Follicular mediated etodolac phosalosomal gel for contact dermatitis alleviation, insights from optimization to in-vivo appraisal.

Despite its long history as a preferential cyclooxygenase-2 inhibitor, the topical application of etodolac in inflammatory disorders does not achieve the desired clinical efficiency because of its poor water solubility and poor skin permeation. In the ongoing study, phosalosomes were designed to mitigate the etodolac drawbacks and to enhance its skin localization. Hyaluronic acid was utilized to prepare a dermal gel for the alleviation of skin inflammation. Etodolac loaded hyaluronic acid phosalosomal gel had a sustainable release profile and 10.59-fold enhanced skin retention compared to free etodolac, with boosted skin tolerability on histopathological examination after acute and chronic applications. Confocal laser microscopy imaging indicated that the etodolac amounts accumulated in the liver and kidney following dermal application were 29 and 5.7-fold lower than those following the systemic dose, respectively. For in vivo studies, etodolac loaded hyaluronic acid phosalosomal gel presented superior anti-oedemic and significant anti-nociception potential. The promising homogenous localization highlighted its potential for the delivery of lipophilic drugs for the targeted treatment of other localized skin disorders.

Does etodolac affect TRPA1 functionality in vivo in human?

OBJECTIVES: In preclinical research, etodolac, a non-steroidal anti-inflammatory drug, affected transient receptor potential ankyrin 1 (TRPA1) activation. Yet, whether the in vitro interaction between etodolac and TRPA1 translates to altered TRPA1 functionality in vivo in human remains to be investigated. METHODS: A randomized, double-blinded, celecoxib-controlled study was conducted to assess the effect of etodolac on TRPA1-mediated dermal blood flow (DBF) changes on the forearm of 15 healthy, male volunteers aged between 18 and 45 years. Over four study visits, separated by at least five days wash-out, a single or four-fold dose of etodolac 200 mg or celecoxib 200 mg was administered orally. Two hours post-dose, TRPA1 functionality was evaluated by assessing cinnamaldehyde-induced DBF changes. DBF changes were quantified and expressed in Perfusion Units (PUs) using laser Doppler imaging during 60 min post-cinnamaldehyde application. The corresponding area under the curve (AUC0-60min) was calculated as summary measure. Statistical analysis was performed using Linear mixed models with post-hoc Dunnett. RESULTS: Neither the single dose of etodolac nor celecoxib inhibited the cinnamaldehyde-induced DBF changes compared to no treatment (AUC0-60min ± SEM of 17,751 ± 1,514 PUs*min and 17,532 ± 1,706 PUs*min vs. 19,274 ± 1,031 PUs*min, respectively, both p=1.00). Similarly, also a four-fold dose of both compounds failed to inhibit the cinnamaldehyde-induced DBF changes (19,235 ± 1,260 PUs*min and 19,367 ± 1,085 PUs*min vs. 19,274 ± 1,031 PUs*min, respectively, both p=1.00). CONCLUSIONS: Etodolac did not affect the cinnamaldehyde-induced DBF changes, suggesting that it does not alter TRPA1 functionality in vivo in human.

Etodolac Enhances the Radiosensitivity of Irradiated HT-29 Human Colorectal Cancer Cells.

BACKGROUND: Radioresistance is found to be the main therapeutic restriction in colorectal radiation therapy. The aim of this study was to investigate the synergistic effect of Etodolac (ET) and ionizing radiation on human colorectal cancer cells. METHODS: Pretreated HT-29 cells with ET were exposed to ionizing radiation. The radiosensitizing effect of ET was evaluated using MTT, flow cytometry, and clonogenic assay. The amount of nitrite oxide (NO) in irradiated cells was also measured with the Griess reagent. RESULTS: The present study found that pretreatment of HT-29 cells with ET decreases their survival and colony formation. Higher concentrations of ET cause total apoptosis and an increase in NO levels in irradiated cells. CONCLUSION: Applying ET in a concentration-dependent manner had an incremental effect on the amount of apoptosis and cell death induced by radiation.

Induced Prostanoid Synthesis Regulates the Balance between Th1- and Th2-Producing Inflammatory Cytokines in the Thymus of Diet-Restricted Mice.

Multiple external and internal factors have been reported to induce thymic involution. Involution involves dramatic reduction in size and function of the thymus, leading to various immunodeficiency-related disorders. Therefore, clarifying and manipulating molecular mechanisms governing thymic involution are clinically important, although only a few studies have dealt with this issue. In the present study, we investigated the molecular mechanisms underlying thymic involution using a murine acute diet-restriction model. Gene expression analyses indicated that the expression of T helper 1 (Th1)-producing cytokines, namely interferon-γ and interleukin (IL)-2, was down-regulated, while that of Th2-producing IL-5, IL-6, IL-10 and IL-13 was up-regulated, suggesting that acute diet-restriction regulates the polarization of naïve T cells to a Th2-like phenotype during thymic involution. mRNAs for prostanoid biosynthetic enzymes were up-regulated by acute diet-restriction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses detected the increased production of prostanoids, particularly prostaglandin D2 and thromboxane B2, a metabolite of thromboxane A2, in the diet-restricted thymus. Administration of non-steroidal anti-inflammatory drugs, namely aspirin and etodolac, to inhibit prostanoid synthesis suppressed the biased expression of Th1- and Th2-cytokines as well as molecular markers of Th1 and Th2 cells in the diet-restricted thymus, without affecting the reduction of thymus size. In vitro stimulation of thymocytes with phorbol myristate acetate (PMA)/ionomycin confirmed the polarization of thymocytes from diet-restricted mice toward Th2 cells. These results indicated that the induced production of prostanoids during diet-restriction-induced thymic involution is involved in the polarization of naïve T cells in the thymus.

Synthesis and biological evaluation of new prodrugs of etodolac and tolfenamic acid with reduced ulcerogenic potential.

Gastric irritation and ulcerogenic effect of the acidic NSAIDs are of the most challenging problems in designing novel anti-inflammatory agents. In this study, the new prodrugs were prepared through Steglich esterification reaction between the carboxylic acid functional group of etodolac or tolfenamic acid and thymol. The structures were confirmed by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. Their chemical stability in addition to a kinetic study of their hydrolysis in 20% liver homogenate and 10% buffered plasma were investigated. In vitro enzymatic hydrolysis showed half-life times 88.84 and 106.61 min for the prodrugs of etodolac and tolfenamic acid, respectively. Their ability to inhibit paw edema and their ulcerogenic potential were assessed in rats and compared to their parent drugs. the prodrugs were found to be stable in different pHs at room and body temperatures. Both prodrugs proved to possess high percentage of inhibition of paw edema (94.68 & 97.1%) in rats comparable to that of the parent drugs (90.33 & 93.23%) and, most importantly with lower ulcerogenic potential. The prodrugs are expected to be converted to their parent drugs rapidly in plasma and liver in vivo and proved to be safer than their parent drugs. The study opens a perspective chance that can be a backbone for further investigations.

A comparative study: the prospective influence of nanovectors in leveraging the chemopreventive potential of COX-2 inhibitors against skin cancer.

INTRODUCTION: This study was conducted to elucidate the chemopreventive potential, cytotoxic, and suppression of cellular metastatic activity of etodolac (ETD)-loaded nanocarriers. METHODS: To esteem the effect of charge and composition of the nanovectors on their performance, four types of vectors namely, negative lipid nanovesicles; phosalosomes (N-Phsoms), positive phosalosomes (P-Phsoms), nanostructured lipid carriers (NLCs) and polymeric alginate polymer (AlgNPs) were prepared and compared. ETD was used as a model cyclo-oxygenase-2 (COX-2) inhibitor to evaluate the potency of these nanovectors to increase ETD permeation and retention through human skin and cytotoxicity against squamous cell carcinoma cell line (SCC). Moreover, the chemopreventive activity of ETD nanovector on mice skin cancer model was evaluated. RESULTS: Among the utilized nanovectors, ETD-loaded N-Phsoms depicted spherical vesicles with the smallest particle size (202.96±2.37 nm) and a high zeta potential of -24.8±4.16 mV. N-Phsoms exhibited 1.5, and 3.6 folds increase in the ETD amount deposited in stratum corneum, epidermis and dermis. Moreover, cytotoxicity studies revealed a significant cytotoxic potential of such nanovector with IC50=181.76 compared to free ETD (IC50=982.75), correlated to enhanced cellular internalization. Its efficacy extended to a reduction in the relative tumor weight with 1.70 and 1.51-fold compared to positive control and free ETD, that manifested by a 1.72-fold reduction in both COX-2 and proliferating cell nuclear antigen mRNA (PCNA-mRNA) levels and 2.63-fold elevation in caspase-3 level in skin tumors relative to the positive control group with no hepato-and nephrotoxicity. CONCLUSION: Encapsulation of ETD in nanovector enhances its in-vitro and in-vivo anti-tumor activity and opens the door for encapsulation of more relevant drugs.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple dysfunctional pathways. Therefore, a sophisticated treatment strategy that simultaneously targets multiple brain cell types and disease pathways could be advantageous for effective intervention. To elucidate an effective treatment, we developed an in vitro high-throughput screening (HTS) assay to evaluate candidate drugs for their ability to enhance the integrity of the blood-brain barrier (BBB) and improve clearance of amyloid-ß (Aß) using a cell-based BBB model. Results from HTS identified etodolac and α-tocopherol as promising drugs for further investigation. Both drugs were tested separately and in combination for the purpose of targeting multiple pathways including neuroinflammation and oxidative stress. In vitro studies assessed the effects of etodolac and α-tocopherol individually and collectively for BBB integrity and Aß transport, synaptic markers and Aß production in APP-transfected neuronal cells, as well as effects on inflammation and oxidative stress in astrocytes. Transgenic 5XFAD mice were used to translate in vitro results of etodolac and α-tocopherol independently and with concurrent administration. Compared to either drug alone, the combination significantly enhanced the BBB function, decreased total Aß load correlated with increased expression of major transport proteins, promoted APP processing towards the neuroprotective and non-amyloidogenic pathway, induced synaptic markers expression, and significantly reduced neuroinflammation and oxidative stress both in vitro and in vivo. Collective findings demonstrated the combination produced mixed interaction showing additive, less than additive or synergistic effects on the evaluated markers. In conclusion, this study highlights the significance of combination therapy to simultaneously target multiple disease pathways, and suggest the repurposing and combination of etodolac and α-tocopherol as a novel therapeutic strategy against AD.

Synthesis, anticancer activity, and molecular modeling of etodolac-thioether derivatives as potent methionine aminopeptidase (type II) inhibitors.

A series of (R,S)-1-{[5-(substituted)sulfanyl-4-substituted-4H-1,2,4-triazole-3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indoles (5a-v) were designed and synthesized using a five-step synthetic protocol that involves substituted benzyl chlorides and (R,S)-5-[(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)methyl]-4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones in the final step. The synthesized derivatives were evaluated for cytotoxicity and anticancer activity in vitro using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method against VERO, HEPG2 (human hepatocellular liver carcinoma), SKOV3 (ovarian carcinoma), MCF7 (human breast adenocarcinoma), PC3 and DU145 (prostate carcinoma) cells at 10-5 M (10 µM) for 24 h. Compounds 5d and 5h showed the best biological potency against the SKOV3 cancer cell line (IC50 = 7.22 and 5.10 µM, respectively) and did not display cytotoxicity toward VERO cells compared to etodolac. Compounds 5k, 5s, and 5v showed the most potent biological activity against the PC3 cancer cell line (IC50 = 8.18, 3.10, and 4.00 µM, respectively) and did not display cytotoxicity. Moreover, these compounds were evaluated for caspase-3, -9, and -8 protein expression and activation in the apoptosis pathway for 6, 12, and 24 h, which play a key role in the treatment of cancer. In this study, we also investigated the apoptotic mechanism and molecular modeling of compounds 5k and 5v on the methionine aminopeptidase (type II) enzyme active site in order to get insights into the binding mode and energy.

Expression of tissue inhibitor of metalloproteinases and matrix metalloproteinases in the ischemic brain of photothrombosis model mice.

Middle cerebral artery occlusion (MCAO) is the most widely used animal model of ischemic stroke. This model well recapitulates the pathological features of most human cases; however, MCAO is technically difficult to achieve in mice and has some disadvantages for investigating the molecular mechanisms of pathological progression in stroke. The recently developed photothrombosis model may be more suitable for research on the molecular mechanisms of ischemic stroke in mice. Yet, similarities and differences between the photothrombosis and MCAO models are not well characterized. In the present study, we examined the expression of tissue inhibitor of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) in the brains of photothrombosis model mice. Our results indicated that the gene expression of TIMP-1 was upregulated in endothelial cells in the pathological area surrounding the infarction, similar to the MCAO model. Yet, pathologically induced changes in TIMP-1 were not affected by treatment with aspirin or etodolac. Whereas MMP-2 and MMP-8 mRNA were upregulated after infarction in both models, MMP-9 expression, which is induced in the infarct area in the MCAO model, was unchanged in the photothrombosis model. These findings suggest that the expression patterns of TIMP-1 and MMP-9 are regulated independently in photothrombosis model mice.

Development of an Enantioselective and Biomarker-Informed Translational Population Pharmacokinetic/Pharmacodynamic Model for Etodolac.

Cyclooxygenase-2 (COX-2) isoform has a critical role in the development of pain. Inhibition of COX-2 in vitro serves as a biomarker for nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAID concentrations yielding 80% COX-2 inhibition (IC80) correlate with therapeutic doses to achieve analgesia across multiple COX-2 inhibitors. However, there are no time-course models relating COX-2 inhibition with decreased pain. This study aimed to characterize the relationship between NSAID concentrations, in vitro COX-2 inhibition, and acute pain decrease in humans over time by a translational approach using clinical pharmacokinetic and literature reported in vitro and clinical pharmacodynamic data. In a two-way cross-over study, eight healthy volunteers received 300 and 400 mg racemic etodolac, a preferential COX-2 inhibitor. R- and S-etodolac were determined by LC-MS/MS and simultaneously modeled. Literature in vitro IC50 data for COX-2 inhibition by S-etodolac were used to fit adjusted pain score profiles from dental patients receiving etodolac. External model qualification was performed using published ibuprofen data. Etodolac absorption was highly variable due to gastric transit kinetics and low aqueous solubility. The disposition parameters differed substantially between enantiomers with a total clearance of 2.21 L/h for R-etodolac and 26.8 L/h for S-etodolac. Volume of distribution at steady-state was 14.6 L for R-etodolac and 45.8 L for S-etodolac. Inhibition of COX-2 by 78.1% caused a half-maximal pain decrease. The time-course of pain decrease following ibuprofen was successfully predicted via the developed translational model. This proposed enantioselective pharmacodynamic-informed approach presents the first quantitative time-course model for COX-2 induced pain inhibition in patients.