Analysis of omarigliptin forced degradation products by ultra-fast liquid chromatography, mass spectrometry, and in vitro toxicity assay.

Omarigliptin (OMG) is an antidiabetic drug indicated for the treatment of type 2 diabetes mellitus. Forced degradation studies are practical experiments to evaluate the stability of drugs and to establish degradation profiles. Herein, we present the investigation of the degradation products (DPs) of OMG formed under various stress conditions. OMG was subjected to hydrolytic (alkaline and acidic), oxidative, thermal, and photolytic forced degradation. A stability-indicating ultra-fast liquid chromatography method was applied to separate and quantify OMG and its DPs. Five DPs were adequately separated and detected in less than 6 min, while other published methods detected four DPs. MS was applied to identify and obtain information on the structural elucidation of the DPs. Three m/z DPs confirmed previously published research, and two novel DPs were described in this paper. The toxicity of OMG and its DPs were investigated for the first time using in vitro cytotoxicity assays, and the sample under oxidative conditions presented significant cytotoxicity. Based on the results from forced degradation studies, OMG was found to be labile to hydrolysis, oxidation, photolytic, and thermal stress conditions. The results of this study contribute to the quality control and stability profile of OMG.

Reductive stress and cytotoxicity in the swollen river mussel (Unio tumidus) exposed to microplastics and salinomycin.

Multistress effects lead to unpredicted consequences in aquatic ecotoxicology and are extremely concerning. The goal of this study was to trace how specific effects of the antibiotic salinomycin (Sal) and microplastics (MP) on the bivalve molluscs are manifested in the combined environmentally relevant exposures. Unio tumidus specimens were treated with Sal (0.6 µg L-1), MP (1 mg L-1, 2 µm size), and both at 18 °C (Mix) and 25 °C (MixT) for 14 days. The redox stress and apoptotic enzyme responses and the balance of Zn/Cu in the digestive gland were analyzed. The shared signs of stress included a decrease in NAD+/NADH and Zn/Cu ratios and lysosomal integrity and an increase in Zn-metallothioneins and cholinesterase levels. MP caused a decrease in the glutathione (GSH) concentration and redox state, total antioxidant capacity, and Zn levels. MP and Mix induced coordinated apoptotic/autophagy activities, increasing caspase-3 and cathepsin D (CtD) total and extralysosomal levels. Sal activated caspase-3 only and increased by five times Cu level in the tissue. Due to the discriminant analysis, the cumulative effect was evident in the combined exposure at 18 °C. However, under heating, the levels of NAD+, NADH, GSH, GSH/GSSG and metallothionein-related thiols were decreased, and coordination of the cytosolic and lysosomal death stimuli was distorted, confirming that heating and pollution could exert unexpected synergistic effects on aquatic life.

Okadaic Acid Is at Least as Toxic as Dinophysistoxin-1 after Repeated Administration to Mice by Gavage.

Okadaic acid (OA) and its analogues cause diarrhetic shellfish poisoning (DSP) in humans, and risk assessments of these toxins require toxicity equivalency factors (TEFs), which represent the relative toxicities of analogues. However, no human death by DSP toxin has been reported, and its current TEF value is based on acute lethality. To properly reflect the symptoms of DSP, such as diarrhea without death, the chronic toxicity of DSP toxins at sublethal doses should be considered. In this study, we obtained acute oral LD50 values for OA and dinophysistoxin-1 (DTX-1) (1069 and 897 µg/kg, respectively) to set sublethal doses. Mice were treated with sublethal doses of OA and DTX-1 for 7 days. The mice lost body weight, and the disease activity index and intestinal crypt depths increased. Furthermore, these changes were more severe in OA-treated mice than in the DTX-1-treated mice. Strikingly, ascites was observed, and its severity was greater in mice treated with OA. Our findings suggest that OA is at least as toxic as DTX-1 after repeated oral administration at a low dose. This is the first study to compare repeated oral dosing of DSP toxins. Further sub-chronic and chronic studies are warranted to determine appropriate TEF values for DSP toxins.

In Vitro Interactions between Okadaic Acid and Rat Gut Microbiome.

Okadaic acid (OA) is a marine biotoxin associated with diarrhetic shellfish poisoning (DSP), posing some threat to human beings. The oral toxicity of OA is complex, and the mechanism of toxicity is not clear. The interaction between OA and gut microbiota may provide a reasonable explanation for the complex toxicity of OA. Due to the complex environment in vivo, an in vitro study may be better for the interactions between OA and gut microbiome. Here, we conducted an in vitro fermentation experiment of gut bacteria in the presence of 0-1000 nM OA. The remolding ability of OA on bacterial composition was investigated by 16S rDNA sequencing, and differential metabolites in fermentation system with different concentration of OA was detected by LC-MS/MS. We found that OA inhibited some specific bacterial genera but promoted others. In addition, eight possible metabolites of OA, including dinophysistoxin-2 (DTX-2), were detected in the fermentation system. The abundance of Faecalitalea was strongly correlated with the possible metabolites of OA, suggesting that Faecalitalea may be involved in the metabolism of OA in vitro. Our findings confirmed the direct interaction between OA and gut bacteria, which helps to reveal the metabolic process of OA and provide valuable evidence for elucidating the complex toxicity of OA.

RIFM fragrance ingredient safety assessment, tetrahydro-6-(3-pentenyl)-2H-pyran-2-one, CAS Registry Number 32764-98-0.

The existing information supports the use of this material as described in this safety assessment. Tetrahydro-6-(3-pentenyl)-2H-pyran-2-one was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data show that tetrahydro-6-(3-pentenyl)-2H-pyran-2-one is not genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class II material, and the exposure to tetrahydro-6-(3-pentenyl)-2H-pyran-2-one is below the TTC (0.009 mg/kg/day, 0.009 mg/kg/day, and 0.47 mg/day, respectively). Data and read-across to 5-hydroxy-7-decenoic acid δ-lactone (CAS # 25,524-95-2) show that there are no safety concerns for tetrahydro-6-(3-pentenyl)-2H-pyran-2-one for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on data and ultraviolet/visible (UV/Vis) spectra; tetrahydro-6-(3-pentenyl)-2H-pyran-2-one is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; tetrahydro-6-(3-pentenyl)-2H-pyran-2-one was found not to be Persistent, Bioaccumulative, and Toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1.

Discovery of Novel Dihydrothiopyrano[4,3-d]pyrimidine Derivatives as Potent HIV-1 NNRTIs with Significantly Reduced hERG Inhibitory Activity and Improved Resistance Profiles.

Enlightened by the available structural biology information, a novel series of dihydrothiopyrano[4,3-d]pyrimidine derivatives were rationally designed via scaffold hopping and molecular hybridization strategies. Notably, compound 20a yielded exceptionally potent antiviral activities (EC50 = 4.44-54.5 nM) against various HIV-1 strains and improved resistance profiles (RF = 0.5-5.6) compared to etravirine and rilpivirine. Meanwhile, 20a exhibited reduced cytotoxicity (CC50 = 284 µM) and higher SI values (SI = 5210-63992). Molecular dynamics simulations were performed to rationalize the distinct resistance profiles. Besides, 20a displayed better solubility (sol. = 12.8 µg/mL) and no significant inhibition of the main CYP enzymes. Furthermore, 20a was characterized for prominent metabolic stability and in vivo safety properties. Most importantly, the hERG inhibition profile of 20a (IC50 = 19.84 µM) was a remarkable improvement. Overall, 20a possesses huge potential to serve as a promising drug candidate due to its excellent potency, low toxicity, and favorable drug-like properties.

Synthesis and Bioactivities of Marine Pyran-Isoindolone Derivatives as Potential Antithrombotic Agents.

2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-teraahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid (FGFC1) is a marine pyran-isoindolone derivative isolated from a rare marine microorganism Stachybotrys longispora FG216, which showed moderate antithrombotic(fibrinolytic) activity. To further enhance its antithrombotic effect, a series of new FGFC1 derivatives (F1-F7) were synthesized via chemical modification at C-2 and C-2' phenol groups moieties and C-1″ carboxyl group. Their fibrinolytic activities in vitro were evaluated. Among the derivatives, F1-F4 and F6 showed significant fibrinolytic activities with EC50 of 59.7, 87.1, 66.6, 82.8, and 42.3 µM, respectively, via enhancement of urokinase activity. Notably, derivative F6 presented the most remarkable fibrinolytic activity (2.72-fold than that of FGFC1). Furthermore, the cytotoxicity of derivative F6 was tested as well as expression of Fas/Apo-1 and IL-1 on HeLa cells. The results showed that, compared to FGFC1, derivative F6 possessed moderate cytotoxicity and apoptotic effect on HeLa cells (statistical significance p > 0.1), making F6 a potential antithrombotic agent towards clinical application.

Phytochemical and pharmacological properties of asperuloside, a systematic review.

Plants are a natural source of bioactive compounds such as secondary metabolites. These molecules, also called phytochemicals, are fundamental for plant survival and often show therapeutic properties used for the treatment of human diseases. Asperuloside is a secondary metabolite which belongs to iridoid glycosides and is commonly present in the plant family Rubiaceae. In this review we aim to summarize the scientific knowledge on asperuloside, with a special emphasis on its pharmacological properties as anti-viral, anti-malarial, anti-protozoal, anti-tumorigenic, anti-hypertensive, anti-obesity, immunomodulatory, anti-inflammatory and antioxidant agent. Preclinical studies in animal models suggest that asperuloside has therapeutic potential that could be evaluated in humans. However, despite its tangible phytochemical characteristics, no clinical trial has been performed so far. Thus, we hope that this review will facilitate scientific dissemination of asperuloside pharmacological properties and encourage researchers to evaluate both pharmacokinetic and toxicity of asperuloside in animal models. This will be the first step towards clinical studies in humans.

Benchmark dose analyses of γH2AX and pH3 endpoints for quantitative comparison of in vitro genotoxicity potential of lipophilic phycotoxins.

The phycotoxins, okadaic acid (OA) and dinophysistoxins 1 and 2 (DTX-1 and -2), are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP) in humans. Data on the in vivo acute toxicity of the OA-group toxins show some differences and the European Food Safety Authority (EFSA) has determined toxicity equivalent factors (TEFs) of one for the reference toxin, OA, as well as for DTX-1 and 0.6 for DTX-2. However, recent in vitro studies indicated that DTX-1 seems to be more toxic than OA. As OA was described as apoptotic and aneugenic compound, we analyzed the DNA damage responses induced by the 3 toxins through γH2AX and pH3 biomarkers on proliferative HepaRG cells using High Content Analysis. We quantitatively examined the responses for γH2AX and pH3 by benchmark dose analyzing (BMD) using PROAST software. We found that the three toxins increased both γH2AX- and pH3-positive cells populations in a concentration-dependent manner. The 3 toxins induced mitotic arrest, characteristic of aneugenic compounds, as well as DNA strand-breaks concomitantly to cytotoxicity. BMD analysis showed that DTX-1 is the most potent inducer of DNA damage, followed by OA and DTX-2. The quantitative genotoxic data provided in this study are additional findings for reconsidering the estimated TEFs of this group of phycotoxins.

Paederus dermatitis - A bibliometric analysis of an emerging disease in travelers.

Not available.

Characterization of scallop midgut gland certified reference material for quantification of diarrhetic shellfish toxins.

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ±â€¯0.061) mg/kg for OA and (0.45 ±â€¯0.11) mg/kg for DTX1.

RIFM fragrance ingredient safety assessment, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, CAS registry number 63500-71-0.

2-Isobutyl-4-methyltetrahydro-2H-pyran-4-ol) was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data show that 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol is not genotoxic, does not have skin sensitization potential, and provided an MOE >100 for the repeated dose, developmental, and reproductive toxicity endpoints. The local respiratory toxicity endpoint was completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class III material (0.47 mg/day). The phototoxicity/photoallergenicity endpoint was completed based on UV spectra and data on 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol. The environmental endpoints were evaluated; 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol was found not to be PBT as per the IFRA Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., PEC/PNEC), are <1.

Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions.

BACKGROUND: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. RESULTS: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10-12-1 × 10-7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl--, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. CONCLUSIONS: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl- ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Inhibition of thymidine phosphorylase expression by Hsp90 inhibitor potentiates the cytotoxic effect of salinomycin in human non-small-cell lung cancer cells.

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells.

Salinomycin decreases feline sarcoma and carcinoma cell viability when combined with doxorubicin.

BACKGROUND: Cancer is a significant health threat in cats. Chemoresistance is prevalent in solid tumors. The ionophore salinomycin has anti-cancer properties and may work synergistically with chemotherapeutics. The purpose of our study was to determine if salinomycin could decrease cancer cell viability when combined with doxorubicin in feline sarcoma and carcinoma cells. RESULTS: We established two new feline injection-site sarcoma cell lines, B4 and C10, and confirmed their tumorigenic potential in athymic nude mice. B4 was more resistant to doxorubicin than C10. Dose-dependent effects were not observed until 92 µM in B4 cells (p = 0.0006) vs. 9.2 µM (p = 0.0004) in C10 cells. Dose-dependent effects of salinomycin were observed at 15 µM in B4 cells (p = 0.025) and at 10 µM in C10 cells (p = 0.020). Doxorubicin plus 5 µM salinomycin decreased viability of B4 cells compared to either agent alone, but only at supra-pharmacological doxorubicin concentrations. However, doxorubicin plus 5 µM salinomycin decreased viability of C10 cells compared to either agent alone at doxorubicin concentrations that can be achieved in vivo (1.84 and 4.6 µM, p < 0.004). In SCCF1 cells, dose-dependent effects of doxorubicin and salinomycin were observed at 9.2 (p = 0.036) and 2.5 (p = 0.0049) µM, respectively. When doxorubicin was combined with either 1, 2.5, or 5 µM of salinomycin in SCCF1 cells, dose-dependent effects of doxorubicin were observed at 9.2 (p = 0.0021), 4.6 (p = 0.0042), and 1.84 (p = 0.0021) µM, respectively. Combination index calculations for doxorubicin plus 2.5 and 5 µM salinomycin in SCCF1 cells were 0.4 and 0.6, respectively. CONCLUSIONS: We have developed two new feline sarcoma cell lines that can be used to study chemoresistance. We observed that salinomycin may potentiate (C10 cells) or work synergistically (SCCF1 cells) with doxorubicin in certain feline cancer cells. Further research is indicated to understand the mechanism of action of salinomycin in feline cancer cells as well as potential tolerability and toxicity in normal feline tissues.

Effects of a spiroketal compound Peniciketal A and its molecular mechanisms on growth inhibition in human leukemia.

Peniciketal A (Pe-A), a spiroketal compound, is isolated from the saline soil-derived fungus Penicillium raistrickii. However, the underlying molecular mechanistic basis for the effects of Pe-A on leukemia is poorly understood. Here, we investigated that Pe-A reduced cell proliferation in three leukemia cell lines (THP-1, K562 and HL60). Importantly, Pe-A showed little cytotoxicity in primary mouse embryonic fibroblast (MEF) cells in a long-duration treatment. For the mechanistic research, we identified 3449 differentially expressed Pe-A-induced proteins through liquid chromatography-tandem mass spectrometry (LC-MS/MS) with TMT label in THP-1 cells. Results showed that many identified proteins were involved in apoptosis and/or autophagy. Then, we confirmed that Pe-A induced not only apoptosis via the mitochondrial pathway but also cytoprotective autophagy by activating the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway indeed. In addition, Pe-A also arrested the cell cycle at the G0-G1 phase by regulating the expressions of checkpoint protein. Collectively, these results provide new insights into the mechanisms that Pe-A may target autophagy-related or apoptosis-related pathways to suppress the development of human leukemia.

Pyrano[3,2-c]quinoline Derivatives as New Class of α-glucosidase Inhibitors to Treat Type 2 Diabetes: Synthesis, in vitro Biological Evaluation and Kinetic Study.

BACKGROUND: Pyrano[3,2-c]quinoline derivatives 6a-n were synthesized via simple two-step reactions and evaluated for their in vitro α-glucosidase inhibitory activity. METHODS: Pyrano[3,2-c]quinoline derivatives 6a-n derivatives were prepared from a two-step reaction: cycloaddition reaction between 1-naphthyl amine 1 and malonic acid 2 to obtain benzo[h]quinoline-2(1H)-one 3 and reaction of 3 with aryl aldehydes 4 and Meldrum's acid 5. The anti- α-glucosidase activity and kinetic study of the synthesized compounds were evaluated using α-glucosidase from Saccharomyces cerevisiae and p-nitrophenyl-a-D-glucopyranoside as substrate. The α-glucosidase inhibitory activity of acarbose was evaluated as positive control. RESULTS: All of the synthesized compounds, except compounds 6i and 6n, showed more inhibitory activity than the standard drug acarbose and were also found to be non-cytotoxic. Among the synthesized compounds, 1-(2-bromophenyl)-1H-benzo[h]pyrano[3,2-c]quinoline-3,12(2H,11H)-dione 6e displayed the highest α-glucosidase inhibitory activity (IC50 = 63.7 ± 0.5 µM). Kinetic study of enzyme inhibition indicated that the most potent compound, 6e, is a non-competitive inhibitor of α-glucosidase with a Ki value of 72 µM. Additionally, based on the Lipinski rule of 5, the synthesized compounds were found to be potential orally active drugs. CONCLUSION: Our results suggest that the synthesized compounds are promising candidates for treating type 2 diabetes.

Protective effects of taurine against inflammation, apoptosis, and oxidative stress in brain injury.

The protective effect of taurine against inflammation, apoptosis and oxidative stress in traumatic brain injury was investigated in the present study. Taurine is a non­proteogenic and essential amino acid in animals. It plays a critical nutritional role in brain cell growth, differentiation, and development. Taurine is involved in regeneration and neuroprotection in the injured nervous system, and is an effective antioxidant against lead­, cadmium­, and exercise­induced oxidative stress. Astrocytes and neuron cells were co­cultured and cells were treated with different concentrations of taurine (100, 200 and 300 mg/l) for 72 h, and the levels of reactive oxygen species, malondialdehyde, reduced glutathione, glutathione peroxidase, superoxide dismutase, catalase, acetylcholinesterase, tumor necrosis factor­α, interleukin­6, caspase­3, p53, B­cell lymphoma 2 and Bcl­2­associated X protein were determined. These inflammatory, apoptotic, and oxidative stress markers were substantially increased in injured cells, and returned to normal levels following taurine supplementation. Thus, taurine supplementation may be effective against oxidative stress, apoptosis, and inflammation in injured brain cells.