261) compared with 2019 (n = 1340), with a high number of cases of intoxication in children between one and four years old in both years (21.2%; n = 152 in 2019; 16.4%; n = 115 in 2020). The drugs belonging to the locomotor system group (paracetamol and ibuprofen) were the main drugs involved, followed by the central nervous system pharmacotherapeutic group, namely benzodiazepines (diazepam and alprazolam). Paracetamol was the main drug responsible for the calls to CIAV (n = 71 in 2019; n = 63 in 2020), while for the remaining drugs there were fluctuations in their positions between both years. In some cases, this swinging may be explained by the possible changes in therapy due to potential interactions with drugs used for the treatment of symptoms of COVID-19 or perhaps related to misleading information released by the media about the use of some drugs, such as ibuprofen, during lockdown periods. Although there has been a decrease in calls to report possible drug intoxication in the Algarve region, the profile of calls was very similar. Paracetamol was the drug with the highest number of reported cases and the group of psychotropic drugs showed the largest increase between 2019 and 2020. Full article

(This article belongs to the Section Pharmacology)

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Characterization of the contact person to the Poison Information Centre in the Algarve region in 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows: doctor (35.96% vs. 35.99%), family (30.01% vs. 26.05%), nurse (3.49% vs. 3.22%), self (13.97% vs. 14.43%), pharmacist (0% vs. 0.28%), CODU (14.75% vs. 18.49%), other (1.81% vs. 1.54%). Abbreviations: CODU—Urgent Patient Orientation Centre, from the Portuguese Centro de Orientação de Doentes Urgentes. Full article ">Figure 2
Characterization of the place of origin of the call to the Poison Information Centre in the Algarve region in 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows: hospital (38.29% vs. 36.41%), saúde24 (37.52% vs. 25.07%), CODU (14.75% vs. 18.63%), home (7.50% vs. 18.07%), workplace (0.52% vs. 0.70%), ambulance (0.26% vs. 0.42%), public place (0.26% vs. 0.14%), other (0.91% vs. 0.56%). Saúde24 (SNS24) is a healthcare hotline, i.e., a telephone and online service of the Portuguese National Health Service (SNS). Abbreviations: CODU—Urgent Patient Orientation Centre, from the Portuguese Centro de Orientação de Doentes Urgentes. Full article ">Figure 3
Distribution of cases by age group and sex during 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows by age group and sex: <1 years old (2.14% F; 3.28% M vs. 0.91% F; 2.93% M), 1–4 (16.45% F; 24.59% M vs. 12.24% F; 22.34% M), 5–9 (3.21%; 6.23% M vs. 3.40% F; 5.13% M), 10–15 (4.91%; 4.26% M vs. 5.90% F; 4.40% M), 16–19 (6.62%; 4.92% M vs. 9.98% F; 3.66% M), 20–29 (10.47%; 7.87% M vs. 10.20% F; 10.99% M), 30–39 (10.26%; 5.90% M vs. 8.84% F; 12.09% M), 40–49 (13.89%; 9.18% M vs. 12.93% F; 11.36% M), 50–59 (13.46%; 8.85% M vs. 13.38% F; 9.52% M), 60–69 (9.62%; 6.56% M vs. 9.98% F; 5.49% M), 70–79 (5.34%; 4.59% M vs. 7.71% F; 5.13% M), 80–89 (3.42%; 3.61% M vs. 3.40% F; 1.10% M), 90–99 (0.21%; 0.66% M vs. 0.68% F; 1.47% M), unknown age (0% F; 9.51% M vs. 0.45% F; 4.40% M). F, females; M, males. Full article ">Figure 4
Characterization of the intoxication circumstances in the Algarve region in 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows: intentional (43.47% vs. 51.19%), therapeutic error (29.11% vs. 21.74%), accidental (25.74% vs. 25.39%), adverse reaction (1.03% vs. 0.84%), unknown (0.65% vs. 0.84%). Full article ">Figure 5
General symptoms grouped by main system affected described in the calls to the Poison Information Centre in the Algarve region in 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows: neurologic (52.54% vs. 64.06%), general/others (23.45% vs. 10.73%), digestive (12.15% vs. 11.88%), psychiatric (6.45% vs. 6.09%), cardiovascular (4.80% vs. 5.51%), respiratory (0.57% vs. 1.74%). Percentages were calculated by the total of symptomatic cases. Full article ">Figure 6
Characterization of the pharmacotherapeutic groups most involved in potential drug intoxications in the Algarve region in 2019 and 2020. Comparing 2019 and 2020, the distributions were as follows: anxiolytics/antidepressants/antipsychotics (38.74% vs. 48.40%), anticonvulsants (11.49% vs. 6.85%), analgesics/antipyretics/anti-inflammatory (7.04% vs. 9.50%), antihypertensives (5.38% vs. 7.29%), antimicrobial agents (3.34% vs. 2.65%), others (34.01% vs. 25.30%). Full article ">

10 pages, 1239 KiB

Open AccessArticle

Nintedanib in Idiopathic Pulmonary Fibrosis: Tolerability and Safety in a Real Life Experience in a Single Centre in Patients also Treated with Oral Anticoagulant Therapy

by Barbara Ruaro, Ilaria Gandin, Riccardo Pozzan, Stefano Tavano, Chiara Bozzi, Michael Hughes, Metka Kodric, Rossella Cifaldi, Selene Lerda, Marco Confalonieri, Elisa Baratella, Paola Confalonieri and Francesco Salton

Pharmaceuticals 2023, 16(2), 307; https://doi.org/10.3390/ph16020307 - 16 Feb 2023

Cited by 11 | Viewed by 3857

Abstract

Idiopathic pulmonary fibrosis (IPF) is a rare and severe disease with a median survival of ~3 years. Nintedanib (NTD) has been shown to be useful in controlling interstitial lung disease (ILD) in IPF. Here we describe the experience of NTD use in IPF [...] Read more.

Idiopathic pulmonary fibrosis (IPF) is a rare and severe disease with a median survival of ~3 years. Nintedanib (NTD) has been shown to be useful in controlling interstitial lung disease (ILD) in IPF. Here we describe the experience of NTD use in IPF in a real-life setting. Objective. Our objective was to examine the safety profile and efficacy of nintedanib even in subjects treated with anticoagulants. Clinical data of patients with IPF treated with NTD at our center were retrospectively evaluated at baseline and at 6 and 12 months after the introduction of NTD. The following parameters were recorded: IPF clinical features, NTD tolerability, and pulmonary function tests (PFT) (i.e., Forced Vital Capacity (FVC) and carbon monoxide diffusing capacity (DLCO)). In total, 56 IPF patients (34% female and 66% male, mean onset age: 71 ± 11 years, mean age at baseline: 74 ± 9 years) treated with NTD were identified. At enrollment, HRCT showed an UIP pattern in 45 (80%) and a NSIP in 11 (20%) patients. For FVC and FEV1 we found no significant change between baseline and 6 months, but for DLCO we observed a decrease (p = 0.012). We identified a significant variation between baseline and 12 months for FEV1 (p = 0.039) and for DLCO (p = 0.018). No significant variation was observed for FVC. In the cohort, 18 (32%) individuals suspended NTD and 10 (18%) reduced the dosage. Among individuals that suspended the dosage, 14 (78%) had gastrointestinal (GI) collateral effects (i.e., diarrhea being the most common complaint (67%), followed by nausea/vomiting (17%) and weight loss (6%). Bleeding episodes have also not been reported in patients taking anticoagulant therapy. (61%). One patient died within the first 6 months and two subjects died within the first 12 months. In a real-life clinical scenario, NTD may stabilize the FVC values in IPF patients. However, GI side effects are frequent and NTD dose adjustment may be necessary to retain the drug in IPF patients. This study confirms the safety of NTD, even in patients treated with anticoagulant drugs. Full article

(This article belongs to the Special Issue Rheumatic Diseases: Pathophysiology, Targeted Therapy, Focus on Vascular and Pulmonary Manifestations 2022)

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22 pages, 12022 KiB

Open AccessArticle

Combinatorial Therapeutic Potential of Stem Cells and Benzimidazol Derivatives for the Reduction of Liver Fibrosis

by Maryam Iqbal, Sulaiman Shams, Huma Rafiq, Momin Khan, Shahid Khan, Umer Sadique Khattak, Sahib Gul Afridi, Fehmida Bibi, Angham Abdulrhman Abdulkareem and Muhammad Imran Naseer

Pharmaceuticals 2023, 16(2), 306; https://doi.org/10.3390/ph16020306 - 15 Feb 2023

Cited by 1 | Viewed by 2230

Abstract

(1) Background: Liver fibrosis is currently one of the top ten causes of death worldwide. Stem cells transplantation using mesenchymal stem cells (MSCs) is an alternative therapy which is used in the place of organ transplant, due to the incapacity of stem cells [...] Read more.

(1) Background: Liver fibrosis is currently one of the top ten causes of death worldwide. Stem cells transplantation using mesenchymal stem cells (MSCs) is an alternative therapy which is used in the place of organ transplant, due to the incapacity of stem cells to endure oxidative stress in the damage site, thus affecting the healing process. The present study aimed to enhance the therapeutic potential of MSCs using combined therapy, along with the novel synthetic compounds of benzimidazol derivatives. (2) Methods: Eighteen compound series (benzimidazol derivatives) were screened against liver fibrosis using an in vitro CCl₄-induced injury model on cultured hepatocytes. IC50 values were calculated on the bases of LDH assay and cell viability assay. (3) Results: Among the eighteen compounds, compounds (10), (14) and (18) were selected on the basis of IC50 value, and compound (10) was the most potent and had the lowest IC50 value in the LDH assay (8.399

\pm

0.23 uM) and cell viability assay (4.73

\pm

0.37 uM). Next, these compounds were combined with MSCs using an in vitro hepatocytes injury culture and in vivo rat fibrotic model. The effect of the MSCs + compounds treatment on injured hepatocytes was evaluated using LDH assay, cell viability assay, GSH assay and real-time PCR analysis and immuno-staining for caspase-3. Significant reductions in LDH level, caspase-3 and apoptotic marker genes were noted in MSCs + compounds-treated injured hepatocytes. In vivo data also showed the increased homing of the MSCs, along with compounds after transplantation. Real-time PCR analysis and TUNEL assay results also support our study. (4) Conclusions: It was concluded that compounds (10), (14) and (18) can be used in combination with MSCs to reduce liver fibrosis. Full article

(This article belongs to the Special Issue Stem Cells Based Modeling with the Application in Tissue Engineering and Biomaterial Evaluation)

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17 pages, 6261 KiB

Open AccessArticle

PHPB Attenuated Cognitive Impairment in Type 2 Diabetic KK-Ay Mice by Modulating SIRT1/Insulin Signaling Pathway and Inhibiting Generation of AGEs

by Jiang Li, Shaofeng Xu, Ling Wang and Xiaoliang Wang

Pharmaceuticals 2023, 16(2), 305; https://doi.org/10.3390/ph16020305 - 15 Feb 2023

Cited by 3 | Viewed by 2279

Abstract

Diabetes mellitus (DM) has been recognized as an increased risk factor for cognitive impairment, known as diabetic encephalopathy (DE). Hyperglycemia and insulin resistance are the main initiators of DE, which is related to the accumulation of advanced glycation end products (AGEs). Potassium 2-(1-hydroxypentyl)-benzoate [...] Read more.

Diabetes mellitus (DM) has been recognized as an increased risk factor for cognitive impairment, known as diabetic encephalopathy (DE). Hyperglycemia and insulin resistance are the main initiators of DE, which is related to the accumulation of advanced glycation end products (AGEs). Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), a derivative of 3-n-butylphthalide (dl-NBP), has emerged various properties including improved mitochondrial function, antioxidant, anti-neuroinflammation, and neuroprotective effects. The present study aimed to investigate the neuroprotective effect of PHPB against AGEs accumulation in type 2 diabetic KK-Ay mice model with DE and further explore the underlying mechanisms. The results showed that PHPB markedly ameliorated the spatial learning ability of KK-Ay mice in the Morris water maze and decreased AD-like pathologic changes (Tau hyperphosphorylation) in the cortex. Furthermore, we found that PHPB treatment significantly reduced AGEs generation via up-regulation of glyoxalase-1 (GLO1) protein and enhancement of methylglyoxal (MG) trapping, while there was no obvious difference in levels of glucose in plasma or brain, contents of total cholesterol (TC), triglycerides (TG), and plasma insulin. Also, PHPB treatment improved the insulin signaling pathway by increasing sirtuin1 (SIRT1) deacetylase activity and attenuated oxidative stress evidenced by elevating glucose-6-phosphate dehydrogenase (G-6-PD) protein expression, promoting the production of reduced glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), restoring mitochondrial membrane potential, increasing adenosine triphosphate (ATP) generation, and reducing malondialdehyde (MDA) levels in the brain. Taken together, PHPB exhibited a beneficial effect on DE, which involved modulating the SIRT1/insulin signaling pathway and reducing oxidative stress by inhibiting the generation of AGEs. Full article

(This article belongs to the Section Pharmacology)

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Graphical abstract

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Full article ">Figure 1
Schematic diagram of the experimental design. (Note: FBG: Fasting Blood Glucose, TC: Total Cholesterol; TG: Triglycerides). Full article ">Figure 2
Effects of PHPB on body weight and metabolic features in mice. KK-Ay mice were treated with PHPB (50 or 150 mg/kg) or rosiglitazone (5 mg/kg) for two months by oral gavage. (A) Body weight, (B) Fasting blood glucose (FBG), (C) Plasma triglycerides (TG), (D) Plasma total cholesterol (TC), and (E) Plasma insulin. Values are represented as mean ± SEM. n = 10 for C57 group, n = 19 for KK-Ay group, n = 17 for PHPB 50 mg/kg group, n = 17 for PHPB 100 mg/kg group, n = 16 for rosiglitazone group. ## p < 0.01 versus C57 group; * p < 0.05, ** p < 0.01 versus KK-Ay group. Full article ">Figure 3
PHPB attenuated cognitive impairment in type 2 diabetic KK-Ay mice. KK-Ay mice were treated with PHPB (50 mg/kg or 150 mg/kg or rosiglitazone (Ros, 5 mg/kg) for two months by oral gavage. The performance of the Morris water maze was tested among all groups. (A) Spatial learning performance was analyzed by escape latency for mice to locate the hidden platform during the 6-days acquisition training. In the probe trial test on the seventh day, (B) the first latency time and (C) the times of crossings over the original platform location, and (D) the swimming velocity were recorded to evaluate the spatial memory maintenance/movement ability, n = 10–19 for each group. Furthermore, (E) the expression of classical hallmarks of AD, p-Tau (S404) protein, was detected in the cortex and hippocampus by western blotting, n = 8 for each group. And then, quantitative analysis of the expression of the p-Tau protein is presented as fold change relative to C57 mice. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05 versus KK-Ay group. Full article ">Figure 4
PHPB increased ATP production in the cortex of KK-Ay mice without any obvious differences in glucose levels. (A,B) Quantification of blood-glucose and cerebral-glucose using a micro-dialysis method in mice treated with PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg) for two months, N = 7–10 for each group. (C,D) Detection of the ATP contents in the cortex and hippocampus by firefly luciferase method, n = 8 for each group. The data are presented as means ± SEM. # p < 0.05 versus C57 group. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05, ** p < 0.01 versus KK-Ay group. Full article ">Figure 5
PHPB decreased the generation of AGEs or MG in the plasma and brain of KK-Ay mice. (A–C) Quantification of AGEs levels in plasma, cortex, and hippocampus by ELISA kits, and its levels were normalized to total protein concentration in the tissue samples of mice treated by PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg) for two months, n = 8 for each group. (D–F) The MG contents (fold increase vs. that of C57 mice) of plasma, cortex, and hippocampus were measured by using the HPLC-UV method, n = 7–10 for each group. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05 versus KK-Ay group. Full article ">Figure 6
PHPB attenuated MG through up-regulating GLO1 protein and enhancing MG trapping. (A) Western blotting was used to detect the protein expressions of GLO1 in the cortex and hippocampus of mice treated with PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg) for two months, and densitometric quantification of them, n = 8 for each group. (B) MG trapping of PHPB was detected by the HPLC method. MG (1 mmol/L) was incubated with vehicle, PHPB (0.1, 1.0, 2.5, 5.0 and 10.0 mg/mL), or aminoguanidine (AG, 0.1 mg/mL or 1.0 mg/mL) at 37 °C for 0, 3, 6, 9, and 24 h, n = 3 independent experiments. The data are presented as means ± SEM. # p < 0.05 versus C57 group; * p < 0.05, ** p < 0.01 versus KK-Ay group, a p < 0.05, b p < 0.01 versus corresponding vehicle value. Full article ">Figure 7
PHPB modulated SIRT1 deacetylase activity and insulin signaling pathway in cortex and hippocampus of KK-Ay mice. (A) The SIRT1 deacetylase activity (fold increase vs. that of C57 mice) was measured by an enzymatic fluorescence method using commercial kits. (B–F) Phosphorylation of IRβTyr1150/1151, IRS-1Tyr859, and PI3Kp85α or PI3KTyr458/p55 Tyr199; Akt or phosphorylated AktSer473; and GSK3β or phosphorylated GSK3βSer9 were determined with corresponding antibodies by western blotting in the cortex and hippocampus of mice treated by PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg), and densitometric quantification of them, n = 5–8 for each group. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05 versus KK-Ay group. Full article ">Figure 7 Cont.
PHPB modulated SIRT1 deacetylase activity and insulin signaling pathway in cortex and hippocampus of KK-Ay mice. (A) The SIRT1 deacetylase activity (fold increase vs. that of C57 mice) was measured by an enzymatic fluorescence method using commercial kits. (B–F) Phosphorylation of IRβTyr1150/1151, IRS-1Tyr859, and PI3Kp85α or PI3KTyr458/p55 Tyr199; Akt or phosphorylated AktSer473; and GSK3β or phosphorylated GSK3βSer9 were determined with corresponding antibodies by western blotting in the cortex and hippocampus of mice treated by PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg), and densitometric quantification of them, n = 5–8 for each group. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05 versus KK-Ay group. Full article ">Figure 8
PHPB restored mitochondrial function and attenuated oxidative stress in KK-Ay mice. (A) Mitochondrial membrane potential (MMP, fold increase vs. that of C57 mice) was detected by JC-1 dye using commercial kits. (B) MDA levels and (C,D) the production of GSH or NADPH in the cortex or hippocampus were quantified using commercial assay kits. (E) Western blotting was used to detect the protein expressions of glucose-6-phosphate dehydrogenase (G-6-PD) in the cortex or hippocampus of mice treated with PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg) for two months, and densitometric quantification of them. n = 8 for each group. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05, ** p < 0.01 versus KK-Ay group. Full article ">

21 pages, 11302 KiB

Open AccessArticle

Discovery of Ureido-Based Apcin Analogues as Cdc20-specific Inhibitors against Cancer

by Yiqin He, Xiangyang Le, Gaoyun Hu, Qianbin Li and Zhuo Chen

Pharmaceuticals 2023, 16(2), 304; https://doi.org/10.3390/ph16020304 - 15 Feb 2023

Cited by 2 | Viewed by 2644

Abstract

Cdc20 is a promising drug target that plays an important role in the mid-anaphase process of cellular mitosis, and Apcin is the only reported core structure of the Cdc20-specific inhibitor. Some potent Apcin derivatives were obtained in our previous research, and a structure–activity [...] Read more.

Cdc20 is a promising drug target that plays an important role in the mid-anaphase process of cellular mitosis, and Apcin is the only reported core structure of the Cdc20-specific inhibitor. Some potent Apcin derivatives were obtained in our previous research, and a structure–activity relationship was determined. In this study, we designed and synthesized a series of ureido-based Apcin derivatives. The proliferation-inhibition experiments on four cancer-cell lines showed that ureido skeleton could promote the anti-proliferation activity of purine-substituted compounds, whereas the ureido analogues with pyrimidine substitutes showed no significant improvement in the inhibitory effect compared with the original ones. Further tests confirmed that ureido-based compounds can enhance the binding affinity to Cdc20 by increasing the levels of Cdc20 downstream proteins. Compound 27 revealed a remarkably antitumor activity pattern against Hela (IC₅₀ = 0.06 ± 0.02 μM) and potent binding affinity to Cdc20. Moreover, compound 20 induced caspase-dependent apoptosis and cell-cycle arrest at the G2/M phase, and compound 27 induced caspase-dependent apoptosis and promoted microtubule polymerization. Finally, a molecular-docking simulation was performed for compounds 20 and 27 to predict the potential ligand–protein interactions with the active sites of the Cdc20 proteins. Full article

(This article belongs to the Topic Advances in Anti-Cancer Drugs)

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15 pages, 3431 KiB

Open AccessArticle

Erythromycin Restores Osteoblast Differentiation and Osteogenesis Suppressed by Porphyromonas gingivalis Lipopolysaccharide

by Hikaru Tamura, Tomoki Maekawa, Hisanori Domon, Kridtapat Sirisereephap, Toshihito Isono, Satoru Hirayama, Takumi Hiyoshi, Karin Sasagawa, Fumio Takizawa, Takeyasu Maeda, Yutaka Terao and Koichi Tabeta

Pharmaceuticals 2023, 16(2), 303; https://doi.org/10.3390/ph16020303 - 15 Feb 2023

Cited by 7 | Viewed by 3011

Abstract

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the [...] Read more.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1. Full article

(This article belongs to the Special Issue Pharmacological Targets of Bone Homeostasis: Osteoclasts and Osteoblasts)

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Full article ">Figure 1
Erythromycin rescued osteoblast mineralization inhibited by Porphyromonas gingivalis LPS. MC3T3-E1 osteoblastic progenitors were incubated in growth medium with P. gingivalis LPS (100 ng/mL) and 20% ethanol (EtOH), erythromycin (ERM, 10 μg/mL), penicillin (PC, 5 unit/mL), or josamycin (JSM, 10 μg/mL). (A) Representative images (entire bottom of each well of a 96-well plate) of mineralized nodule formation detected by Alizarin Red S staining after 15 days. (B) The total mineralization area in each culture was quantified and expressed in % relative to the total area. (C) Cell viability was assessed using the MTT assay. * p < 0.05 compared to the EtOH + LPS group; values are shown as the mean ± SD (n = 5 per group). Full article ">Figure 2
Erythromycin promoted the expression of DEL-1 and osteoblast differentiation-related factors. MC3T3-E1 osteoblastic progenitors were incubated in growth medium with P. gingivalis LPS (100 ng/mL) and EtOH, ERM (10 μg/mL), PC (5 unit/mL), or JSM (10 μg/mL). (A–D) The mRNA transcription levels of osteoblast differentiation-related factors and Del1 were quantified using real-time qPCR on day 12; * p < 0.05 compared to the EtOH + LPS group; values are shown as the mean ± SD (n = 4 per group). (E) Intracellular expression of Runx2 protein was determined using western blot analysis on day 12. Full article ">Figure 3
Peritoneal injection of antibiotics inhibits bone resorption in Porphyromonas gingivalis LPS-induced periodontitis. Periodontal bone loss was induced by the administration of P. gingivalis LPS to maxillary molars. The untreated (UT) group was set as a baseline control. The groups of mice were administered intraperitoneally 20% ethanol (EtOH; control), erythromycin (ERM), josamycin (JSM), or penicillin (PC). (A) The distance from the cement–enamel junction to the pinnacle of the alveolar bone was measured. Negative values (in mm) indicate bone loss relative to the UT control. (B) Representative images of the mouse maxillary bone from the indicated groups (scale bar of 0.5 mm). * p < 0.05 compared to the EtOH group; † p < 0.05 compared to the ERM group; values are shown as the mean ± SD (n = 5 per group). Full article ">Figure 4
Effect of antibacterial drugs on the transcription of osteoblast-related factors in the gingiva, using the Porphyromonas gingivalis LPS-induced periodontitis model. (A–F) Real-time qPCR was performed to quantify the mRNA transcription levels of osteoblast-related factors, DEL-1, and osteoclast-related factors. * p < 0.05 compared to the indicated group; values are shown as the mean ± SD (n = 5 per group). N.S., not significant. Full article ">Figure 5
Erythromycin increases DEL-1 expression in the periodontal ligament. Frozen maxillae sections were stained for DEL-1 (green) and nuclei using DAPI (blue). Representative images obtained by optical microscopy are shown. C: cementum; PDL: periodontal ligament; B: alveolar bone. Scale bars, 50 µm. Full article ">Figure 6
Erythromycin increases the alkaline phosphatase activity of cells in periodontal ligament tissue. Alkaline phosphatase staining was performed on frozen maxillary sections. (A) Representative images obtained by optical microscopy are shown. C: cementum; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 µm. (B) The total active region of alkaline phosphatase in each field was quantified and expressed as % relative to the total area. * p < 0.05 as compared to EtOH + LPS group, means ± SD (n = 3 per group). Full article ">

19 pages, 1375 KiB

Open AccessReview

Autophagy Induction by Scutellaria Flavones in Cancer: Recent Advances

by Hardeep Singh Tuli, Sakshi Bhushan, Ajay Kumar, Poonam Aggarwal, Katrin Sak, Seema Ramniwas, Kanupriya Vashishth, Tapan Behl, Rashmi Rana, Shafiul Haque and Miguel A. Prieto

Pharmaceuticals 2023, 16(2), 302; https://doi.org/10.3390/ph16020302 - 15 Feb 2023

Cited by 9 | Viewed by 3731

Abstract

In parallel with a steady rise in cancer incidence worldwide, the scientific community is increasingly focused on finding novel, safer and more efficient modalities for managing this disease. Over the past decades, natural products have been described as a significant source of new [...] Read more.

In parallel with a steady rise in cancer incidence worldwide, the scientific community is increasingly focused on finding novel, safer and more efficient modalities for managing this disease. Over the past decades, natural products have been described as a significant source of new structural leads for novel drug candidates. Scutellaria root is one of the most studied natural products because of its anticancer potential. Besides just describing the cytotoxic properties of plant constituents, their molecular mechanisms of action in different cancer types are equally important. Therefore, this review article focuses on the role of the Scutellaria flavones wogonin, baicalein, baicalin, scutellarein and scutellarin in regulating the autophagic machinery in diverse cancer models, highlighting these molecules as potential lead compounds for the fight against malignant neoplasms. The knowledge that autophagy can function as a dual-edged sword, acting in both a pro- and antitumorigenic manner, further complicates the issue, revealing an amazing property of flavonoids that behave either as anti- or proautophagic agents. Full article

(This article belongs to the Special Issue Anticancer Compounds in Medicinal Plants 2023)

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20 pages, 6268 KiB

Open AccessArticle

Hesperidin Mitigates Cyclophosphamide-Induced Testicular Dysfunction via Altering the Hypothalamic Pituitary Gonadal Axis and Testicular Steroidogenesis, Inflammation, and Apoptosis in Male Rats

by Tarek Khamis, Abdelmonem Awad Hegazy, Samaa Salah Abd El-Fatah, Eman Ramadan Abdelfattah, Marwa Mohamed Mahmoud Abdelfattah, Liana Mihaela Fericean and Ahmed Hamed Arisha

Pharmaceuticals 2023, 16(2), 301; https://doi.org/10.3390/ph16020301 - 15 Feb 2023

Cited by 15 | Viewed by 3841

Abstract

Cyclophosphamide (CP) is a cytotoxic, cell cycle, non-specific, and antiproliferative drug. This study aimed to address the toxic effects of CP on male fertility and the possible ameliorative role of hesperidin (HSP). Thirty-two adult albino rats were randomly divided into four groups, namely, [...] Read more.

Cyclophosphamide (CP) is a cytotoxic, cell cycle, non-specific, and antiproliferative drug. This study aimed to address the toxic effects of CP on male fertility and the possible ameliorative role of hesperidin (HSP). Thirty-two adult albino rats were randomly divided into four groups, namely, the negative control, HSP, CP-treated, and CP+HSP-treated groups. The CP-treated rats showed a significant reduction in the levels of serum LH, FSH, testosterone, prolactin, testicular glutathione peroxidase (GPx), and total antioxidant capacity (TAC) with an elevation in levels of malondialdehyde (MDA), and p53, and iNOS immune expression, compared to the control group. A significant downregulation in hypothalamic KISS-1, KISS-1r, and GnRH, hypophyseal GnRHr, and testicular mRNA expression of steroidogenesis enzymes, PGC-1α, PPAR-1, IL10, and GLP-1, as well as a significant upregulation in testicular mRNA of P53 and IL1β mRNA expression, were detected in the CP-treated group in comparison to that in the control group. The administration of HSP in CP-treated rats significantly improved the levels of serum LH, FSH, testosterone, prolactin, testicular GPx, and TAC, with a reduction in levels of MDA, and p53, and iNOS immune expression compared to the CP-treated group. A significant upregulation in hypophyseal GnRHr, and testicular mRNA expression of CYP19A1 enzymes, PPAR-1, IL10, and GLP-1, as well as a significant downregulation in testicular mRNA of P53 and IL1β mRNA expression, were detected in the CP+HSP-treated group in comparison to that in the CP-treated group. In conclusion, HSP could be a potential auxiliary agent for protection from the development of male infertility. Full article

(This article belongs to the Special Issue Pharmacological Activities of Flavonoids and Its Analogues)

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21 pages, 3093 KiB

Open AccessArticle

Targeting the Endoplasmic Reticulum Stress-Linked PERK/GRP78/CHOP Pathway with Magnesium Sulfate Attenuates Chronic-Restraint-Stress-Induced Depression-like Neuropathology in Rats

by Hany H. Arab, Ali Khames, Shuruq E. Alsufyani, Azza A. K. El-Sheikh and Amany M. Gad

Pharmaceuticals 2023, 16(2), 300; https://doi.org/10.3390/ph16020300 - 15 Feb 2023

Cited by 6 | Viewed by 3057

Abstract

Magnesium sulfate has demonstrated marked neuroprotection in eclampsia, hypoxia, stroke, and post-traumatic brain injury rodent models. However, its potential impact against chronic-restraint-stress (CRS)-induced depression-like neuropathology and associated alterations in endoplasmic reticulum (ER) stress have not been adequately examined. The present study aimed to [...] Read more.

Magnesium sulfate has demonstrated marked neuroprotection in eclampsia, hypoxia, stroke, and post-traumatic brain injury rodent models. However, its potential impact against chronic-restraint-stress (CRS)-induced depression-like neuropathology and associated alterations in endoplasmic reticulum (ER) stress have not been adequately examined. The present study aimed to investigate the neuroprotective potential of magnesium sulfate in a rat model of CRS-triggered depression-like behavioral disturbance and the underlying molecular mechanisms. Herein, CRS was induced by placing rats into restraining tubes for 6 h/day for 21 days and the animals were intraperitoneally injected with magnesium sulfate (100 mg/kg/day) during the study period. After stress cessation, the depression-like behavior was examined by the open-field test, sucrose preference test, and forced swimming test. The present data demonstrated that CRS triggered typical depression-like behavioral changes which were confirmed by the Z-normalization scores. Mechanistically, serum circulating corticosterone levels spiked, and the hippocampi of CRS-exposed animals demonstrated a significant decline in serotonin, norepinephrine, and dopamine neurotransmitters. At the molecular level, the hippocampal pro-inflammatory TNF-alpha and IL-1β cytokines and the oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-HG) increased in stressed animals. In tandem, enhancement of hippocampal ER stress was evidenced by the activation of iNOS/PERK/GRP78/CHOP axis seen by increased protein expression of iNOS, PERK, GRP78, and CHOP signal proteins in the hippocampi of stressed rats. Interestingly, magnesium sulfate administration attenuated the depression-like behavioral outcomes and the histopathological changes in the brain hippocampi. These favorable actions were driven by magnesium sulfate’s counteraction of corticosterone spike, and hippocampal neurotransmitter decline, alongside the attenuation of neuroinflammation, pro-oxidation, and ER stress. In conclusion, the current results suggest the promising neuroprotective/antidepressant actions of magnesium sulfate in CRS by dampening inflammation, ER stress, and the associated PERK/GRP78/CHOP pathway. Full article

(This article belongs to the Special Issue Advances in Behavioral Psychopharmacology)

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68 pages, 12431 KiB

Open AccessReview

Nitrogen Containing Heterocycles as Anticancer Agents: A Medicinal Chemistry Perspective

by Adarsh Kumar, Ankit Kumar Singh, Harshwardhan Singh, Veena Vijayan, Deepak Kumar, Jashwanth Naik, Suresh Thareja, Jagat Pal Yadav, Prateek Pathak, Maria Grishina, Amita Verma, Habibullah Khalilullah, Mariusz Jaremko, Abdul-Hamid Emwas and Pradeep Kumar

Pharmaceuticals 2023, 16(2), 299; https://doi.org/10.3390/ph16020299 - 14 Feb 2023

Cited by 118 | Viewed by 11163

Abstract

Cancer is one of the major healthcare challenges across the globe. Several anticancer drugs are available on the market but they either lack specificity or have poor safety, severe side effects, and suffer from resistance. So, there is a dire need to develop [...] Read more.

Cancer is one of the major healthcare challenges across the globe. Several anticancer drugs are available on the market but they either lack specificity or have poor safety, severe side effects, and suffer from resistance. So, there is a dire need to develop safer and target-specific anticancer drugs. More than 85% of all physiologically active pharmaceuticals are heterocycles or contain at least one heteroatom. Nitrogen heterocycles constituting the most common heterocyclic framework. In this study, we have compiled the FDA approved heterocyclic drugs with nitrogen atoms and their pharmacological properties. Moreover, we have reported nitrogen containing heterocycles, including pyrimidine, quinolone, carbazole, pyridine, imidazole, benzimidazole, triazole, β-lactam, indole, pyrazole, quinazoline, quinoxaline, isatin, pyrrolo-benzodiazepines, and pyrido[2,3-d]pyrimidines, which are used in the treatment of different types of cancer, concurrently covering the biochemical mechanisms of action and cellular targets. Full article

(This article belongs to the Special Issue Nitrogen Containing Scaffolds in Medicinal Chemistry)

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15 pages, 2107 KiB

Open AccessArticle

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer

by Salvatore Villani, Giulia Dematteis, Laura Tapella, Mara Gagliardi, Dmitry Lim, Marco Corazzari, Silvio Aprile and Erika Del Grosso

Pharmaceuticals 2023, 16(2), 298; https://doi.org/10.3390/ph16020298 - 14 Feb 2023

Cited by 5 | Viewed by 3270

Abstract

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography–High Resolution Mass Spectrometry [...] Read more.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco’s Modified Eagle widely used cell culture media. Samples were injected on a Luna^® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38–24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer’s disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines. Full article

(This article belongs to the Section Medicinal Chemistry)

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Figure 1

Figure 1
Chemical structures and chromatograms of 4-phenylbutyric acid (4-PBA) and 4-(4-nitrophenyl)butyric acid (NPBA, IS). (A,B) allow to compare the signal to noise ratio of 4-PBA in SIM and PRM acquisition modes, respectively. (C) shows IS (NPBA) chromatogram recorded in SIM. Full article ">Figure 2
Treatment of Alzheimer’s astrocytes at various 4-PBA concentrations in conditioned medium. (A) WT and 3Tg-iAstro were treated with astrocyte conditioned medium (ACM), astrocyte conditioned medium with 4-PBA 3 µM freshly added (ACM + fresh 4-PBA), or astrocyte conditioned medium collected form astrocytes treated with 4-PBA for 48 h (ACM + 4-PBA 48 h). After 48 h, cells were pulsed with 4 µM puromycin and lysate for 1 h, then protein synthesis rate was accessed via WB. Concentrations of 4-PBA were assessed by LC-HRMS. (B) Representative WB of protein synthesis analysis, (C) data are expressed as mean ± SEM of 3 independent experiments, (D) 4-PBA absorption determined by LC-HRMS; concentrations are expressed as µM ± SEM of 3 independent experiments repeated twice. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; by one-way ANOVA, Sidak’s multiple comparison.). Full article ">Figure 3
Thapsigargin-induced ER stress markers monitoring related to 4-PBA absorption from DMEM. (A,B) UPR analysis following TG and 4-PBA treatment: ATF4 and ATF6 gene expression was evaluated in CHL-1 and A375 cell lines after 24 h, by qPCR analysis. The expression of the ribosomal factor L34 was used as an internal control. Absorption data were determined in CHL-1 and A375 cell lines after 24 h, by LC-HRMS (right histograms). CTRL+ represents medium + 4-PBA not exposed to cells. 4-PBA concentrations are expressed as µM ± SE. Experiments were performed in triplicate and repeated three times. The histograms represent the mean ± SD; *** p < 0.001; **** p < 0.0001. Full article ">Figure 4
Adsorption kinetics of 4-PBA in cell culture plates. (A,B) shows the adsorption curves as a function of time, respectively in NBA and DMEM. X axes represent time expressed in hours [h] and Y axes the concentration of free 4-PBA in solution [µM]. (C) Glass petri (G), non-treated polystyrene petri dishes (NTP) and plasma-treated polystyrene plasticware (PTP) showed a similar adsorption with a decrease of about 40%, which is in line with the adsorption curves (A,B). (*** p < 0.001; by one-way ANOVA, Sidak’s multiple comparison). Full article ">

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Pharmaceuticals, Volume 16, Issue 2 (February 2023) – 194 articles

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