Withdrawal from 3-Fluoroethamphetamine induces hyperactivity and depression-like behaviors in male mice.

3-Fluoroethamphetamine (3-FEA) belongs to the amphetamine class of stimulant drugs and functions as a releasing agent for the monoamine neurotransmitters norepinephrine, dopamine, and serotonin. 3-FEA acts on the central nervous system and elicits physical and mental side effects, such as euphoria, increased heart rate, and excitement. However, little is known about the withdrawal symptoms and behavioral changes induced by 3-FEA administration. This study aimed to evaluate the short-term consequences of 3-FEA administration (twice a day, 7 days, i.p.; 1 and 10 mg/kg) in C57BL/6J mice (male, 7 weeks old) at three behavioral levels following 1-4 days of withdrawal. The evaluation included (1) withdrawal score, (2) hyperactivity (open field [OF], elevated plus maze [EPM], and cliff avoidance [CA] test), and (3) depression-like behavior (forced-swim test). In the withdrawal score test, withdrawal behavior increased in all 3-FEA groups at 16 and 40 h after withdrawal. In the OF, EPM, and CA tests, the 3-FEA administration group showed significant changes in terms of hyperactivity. In addition, in the forced-swim test, both the 1 mg/kg and 10 mg/kg 3-FEA groups showed increased immobility time. These findings indicate that 3-FEA administration may lead to physical dependence, demonstrated by the withdrawal score increase and significant changes in hyperactivity and depression-like behavior following repeated administration and drug cessation. In conclusion, this study reveals the adverse consequences of 3-FEA administration and highlights the need for awareness raising and regulatory action to control the use of this new psychoactive substance.

Rewarding and Reinforcing Effects of 25H-NBOMe in Rodents.

The drug 25H-NBOMe is a new psychoactive substance (NPS). The use of these substances is likely to pose a threat to public health because they elicit effects similar to those of known psychoactive substances with similar chemical structures. However, data regarding the abuse potential of 25H-NBOMe are lacking. Here, we evaluated the abuse liability of 25H-NBOMe in rodents. The rewarding and reinforcing effects were evaluated through conditioned place preference (CPP) and self-administration (SA) tests after administration of 25H-NBOMe. To investigate the effects of 25H-NBOMe on the central nervous system, we determined the changes in dopamine levels by in vivo microdialysis. In the locomotor activity test, 25H-NBOme significantly increased locomotor activity in mice. In the place conditioning test, the 25H-NBOMe (0.1 and 0.5 mg/kg) groups showed a significantly increase in CPP in mice. In the SA test, the 25H-NBOMe (0.01 mg/kg) administered group showed a significant increased number of infusions and active lever presses. In microdialysis, the 25H-NBOMe (10 mg/kg) administered group was significantly increased in rats.

Intercomparison study of fragmentation pathways and fragment ion structures for screening of illegal drugs and their novel analogues used to adulterate dietary supplements using liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: As public interest in health and immunity has increased in recent years, so has the demand for dietary supplements. However, supplements adulterated with illegal drugs and their novel analogues are being sold even as the pharmacological efficacies of these drugs are being advertised. Since the use of these illegal compounds can have serious side effects, they pose a risk to public health. Hence, in this study, we propose a strategy for proactively testing drugs and novel analogues that may be added to dietary supplements illegally. METHODS: The optimal conditions for liquid chromatography/quadrupole time-of-flight mass spectrometry were explored to determine the fragmentation patterns for 60 compounds. The optimal conditions were established by comparing the areas and heights of the precursor ion peaks at a fragmentor voltage of 125 or 175 V. Furthermore, the optimized spectra were acquired using collision energies of 1 to 50 eV. The energy value was selected based on the condition that the mass error of the precursor ions is 10 ppm or lower. RESULTS: The fragmentation pathway of each product ion and its chemical structure were predicted and determined. In addition, the obtained structural information was used to screen 18 seized samples. Based on the precursor ions and the corresponding fragmentation patterns, the unknown compounds present in the samples were identified as desulfonylchlorosildenafil and propoxyphenylthiohydroxy homosildenafil. CONCLUSIONS: We obtained mass spectrometry-based information for various compounds by predicting the fragmentation pathways and chemical structures of their fragment ions. Subsequently, based on the obtained structural information, we tested several seized samples and were able to detect two novel analogues in four of the samples. Therefore, the proposed approach is suitable for quickly and accurately identifying the unknown compounds detected in real-world samples.

Role of the JAK/STAT pathway in a streptozotocin-induced diabetic retinopathy mouse model.

PURPOSE: The Janus tyrosine kinase and signal transducers and activators of transcription (JAK/STAT) pathway is involved in vascular endothelial growth factor (VEGF) expression, but the role of this pathway in diabetic retinopathy (DR) remains unclear. We investigated the role of the JAK/STAT pathway on DR and VEGF expression using a streptozotocin (STZ)-induced DR mouse model. METHODS: Cultured ARPE-19 cells were exposed to high-glucose conditions and treated with JAK/STAT inhibitors (JAK inhibitor I [JAKiI], tofacitinib, STAT3 inhibitor [STAT3i]) for 48 h. Reverse-transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to investigate p-JAK/STAT and VEGF expression. Diabetes was induced by intraperitoneal injection of STZ (50 mg/kg) in C57BL/6 mice for 5 days. DR development was evaluated every 4 weeks. JAK/STAT inhibitors were administered for 8 weeks. Immunofluorescence was used to measure the activation status of the JAK/STAT pathway and VEGF production in the retinal tissue. RESULTS: In ARPE-19 cells exposed to high-glucose conditions, the mRNA and secretory protein levels of VEGF, p-JAK1, p-JAK2, p-STAT3, and p-STAT5 levels were significantly increased. Treatment with JAKiI, tofacitinib, and STAT3i significantly suppressed VEGF to basal levels at both the mRNA and secretory levels in vitro. In STZ-induced mice, retinal vascular leakage, p-JAK1, p-JAK2, p-JAK3, p-STAT3, and VEGF were significantly increased after diabetes induction. Diabetes-induced retinal vascular leakage was significantly reduced by treatment with JAKiI and tofacitinib. Increased p-JAK1 and VEGF in STZ-induced mice were significantly reduced by JAKiI (p < 0.05, p < 0.001) and tofacitinib (p < 0.001, respectively). CONCLUSION: JAK1 may be more involved in VEGF production and DR progression in mice than JAK2, JAK3, and STAT3.

Application of predicted fragmentation pathways and fragment ion structures for detecting steroids and selective androgen receptor modulators in dietary supplements using liquid chromatography-quadrupole time-of-flight mass spectrometry.

RATIONALE: Dietary supplements advertised to strengthen muscles have earned fame among athletes. However, several products containing unauthorized compounds are often detected, which can cause a public health risk. Particularly, steroids and selective androgen receptor modulators (SARMs) can cause serious side effects as hormone modulators. In this study, we analyzed 15 steroids and 20 SARMs using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/QTOFMS) to provide fundamental information about fragmentation pathways and fragment ion structures. METHODS: The optimal conditions of LC/QTOFMS were explored to obtain fragmentation patterns for each compound. The optimal conditions were established by comparing the area and height of the precursor ion peak at 125 or 175 V as a fragmentor energy. Furthermore, the optimized spectra were acquired by applying collision energy ranging from 1 to 50 eV. The energy value was selected under the condition that the mass error of precursor ions was less than 10 ppm. RESULTS: The 35 compounds were classified on the basis of their chemical core structures: arylpropionamide (3 compounds), quinolinone (2), pyrrolidinylbenzonitrile (1), indole (2), tropanol (2), phenylaxadaizole (1), hydantoin (2), phenylthiazole (1), nitrothiophene (1) and steroidal derivative (20). Fragmentation pathways and the chemical structure of each product ion were predicted and identified. Furthermore, the obtained structural information was applied to screen seized samples. As a result, 10 seized samples were confirmed to contain one or more SARMs by comparing each precursor ion and fragmentation pattern. CONCLUSIONS: The application to real samples for accurate screening indicated that the same fragmentation patterns and product ions as one or more SARM standards were detected and identified in the seized samples advertised as muscle building. Therefore, this study can contribute to ensuring the safety of public health through providing fundamental information about the risk of illegal adulteration.

Simultaneous screening of 21 potential adulterants in dietary supplements for the treatment of prostate diseases using ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry.

Reports of the number of adulteration cases using illegal therapeutic substances in dietary supplements have increased. In recent years, various dietary supplements are being distributed that exaggerate the efficacy of treatment for prostate-related diseases. To develop the preemptive monitoring method, we selected 21 prostate-related therapeutic substances and optimized the simultaneous ultra-performance liquid chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry and pretreatment procedures for various types of matrices including solid, liquid, and soft capsule samples. The methods were validated by determining the specificity, linearity, limit of detection, limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, stability, and matrix effect. The simultaneous methods were validated according to the international guidelines. In addition, using the validated methods, 81 real samples, which were searched and purchased by focusing on promotional phrases, such as prostate and prostatic hyperplasia, were successfully screened. As a result, sildenafil and tadalafil were detected in one seized capsule sample (5.15 and 14.6 mg/g, respectively). Synthetically, our approach could be useful for the determination of illegal therapeutic substances potentially adulterated in various types of dietary supplements.

Application of a simultaneous screening method for the detection of new psychoactive substances in various matrix samples using liquid chromatography/electrospray ionization tandem mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Recently, new psychoactive substances (NPS) have emerged as a public health risk. Particularly, their chemical structures are modified to avoid detection. Synthetic NPS with effects similar to those of illegal drugs have been recently detected and synthesized worldwide, including MDMB-FUBINACA and APINAC, making it essential to rapidly and accurately detect NPS. METHODS: Fourteen NPS with similar structures were selected and their structures identified using 1 H and 13 C NMR spectroscopy. Additionally, we proposed the fragmentation pattern of each compound using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). A simultaneous analytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was also developed and applied to real samples to detect the 14 NPS. The method was validated based on the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and stability according to international validation guidelines. RESULTS: The established method was used to screen 65 different matrix samples using LC/ESI-MS/MS. By comparing the calculated product ion ratios with those of standards, 2C-B in one of the real samples and 5F-MDMB-PICA in 20 samples were identified. For re-confirmation of detected compounds, the fragmentation pattern of each compound was compared with that of each standard using LC/QTOF-MS. CONCLUSIONS: In this study, LC/QTOF-MS data were used to elucidate the structures and fragmentation patterns of 14 NPS. A simultaneous method was developed using LC/ESI-MS/MS, which was applied to 65 real samples. The presented method and results can assist in ensuring the safety of public health from illegal adulteration.

Simultaneous screening of dietary supplements for 25 anti-hyperlipidemic substances using ultra-performance liquid chromatography and liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Recently, illegal dietary supplements containing unauthorized compounds have been seized and internationally publicized with a warning to avoid their consumption. This adulteration can be a serious threat to public health because of insufficient and reliable safety data as well as their undesirable side effects. It is, therefore, essential to rapidly and accurately develop and simultaneously validate analytical methods for these unauthorized anti-hyperlipidemic substances. METHODS: Dietary supplements were screened simultaneously for 25 anti-hyperlipidemic drugs using an ultra-high-performance liquid chromatography (UPLC) system with a photodiode array (PDA) detector and LC/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The method validation, according to ICH guidelines, considered specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effects, and stability for solid and liquid blank samples. The established UPLC-PDA and LC/ESI-MS/MS methods were applied to screen 103 dietary supplements for 25 anti-hyperlipidemic substances. RESULTS: Using the validated methods, the screened samples were found to contain peaks with similar retention times and PDA spectra. By comparing the calculated precursor-product ion ratios with those of standards, lovastatin and lovastatin acid were detected at concentrations from LOQ to 4.12 mg/g and LOQ to 9.65 mg/g, respectively. CONCLUSIONS: The developed UPLC-PDA and LC/ESI-MS/MS analytical methods were applied to 103 real samples, of which 13 samples were found to contain lovastatin and lovastatin acid. In view of the increasing demand for dietary supplements in the treatment of hyperlipidemic diseases, widespread use of these methods will contribute to consumer health by ensuring the safety of dietary supplements.

Simultaneous determination of illegal drug substances in dietary supplements for gout and osteoporosis using ultra-performance liquid chromatography and liquid chromatography-quadrupole-time-of-flight mass spectrometry.

The aim of this study was to simultaneously determine the presence of unauthorized drug substances in health foods and herbal products used in the treatment of conditions such as gout and anti-osteoporosis. Therefore, we developed and optimised a rapid and accurate method to simultaneously measure 20 anti-gout and anti-osteoporosis drug substances using an ultra-high-performance liquid chromatography (UPLC) system equipped with a photodiode array (PDA) detector. The method was validated to fully meet internationally accepted standards. LODs and LOQs spiked in solid and liquid negative samples were ranged from 0.12 to 1.50 µg/mL, and ranged from 0.36 to 4.50 µg/mL. Linearities (R2> 0.999), stabilities (RSD ≤ 2.92%), accuracies (84.25∼106.62%, intra-day; 84.56∼105.85%, inter-day), precisions (RSD ≤ 3.71% on the intra-day; RSD ≤ 3.47% on the inter-day), recoveries spiked in various type of blank samples such as powder, liquid, tablet, and capsule were determined within 81.20-116.20 %, respectively. From a confirmation of matrix effects (88.06∼110.50% in solid blank; 89.16∼110.52% in liquid blank), it was confirmed that this method was not significantly affected by a sample matrix. The validated method was used to analyse 116 samples containing health foods, herbal products, and seized forensic samples advertised to be effective anti-gout and anti-osteoporosis agents. Of the 20 drug substances screened, dexamethasone was detected and confirmed by comparing the tandem mass spectrometry (MS/MS) fragment ion patterns of a reference standard and the sample using LC-quadrupole-time-of-flight (Q-TOF)/MS. The concentrations of adulterants in seized forensic samples ranged from 0.013 to 0.022 %.

Terpyridine-based complex nanofibers with Eu3+ as a highly selective chemical probes for UO22.

Uranyl is a radioactive, toxic pollutant commonly found in the waste remaining after nuclear fuel reprocessing, and it poses several types of risks to human health; therefore, developing absorbents and chemical probes for this compound is crucial to overcoming these issues. This study examined the sensing abilities of terpyridine-appended benzenetricarboxyamide (T-BTA) as a chromogenic probe for detecting uranyl ions (UO22+). The complex with Eu3+ (1-Eu) spontaneously formed nanostructured fibers in H2O owing to the triamide groups of T-BTA, which induced intermolecular hydrogen-bonding interactions. The strong blue emission of these nanofibers in H2O was quenched upon adding UO22+ but not upon adding any other metal ion. This high selectivity was probably because of the interactions between the nitrigen atoms of the terpyridine moieties of 1 and UO22+. Furthermore, the 1-Eu nanofibers assumed spherical morphologies when UO22+ was added. To develop a convenient UO22+ sensor, an electrospun film incorporating 1-Eu (ESF-1-Eu) was manufactured, and it exhibited high selectivity for UO22+ over a variety of rival metal ions. The plot for luminescence change of ESF-1-Eu vs UO22+ concentrations in seawater samples showed a good linearty. Thus, the ESF-1-Eu shows potential as a useful sensor for detecting and removing UO22+ in H2O.

Type I pig collagen enhances the efficacy of PEDF 34-mer peptide in a mouse model of laser-induced choroidal neovascularization.

BACKGROUND: Pigment epithelium-derived factor (PEDF)-derived 34-mer peptide (PEDF34, Asp44-Asn77) has anti-angiogenic activity but has limitations in clinical application because of an inverted bell-shaped dose-effect relationship and a short half-life. In this study, we attempted to mitigate these problems by mixing PEDF34 with type I collagen. METHODS: The anti-angiogenic activity of the PEDF34/atelocollagen mixture was evaluated by HUVEC tube formation assay and in a laser-induced choroidal neovascular (CNV) mouse model. PEDF34 and/or collagen were administrated using intravitreal injections or eye drops. CNV lesion size was quantified using FITC-dextran-perfused retinal whole mounts. Western blot analysis and inhibitor assays were used to define the action mechanisms of PEDF34 and the mixture. RESULTS: Collagen broadened the effective dose range of PEDF34 in the tube formation assay by > 250 times (from 0.2 to 50 nM). In the CNV model, five intravitreal injections of PEDF34 were required for therapeutic effect, whereas the mixture had a significant therapeutic effect following a single injection. Eye drops of the mixture showed significantly stronger CNV-suppressive effects than drops of PEDF34 alone. The anti-angiogenic activity of PEDF34 might be mediated by inhibition of ERK and JNK activation by VEGF, and collagen potentiated these effects. CONCLUSIONS: Collagen can serve as a carrier and reservoir of PEDF34. PEDF peptide/collagen mixture is easy to prepare than conventional methods for maintaining the therapeutic effect of PEDF peptide.

Novel Cyanostilbene-Based Fluorescent Chemoprobe for Hydroxyl Radicals and Its Two-Photon Bioimaging in Living Cells.

A novel cyanostilbene derivative as a selective fluorescent chemoprobe for hydroxyl radicals was synthesized. The chemoprobe shows strong green emission in aqueous solution with the addition of hydroxyl radicals. Conversely, negligible emission changes are observed upon addition of other reactive oxygen species. The chemoprobe 1 shows high sensitivity, having the low detection limit of ∼1.0 × 10-7 M. Furthermore, the fluorescent chemoprobe exhibits low cytotoxicity and is effectively applied to bioimaging of hydroxyl radicals by two-photon confocal microscopy in HeLa cells. These results indicate that the new chemoprobe has great potential for bioimaging in vivo and in vitro systems.

Self-Assembled Triphenylphosphonium-Conjugated Dicyanostilbene Nanoparticles and Their Fluorescence Probes for Reactive Oxygen Species.

We report self-assembled novel triphenylphosphonium-conjugated dicyanostilbene-based as selective fluorescence turn-on probes for ¹O2 and ClO-. Mono- or di-triphenylphosphonium-conjugated dicyanostilbene derivatives 1 and 2 formed spherical structures with diameters of ca. 27 and 56.5 nm, respectively, through π-π interaction between dicyanostilbene groups. Self-assembled 1 showed strong fluorescent emission upon the addition of ¹O2 and ClO- compared to other ROS (O2-, •OH, NO, TBHP, H2O2, GSH), metal ions (K⁺, Na⁺), and amino acids (cysteine and histidine). Upon addition of ¹O2 and ClO-, the spherical structure of 1 changed to a fiber structure (8-nm wide; 300-nm long). Upon addition of ¹O2 and ClO-, the chemical structural conversion of 1 was determined by FAB-Mass, NMR, IR and Zeta potential analysis, and the strong emission of the self-assembled 1 was due to an aggregation-induced emission enhancement. This self-assembled material was the first for selective ROS as a fluorescence turn-on probe. Thus, a nanostructure change-derived turn-on sensing strategy for ¹O2 or ClO- may offer a new approach to developing methods for specific guest molecules in biological and environmental subjects.

Non-peptidic guanidinium-functionalized silica nanoparticles as selective mitochondria-targeting drug nanocarriers.

We report on the design and fabrication of a Fe3O4 core-mesoporous silica nanoparticle shell (Fe3O4@MSNs)-based mitochondria-targeting drug nanocarrier. A guanidinium derivative (GA) was conjugated onto the Fe3O4@MSNs as the mitochondria-targeting ligand. The fabrication of the Fe3O4@MSNs and their functionalization with GA were carried out by the sol-gel polymerization of alkoxysilane groups. Doxorubicin (DOX), an anti-cancer drug, was loaded into the pores of a GA-attached Fe3O4@MSNs due to both its anti-cancer properties and to allow for the fluorescent visualization of the nanocarriers. The selective and efficient mitochondria-targeting ability of a DOX-loaded GA-Fe3O4@MSNs (DOX/GA-Fe3O4@MSNs) was demonstrated by a co-localization study, transmission electron microscopy, and a fluorometric analysis on isolated mitochondria. It was found that the DOX/GA-Fe3O4@MSNs selectively accumulated into mitochondria within only five minutes; to the best of our knowledge, this is the shortest accumulation time reported for mitochondria targeting systems. Moreover, 2.6 times higher amount of DOX was accumulated in mitochondria by DOX/GA-Fe3O4@MSNs than by DOX/TPP-Fe3O4@MSNs. A cell viability assay indicated that the DOX/GA-Fe3O4@MSNs have high cytotoxicity to cancer cells, whereas the GA-Fe3O4@MSNs without DOX are non-cytotoxic; this indicates that the DOX/GA-Fe3O4@MSNs have great potential for use as biocompatible and effective mitochondria-targeting nanocarriers for cancer therapy.