Nanoemulgel, an Innovative Carrier for Diflunisal Topical Delivery with Profound Anti-Inflammatory Effect: in vitro and in vivo Evaluation.

PURPOSE: Rheumatoid arthritis is an autoimmune disorder that directly affects joints. However, other body organs including heart, eyes, skin, blood vessels and lungs may also be affected. The purpose of this study was to design and evaluate a nanoemulgel formulation of diflunisal (DIF) and solubility enhanced diflunisal (DIF-IC) for enhanced topical anti-inflammatory activity. METHODOLOGY: Nanoemulsion formulations of both DIF and DIF-IC were prepared and incorporated in three different gelling agents, namely carboxymethylcellulose sodium (CMC-Na), sodium alginate (Na-ALG) and xanthan gum (XG). All the formulations were evaluated in term of particle size, pH, conductivity, viscosity, zeta potential and in vitro drug release. The formulation 2 (NE2) of both DIF and DIF-IC which expressed optimum release and satisfactory physicochemical properties was incorporated with gelling agents to produce final nanoemulgel formulations. The optimized nanoemulgel formulation was subjected to three different in vivo anti-inflammatory models including carrageenan-induced paw edema model, histamine-induced paw edema model and formalin-induced paw edema model. RESULTS: DIF-IC-loaded nanoemulgel formulations yielded significantly enhanced in vitro skin permeation than DIF-loaded nanoemulgel. The nanoemulgel formulation of DIF-IC formulated with XG produced improved in vivo anti-inflammatory activity. CONCLUSION: It was recommended that DIF-IC-based nanoemulgel formulation prepared with XG could be a better option for effective topical treatment of inflammatory conditions.

Diflunisal-loaded poly(propylene sulfide) nanoparticles decrease S. aureus-mediated bone destruction during osteomyelitis.

Osteomyelitis is a debilitating infection of bone that results in substantial morbidity. Staphylococcus aureus is the most commonly isolated pathogen causing bone infections and features an arsenal of virulence factors that contribute to bone destruction and counteract immune responses. We previously demonstrated that diflunisal, a nonsteroidal anti-inflammatory drug, decreases S. aureus-induced bone destruction during osteomyelitis when delivered locally from a resorbable drug delivery depot. However, local diflunisal therapy was complicated by bacterial colonization of the depot's surface, highlighting a common pitfall of devices for local drug delivery to infected tissue. It is, therefore, critical to develop an alternative drug delivery method for diflunisal to successfully repurpose this drug as an antivirulence therapy for osteomyelitis. We hypothesized that a nanoparticle-based parenteral delivery strategy would provide a method for delivering diflunisal to infected tissue while circumventing the complications associated with local delivery. In this study, we demonstrate that poly(propylene sulfide) (PPS) nanoparticles accumulate at the infectious focus in a murine model of staphylococcal osteomyelitis and are capable of efficaciously delivering diflunisal to infected bone. Moreover, diflunisal-loaded PPS nanoparticles effectively decrease S. aureus-mediated bone destruction, establishing the feasibility of systemic delivery of an antivirulence compound to mitigate bone pathology during osteomyelitis.

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-ß Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study1.

BACKGROUND: Transthyretin (TTR) is a tetrameric, amyloid-ß (Aß)-binding protein, which reduces Aß toxicity. The TTR/Aß interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease. OBJECTIVE: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aß interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET). METHODS: Female mice (AßPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aß in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months. RESULTS: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months. CONCLUSION: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aß deposition in certain brain regions.

Stabilization of Cardiac Function With Diflunisal in Transthyretin (ATTR) Cardiac Amyloidosis.

BACKGROUND: Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is an underappreciated cause of heart failure that results from misfolded TTR (prealbumin) protein. Diflunisal is an approved non-steroidal anti-inflammatory drug that stabilizes TTR, with limited data available regarding effects on cardiac structure and function. METHODS AND RESULTS: ATTR-CM patients (n=81, 41% treated with 250 mg twice-daily diflunisal by clinical practice) were retrospectively identified with baseline and follow-up (median interval 1 year) serum biomarker and echocardiographic data compared, including global longitudinal strain (GLS). Chi-squared and Wilcoxon tests assessed differences between subjects, divided by treatment group, and univariable and multivariable linear regression was performed. At baseline, patients treated with diflunisal were younger (68 vs 77 years, P = .0001), with lower B-type natriuretic peptide (BNP; 249 vs 545 pg/mL, P = .009) and serum creatinine (1.1 vs 1.2 mg/dL, P = .04), but similar TTR concentration (P = .31), cardiac troponin I (P = .06), and GLS (P = .67). At follow-up, diflunisal untreated versus treated patients showed differences in TTR concentration (19 vs 33 mg/dL, P = .01) and favorable differences in left atrial volume index (+4.6 vs -1.4 mL/m2, P = .002) and cardiac troponin I (+0.03 vs -0.01 ng/mL, P = .01) for the entire cohort. Among the subset with wild-type ATTR (n=53), diflunisal treatment was associated with differences in GLS (+1.2% untreated vs +0.1% treated, P = .03). Changes in wall thickness (P = .2), left ventricular ejection fraction (P = .71), and BNP (P = .42) were similar between groups. CONCLUSIONS: In ATTR-CM, diflunisal treatment resulted in measurable differences in some parameters of cardiac structure and function after only 1 year of administration. Further longer-term analysis is warranted.

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD.

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aß-peptides and prevent Aß aggregation. We have previously shown that treatment of Alzheimer's Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aß-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice.

Comparative study of liposomes, ethosomes and transfersomes as carriers for enhancing the transdermal delivery of diflunisal: In vitro and in vivo evaluation.

The current study aimed to develop an effective transdermal nanovesicular carrier of diflunisal that provides enhanced delivery through the skin. Two types of nanovesicles, ethosomes and transfersomes, were investigated and compared to conventional liposomes. Ethosomes with variable ethanol contents (10, 30 and 50%) and transfersomes using different edge activators, including sodium deoxycholate, sodium cholate and sodium taurocholate, were prepared and characterized. The obtained vesicles revealed good entrapment efficiencies (46.73-65.99%), nanometric vesicle sizes (453.10-796.80 nm) and negative zeta potential values (-45.40 to -86.90 mV). Ethosomes with 30% ethanol and sodium deoxycholate-containing transfersomes were incorporated into hydrogels to evaluate their in vitro release and permeation patterns. Nanovesicular hydrogels exhibited more sustained diflunisal release than did corresponding dispersions. Compared to liposomal hydrogel, both carriers proved the superiority of diflunisal permeation and flux across the skin. Confocal laser scanning microscopy showed improved penetration of rhodamine-loaded nanovesicles through skin layers with a wider distribution and higher fluorescence intensity. Compared to liposomes, selected nanovesicles exhibited remarkable antinociceptive and anti-inflammatory effects manifested by significant reduction in number of writhings and significantly higher inhibition of paw oedema. Hence, the developed nanovesicles could be considered promising carriers for transdermal delivery of diflunisal for pain and inflammation management.

Supramolecular nano-engineered lipidic carriers based on diflunisal-phospholipid complex for transdermal delivery: QbD based optimization, characterization and preclinical investigations for management of rheumatoid arthritis.

Diflunisal (DIF) is used for treatment of rheumatoid arthritis, osteoarthritis etc. DIF-phospholipid complex (DIF-PL complex) was prepared by solvent-evaporation method and characterized by molecular docking studies, SEM, FTIR, DSC, PXRD studies. Further, the DIF-PL complex was incorporated into supramolecular nano-engineered lipidic carriers (SNLCs) for transdermal delivery. The optimization exercise was done using Face centered cubic design (FCCD) after screening of variables by L8 Taguchi orthogonal array design. The optimized SNLC formulation depicted average particle size (188.1nm), degree of entrapment (86.77±3.33%), permeation flux (5.47±0.48µg/cm2/h) and skin retention (17.72±0.68µg/cm2). The dermatokinetic studies revealed the higher concentration of DIF in dermis. The Confocal laser scanning microscopy (CSLM) studies revealed penetration of SNLCs into the deeper layers of skin. The results of mice ear edema depicted significant inhibition of ear edema (76.37±12.52%; p<0.05). In CFA induced rheumatoid arthritis model, the inhibition of paw edema was significantly higher (73.85±14.5%). The levels of TNF-α were reduced in synovial fluid (146.74±1.69pg/mL) and serum (132.43±2.70pg/mL). Furthermore, the licking and biting time was reduced in formalin induced hyperalgesia model. Hence, it can be concluded that dual formulation strategy based SNLCs were promising in treatment of pain and inflammation associated with rheumatoid arthritis.

Safety and efficacy of long-term diflunisal administration in hereditary transthyretin (ATTR) amyloidosis.

BACKGROUND: A recent 2-year randomized controlled trial indicated that the transthyretin (TTR) tetramer stabilizer, diflunisal, inhibits polyneuropathy progression and preserves quality of life in hereditary ATTR amyloidosis. However, its long-term outcomes are unknown. Here, we report tolerance and efficacy of long-term diflunisal administration in hereditary ATTR amyloidosis. METHODS: Diflunisal was administered orally at 500 mg/day to 40 Japanese hereditary ATTR amyloidosis patents who were not candidates for liver transplantation. The observation period ranged from 2 to 116 months (mean ± SD: 38.0 ± 31.2 months). RESULTS: Diflunisal-related adverse events included deterioration of renal function and thrombocytopenia resulting in discontinuation of the drug in three patients. Orally administered diflunisal significantly increased serum TTR concentration (p = 0.001) and stabilized TTR tetramer structure in each patient. Longitudinal analyses of data collected at baseline, 24 months, and after 24 months confirmed sustaining effects of diflunisal on both neurological and cardiac functions. Notably, ulnar compound muscle action potential amplitude, cardiac wall thickness, and ejection fraction were not deteriorated after 24 months of treatment. CONCLUSIONS: Diflunisal was tolerated well by most hereditary ATTR amyloidosis patients, although renal function and blood cell counts must be carefully monitored. Clinical effects of diflunisal were sustained after 2 years of treatment.

An insight on human skin penetration of diflunisal: lipogel versus hydrogel microemulsion.

Diflunisal is a NSAID used in acute and long term management of pain and inflammation associated with osteoarthritis, rheumatoid arthritis and symptoms of primary dysmenorrhea. However, its oral use is associated with side effects such as peptic ulceration, dyspepsia, gastrointestinal disturbances and bleeding. The aim of this work was to develop lecithin organogels (LO) transdermal delivery system for diflunisal and to study its human skin penetration ability in comparison with an optimized microemulsion-based hydrogel. Ternary phase diagrams were constructed using butyl lactate as an organic solvent and two commercial grades of lecithin. The formation of gel phase was lecithin concentration dependent with Phosholipion 85 G being capable of forming organogels at lower lecithin concentration than Lipoid S75. The gels prepared using butyl lactate were able to tolerate higher amounts of water than could be incorporated in the lipogels prepared with other organic solvents. All the investigated gels possessed acceptable physical properties and were able to deliver diflunisal through human skin. The lipogels delivered higher total drug amount through the skin than the hydrogel. The composition of lecithin seemed to have some effect on the skin permeability enhancement ability of the lipogel. Lecithin containing higher amount of phosphatidyl ethanolamine could provide better transdermal delivery. The elaborated lecithin organogels are potential carriers that create a good opportunity for transdermal delivery of diflunisal overcoming the side effects associating its oral route.

The utilization of drug-polymer interactions for improving the chemical stability of hot-melt extruded solid dispersions.

OBJECTIVE: Interactions between drugs and polymers were utilized to lower the processing temperature of hot-melt extrusion (HME), and thus minimize the thermal degradation of heat-sensitive drugs during preparation of amorphous solid dispersions. METHODS: Diflunisal (DIF), which would degrade upon melting, was selected as a model drug. Hydrogen bonds between DIF and polymeric carriers (PVP K30, PVP VA64, hydroxypropyl methylcellulose and Soluplus) were revealed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The hot-melt extruded solid dispersion was characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). KEY FINDINGS: The results of hot-stage polar microscopy indicated that DIF was dissolved in molten polymers at 160°C, much lower than the melting point of DIF (215°C). At this temperature, amorphous solid dispersions were successfully produced by HME, as confirmed by XRD and SEM. The related impurities in amorphous solid dispersions detected by HPLC were lower than 0.3%, indicating that thermal degradation was effectively minimized. The dissolution of DIF from amorphous solid dispersions was significantly enhanced as compared with the pure crystalline drug. CONCLUSION: This technique based on drug-polymer interactions to prepare chemically stable amorphous solid dispersions by HME provides an attractive opportunity for development of heat-sensitive drugs.

Solubility enhancement of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) using polypolypropylene oxide core PAMAM dendrimers.

The aim of the present study was to evaluate the aqueous solubility enhancement properties of polypropylene oxide cored PAMAM (PPO@PAMAM) dendrimers. The solubility of NSAIDs (Ketoprofen, Ibuprofen and Diflunisal) was investigated in the presence of PPO@PAMAM dendrimers at room temperature in buffer solution. The effects of dendrimer concentration, generation and core size on the solubility of NSAIDs have been investigated. The experimental results showed that the solubility of the NSAIDs was approximately proportional to dendrimer concentration and generation. In addition, the effect of core size on the solubility of NSAIDs in constant generation and concentration of PPO@PAMAM dendrimer was Ketoprofen>Diflunisal>Ibuprofen. Under optimized conditions, PPO@PAMAM dendrimers are highly effective solubility enhancer for NSAIDs due to its new polypropylene oxide core.

Comparative in vitro dissolution and in vivo bioavailability of diflunisal/naproxen fixed-dose combination tablets and concomitant administration of diflunisal and naproxen in healthy adult subjects.

A simple validated high-performance liquid chromatography (HPLC) assay was developed for determination of diflunisal and naproxen in human plasma samples. This is to compare the bioavailability of diflunisal-naproxen fixed-dose combination (FDC) with their separate dosage forms. The in vitro dissolution study was adopted to compare the dissolution behavior of FDC with respect to separate marketed tablets. In vivo study was conducted according to a single-center, randomized, single-dose, laboratory-blinded, 2 Way, Cross-Over Study with a washout period of 10 days. Under fasting conditions, 24 healthy Egyptian male volunteers were randomly allocated to receive a single oral dose of either one FDC tablet or co-administration of two separate diflunisal and naproxen marketed tablets. Plasma samples were obtained over a 72-h interval and analyzed for diflunisal and naproxen by reversed phase liquid chromatography with UV detection. The pharmacokinetic parameters Cmax, AUC0-t, AUC0-∞, tmax, and t1/2 were determined from plasma concentration-time profiles. The 90% confidence intervals for the ratio of log transformed values of Cmax, AUC0-t, and AUCt-∞ of the 2 treatments were within the acceptable range (0.8-1.25) for bioequivalence. From pharmacokinetic and in vitro studies perspectives, 1 FDC tablet demonstrated similar relative bioavailability with the 2 individual -reference tablets.

Cyclodextrin-crosslinked poly(acrylic acid): adhesion and controlled release of diflunisal and fluconazole from solid dosage forms.

The controlled release of diflunisal and fluconazole from tablets made of novel polymers, poly(acrylic acid) (PAA) crosslinked with either ß-cyclodextrin (ßCD) or hydroxypropyl-ßCD (HPßCD), was investigated and Carbopol 934P (Carbopol) was used as a highly crosslinked PAA for comparison. Diflunisal strongly associates with ßCD-PAA and HPßCD-PAA polymers (Ka of 486 and 6,055 M(-1) respectively); thus, it was physically mixed into the conjugates and also precomplexed to identify whether decomplexation has any influence on release kinetics. Fluconazole has poor complexing ability (Ka of 34 M(-1) with HPßCD-PAA); thus, it was only tested as a physical mixture. Swelling and adhesion studies were conducted on all tablet combinations and adhesivity of the CD-PAA polymer tablets was maintained. Diflunisal release was much slower from HPßCD-PAA tablets than from ßCD-PAA, suggesting that a higher degree of complexation retards release. The precomplexed diflunisal release was also slower than the physically mixed diflunisal of the corresponding conjugate. The release closely followed zero-order kinetics for HPßCD-PAA, but was more sigmoidal for ßCD-PAA and especially Carbopol. Conversely, poorly associating fluconazole released in almost exactly the same way across both polymers and Carbopol, indicating that the release kinetics of poorly associating drugs are not influenced by the presence of cyclodextrins. In view of the varying profiles and release rates shown with diflunisal for the different polymers, the fluconazole data support the concept that adequate complexation can indeed modulate the release kinetics of drugs.

A modern approach for controlled transdermal delivery of diflunisal: optimization and in vivo evaluation.

The purpose of the present work was to elaborate an optimized transdermal therapeutic system for diflunisal. Selection of suitable ingredients was done via solubility and phase behavior studies. Composition of microemulsion (ME) systems consisting of butyl lactate, Brij(®) 97, Transcutol(®) and water was optimized using augmented simplex lattice mixture design. The independent variables selected were the percentages of butyl lactate, surfactant mixture and water. The dependent variables were refractive index, pH, conductivity, viscosity, drug solubility in the ME formulation and the ex vivo skin permeation flux. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The statistical validity of the polynomials was established. Optimized formulation factors were selected by desirability approach. The optimized ME formulation was converted into gel using Carbomer(®) 934. The microemulsion based gel (MBG) showed better spreadability and 5.07-fold increase in the transdermal flux than Carbomer(®) 934 gel. The in vivo antihyperalgesia assay performed on mice showed significant reduction of the licking time in the treated group compared to the control group. This demonstrated the reliability of the simplex lattice statistical design for predicting optimum ME formulation. The developed MBG proved its in vivo efficiency for transdermal delivery of diflunisal.

Single dose oral diflunisal for acute postoperative pain in adults.

BACKGROUND: Diflunisal is a long-acting non-steroidal anti-inflammatory drug (NSAID) most commonly used to treat acute postoperative pain or chronic joint pain from osteoarthritis and rheumatoid arthritis. This review analyses the effectiveness and harm of different doses of diflunisal in the context of moderate to severe postoperative pain. OBJECTIVES: To assess efficacy, duration of action, and associated adverse events of single dose oral diflunisal in acute postoperative pain in adults. SEARCH STRATEGY: We searched Cochrane CENTRAL, MEDLINE, EMBASE and the Oxford Pain Relief Database for studies to January 2010. SELECTION CRITERIA: Randomised, double blind, placebo-controlled trials of single dose orally administered diflunisal in adults with moderate to severe acute postoperative pain. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial quality and extracted data. Pain relief or pain intensity data were extracted and converted into the dichotomous outcome of number of participants with at least 50% pain relief over 4 to 6 hours, from which relative risk and number-needed-to-treat-to-benefit (NNT) were calculated. Numbers of participants using rescue medication over specified time periods, and time to use of rescue medication, were sought as additional measures of efficacy. Information on adverse events and withdrawals were collected. MAIN RESULTS: Nine studies in dental, orthopedic and gynaecological surgery met the inclusion criteria, testing doses of diflunisal from 125 mg to 1000 mg.For diflunisal 1000 mg, the NNT for at least 50% pain relief over 4 to 6 hours was 2.1 (1.8 to 2.6) (6 studies, 391 participants); the NNT to prevent remedication within 6 hours was 1.9 (1.7 to 2.3), and within 12 hours was 2.2 (1.9 to 2.7) (6 studies, 409 participants). More participants experienced adverse events with diflunisal 100 mg than with placebo, but none were serious or led to withdrawal.For diflunisal 500 mg, the NNT for at least 50% pain relief over 4 to 6 hours was 2.6 (2.1 to 3.3) (6 studies, 357 participants); the NNT to prevent remedication within 6 hours was 2.6 (2.1 to 3.4) (6 studies, 390 participants), and within 12 hours was 2.9 (2.3 to 4.0) (5 studies, 329 participants). Adverse events did not differ significantly from placebo. AUTHORS' CONCLUSIONS: Diflunisal has an analgesic effect similar to other NSAIDs in single dose, but benefits from providing significant analgesia for about twelve hours. This property may be useful when regular dosing is needed, or when taking several doses of a shorter acting analgesic is impractical.

Evaluation of the drug-polymer interaction in calcium alginate beads containing diflunisal.

Calcium alginate gel beads have been developed in recent years as a unique vehicle for oral drug delivery due to their excellent biocompatibility, biodegradability, simple method of preparation, abundant sources, low cost and minimal processing requirements. The objective of this study was to evaluate the drug-polymer interaction in calcium alginate beads containing diflunisal. Diflunisal loaded calcium alginate beads were successfully prepared by ionotropic gelation from solution of sodium alginate and diflunisal into calcium chloride solution. The weight ratio of drug to polymer was selected as 1:1. The calcium alginate beads were characterized by size, Scanning Electron Microscopy (SEM), weight uniformity and drug entrapment efficiency. The existence of a possible interaction between diflunisal and the calcium alginate was investigated by Differential Scanning Calorimetry (DSC), Powder X-Ray Diffraction (PXRD) and Fourier Transform Infra-Red (FTIR) analysis. Drug loaded beads were spherical to oval in shape with low drug entrapment efficiency. The drug was found to be present inside the beads as crystalline to semicrystalline form with no significant physical or chemical interaction between drug and excipients. The results implied that calcium alginate beads can be used as a suitable controlled release carrier for diflunisal.