Synthesis of Copper Oxide-Based Nanoformulations of Etoricoxib and Montelukast and Their Evaluation through Analgesic, Anti-Inflammatory, Anti-Pyretic, and Acute Toxicity Activities.

Copper oxide nanoparticles (CuO NPs) were synthesized through the coprecipitation method and used as nanocarriers for etoricoxib (selective COX-2 inhibitor drug) and montelukast (leukotriene product inhibitor drug) in combination therapy. The CuO NPs, free drugs, and nanoformulations were investigated through UV/Vis spectroscopy, FTIR spectroscopy, XRD, SEM, and DLS. SEM imaging showed agglomerated nanorods of CuO NPs of about 87 nm size. The CE1, CE2, and CE6 nanoformulations were investigated through DLS, and their particle sizes were 271, 258, and 254 nm, respectively. The nanoformulations were evaluated through in vitro anti-inflammatory activity, in vivo anti-inflammatory activity, in vivo analgesic activity, in vivo anti-pyretic activity, and in vivo acute toxicity activity. In vivo activities were performed on albino mice. BSA denaturation was highly inhibited by CE1, CE2, and CE6 as compared to other nanoformulations in the in vitro anti-inflammatory activity. The in vivo bioactivities showed that low doses (5 mg/kg) of nanoformulations were more potent than high doses (10 and 20 mg/kg) of free drugs in the inhibition of pain, fever, and inflammation. Lastly, CE2 was more potent than that of other nanoformulations.

Potent Phenylpyridine and Oxodihydrofuran Inhibitors of Cyclooxygenase-2: Optimization toward a Long Residence Time with Balanced Internal Energetics.

Long residence time enzyme inhibitors with a two-step binding mechanism are characterized by a high internal energy barrier for target association. This raises the question of whether optimizing residence time via further increasing this internal energy barrier would inevitably lead to insufficient target occupancy in vivo due to slow, time-dependent binding. We attempted to address this question during optimization of cyclooxygenase-2 (COX-2) inhibitors. Defining long residence time drugs with acceptable association and dissociation rate constants required for sufficient target occupancy and sustained efficacy, which we termed "balanced internal energetics", provides an important criterion for successful progression during lead optimization. Despite the advancement of several COX-2 inhibitors to marketed drugs, their detailed inhibition kinetics have been surprisingly limiting especially during the structure-activity relationship process mainly due to the lack of robust kinetic assays. Herein, we describe a reoptimized COX enzymatic assay and a novel MS-based assay enabling detailed mechanistic studies for identifying long residence time COX-2 inhibitors with balanced internal energetics. These efforts led to the discovery of promising leads possessing dissociation half-lives of ≤40 h, much greater than the values of 6 and 0.71 h for two marketed drugs, etoricoxib and celecoxib, respectively. Importantly, the inhibition rate constants remain comparable to those of the marketed drugs and above the lower limits set by the criteria of balanced internal energetics, predicting sufficient target occupancy required for efficacy. Taken together, this study demonstrates the feasibility of increasing the internal energy barrier as a viable approach for lead optimization toward discovering long residence time drug candidates.

Gut-Restricted Selective Cyclooxygenase-2 (COX-2) Inhibitors for Chemoprevention of Colorectal Cancer.

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.

Etoricoxib-loaded bio-adhesive hybridized polylactic acid-based nanoparticles as an intra-articular injection for the treatment of osteoarthritis.

Osteoarthritis is a major problem in elder people. Etoricoxib-loaded bio-adhesive hybridized nanoparticles were prepared using polylactic acid (PLA) and chitosan hydrochloride (CS-HCl) in presence of Captex®200 as a liquid oil, polyvinyl alcohol (PVA) and Tween®80 as surfactants. The study aimed to present a new intra-articular treatment of osteoarthritis with anti-inflammatory as well as bone rebuilding effects. Hybridized nanoparticles were fabricated applying the emulsion solvent evaporation technique then assessed for particle size, zeta potential, entrapment efficiency and in-vitro drug release. Furthermore, FT-IR and DSC in addition to morphological examination were done. Results revealed that the formulation composed of PLA:Captex®200 in ratio 1:2 (w/w), 1%w/v Tween®80, 0.3% w/v CS-HCl and 3%w/v PVA possessed the smallest particle size and the most sustained drug release, thus was sorted for further analyses. The selected formulation ability to interact with the negatively charged sodium fluroscein was evaluated to predict its binding with the naturally occurring hyaluronic acid in the knee joint where promising results were obtained. Results showed the cytocompatibility of the formulation when tested using MC3T3-E1 normal bone cell line, enhanced ALP activity and increased calcium ion deposition and binding. Results suggested that the presented formulation can be considered as an innovative approach for osteoarthritis.

Design and optimization of film-forming gel of etoricoxib using research surface methodology.

The present investigation is focused on the development of transdermal film-forming gel (FFG) loaded with etoricoxib employing research surface methodology (RSM). Box-Behnken surface design method was used to develop experimental run using different concentrations of etoricoxib, hydroxypropyl methylcellulose (HPMC K100M), and eudragit RL100 as independent variables, and Derringer's optimization tool was employed to optimize best possible formulation. The dependent variables considered in this study were viscosity and drug permeation at 24 h (Q24, µg/cm2). Anti-inflammatory study was performed on Wistar albino rats for 8 h. Skin irritation studies and accelerated stability studies were performed for validated FFG formulations. Quadratic model was found to be best fit model (p < 0.0001) for both the responses. The influence of HPMC concentration on the viscosity was found to be highest whereas concentration of etoricoxib was maximum for Q24. The optimum composition of the FFG was observed to be 4% of etoricoxib, 1.1246% of HPMC, and 0.4% of eudragit. Above composition resulted in viscosity of 1549.5 mPa.s and maximum Q24 of 4639.11 µg/cm2 with desirability 0.918. The in vivo anti-inflammatory study demonstrated better sustained release effect (for 8 h) of optimized FFG compared to orally administered drug suspension. An average irritation score of 0.555 was observed on Draize scoring system. The validated FFG formulation was found to be stable for the 3 months in accelerated conditions. It can be concluded from the above investigations that the validated FFG formulation of etoricoxib is well tolerated and could provide sustained drug release for 8 h. Graphical abstract.

Exploring the binding dynamics of etoricoxib with human hemoglobin: A spectroscopic, calorimetric, and molecular modeling approach.

Etoricoxib, widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and related conditions has ample affinity to bind with globular proteins. Here, the molecular interaction between purified human hemoglobin (HHb), a major heme protein and etoricoxib, a cyclooxygenase-2 inhibitor was studied by various spectroscopic, calorimetric, and molecular modeling techniques. The binding affected hypochromic changes in the Soret band of hemoglobin (Hb) and induced remarkable quenching of the intrinsic fluorescence property of protein molecules. Synchronous fluorescence studies revealed alterations in tryptophan (Trp) and tyrosine (Tyr) microenvironments of HHb molecule in presence of etoricoxib. Flouremetric and isothermal titration calorimetric studies suggested two binding sites in HHb for etoricoxib at three different temperatures (298.15, 303.15, and 310.15 K). Negative values of Gibbs energy change (ΔG0) and enthalpy change (ΔH0) strongly suggest that it is spontaneous and exothermic reaction, mainly stabilized by hydrogen bonding as evidenced by sucrose binding assay. These findings support our in silico molecular docking study, which specified the binding site and the non-covalent interactions involved in the association. Moreover, the interaction impacts on structural integrity and functional aspects of HHb as confirmed by decreased α helicity, increased free iron release, increased rate of co-oxidation, and decreased rate of esterase activity. Overall, these studies conclude that etoricoxib leads to a remarkable alteration in the conformational aspects of binding to HHb. Communicated by Ramaswamy H. Sarma.

Rapid quantitative analysis of etoricoxib in human plasma by UPLC-MS/MS and application to a pharmacokinetic study in Chinese healthy volunteers.

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of etoricoxib in human plasma. Chromatography was performed on an Acquity UPLC HSS T3 column (1.8 µm, 50 × 2.1 mm), with a flow rate of 0.600 mL/min, using a gradient elution with acetonitrile and water which contained 2 mm ammonium acetate as the mobile phase. Detection was carried out on Triple QuadTM 5500 mass spectrometer under positive-ion multiple reaction monitoring mode. The respective mass transitions used for quantification of etoricoxib and etoricoxib-d3 were m/z 359.0 → 280.1 and m/z 362.0 → 280.2. Calibration curves were linear over the concentration range of 5-5000 ng/mL. The validated method was applied in the pharmacokinetic study of etoricoxib in Chinese healthy volunteers under fed and fasted conditions. After a single oral dose of 120 mg, the main pharmacokinetic parameters of etoricoxib in fasted and fed groups were respectively as follows: peak concentration, 2364.78 ± 538.01 and 1874.55 ± 367.90 ng/mL; area under the concentration-time curve from 0 to 120 h, 44,605.53 ± 15,266.66 and 43,516.33 ± 12,425.91 ng h/mL; time to peak concentration, 2.00 and 2.50 h; and half-life, 24.08 ± 10.06 and 23.64± 6.72 h. High-fat food significantly reduced the peak concentration of etoricoxib (p = 0.001) but had no effect on the area under the concentration-time curve.

Changes in Physical Stability of Supercooled Etoricoxib after Compression.

In the case of formulations with amorphous active pharmaceutical ingredients the risk of pressure-induced recrystallization should be carefully considered. We reported here that supercooled etoricoxib (ETB), which was found as a relatively stable system with low crystallization tendency at atmospheric pressure, crystallized quickly after compression. The observed strong pressure-dependence of the induction period suggests that during compression the first step of crystallization that is nucleation may be accelerated. To overcome the experimental challenge associated with studies at elevated temperatures and high pressures we applied broadband dielectric spectroscopy. Dielectric measurements gave us detailed insight into crystallization kinetics of ETB at varying ( T, p) conditions corresponding to the supercooled liquid state of a drug. We found that pressure-induced recrystallization of supercooled ETB, constituting a serious impediment from a technological point of view, can be efficiently inhibited when amorphous solid dispersion containing ETB and polymer polyvinylpyrrolidone PVP (10% w/w) was prepared. Besides, we performed the comprehensive analysis of molecular dynamics of both systems at elevated pressure to address some fundamental issues related to the pressure sensitivity of their supercooled dynamics.

Confirmation of More Stable Polymorphic Form of Etoricoxib at Room Temperature.

Polymorphic forms of etoricoxib have been reported in the literature, and form I was considered to be the most stable one. However, in this work, it was found that form I and form V are enantiotropic by differential scanning calorimetry analysis, solubility measurements, and solution-mediated polymorphic transformation experiments with form V being more stable than form I at room temperature. Thermodynamic transition temperature is determined as (353.45 ± 0.10) K. Besides, form V would transform to form I with the seeding effect of form I at high temperature below the melting point of form V. The crystal structure of form V was solved for the first time. The molecules in form V are linked by weak hydrogen bond C-H⋯O to form ring motif, which is nonexistent in form I.