In vitro hepatotoxicity evaluation of methotrexate-loaded niosome formulation: fabrication, characterization and cell culture studies.

BACKGROUND: Methotrexate (MTX) is a folic acid antagonist that is widely used to treat osteosarcoma, leukemia, breast cancer, and autoimmune and inflammatory diseases. The most important concerns with MTX are its poor solubility and high toxicity, particularly in liver cells. To enhance its solubility and to minimize its toxicity, we encapsulated MTX in niosomes and investigated its hepatotoxicity mechanisms using genetic biomarkers. METHODS: Niosomes were successfully prepared using a modified thin film method, and the prepared monodisperse smallsized formulation was subsequently characterized. In vitro cytotoxicity studies were performed both in hepatocarcinoma (HEP3G) and healthy liver (AML12) cell lines. Specifically, immunofluorescence assay and evaluation of the expression levels of apoptotic, antioxidant, heat shock protein, and oxidative stress genes were performed. RESULTS: The formulation had a particle size of 117.1 ± 33 nm, a surface charge of -38.41 ± 0.7 mV, and an encapsulation efficiency of 59.7% ± 2.3%. The results showed that the niosomal formulation exhibited significantly higher cytotoxic effects in HEP3G than in AML12. The immunofluorescence and genetic analyses showed that the increased cytotoxicity of niosomes resulted mainly from oxidative stress and slight apoptosis. DISCUSSION: These results demonstrated that niosomal drug delivery systems could be a new potential formulation for minimizing MTX-related hepatotoxicity.

Improving the Bioavailability and Efficacy of Coenzyme Q10 on Alzheimer's Disease Through the Arginine Based Proniosomes.

Coenzyme Q10 (CoQ10) is a fat-soluble vitamin-with a benzoquinone-like structure. CoQ10 plays a role in membrane stability, energy conversion, and ATP production. It is also one of the important antioxidants in the body. The bioavailability of exogenous CoQ10 is extremely low due to its poor aqueous solubility and large molecular mass. In this study, mixed proniosomal drug delivery systems have been used to increase solubility and bioavailability of CoQ10. Arginine (semi-essential amino acid) was incorporated in the formulation composition to achieve higher efficacy by boosting nitric oxide presence, endothelial dysfunction, and cellular uptake. Proniosomes were investigated in terms of particle size, polydispersity index, zeta potential, encapsulation efficiency, and process yield, and optimization studies were carried on by utilizing STATISTICA 8.0 software considering dependent factors (carrier amount, drug amount, and surfactant ratio). Optimum proniosome formulation (particle size 187.5 ± 16.35 nm, zeta potential: -44.7 ± 12.8 mV, encapsulation efficiency 99.05±0.30%, and product yield: 90.55%) was evaluated for thermal analysis, in-vitro drug release using microcentrifuge method. In-vitro cytotoxicity studies of proniosomes were performed on intestinal Epithelial Cells (Cellartis®, ChiPSC18) and no cytotoxic effects was seen during the 72 h. Besides, anti Alzheimer effect was investigated on APPSL-GFP lentivirus-infected human neural cells (APPSL-GFP-l-HNC) and Alzheimer biomarkers (p-tau181 and p-tau217). While CoQ10's relative bioavailability was statistically increased by proniosome compared to CoQ10 suspension (p<0.01, Grubb test). PK parameters of proniosome formulation, obtained with non-compartmental modeling, were fitting to the data (R2=0.956±0.026). The study results proved that proniosomal formulation has a high potential drug delivery system for both increasing bioavailability and anti-Alzheimer effect of CoQ10.

Enhanced Cytotoxic Activity of 6-Mercaptopurine-Loaded Solid Lipid Nanoparticles in Hepatic Cancer Treatment.

6-Mercaptopurine (6-MCP) is an antiproliferative purine analog used in acute lymphoblastic leukemia, non-Hodgkin lymphoma, and inflammatory bowel disease (Crohn's disease, ulcerative colitis). Although 6-MCP has the great therapeutic potential for cancer and immunosuppressant-related diseases, 6-MCP is not readily soluble in water, presents a high first-pass effect, short half-life (0.5-1.5 h), and implies a low bioavailability (16%). On the contrary, solid lipid nanoparticles (SLNs) are prepared from solid lipids at room temperature and body temperature. In this study, SLNs were prepared w/o/w double emulsion-solvent evaporation method using Precirol ATO5 as matrix lipid. In the emulsion stabilization, surfactant (Tween 80) and polymeric stabilizer (polyvinyl alcohol [PVA]) were used. Two group formulations using Tween 80 and PVA were compared in terms of particle size, polydispersity index, zeta potential encapsulation efficiency%, and process yield%. Differential calorimetric analysis and release properties were examined for optimum formulation, and release kinetics were calculated. According to studies, sustained release was obtained with SLNs by the Korsmayer-Peppas kinetic model. The in vitro cytotoxicity studies were performed on the hepatocarcinoma (HEP3G) cell line. According to the results, successful SLN formulations were produced, and PVA was found best stabilizer. Optimum formulation exhibited significantly higher cytotoxic effects on HEP3G than on pure 6-MCP. These results demonstrated that solid lipid nanodrug delivery systems have great potential for formulation of 6-MCP.

Evaluation of the cytotoxic effect of titanium dioxide nanoparticles in human embryonic lung cells.

Background/aim: Titanium dioxide nanoparticles are widely used in a variety of products, including sunscreens, paints, and ceramics. However, their increasing use has raised concerns about their potential health risks. Titanium dioxide nanoparticles have been shown to have the ability to enter the bloodstream and accumulate in various tissues, reaching the fetus via the placenta. The aim of this study was to investigate the cytotoxic effects of titanium dioxide nanoparticles on a human embryonic lung cell line (HEL 299/An1) and the formation of oxidative DNA damage. Materials and methods: The cytotoxic effects of brookite-based titanium dioxide nanoparticles (<100 nm) were assessed using the 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay for 24 and 48 h. Cell titanium levels were determined using inductively coupled plasma mass spectrometry. Oxidative DNA damage was assessed by measuring the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) as a biomarker. Results: Titanium dioxide nanoparticles caused dose-dependent cytotoxicity in HEL 299/An1 cells. The IC50 values were 25.93 µM and 0.054 µM after 24 h and 48 h of exposure, respectively. Cell titanium levels were found to be 25,967 ppb after 24 h and 210,353 ppb after 48 h (p < 0.01). 8-OHdG was detected at 32.96 ng/mL after 24 h of exposure and 17.89 ng/mL after 48 h of exposure. Conclusion: In our study, it was shown that titanium nanoparticles caused dose-dependent cytotoxicity and oxidative DNA damage in human embryonic lung cells. The nanoparticles also accumulated in cells and were taken up in higher amounts after 48 h of exposure. These findings suggest that titanium dioxide nanoparticles may pose a health risk, especially for pregnant women who may not be aware of their pregnancy. Therefore, it is important to take preventive measures to reduce exposure to these nanoparticles.

Effects of in vitro Amitriptyline, Fluoxetine, Tranylcypromine and Venlafaxine on Saphenous Vein Grafts.

OBJECTIVE: In this study, we aimed to examine the effects of amitriptyline, fluoxetine, tranylcypromine and venlafaxine on saphenous vein grafts in coronary artery bypass graft surgeries. METHODS: 59 patients (40 males and 19 females; mean age 65.1 years, distribution: 45-84 years) who had coronary artery bypass graft surgery between February 2014 and May 2016 were included in the study. After the saphenous vein grafts with intact and denuded endothelium were precontracted with 3×10-6M phenylephrine, amitriptyline, fluoxetine and tranylcypromine were cumulatively added to isolated organ baths in the range of 10-11-3x10-5M, while venlafaxine was added in the range of 10-9-3×10-5M. Then, the antidepressant-induced relaxation responses were recorded isometrically. RESULTS: While the relaxation response of amitriptyline at -6.42 (Log M) was 74.6%, the response at -6.32 (Log M) was 75.5%. While the relaxation response at -6.46 (Log M) of fluoxetine was 68.02%, the response at -6.02 (Log M) was 72.12%. While the relaxation response of tranylcypromine at -7.53 (Log M) was 61.13%, the response at -7.23 (Log M) was 65.53%. While the relaxation response of venlafaxine at -6.21 (Log M) was 29.98%, the response at -5.90 (Log M) was 32.96%. CONCLUSION: The maximum relaxation at minimum and maximum therapeutic concentrations was obtained with amitriptyline, fluoxetine and tranylcypromine, and the minimum relaxation was obtained with venlafaxine. The relaxation responses were independent of the endothelium.

Effects of in vitro amitriptyline, fluoxetine, tranylcypromine and venlafaxine on saphenous vein grafts

Abstract Objective: In this study, we aimed to examine the effects of amitriptyline, fluoxetine, tranylcypromine and venlafaxine on saphenous vein grafts in coronary artery bypass graft surgeries. Methods: 59 patients (40 males and 19 females; mean age 65.1 years, distribution: 45-84 years) who had coronary artery bypass graft surgery between February 2014 and May 2016 were included in the study. After the saphenous vein grafts with intact and denuded endothelium were precontracted with 3×10-6M phenylephrine, amitriptyline, fluoxetine and tranylcypromine were cumulatively added to isolated organ baths in the range of 10-11-3x10-5M, while venlafaxine was added in the range of 10-9-3×10-5M. Then, the antidepressant-induced relaxation responses were recorded isometrically. Results: While the relaxation response of amitriptyline at -6.42 (Log M) was 74.6%, the response at -6.32 (Log M) was 75.5%. While the relaxation response at -6.46 (Log M) of fluoxetine was 68.02%, the response at -6.02 (Log M) was 72.12%. While the relaxation response of tranylcypromine at -7.53 (Log M) was 61.13%, the response at -7.23 (Log M) was 65.53%. While the relaxation response of venlafaxine at -6.21 (Log M) was 29.98%, the response at -5.90 (Log M) was 32.96%. Conclusion: The maximum relaxation at minimum and maximum therapeutic concentrations was obtained with amitriptyline, fluoxetine and tranylcypromine, and the minimum relaxation was obtained with venlafaxine. The relaxation responses were independent of the endothelium.

5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice.

The role of 5-HT7 receptors in the nociceptive processing received most attention during the last few years. The involvement of 5-HT7 receptors in nerve injury-induced neuropathic pain states have been reported only recently; however, there are no reports on its contribution in diabetic neuropathic pain. We therefore planned to investigate the effect of 5-HT7 receptor activation on the changes of nociceptive threshold in diabetic mice. Diabetes was induced by a single intraperitoneal injection of streptozocin (150 mg/kg, i.p.). The nociceptive responses in normal and diabetic animals were tested in the hot-plate and tail-flick assays. Both hot-plate and tail-flick latencies significantly shortened at 1-3/4 weeks (thermal hyperalgesia) and prolonged at 6-7 weeks (thermal hypoalgesia) after streptozocin administration. At the dose of 10 mg/kg, systemic injections of AS-19, a selective 5-HT7 receptor agonist, reduced thermal hyperalgesia at early stage of diabetes, but did not influence thermal hypoalgesia at late stage. Co-administration of SB-258719, a selective 5-HT7 receptor antagonist, at a dose that had no effect on its own (10 mg/kg), reversed the anti-hyperalgesic effect of AS-19. Our results indicate that systemic administration of 5-HT7 receptor agonists may have clinical utility in treating diabetic neuropathic pain.

Pharmacological and behavioral characterization of the saphenous chronic constriction injury model of neuropathic pain in rats.

The aim of the present study was to develop a new experimental pain model by adapting the chronic constriction injury (CCI) model of the sciatic nerve to the exclusively sensory saphenous nerve in rats. Animals were divided into naïve, sham, and two experimental groups, in which two or four 4-0 chromic gut ligatures were loosely ligated around the saphenous nerve. Then, behavioral signs of neuropathic pain were observed for 8 weeks. In rats with four ligatures, prominent mechanical allodynia and thermal hyperalgesia developed; these behavioral signs were not prominent in rats with two ligatures. Pharmacological analysis was made in rats with four loose ligations; morphine and WIN 55,212-2, a cannabinoid agonist, reversed all of the modalities tested, whereas gabapentin only suppressed mechanical allodynia and amitriptyline only reduced mechanical hyperalgesia. Our data establish a rat model of saphenous CCI with significant allodynia and hyperalgesia, which is sensitive to a number of analgesic compounds.

Role of GLT-1 transporter activation in prevention of cannabinoid tolerance by the ß-lactam antibiotic, ceftriaxone, in mice.

Recently, it has been indicated that beta lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Furthermore, these antibiotics have been shown to prevent the development of tolerance and dependence to opioids. Since cannabinoid tolerance is known to be similar to opioids, our purpose was to examine the effect of ceftriaxone on the development of tolerance to WIN 55,212-2, a cannabinoid agonist. The tail flick test, a rectal thermometer, and the ring test were used for evaluating the degree of tolerance to the analgesic, hypothermic, and cataleptic effects of WIN 55,212-2, respectively. Within one week, animals became completely tolerant to analgesic, hypothermic and cataleptic effects of WIN 55,212-2 (6mg/kg). Ceftriaxone, with its higher doses (100-200mg/kg), attenuated the development of tolerance to the analgesic and hypothermic effects of WIN 55,212-2, but had no effect on its cataleptic action. Dihydrokainic acid (10mg/kg), a GLT-1 transporter inhibitor, prevented this effect of ceftriaxone. Our results suggest that repeated treatment with ceftriaxone prevents the development of tolerance to the analgesic and hypothermic effects of cannabinoids, and GLT-1 activation appears to play a key role in this preventive effect of beta-lactam antibiotics.

Anti-allodynic and anti-hyperalgesic effects of ceftriaxone in streptozocin-induced diabetic rats.

Glutamate is the principal excitatory neurotransmitter in the central nervous system. Recent evidence suggests that beta lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Moreover, these antibiotics have been shown to prevent the development of tolerance and dependence to opioids, and reduce visceral and nerve injury-induced neuropathic nociceptive responses. The aim of this study is to observe the effect of a beta lactam antibiotic, ceftriaxone, on mechanical allodynia and mechanical hyperalgesia in diabetic rats. Diabetes was produced with the injection of a single dose of streptozocin (50 mg/kg, i.p.) and this procedure resulted in neuropathic pain behaviors in the hindpaws. Mechanical allodynia was detected with an electronic aesthesiometer, and mechanical hyperalgesia was studied using the method of Randall-Selitto. With its higher doses, ceftriaxone (100, 200 mg/kg, i.p.) reduced both mechanical allodynia and hyperalgesia. Dihydrokainic acid (10 mg/kg, i.p.), a selective GLT-1 transporter inhibitor, reversed the anti-allodynic and anti-hyperalgesic effects of ceftriaxone, at doses that produced no effect on its own. Our results indicate that ceftriaxone exerts an antinociceptive effect in streptozocin-induced diabetic rats and GLT-1 activation by beta lactam antibiotics may be a promising option in the treatment of diabetic neuropathy.