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Context: Peucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy., Objective: The interaction between peucedanol and CYP450s was investigated., Materials and Methods: Peucedanol (0, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro ., Results: Peucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC 50 values of 6.03, 13.57, and 7.58 μM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a K i value of 4.07 μM and a competitive inhibitor of CYP1A2 and 2D6 with a K i values of 3.39 and 6.77 μM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the K i / K inact value of 5.44/0.046 min/μM., Discussion and Conclusions: In vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.

At present, thiamine deficiency (TD) is managed with administration of high doses of thiamine. Even so, severe and permanent neurological disorders can occur in recurrent episodes of TD. In this study, we used a murine model to assess the efficacy of TD recovery treatments using thiamine with or without additional administration of the antioxidant Trolox or the anti-inflammatory dimethyl sulfoxide (DMSO) after a single or recurrent episode of TD. TD was induced for 9 days with deficient chow and pyrithiamine, and the recovery period was 7 days with standard amounts of chow and thiamine, Trolox, and/or DMSO. After these periods, we evaluated behavior, histopathology, and ERK1/2 modulation in the brain. Deficient animals showed reductions in locomotor activity, motor coordination, and spatial memory. Morphologically, after a single episode of TD and recovery, deficient mice showed neuronal vacuolization in the dorsal thalamus and, after two episodes, a reduction in neuronal cell number. These effects were attenuated or reversed by the recovery treatments, mainly in the treatments with thiamine associated with Trolox or DMSO. Deficient animals showed a strong increase in ERK1/2 phosphorylation in the thalamus, hippocampus, and cerebral cortex after one deficiency episode and recovery. Interestingly, after recurrent TD and recovery, ERK1/2 phosphorylation remained high only in the deficient mice treated with thiamine and/or Trolox or thiamine with DMSO. Our data suggest that a protocol for TD treatment with thiamine in conjunction with Trolox or DMSO enhances the recovery of animals and possibly minimizes the late neurological sequelae., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

With the successful development and increased use of targeted radionuclide therapy for treating cancer comes the increased risk of radiation injury to bone marrow-both direct suppression and stochastic effects, leading to neoplasia. Herein, we report a novel radioprotector drug, a liposomal formulation of γ-tocotrienol (GT3), or GT3-Nano for short, to mitigate bone marrow radiation damage during targeted radionuclide therapy. Methods: GT3 was loaded into liposomes using passive loading. 64 Cu-GT3-Nano and 3 H-GT3-Nano were synthesized to study the in vivo biodistribution profile of the liposome and GT3 individually. The radioprotection efficacy of GT3-Nano was assessed after acute 137 Cs whole-body irradiation at a sublethal (4 Gy), a lethal (9 Gy), or a single high-dose administration of 153 Sm-ethylenediamine- N,N,N',N' -tetrakis(methylene phosphonic acid) (EDTMP). Flow cytometry and fluorescence microscopy were used to analyze hematopoietic cell population dynamics and the cellular site of GT3-Nano localization in the spleen and bone marrow, respectively. Results: Bone marrow uptake and retention (percentage injected dose per gram of tissue) at 24 h was 6.98 ± 2.34 for 64 Cu-GT3-Nano and 7.44 ± 2.52 for 3 H-GT3-Nano. GT3-Nano administered 24 h before or after 4 Gy of total-body irradiation (TBI) promoted rapid and complete hematopoietic recovery, whereas recovery of controls stalled at 60%. GT3-Nano demonstrated dose-dependent radioprotection, achieving 90% survival at 50 mg/kg against lethal 9-Gy TBI. Flow cytometry of the bone marrow indicated that progenitor bone marrow cells MPP2 and CMP were upregulated in GT3-Nano-treated mice. Immunohistochemistry showed that GT3-Nano accumulates in CD105-positive sinusoid epithelial cells. Conclusion: GT3-Nano is highly effective in mitigating the marrow-suppressive effects of sublethal and lethal TBI in mice. GT3-Nano can facilitate rapid recovery of hematopoietic components in mice treated with the endoradiotherapeutic agent 153 Sm-EDTMP., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Tumor necrosis factor α (TNF-α) is an important therapeutic target for rheumatoid arthritis, inflammatory bowel disease, and septic hepatitis. In this study, structure-based virtual ligand screening combined with in vitro and in vivo assays were applied. A lead compound, benpyrine, could directly bind to TNF-α and block TNF-α-trigged signaling activation. Furthermore, the endotoxemic murine model showed that benpyrine could attenuate TNF-α-induced inflammation, thereby reducing liver and lung injury. Meanwhile, administration of benpyrine by gavage significantly relieved the symptoms of collagen-induced arthritis and imiquimod-induced psoriasiform inflammation in mice. Thus, our study discovered a novel, highly specific, and orally active small-molecule TNF-α inhibitor that is potentially useful for treating TNF-α-mediated inflammatory and autoimmune disease.

Purpose: The mechanism of cardioprotection by Kv7.1-5 (KCNQ1-5) channels inhibition by XE991 is unclear. We examined the impact of administration time on the cardioprotective efficacy of XE991, the involvement of key pro-survival kinases, and the importance of the Kv7 subchannels., Methods: Isolated perfused rat hearts were divided into five groups: 1) vehicle, 2) pre-, 3) post- or 4) pre- and post-ischemic administration of XE991 or 5) chromanol 293B (Kv7.1 inhibitor) followed by infarct size quantification. HL-1 cells undergoing simulated ischemia/reperfusion were exposed to either a) vehicle, b) pre-, c) per-, d) post-ischemic administration of XE991 or pre-, per- and post-ischemic administration of e) XE991, f) Chromanol 293B or g) HMR1556 (Kv7.1 inhibitor). HL-1 cell injury was evaluated by propidium iodide/Hoechst staining. Pro-survival kinase activation of Akt, Erk and STAT3 in XE991-mediated HL-1 cell protection was evaluated using phosphokinase inhibitors. Kv7 subtype expression was examined by RT-PCR and qPCR., Results: XE991, but not Chromanol 293B, reduced infarct size and improved hemodynamic recovery in all isolated heart groups. XE991 protected HL-1 cells when administered during simulated ischemia. Minor activation of the survival kinases was observed in cells exposed to XE991 but pharmacological inhibition of kinase activation did not reduce XE991-mediated protection. Kv7 subchannels 1-5 were all present in rat hearts but predominately Kv7.1 and Kv7.4 were present in HL-1 cells and selective Kv7.1 did not reduce ischemia/reperfusion injury., Conclusion: The cardioprotective efficacy of XE991 seems to depend on its presence during ischemia and early reperfusion and do not rely on RISK (p-Akt and p-Erk) and SAFE (p-STAT3) pathway activation. The protective effect of XE991 seems mainly mediated through the Kv7.4 subchannel., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Hansen et al.)

The orally available novel small molecule SHetA2 is the lead sulfur-containing heteroarotinoid that selectively inhibits cancer cells over normal cells, and is currently under clinical development for anticancer treatment and cancer prevention. The objective of this study was to assess and characterize the tissue distribution of SHetA2 in tumor-bearing mice by developing a physiologically based pharmacokinetic (PBPK) model. An orthotopic SKOV3 ovarian cancer xenograft mouse model was used to most accurately mimic the ovarian cancer tumor microenvironment in the peritoneal cavity. SHetA2 concentrations in plasma and 14 different tissues were measured at various time points after a single intravenous dose of 10 mg/kg and oral dose of 60 mg/kg, and these data were used to develop a whole-body PBPK model. SHetA2 exhibited a multi-exponential plasma concentration decline with an elimination half-life of 4.5 h. Rapid and extensive tissue distribution, which was best described by a perfusion rate-limited model, was observed with the tissue-to-plasma partition coefficients (k p  = 1.4-21.2). The PBPK modeling estimated the systemic clearance (76.4 mL/h) from circulation as a main elimination pathway of SHetA2. It also indicated that the amount absorbed into intestine was the major determining factor for the oral bioavailability (22.3%), while the first-pass loss from liver and intestine contributed minimally (< 1%). Our results provide an insight into SHetA2 tissue distribution characteristics. The developed PBPK model can be used to predict the drug exposure at tumors or local sites of action for different dosing regimens and scaled up to humans to correlate with efficacy.

Background: There is a growing interest on women' sexual function improvement provided by topical vulvar application of Visnadine, a natural extractive substance with putative vasodilatory properties. Aims of this study were to evaluate: 1) the vasokinetic activity of a Visnadine Emulgel on mucosal genitalia of 15 healthy postmenopausal women clinically and by instrumental non-invasive analysis; 2) the treatment efficacy by volunteers' judgment regarding to subjective comfort, pleasant warmth, lubrication grade, pinching and burning vulvar sensations., Methods: Fifteen informed healthy female volunteers with menopause were enrolled in the study, with a single blind controlled study versus placebo corresponding to one single application of the emulgel product (active or placebo) on external genitalia., Results: Visnadine Emulgel single application determined a significant increase of vulvar hyperemia, evaluated both clinically and instrumentally, accompanied by a significant increase of local turgor versus placebo. The volunteers reported a pleasant comfort sensation., Conclusions: The topical use of Visnadine on female external genitalia may increase regional vascularization affecting turgidity and sensorial threshold of the area of application.

Although G protein-coupled receptors (GPCRs) are recognized as pivotal drug targets involved in multiple physiological and pathological processes, the majority of GPCRs including orphan GPCRs (oGPCRs) are unexploited. GPR88, a brain-specific oGPCR with particularly robust expression in the striatum, regulates diverse brain and behavioral functions, including cognition, mood, movement control, and reward-based learning, and is thus emerging as a novel drug target for central nervous system disorders including schizophrenia, Parkinson's disease, anxiety, and addiction. Nevertheless, no effective GPR88 synthetic ligands have yet entered into clinical trials, and GPR88 endogenous ligands remain unknown. Despite the recent discovery and early stage study of several GPR88 agonists, such as 2-PCCA, RTI-13951-33, and phenylglycinol derivatives, further research into GPR88 pharmacology, medicinal chemistry, and chemical biology is urgently needed to yield structurally diversified GPR88-specific ligands. Drug-like pharmacological tool function and relevant signaling elucidation will also accelerate the evaluation of this receptor as a viable neurotherapeutic target.

γ-Tocotrienol (γ-T3) exhibits the activity of anticancer via regulating cell signaling pathways. Nuclear factor-κB (NF-κB), one of the crucial pro-inflammatory factors, is involved in the regulation of cell proliferation, apoptosis, invasion, and migration of tumor. In the present study, NF-κB activity inhibited by γ-T3 was investigated in gastric cancer cells. Cell proliferation, NF-κB activity, active protein phosphatase type 2A (PP2A), and ataxia-telangiectasia mutated (ATM) protein were explored using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), methylene blue, enzyme-linked immunosorbent assay (ELISA), malachite green, luciferase, and Western blotting assays. The effects of γ-T3 on tumor growth and the expression of NF-κB and PP2A proteins were also further examined by implanting human gastric cancer cells in a BALB/c nude mouse model. The results showed that γ-T3 significantly inhibited the cell proliferation and attenuated the NF-κB activity in vitro and in vivo. γ-T3 dramatically increased PP2A activity and protein expression, which suppressed ATM phosphorylation and its translocation to the cytoplasm in gastric cancer cells. Thus, our findings may provide mechanistic insight into effects of γ-T3 on the regulation of NF-κB activity by a PP2A-dependent mechanism and suggest that PP2A may serve as a molecular target for a potential chemopreventive agent.

SHetA2 is a novel anticancer drug with poor aqueous solubility. In formal toxicological studies, Kolliphor HS 15 was used as a solubilizing agent to increase the oral bioavailability of SHetA2. The purpose of this study was to formulate SHetA2 and Kolliphor HS 15 as solid powders to facilitate their filling in hard gelatin capsules for clinical trials. Two manufacturing processes, ultra-rapid freeze-drying (URFD) and spray freeze drying (SFD), were employed to fabricate solid powders of SHetA2-Kolliphor HS 15 and trehalose. The morphology, size, flowability, and compressibility of URFD-SHetA2 and SFD-SHetA2 powders were characterized. The crystallinity and apparent maximum solubility of SHetA2 in both powders were also determined. SFD-SHetA2 powders were spherical in shape, small, and with a wide size distribution while the URFD-SHetA2 powders were irregularly shaped and big but with a narrower distribution. DSC and XRD analyses indicated that SHetA2 was mostly amorphous in both powders. The flow of both powders was categorized as "good" (angle of repose < 35°). The uniformity of drug content in URFD-SHetA2 powders was more variable than that in SFD-SHetA2 powders. The solubility profile of SHetA2 in both powders SGF exhibited a transient supersaturation "spring effect" due to the drug's amorphousness followed by extended supersaturation "parachute effect" at approximately 6 μg/ml for both powders compared to 0.02 ± 0.01 μg/ml for unprocessed drug. In conclusion, both URFD and SFD formed solid SHetA2 Kolliphor powders that are possible formulation candidates to be filled in hard gelatin capsules for clinical trials.

Introduction: One of the serious complications of stroke is memory impairment, which is considered as one of the complications of reperfusion of tissue. The present study was designed to compare the effect of administration of Trolox, carnosic acid and Human Chorionic Gonadotropin (HCG) immediately after reperfusion of the stroke tissue on the memory and hippocampal histology., Method: Ischemia-Reperfusion Model (IRI) was created by bilateral occlusion of the common carotid artery for 15 minutes and the first dose was administered immediately after reperfusion. 10 days after ischemia, passive avoidance memory test and apoptotic protein levels were evaluated., Results: Cerebral Ischemia perfusion reduced the time of latency in entering the dark box in the ischemic group. Administration of Trolox and HCG increased this latency time, while treatment with carnosic acid had no effect. Also, IRI significantly reduced the number of healthy cells in the hippocampus. Administration of Trolox, carnosic acid and HCG increased the number of healthy cells and decreased the expression of Caspase-3 and Bax, but significantly increased the expression of Bcl-2 compared to the ischemic group., Conclusion: Findings indicate the beneficial effects of HCG and Trolox on the improvement of memory and the number of healthy cells in the hippocampal region. It is worth noting that the amount of apoptosis in the hippocampus was significantly reduced by Trolox, HCG and Carnosic acid., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 μmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 μmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

KH176 is a potent intracellular reduction-oxidation-modulating compound developed to treat mitochondrial disease. We studied tolerability, safety, pharmacokinetics, pharmacodynamics, and efficacy of twice daily oral 100 mg KH176 for 28 days in a double-blind, randomized, placebo-controlled, two-way crossover phase IIA study in 18 adult m.3243A>G patients without cardiovascular involvement. Efficacy parameters included clinical and functional outcome measures and biomarkers. The trial was registered within ClinicalTrials.gov (NCT02909400), the European Clinical Trials Database (2016-001696-79), and ISRCTN (43372293) (The KHENERGY study). Twice daily oral 100 mg KH176 was well tolerated and appeared safe. No serious treatment-emergent adverse events were reported. No significant improvements in gait parameters or other outcome measures were obtained, except for a positive effect on alertness and mood, although a coincidence due to multiplicity cannot be ignored. The results of the study provide first data on safety and efficacy of KH176 in patients with mitochondrial disease and will be instrumental in designing future clinical trials., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

SHetA2 is a novel compound with strong potential to treat cervical dysplasia, but its low aqueous solubility limits its oral bioavailability. A vaginal suppository achieved SHetA2 cervix concentrations that were severalfold above the predicted therapeutic levels. Thus, we aimed at determining the minimum dose that would achieve SHetA2 therapeutic levels while reducing cyclin D1 levels, the pharmacodynamic end point. The disposition of SHetA2 after vaginal administration of escalating SHetA2 doses and the corresponding reduction in cyclin D1 levels was compared to that after the conventional oral treatment. Vaginal administration of a 15-mg/kg dose achieved an area under the cervix concentration versus time curve (AUC cervix ) that was ∼120 times larger than that after a 60 mg/kg administered orally. AUC cervix and C max-cervix did not increase proportionally with respect to the dose, with the 30-mg/kg dose resulting in higher AUC cervix and C max-cervix (1368.53 μg.mL/h and 155.38 μg/g, respectively) compared to the 15 mg/kg (334.98 μg.mL/h and 121.78 μg/g, respectively) or 60 mg/kg (1178.55 μg.mL/h and 410.38 μg/g, respectively). Likewise, the 30-mg/kg dose caused a larger reduction in cyclin D1 levels than the other doses. Thus, the 30-mg/kg dose was selected for future efficacy studies in a mouse model of cervical neoplasia., (Copyright © 2018. Published by Elsevier Inc.)

SHetA2 is a novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stages of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUC cervix (335 μg mL h -1 ), higher C max (121.8 ± 38.7 µg/g) and longer t 1/2-cervix (30.3 h) compared to mice dosed during estrus (120 μg mL h -1 , 44.6 ± 29.5 µg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the present study, the anticancer activity of 1‑[(3S,4R)‑2,2‑dimethyl‑3‑oxo‑4‑(2‑piperidonyl)chroman‑6‑yl]‑3‑phenylurea (S32) was investigated by testing its effect in vitro on the growth of HeLa cells. First, we showed that the IC50 value of S32 was ~70 µM by using WST‑8 assay, and that it significantly inhibited the proliferation and viability of HeLa cells in a dose‑dependent manner after 48 h. Morphological changes in apoptotic cells included cellular shrinkage and nuclear condensation. The results of [3H]‑thymidine incorporation and flow cytometric analysis indicated that S32 induced inhibition of DNA replication and G2‑phase cell cycle arrest. Moreover, S32 induced the levels of reactive oxygen species (ROS) and decreased the mitochondrial membrane potential (MMP) in a time‑dependent manner. Using Annexin V‑FITC/propidium iodide (PI) dual staining assay, we found that S32 noticeably increased early apoptosis in HeLa cells in a time‑dependent manner. The result of western blot analysis showed that the apoptotic induction was associated with an increase in Bax levels and a decrease in Bcl‑2 levels, which led to activation of caspase‑8, ‑9 and ‑3. Taken together, our findings demonstrated that S32 induces mitochondrial‑mediated apoptosis in HeLa cells and suggest that S32 has potential as an anticancer drug.

Anaplastic thyroid cancer (ATC) is a malignant subtype of thyroid cancers and its mechanism of development remains inconclusive. Importantly, there is no effective strategy for treatment since ATC is not responsive to conventional therapies, including radioactive iodine therapy and thyroid-stimulating hormone suppression. Here, we report that a combinational approach consisting of drugs designed for targeting lipid metabolism, lovastatin (an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR) and troglitazone (an agonist of peroxisome proliferator-activated receptor gamma, PPARγ), exhibits anti-proliferation in cell culture systems and leads to tumor regression in a mouse xenograft model. The composition contains a sub-lethal concentration of both drugs and exhibits low toxicity to certain types of normal cells. Our results support a hypothesis that the inhibitory effect of the combination is partly through a cell cycle arrest at G0/G1 phase, as evidenced by the induction of cyclin-dependent kinase inhibitors, p21 cip and p27 kip , and the reduction of hyperphosphorylated retinoblastoma protein (pp-Rb)-E2F1 signaling. Therefore, targeting two pathways involved in lipid metabolism may provide a new direction for treating ATC.

SHetA2 is a small molecule drug with promising cancer prevention and therapeutic activity and a high preclinical safety profile. The study objectives were to perform interspecies scaling and pharmacokinetic (PK) modeling of SHetA2 for human PK prediction. The PK data obtained from mice, rats, and dogs after intravenous and oral doses were used for simultaneous fitting to PK models. The disposition of SHetA2 was best described by a two-compartment model. The absorption kinetics was well characterized with a first-order absorption model for mice and rats, and a gastrointestinal transit model for dogs. Oral administration of SHetA2 showed a relatively fast absorption in mice, prolonged absorption (i.e., flip-flop kinetics) toward high doses in rats, and an early peak followed by a secondary peak at high doses in dogs. The oral bioavailability was 17.7-19.5% at 20-60 mg/kg doses in mice, <1.6% at 100-2000 mg/kg in rats, and 11.2% at 100 mg/kg decreasing to 3.45% at 400 mg/kg and 1.11% at 1500 mg/kg in dogs. The disposition parameters were well correlated with the body weight for all species using the allometric equation, which predicted values of CL (17.3 L/h), V1 (36.2 L), V2 (68.5 L) and CLD (15.2 L/h) for a 70-kg human. The oral absorption rate and bioavailability of SHetA2 was highly dependent on species, doses, formulations, and possibly other factors. The limited bioavailability at high doses was taken into consideration for the suggested first-in-human dose, which was much lower than the dose estimated based on toxicology studies. In summary, the present study provided the PK model for SHetA2 that depicted the disposition and absorption kinetics in preclinical species, and computational tools for human PK prediction.

The aim of the study was to verify the efficacy of vulvar Visnadine spray in premenopausal women affected by female sexual arousal disorder (FSAD). Thirty-eight women aged 25-40 years affected by FSAD were enrolled in the randomized crossover study, by two possible sequences: on-demand, washout, daily (A sequence); and daily, washout, on-demand (B sequence). The Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale (FSDS) were used to assess sexual function and sexual distress, respectively. Color Doppler ultrasonography was used to measure clitoral blood flow. The study had two follow-ups at 30 (T1) and 60 days (T2). Thirty-one women completed the study. Mean (SD) sexual activity and vulvar Visnadine spray usage was 1 ± 0.9 weekly during on-demand administration for both the sequences (Vs T0, p = NS). The mean sexual activity during daily usage was 2 ± 0.9 (Vs T0, p < .004) and 2 ± 0.8 (Vs T0, p < .001) for A and B sequences, respectively. FSFI total score, particularly genital arousal, improved more during the daily than during on-demand phases of both sequences (p < .001). Finally, clitoral blood flow improved significantly during daily usage of both the sequences (p < .001). Our study suggests that vulvar Visnadine spray could improve sexual performance of women affected by FSAD, producing changes in subjective and objective sexual aspects.

Cervical dysplasia induced by the human papilloma virus unpredictably progresses to cervical cancer. Therapeutic options are invasive and affect the patient's quality of life. SHetA2 has demonstrated therapeutic efficacy against human and murine human papilloma virus-induced tumors, but its oral bioavailability is <1%. An optimized vaginal suppository formulation can deliver SHetA2 in sufficient doses to prevent cervical dysplasia. The quality by design approach was employed to optimize the suppository formulation consisting of cocoa butter as base with 5% Kolliphor and 40% SHetA2. The suppository had a content uniformity of 105.44 ± 0.42%, melted in <8 min, and had a complete release of SHetA2 in water. Administration of the suppository to mice-achieved cervix concentrations that were significantly higher than the SHetA2 therapeutic concentration, with the maximum concentration (C max-cervix  = 336.78 μg/g) being more than 100-fold the therapeutic SHetA2 concentration. Furthermore, the levels of cyclin D1 protein decreased 9-fold indicating a correlation of drug concentrations with the pharmacodynamic endpoint. These proof-of-concept studies suggest that the SHetA2 optimized vaginal suppository formulation may have a potential use in the prevention of cervical dysplasia, but detailed efficacy studies are required to confirm this assumption., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Asthma is an inflammatory disorder with multiple mediators involved in the inflammatory response. Despite several attempts, no new anti-inflammatory drugs have been registered for asthma treatment for several years. However, thiazolidinediones, peroxisome proliferator-activated receptor agonists, have demonstrated some anti-inflammatory properties in various experimental settings. The aim of this study was to assess the influence of troglitazone on LTC 4 and 15-HETE concentrations. It also evaluates TNF-induced eotaxin synthesis in peripheral blood mononuclear cells from 14 patients with mild asthma and 13 healthy controls., Methods: PBMCs were isolated from the whole blood of the asthmatics and healthy subjects and pretreated with 0.1, 1 or 10μM of Troglitazone. The cells were then exposed to 10 -6 M calcium jonophore or 10ng/ml TNF. The production and release of LTC 4 , 15-HETE and eotaxin were then assessed., Results: Troglitazone caused a dose-dependent inhibition in LTC 4 synthesis in both asthmatics and healthy subjects. Troglitazone did not influence 15-HETE or eotaxin production in either asthmatic patients or in healthy individuals., Conclusion: Due to its inhibition of LTC 4 synthesis, troglitazone therapy is an interesting potential therapeutic approach in asthma and other LTC 4 related inflammatory disorders., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Vitamin E is a fat-soluble compound and powerful antioxidant that have been shown to protect the cell membranes against damage caused by free radicals. Human vitamin E supplementation studies are usually limited to α-tocopherol but currently tocotrienols are also available. This study aims to compare the effects of tocotrienol rich fraction (TRF) with α-tocopherol (α-TF) supplementation on oxidative stress in healthy male and female older adults aged 50-55 years old. A total of 71 subjects both male and female aged between 50 and 55 years were divided into groups receiving placebo (n = 23), α-TF (n = 24) and TRF (n = 24) for six months. Blood was taken at baseline (month 0), 3 months and 6 months osf supplementation for determination of plasma malondialdehyde (MDA), protein carbonyl, total DNA damage, vitamin D concentration and vitamin E isomers. α-TF supplementation reduced plasma MDA and protein carbonyl in female subjects after 3 and 6 months. TRF supplementation reduced MDA levels in both males and females as early as 3 months while DNA damage was reduced in females only at 6 months. Supplementation with α-TF and TRF increased plasma vitamin D concentration in both males and females after 6 months, but vitamin D concentration in male subjects were significantly higher compared to female subjects in TRF group. Vitamin E isomer determination showed α-TF, α-tocotrienol and γ-tocotrienol were increased in both male and female subjects. In conclusion, TRF supplementation effects were different from α-TF in reducing oxidative stress markers and vitamin D levels with a more pronounced effect in female subjects., (Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Diabetic nephropathy is still a common complication of type 2 diabetes mellitus (T2DM) and improvement of endothelial dysfunction (ED) and inhibition of reactive oxygen species (ROS) are considered important targets for new therapies. Recently, we developed a new class of compounds (Sul compounds) which inhibit mitochondrial ROS production. Here, we tested the therapeutic effects of Sul-121 on ED and kidney damage in experimental T2DM. Diabetic db/db and lean mice were implanted with osmotic pumps delivering Sul-121 (2.2 mg/kg/day) or vehicle from age 10 to 18 weeks. Albuminuria, blood pressure, endothelial mediated relaxation, renal histology, plasma creatinine, and H 2 O 2 levels were assessed. Sul-121 prevented progression of albuminuria and attenuated kidney damage in db/db, as evidenced by lower glomerular fibronectin expression (~50%), decreased focal glomerular sclerosis score (~40%) and normalization of glomerular size and kidney weight. Further, Sul-121 restored endothelium mediated vasorelaxation through increased production of Nitric Oxide production and normalized plasma H 2 O 2 levels. Sul-121 treatment in lean mice demonstrated no observable major side-effects, indicating that Sul-121 is well tolerated. Our data show that Sul-121 inhibits progression of diabetic kidney damage via a mechanism that involves restoration of endothelial function and attenuation of oxidative stress.

Background: γ-Tocotrienol, a vitamin E isomer possesses pronounced in vitro anticancer activities. However, the in vivo potency has been limited by hardly achievable therapeutic levels owing to inefficient high-dose oral delivery which leads to subsequent metabolic degradation. Jerantinine A, an Aspidosperma alkaloid, originally isolated from Tabernaemontana corymbosa, has proved to possess interesting anticancer activities. However, jerantinine A also induces toxicity to non-cancerous cells., Purpose: We adopted a combinatorial approach with the joint application of γ-tocotrienol and jerantinine A at lower concentrations in order to minimize toxicity towards non-cancerous cells while improving the potency on brain cancer cells., Methods: The antiproliferative potency of individual γ-tocotrienol and jerantinine A as well as combined in low-concentration was firstly evaluated on U87MG cancer and MRC5 normal cells. Morphological changes, DNA damage patterns, cell cycle arrests and the effects of individual and combined low-concentration compounds on microtubules were then investigated. Finally, the potential roles of caspase enzymes and apoptosis-related proteins in mediating the apoptotic mechanisms were investigated using apoptosis antibody array, ELISA and Western blotting analysis., Results: Combinatorial study between γ-tocotrienol at a concentration range (0-24µg/ml) and fixed IC 20 concentration of jerantinine A (0.16µg/ml) induced a potent antiproliferative effect on U87MG cells and led to a reduction on the new half maximal inhibitory concentration of γ-tocotrienol (i.e. t IC 50 =1.29µg/ml) as compared to that of individual γ-tocotrienol (i.e. IC 50 =3.17µg/ml). A reduction on undesirable toxicity to MRC5 normal cells was also observed. G0/G1 cell cycle arrest was evident on U87MG cells receiving IC 50 of individual γ-tocotrienol and combined low-concentration compounds (1.29µg/ml γ-tocotrienol + 0.16µg/ml jerantinine A), whereas, a profound G2/M arrest was evident on cells treated with IC 50 of individual jerantinine A. Additionally, individual jerantinine A and combined compounds (except individual γ-tocotrienol) caused a disruption of microtubule networks triggering Fas- and p53-induced apoptosis mediated via the death receptor and mitochondrial pathways., Conclusions: These findings demonstrated that the combined use of lower concentrations of γ-tocotrienol and jerantinine A induced potent cytotoxic effects on U87MG cancer cells resulting in a reduction on the required individual concentrations and thereby minimizing toxicity of jerantinine A towards non-cancerous MRC5 cells as well as probably overcoming the high-dose limiting application of γ-tocotrienol. The multi-targeted mechanisms of action of the combination approach have shown a therapeutic potential against brain cancer in vitro and therefore, further in vivo investigations using a suitable animal model should be the way forward., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N -acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression., Competing Interests: The authors declare no conflict of interest.

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

The purpose of this study was two-fold: (1) to formulate γ-tocotrienol (GT3) in a nanoemulsion formulation as a prophylactic orally administered radioprotective agent; and (2) to optimize the storage conditions to preserve the structural integrity of both the formulation and the compound. γ-tocotrienol was incorporated into a nanoemulsion and lyophilized with lactose. Ultra performance liquid chromatography-mass spectroscopy (UPLC-MS) was used to monitor the chemical stability of GT3 over time, the particle size and ζ potential, and scanning electron microscopy (SEM) were used to study the physical stability of the nanoemulsion. Radioprotective and toxicity studies were performed in mice. The liquid formulation exhibited GT3 degradation at all storage temperatures. Lyophilization, in the presence of lactose, significantly reduced GT3 degradation. Both the liquid and lyophilized nanoemulsions had stable particle size and ζ potential when stored at 4 °C. Toxicity studies of the nanoemulsion resulted in no observable toxicity in mice at an oral dose of 600 mg/kg GT3. The nano-formulated GT3 (300 mg/kg) demonstrated enhanced survival efficacy compared to GT3 alone (200 and 400 mg/kg) in CD2F1 mice exposed to total body gamma radiation. The optimal long-term storage of formulated GT3 is as a powder at -20 °C to preserve drug and formulation integrity. Formulation of GT3 as a nanoemulsion for oral delivery as a prophylactic radioprotectant shows promise and warrants further investigation., Competing Interests: The authors declare no conflict of interest.

This study was designed to evaluate the possible potentiation of survival protection afforded by relatively low-dose amifostine prophylaxis against total body irradiation in combination with a protective, less toxic agent, gamma-tocotrienol (GT3). Mice were administered amifostine and/or GT3, then exposed to 9.2 Gy 60 Co γ-irradiation and monitored for survival for 30 days. To investigate cytokine stimulation, mice were administered amifostine or GT3; serum samples were collected and analyzed for cytokines. Survival studies show single treatments of GT3 or amifostine significantly improved survival, compared to the vehicle, and combination treatments resulted in significantly higher survival compared to single treatments. In vivo studies with GT3 confirmed prior work indicating GT3 induces granulocyte colony-stimulating factor (G-CSF). This approach, the prophylactic combination of amifostine and GT3, which act through different mechanisms, shows promise and should be investigated further as a potential countermeasure for acute radiation syndrome., (© The Author 2016. Published by Oxford University Press.)

This article reviews studies of progenitor mobilization with gamma-tocotrienol (GT3), a tocol under advanced development as a radiation countermeasure for acute radiation syndrome (ARS). GT3 protects mice against high doses of ionizing radiation and induces high levels of granulocyte colony-stimulating factor (G-CSF). GT3-induced G-CSF in conjunction with AMD3100 (a chemokine receptor antagonist clinically used to improve the yield of mobilized progenitors) mobilizes progenitors; these mobilized progenitors mitigate injury when infused to mice exposed to acute, high-dose ionizing radiation. The administration of a G-CSF antibody to GT3-injected donor mice abrogated the radiomitigative efficacy of blood or peripheral blood mononuclear cells (PBMC) in irradiated recipient mice. The efficacy of GT3-injected donor mice blood or PBMC was comparable to a recently published article involving blood or mononuclear cells obtained from mice injected with G-CSF. The injected progenitors were found to localize in various tissues of irradiated hosts. The authors demonstrate the efficacy of a bridging therapy in a preclinical animal model that allows the lymphohematopoietic system of severely immunocompromised mice to recover. This suggests that GT3 is a highly effective agent for radioprotection and mobilizing progenitors with significant therapeutic potential. Therefore, GT3 may be considered for further translational development and ultimately for use in humans.

This study investigated possible role of U-II and its receptor expression in inflammation by using UTR agonist and antagonist in carrageenan induced acute inflammation. Rats were divided into 5 groups as (1) Healthy control, (2) Carrageenan control, (3) Carrageenan +Indomethacin 20mg/kg, orally, (4) Carrageenan +AC7954 (U-II receptor agonist, intraperitoneally) 30mg/kg and (5) Carrageenan +SB657510 (UTR antagonist, intraperitoneally) 30mg/kg. 1h after drug administration, carrageenan was injected. At the 3rd hour after carrageenan injection, agonist produced no effect while antagonist 63% anti-inflammatory effect respectively. UTR and UT-II expression increased in carrageenan induced paw tissue. Antagonist administration prevented the decrease in an antioxidant system and also capable to decrease TNF-α and IL-6 mRNA expressions. This study showed the role of urotensin II receptors in the physiopathogenesis of acute inflammatory response that underlying many diseases accompanied by inflammation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13'-COOH and δTE-13'-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOH-induced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility., (© 2016 Blackwell Verlag GmbH.)

The hazard of ionizing radiation exposure due to nuclear accidents or terrorist attacks is ever increasing. Despite decades of research, still, there is a shortage of non-toxic, safe and effective medical countermeasures for radiological and nuclear emergency. To date, the U.S. Food and Drug Administration (U.S. FDA) has approved only two growth factors, Neupogen (granulocyte colony-stimulating factor (G-CSF), filgrastim) and Neulasta (PEGylated G-CSF, pegfilgrastim) for the treatment of hematopoietic acute radiation syndrome (H-ARS) following the Animal Efficacy Rule. Promising radioprotective efficacy results of γ-tocotrienol (GT3; a member of the vitamin E family) in the mouse model encouraged its further evaluation in the nonhuman primate (NHP) model. These studies demonstrated that GT3 significantly aided the recovery of radiation-induced neutropenia and thrombocytopenia compared to the vehicle controls; these results particularly significant after exposure to 5.8 or 6.5 Gray (Gy) whole body γ-irradiation. The stimulatory effect of GT3 on neutrophils and thrombocytes (platelets) was directly and positively correlated with dose; a 75 mg/kg dose was more effective compared to 37.5 mg/kg. GT3 was also effective against 6.5 Gy whole body γ-irradiation for improving neutrophils and thrombocytes. Moreover, a single administration of GT3 without any supportive care was equivalent, in terms of improving hematopoietic recovery, to multiple doses of Neupogen and two doses of Neulasta with full supportive care (including blood products) in the NHP model. GT3 may serve as an ultimate radioprotector for use in humans, particularly for military personnel and first responders. In brief, GT3 is a promising radiation countermeasure that ought to be further developed for U.S. FDA approval for the ARS indication.

Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10(-5) M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The search for treatments to counter potentially lethal radiation-induced injury over the past several decades has led to the development of multiple classes of radiation countermeasures. However, to date only granulocyte colony-stimulating factor (G-CSF; filgrastim, Neupogen)and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved by the United States Food and Drug Administration (FDA) for the treatment of hematopoietic acute radiation syndrome (ARS). Gamma-tocotrienol (GT3) has demonstrated strong radioprotective efficacy in the mouse model, indicating the need for further evaluation in a large animal model. In this study, we evaluated GT3 pharmacokinetics (PK) and efficacy at different doses of cobalt-60 gamma radiation (0.6 Gy/min) using the nonhuman primate (NHP) model. The PK results demonstrated increased area under the curve with increasing drug dose and half-life of GT3. GT3 treatment resulted in reduced group mean neutropenia by 3-5 days and thrombocytopenia by 1-5 days. At 5.8 and 6.5 Gy total-body irradiation, GT3 treatment completely prevented thrombocytopenia. The capability of GT3 to reduce severity and duration of neutropenia and thrombocytopenia was dose dependent; 75 mg/kg treatment was more effective than 37.5 mg/kg treatment after a 5.8 Gy dose. However, the higher GT3 dose (75 mg/kg) was associated with higher frequency of adverse skin effects (small abscess) at the injection site. GT3 treatment of irradiated NHPs caused no significant difference in animal survival at 60 days postirradiation, however, low mortality was observed in irradiated, vehicle-treated groups as well. The data from this pilot study further elucidate the role and pharmacokinetics of GT3 in hematopoietic recovery after irradiation in a NHP model, and demonstrate the potential of GT3 as a promising radioprotector.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10(3) antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0-360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood-brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified., (Copyright © 2015 John Wiley & Sons, Ltd.)

DILI is a major safety issue during drug development and one of the leading causes for market withdrawal. Despite many efforts made in the past, the prediction of DILI using in vitro models remains very unreliable. In the present study, the well-established hepatocyte Collagen I-Matrigel™ sandwich culture was used, mimicking chronic drug treatment after multiple incubations for 14 days. Ten drugs associated with different types of specific preclinical and clinical liver injury were evaluated at non-cytotoxic concentrations. Mrp2-mediated transport, intracellular accumulation of neutral lipids and phospholipids were selected as functional endpoints by using Cellomics™ Arrayscan® technology and assessed at five timepoints (day 1, 3, 7, 10, 14). Liver specific functional impairments after drug treatment were enhanced over time and could be monitored by HCI already after few days and before cytotoxicity. Phospholipidosis-inducing drugs Chlorpromazine and Amiodarone displayed the same response as in vivo. Cyclosporin A, Chlorpromazine, and Troglitazone inhibited Mrp2-mediated biliary transport, correlating with in vivo findings. Steatosis remained difficult to be reproduced under the current in vitro testing conditions, resulting into false negative and positive responses. The present results suggest that the repeated long-term treatment of rat hepatocytes in the Collagen I-Matrigel™ sandwich configuration might be a suitable tool for safety profiling of the potential to induce phospholipidosis and impair Mrp2-mediated transport processes, but not to predict steatosis., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: This study was designed to examine the differential protection of pre- versus post-treatment with three different antioxidants, curcumin (CUR), Trolox, and N-acetylcysteine (NAC), on acrylonitrile (AN)-induced cytotoxicity in primary rat astrocytes., Methods: Primary astrocyte cultures were treated with CUR, Trolox and NAC for 4h prior to, or following 24h treatment with AN (2.5mM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) release, lipid peroxidation, glutathione, reactive oxygen species (ROS) and mitochondrial membrane potential were measured to evaluate protection associated with the three antioxidants. Knockdown of Nrf2 expression by liposome transfection with siRNA was used to confirm the role of Nrf2 activation in the protection associated with the three antioxidants., Results: Compared with AN treatment alone, pre-treatment with CUR at either concentration significantly increased cell viability and mitochondrial membrane potential, and reduced glutathione levels; lipid peroxidation and ROS production were significantly decreased as well. NAC also showed significant efficacy in attenuating AN-induced toxicity at higher concentration. However, pre-treatment with Trolox failed to ameliorate the AN-induced toxicity. When post-treatment with Trolox, this antioxidant led to significant protective effects at both concentrations, while CUR and NAC were efficacious only at the higher concentrations. Knockdown of Nrf2 only abolished the protective effects of CUR pre-treatment on AN-induced cytotoxicity, while the protective effects of NAC and Trolox pre-treatment groups showed no differences between the Nrf2-knockdown and non-knockdown treatments., Conclusions: The selected antioxidants exert differential cellular protection when administered prior or subsequent to AN-induced cytotoxic events in decreasing cellular viability, antioxidative capacity and mitochondrial function, enhanced cytotoxicity and ROS production. These results suggest that antioxidants should be carefully chosen for their efficacy in preventing or diminishing oxidative damage caused by AN. The differential effect of pre- and post-treatment may be attributed to activation of the Nrf2 signaling pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The impact of supplementing laying-hen feed with annatto tocotrienols (T3s) and alpha-tocopherol on the distribution of various forms of vitamin E and cholesterol throughout the hen's body was evaluated. A total of 18 organs or tissues (skin, fat pad, liver and gall bladder, heart, oviduct, forming yolk, laid yolk, lungs, spleen, kidney, pancreas, gizzard, digestive tract, brain, thigh, breast, manure, and blood) were collected after 7 wk of feeding on diets enriched with various levels of alpha-tocopherol and annatto extract that contained gamma-T3 and delta-T3. Tissue weights, contents of lipid, alpha-tocopherol, gamma-T3, delta-T3, cholesterol, and fatty acid composition of extracted lipids from the collected organs and tissues were determined. Tissue weight and lipid content did not change significantly with feed supplementation treatments, except that the liver became heavier with increased levels of supplementation. Overall, the main organs that accumulated the supplemented vitamin E were fat pad, liver and gall bladder, oviduct, forming yolks, laid yolks, kidney, brain, thigh, and breast. Much of annatto gamma-T3 and delta-T3 (> 90%) was found in the manure, indicating poor uptake. In some tissues (brain and oviduct,) a significant increase in polyunsaturated fatty acids was seen with increased supplementation. Alpha-tocopherol impacted the transfer of gamma-T3 to forming and laid yolks, but did not impact delta-T3 transfer. No significant differences were found in most of the tissues in cholesterol, except a reduction in heart, based on tissue as-is. Blood samples showed large variations in individual hens with no significant differences in total and HDL cholesterol, or total triacylglycerols. Supplementing feed with annatto T3s and alpha-tocopherol showed that the vitamin E profile and distribution of the laying-hen body can be altered, but to different extents depending on tissue. The result of this research has significance in enhancing meat nutrient content., (© 2015 Poultry Science Association Inc.)

Gamma and delta tocotrienols are isomers of Vitamin E with established potency in pre-clinical anti-cancer research. This single-dose, randomized, crossover study aimed to compare the safety and bioavailability of a new formulation of Gamma Delta Tocotrienol (GDT) in comparison with the existing Tocotrienol-rich Fraction (TRF) in terms of gamma and delta isomers in healthy volunteers. Subjects were given either two 300 mg GDT (450 mg γ-T3 and 150 mg δ-T3) capsules or four 200 mg TRF (451.2 mg γ-T3 &102.72 mg δ-T3) capsules and blood samples were taken at several time points over 24 hours. Plasma tocotrienol concentrations were determined using HPLC method. The 90% CI for gamma and delta tocotrienols for the ratio of log-transformation of GDT/TRF for Cmax and AUC0-∞ (values were anti-logged and expressed as a percentage) were beyond the bioequivalence limits (106.21-195.46, 154.11-195.93 and 52.35-99.66, 74.82-89.44 respectively). The Wilcoxon Signed Rank Test for Tmax did not show any significant difference between GDT and TRF for both isomers (p > 0.05). No adverse events were reported during the entire period of study. GDT was found not bioequivalent to TRF, in terms of AUC and Cmax. Gamma tocotrienol in GDT showed superior bioavailability whilst delta tocotrienol showed less bioavailability compared to TRF.

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures., (Copyright © 2015. Published by Elsevier B.V.)

The aim of this study was to formulate and characterize α-tocopherol (α-T) and tocotrienol-rich fraction (TRF) entrapped in poly (lactide-co-glycolide) (PLGA) and chitosan covered PLGA (PLGA-Chi) based nanoparticles. The resultant nanoparticles were characterized and the effect of nanoparticles entrapment on the cellular uptake, antioxidant, and antiproliferative activity of α-T and TRF were tested. In vitro uptake studies in Caco2 cells showed that PLGA and PLGA-Chi nanoparticles displayed a greater enhancement in the cellular uptake of α-T and TRF when compared with the control without causing toxicity to the cells (p<0.0001). Furthermore, the cellular internalization of both PLGA and PLGA-Chi nanoparticles labeled with FITC was investigated by fluorescence microscopy; both types of nanoparticles were able to get internalized into the cells with reasonable amounts. However, PLGA-Chi nanoparticles showed significantly higher (3.5-fold) cellular uptake compared to PLGA nanoparticles. The antioxidant activity studies demonstrated that entrapment of α-T and TRF in PLGA and PLGA-Chi nanoparticles exhibited greater ability in inhibiting cholesterol oxidation at 48 h compared to the control. In vitro antiproliferative studies confirmed marked cytotoxicity of TRF on MCF-7 and MDA-MB-231 cell lines when delivered by PLGA and PLGA-Chi nanoparticles after 48 h incubation compared to control. In summary, PLGA and PLGA-Chi nanoparticles may be considered as an attractive and promising approach to enhance the bioavailability and activity of poorly water soluble compounds such as α-tocopherol and tocotrienols., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Objectives: It has been shown that certain vitamins can significantly enhance the effect of photodynamic anti-tumor therapy. Unfortunately, there is no sufficient information available about the impact of those antioxidants on antimicrobial Photodynamic Therapy (aPDT). The present study is aimed at investigating the antimicrobial effect of the dye indocyanine green (ICG) in the presence of Trolox(TM) , a vitamin E analogue, upon irradiation with near-infrared (NIR) laser light (808 nm) on the gramnegative periodontopathogenic bacteria Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.) and Fusobacterium nucleatum (F.n.)., Methods: Bacteria solved in PBS were incubated with ICG (50-500 μg/ml) in the presence and absence of Trolox(TM) (2 mM). Irradiation was performed after 10 minutes of dark-incubation with NIR-laser-light (25-100 J/cm(2) , 810 nm). During treatment, temperature was also recorded inside the bacterial solutions. The treated suspensions were serial diluted and plated onto blood agar plates. After anaerobe cultivation for 5 days the colony-forming units (CFU/ml) were determined., Results: The antibacterial effect was ICG-concentration and exposure dependent. It was found that high ICG-concentrations and light fluence rates caused bacterial reduction due to hyperthermia. Where low ICG-concentrations (<250 μg/ml) and fluence rates only induced minor regression, additional Trolox(TM) -administration significantly enhanced the photodynamic effect. While treatment of A.a. (250 μg/ml ICG, 100 J/cm(2) ) without Trolox(TM) caused no bacterial reduction, additional administration led to total eradication. In the presence of Trolox(TM) reduction to one-fifth of the original ICG-concentration (50 μg/ml) still induced total suppression of P.g. and F.n. at identical fluence (100 J/cm(2) ). Treatment with ICG, NIR-light or Trolox(TM) alone showed no remarkable bactericidal effect. Application of high ICG-concentrations (500 μg/ml) and exposure values (100 J/cm(2) ) caused peak temperatures of 64.53°C., Conclusions: The results clearly show that Trolox(TM) significantly enhanced the antibacterial effect of ICG upon irradiation with NIR-laser-light. Additional administration of Trolox(TM) may also increase the efficiency of other aPDT systems., (© 2015 Wiley Periodicals, Inc.)

Background: The objective of the present study was to investigate how oxidative status influences the effectiveness of cytotoxicity of artemisinin towards cancer cells. It is hypothesized that antioxidants would reduce, whereas pro-oxidants would enhance, cytotoxicity., Materials and Methods: Molt-4 human leukemia cells were incubated with vitamins C, E, D3, dexamethasone, or hydrogen peroxide alone or in combination with dihydroartemisinin (DHA). Concentrations of these compounds studied were similar to those achievable by oral administration. Viable cell counts were performed before (0 h) and at, 24 and 48 h after treatment., Results: Vitamin C, vitamin D3, dexamethasone, and H2O2 caused significant Molt-4 cell death. Vitamin E caused an increase in Molt-4 cell growth. Vitamin C and vitamin D3 significantly interacted with DHA at the 48-h time point and with H2O2 at both 24-h and 48-h time points., Conclusion: Cellular oxidative status could alter the potency of artemisinin in killing cancer cells., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

In this study a brushite cement was doped with the chain-breaking antioxidant Trolox. The effect of the antioxidant on the physical properties of the cement was evaluated and the release of Trolox was monitored by UV spectroscopy. The ability of the Trolox set free to scavenge reactive oxygen species (ROS) released by macrophages was determined in vitro using a luminol-amplified chemiluminescence assay. Trolox did not modify the crystalline phases of the set cement, which mainly formed crystalline brushite after 7 days in humid conditions. The setting time, compressive strength and morphology of the cement also remained unaltered after the addition of the antioxidant. Trolox was slowly released from the cement following a non-Fickian transport mechanism and nearly 64% of the total amount was released after 3 days. Moreover, the capacity of Trolox to scavenge the ROS released by macrophages increased in a dose-dependent manner. Trolox-loaded cements are expected to reduce some of the first harmful effects of acute inflammation and can thus potentially protect the surrounding tissue during implantation of these as well as other materials used in conjunction., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A new strategy was designed to evaluate the antioxidant effectiveness of five topically applied polyphenols following skin penetration profiles. The antioxidants were the following polyphenol derivatives: epicatechin, resveratrol, rutin, quercetin, and trolox, which was used as the reference antioxidant. The hydrophilic/lipophilic character of these compounds was evaluated, and their antioxidant activity was measured by the DPPH method. The percutaneous absorption of these polyphenols was obtained by an in vitro methodology using porcine skin biopsies. This methodology involves the quantification of the antioxidants present in each specific skin layer to evaluate antioxidant effectiveness. The antioxidant activity in each skin layer was also determined by the DPPH method. The results indicated that lipophilic antioxidants (epicatechin, resveratrol, quercetin, and trolox) penetrated deeper into the skin layers, whereas a more hydrophilic compound, rutin, remained on the skin surface. The antioxidant evaluation of each skin compartment suggested that resveratrol and rutin were the most effective topically applied compounds in view of their antioxidant activity and their skin penetration profile., (Copyright © 2014 Elsevier Inc. All rights reserved.)