A Proteomics Investigation of Cigarette Smoke Exposed Wistar Rats Revealed Improved Anti-Inflammatory Effects of the Cysteamine Nanoemulsions Delivered via Inhalation.

Cigarette smoking is the major cause of chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It is paramount to develop pharmacological interventions and delivery strategies against the cigarette smoke (CS) associated oxidative stress in COPD. This study in Wistar rats examined cysteamine in nanoemulsions to counteract the CS distressed microenvironment. In vivo, 28 days of CS and 15 days of cysteamine nanoemulsions treatment starting on 29th day consisting of oral and inhalation routes were established in Wistar rats. In addition, we conducted inflammatory and epithelial-to-mesenchymal transition (EMT) studies in vitro in human bronchial epithelial cell lines (BEAS2B) using 5% CS extract. Inflammatory and anti-inflammatory markers, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, IL-8, IL-10, and IL-13, have been quantified in bronchoalveolar lavage fluid (BALF) to evaluate the effects of the cysteamine nanoemulsions in normalizing the diseased condition. Histopathological analysis of the alveoli and the trachea showed the distorted, lung parenchyma and ciliated epithelial barrier, respectively. To obtain mechanistic insights into the CS COPD rat model, "shotgun" proteomics of the lung tissues have been carried out using high-resolution mass spectrometry wherein genes such as ABI1, PPP3CA, PSMA2, FBLN5, ACTG1, CSNK2A1, and ECM1 exhibited significant differences across all the groups. Pathway analysis showed autophagy, signaling by receptor tyrosine kinase, cytokine signaling in immune system, extracellular matrix organization, and hemostasis, as the major contributing pathways across all the studied groups. This work offers new preclinical findings on how cysteamine taken orally or inhaled can combat CS-induced oxidative stress.

Ion-Triggered In Situ Gelling Nanoemulgel as a Platform for Nose-to-Brain Delivery of Small Lipophilic Molecules.

Background: Intranasal route offers a direct nose-to-brain delivery via olfactory and trigeminal nerves and minimizes the systemic exposure of the drug. Although reliable and non-invasive, intranasal administration of lipophilic neuroprotective agents for brain targeting is still challenging. Literature focuses on naturally-derived compounds as a promising therapeutics for chronic brain diseases. Naringin, a natural flavonoid obtained from citrus fruits possesses neuroprotective effects. By regulating multiple crucial cellular signaling pathways, naringin acts on several therapeutic targets that make it suitable for the treatment of neurodegenerative diseases like Alzheimer's disease and making it a suitable candidate for nasal administration. However, the hydrophobicity of naringin is the primary challenge to formulate it in an aqueous system for nasal administration. Method: We designed a lipid-based nanoemulsifying drug delivery system of naringin using Acrysol K140 as an oil, Tween 80 as a surfactant and Transcutol HP as a cosolvent, to improve solubility and harness the benefits of nanosizing like improved cellular penetration. Intranasal instillations of therapeutic agents have limited efficacy due to drug washout and inadequate adherence to the nasal mucosa. Therefore, we reconstituted the naringin self-emulsifying system in a smart, biodegradable, ion-triggered in situ gelling hydrogel and optimized for desirable gel characteristics. The naringin-loaded composition was optimized and characterized for various physicochemical and rheological properties. Results: The formulation showed a mean droplet size 152.03 ± 4.6 nm with a polydispersity index <0.23. Ex vivo transmucosal permeation kinetics of the developed formulation through sheep nasal mucosa showed sustained diffusion and enhanced steady-state flux and permeability coefficient. Scanning and transmission electron microscopy revealed the spherical shape of emulsion droplets and entrapment of droplets in a gel structure. The formulation showed excellent biocompatibility as analyzed from the viability of L929 fibroblast cells and nasal mucosa histopathology after treatment. In vivo biodistribution studies revealed significantly higher drug transport and brain targeting efficiency. Conclusion: In situ gelling system with nanoemulsified naringin demonstrated a safe nasal delivery providing a new dimension to the treatment of chronic neurodegenerative diseases using small hydrophobic phytoconstituents with minimization of dose and related systemic adverse effects.

Topical Nanoemulgel for the Treatment of Skin Cancer: Proof-of-Technology.

The present study is a mechanistic validation of 'proof-of-technology' for the effective topical delivery of chrysin nanoemulgel for localized, efficient treatment of melanoma-affected skin. BACKGROUND: Currently available treatments for skin cancer are inefficient due to systemic side effects and poor transcutaneous permeation, thereby presenting a formidable challenge for the development of novel nanocarriers. METHODS: We opted for a novel approach and formulated a nanocomplex system composed of hydrophobic chrysin dissolved in a lipid mix, which was further nanoemulsified in Pluronic® F-127 gel to enhance physicochemical and biopharmaceutic characteristics. Chrysin, a flavone extracted from passion flowers, exhibits potential anti-cancer activities; however, it has limited applicability due to its poor solubility. Pseudo-ternary phase diagrams were constructed to identify the best self-nanoemulsifying region by varying the compositions of oil, Caproyl® 90 surfactant, Tween® 80, and co-solvent Transcutol® HP. Chrysin-loaded nanoemulsifying compositions were characterized for various physicochemical properties. RESULTS: This thermodynamically stable, self-emulsifying drug delivery system showed a mean droplet size of 156.9 nm, polydispersity index of 0.26, and viscosity of 9100 cps after dispersion in gel. Mechanical characterization using Texture Analyzer exhibited that the gel had a hardness of 487 g and adhesiveness of 500 g. Ex vivo permeation through rat abdominal skin revealed significant improvement in percutaneous absorption measured as flux, the apparent permeability coefficient, the steady-state diffusion coefficient, and drug deposition. In vitro cytotoxicity on A375 and SK-MEL-2 cell lines showed a significantly improved therapeutic effect, thus ensuring reduction in dose. The safety of the product was established through biocompatibility testing on the L929 cell line. CONCLUSION: Aqueous, gel-based, topical, nanoemulsified chrysin is a promising technology approach for effective localized transcutaneous delivery that will help reduce the frequency and overall dose usage and ultimately improve the therapeutic index.

Risk management and statistical multivariate analysis approach for design and optimization of satranidazole nanoparticles.

Rapidly evolving technical and regulatory landscapes of the pharmaceutical product development necessitates risk management with application of multivariate analysis using Process Analytical Technology (PAT) and Quality by Design (QbD). Poorly soluble, high dose drug, Satranidazole was optimally nanoprecipitated (SAT-NP) employing principles of Formulation by Design (FbD). The potential risk factors influencing the critical quality attributes (CQA) of SAT-NP were identified using Ishikawa diagram. Plackett-Burman screening design was adopted to screen the eight critical formulation and process parameters influencing the mean particle size, zeta potential and dissolution efficiency at 30min in pH7.4 dissolution medium. Pareto charts (individual and cumulative) revealed three most critical factors influencing CQA of SAT-NP viz. aqueous stabilizer (Polyvinyl alcohol), release modifier (Eudragit® S 100) and volume of aqueous phase. The levels of these three critical formulation attributes were optimized by FbD within established design space to minimize mean particle size, poly dispersity index, and maximize encapsulation efficiency of SAT-NP. Lenth's and Bayesian analysis along with mathematical modeling of results allowed identification and quantification of critical formulation attributes significantly active on the selected CQAs. The optimized SAT-NP exhibited mean particle size; 216nm, polydispersity index; 0.250, zeta potential; -3.75mV and encapsulation efficiency; 78.3%. The product was lyophilized using mannitol to form readily redispersible powder. X-ray diffraction analysis confirmed the conversion of crystalline SAT to amorphous form. In vitro release of SAT-NP in gradually pH changing media showed <20% release in pH1.2 and pH6.8 in 5h, while, complete release (>95%) in pH7.4 in next 3h, indicative of burst release after a lag time. This investigation demonstrated effective application of risk management and QbD tools in developing site-specific release SAT-NP by nanoprecipitation.

Mechanistic investigation of biopharmaceutic and pharmacokinetic characteristics of surface engineering of satranidazole nanocrystals.

The designing of surface engineered nanocrystals for improved stability and bioavailability is a multivariate process depending on several critical formulation and process variables. The present investigation deals with formulation of stable nanocrystals of poorly soluble satranidazole (SAT) for improving dissolution rate and pharmacokinetic profiling. SAT has low polar surface area, high dose and dosing frequency. Based on goniometric and stability studies of formulations prepared with various stabilizers, a unique combination of Span 20 and HPMC E-5 was selected for detailed investigation. Lyophilization of SAT nanosuspension was explored with nine different cryoprotectants in varying amounts to obtain easily redispersible nanocrystals (SAT-NC). The mean particle size and zeta potential of SAT-NC were found to be 208.8nm and -41.3mV respectively. DSC and XRPD confirmed the crystalline state of SAT. In vitro release studies of SAT-NC showed almost complete dissolution within 20min in water. Extravascular, one compartment pharmacokinetic modeling of in vivo plasma concentration versus time studies in male Wistar rats revealed twofold increase in Cmax, and AUC0-∞. Method of residuals was employed to calculate rate of absorption Ka and lag time. Nanosizing with appropriate stabilizers and programmed processing conditions successfully produced SAT-NC with improved pharmaceutic and pharmacokinetic characteristics.

Transcutaneous delivery of leflunomide nanoemulgel: Mechanistic investigation into physicomechanical characteristics, in vitro anti-psoriatic and anti-melanoma activity.

The present study is a mechanistic validation of 'proof of concept' of effective topical delivery of leflunomide (LFD) nanoemulgel for localized efficient treatment of psoriatic lesions as well as melanoma affected skin regions. Hyperproliferation of keratinocytes in psoriasis and symbiotic relationship between keratinocytes and melanocytes, justifies the need of dual acting treatment. LFD is recently introduced significantly effective disease modifying anti-rheumatic drug and has been considered valuable for the treatment of psoriatic arthritis as well as melanoma. Current available treatments for psoriasis and melanoma are inefficient due to systemic side effects, poor transcutaneous permeation and thus present a challenge for development of novel colloidal carriers. We newly reformulated LFD as a nanoemulgel based on self nanoemulsifying technique using Capryol 90, Cremophor EL, Transcutol HP as nanoemulsifying components and Pluronic F127 as a gelling agent. This thermodynamically stable nanoemuslsifying preconcentrate after gelation showed mean globule size, 123.7 nm and viscosity 9620 ± 93 cp. Complete mechanical characterization was carried out using Texture Analyzer and hardness, adhesiveness and springiness index were found to be 523 gms, 431 gms and 1.02, respectively. Ex vivo permeation through rat abdominal skin revealed significant improvement in flux, apparent permeability coefficient, steady state diffusion coefficient and drug deposition in skin due to nanoemulsification of LFD. The in vitro cytoxicity of LFD nanoemulgel in human HaCaT, melanoma A375 and SK-MEL-2 cell lines showed significantly enhanced therapeutic response. In gist, LFD nanoemulgel for trancutaneous delivery will reduce the overall dose and drug consumption, by effectively localizing at the applied target site and will ultimately minimize systemic side effects.

Lipid based nanoemulsifying resveratrol for improved physicochemical characteristics, in vitro cytotoxicity and in vivo antiangiogenic efficacy.

Resveratrol, a dietary non-flavonoid polyphenolic phytoalexin, has gained attention in cancer chemoprevention. However, poor aqueous solubility and cellular bioavailability has limited its therapeutic application. We formulated a lipid based delivery system of resveratrol with self nanoemulsifying ability. Several edible and safe lipids, surfactants and cosolvents were screened for solubilization of resevratrol. Developed formulation comprised of Acrysol K 150 as a lipid and mixture of Labrasol and Transcutol HP as the surfactant system, as these components showed higher solubility. Pseudoternary phase diagram was constructed to identify the region of nanoemulsification. The formulations showed rapid emulsification with an average globule diameter; 85nm to 120nm and slight negative zeta potential. The nanocompositions exhibited cloud point above 55°C and were stable toward the gastrointestinal pH and thermodynamic stress testing. As compared to pristine resveratrol, the developed delivery system showed significant increase in vitro cytotoxicity in MCF-7 breast cancer cells. In vivo chick chorioallantoic membrane assay revealed enhanced antiangiogenic activity of composition with high lipid level. Briefly, lipid based nanoemulsifying resveratrol dramatically enhanced the anticancer and antiangiogenic activities, thus increasing its potential application in cancer chemotherapy.

Multivariate analysis of physicochemical characteristics of lipid based nanoemulsifying cilostazol--quality by design.

Simultaneous analysis of the effect of multiple formulation ingredients on the critical physico-chemical properties of lipid based nanoemulsifying cilostazol was studied using integrated quality by design approach. Cilostazol is a poorly soluble drug belonging to class II of the biopharmaceutics classification system. To improve the solubility and in turn bioavailability of cilostazol, a lipid based nanoemulsifying cilostazol was developed. Self nanoemulsifying system comprising of Capmul MCM (oily solubilizer), Tween 80 (surfactant); and Transcutol HP (cosolvent) was developed. A 2(3) full factorial experimental design was employed to optimize simultaneously the effect of levels of these three components on physico-chemical responses (viz. globule size, span, zeta potential, solubility, and dissolution efficiency at 30 min) of nanoemulsifying cilostazol. Graphical analysis using Pareto charts and Bayesian analysis along with mathematical modelling of the results allowed the identification and quantification of the formulation variables active on the selected responses. A polynomial equation fitted to the data was used to predict the composition with optimum responses. The optimum formulation was a mixture of Capmul MCM, Tween 80 and Transcutol HP; 3:5:5 parts by weight. Optimized composition on dilution with water showed globule size; 215.2 nm with a span of 0.42. The nanoemulsifying formulation showed equilibrium solubility and dissolution efficiency; 9.82 mg/ml and 83.3% respectively, indicating significant improvement in comparison to pristine cilostazol. Interaction between oil and the cosolvent significantly affected the globule size and the span of the resultant nanoemulsion. Zeta potential was independent of selected formulation variables. The optimized formulation was adsorbed onto Neusilin US2 without affecting nanoemusifying ability of lipid based cilostazol composition.

Improvement of anti-inflammatory and anti-angiogenic activity of berberine by novel rapid dissolving nanoemulsifying technique.

Berberine, an isoquinoline alkaloid, has wide biological and pharmacological actions. Despite the promising pharmacological effects and safety of berberine, poor oral absorption due to its extremely low aqueous solubility results in poor oral systemic bioavailability. This limits its clinical usage. This study describes the development and characterization of self-nanoemulsifying drug delivery system (SNEDDS) of berberine in liquid as well as solid form with improved solubility, dissolution and in vivo therapeutic efficacy. The SNEDDS of berberine were prepared using Acrysol K-150, Capmul MCM and polyethylene glycol 400. The formulations were characterized for various in vitro physicochemical characteristics. In vivo efficacy was evaluated in acetic acid induced inflammatory bowel model in rats. Anti-angiogenic activity of the developed SNEDDS of berberine was studied using chick chorioallantoic membrane assay. SNEDDS of berberine rapidly formed nanoemulsions with globule size of 17-45 nm. The in vitro rate and extent of release of berberine from SNEDDS was significantly higher than berberine alone. Chick chorioallantoic membrane assay revealed potent anti-angiogenic activity of SNEDDS of berberine. These studies demonstrate that the SNEDDS of berberine is a promising strategy for improving its therapeutic efficacy and have potential application in the treatment of chronic inflammatory conditions and cancer.

Ex vivo permeation characteristics of venlafaxine through sheep nasal mucosa.

Venlafaxine, a dual acting antidepressant is a new therapeutic option for chronic depression. Depression is a common mental disorder associated with the abnormalities in neuronal transport in the brain. Since the nose-to-brain pathway has been indicated for delivering drugs to the brain, we analyzed the transport of venlafaxine through sheep nasal mucosa. Transmucosal permeation kinetics of venlafaxine were examined using sheep nasal mucosa mounted onto static vertical Franz diffusion cells. Nasal mucosa was treated with venlafaxine in situ gel (100 µl; 1% w/v) for 7h. Amount of venlafaxine diffused through mucosa was measured using validated RP-HPLC method. After the completion of the study histopathological investigation of mucosa was carried out. Ex vivo studies through sheep nasal mucosa showed sustained diffusion of venlafaxine with 66.5% permeation in 7h. Transnasal transport of venlafaxine followed a non-Fickian diffusion process. Permeability coefficient and steady state flux were found to be 21.11×10(-3) cmh(-1) and 21.118 µg cm(-2)h(-1) respectively. Cumulative amount permeated through mucosa at 7h was found to be 664.8 µg through an area of 3.14 cm(2). Total recovery of venlafaxine at the end of the permeation study was 87.3% of initial dose distributed (i) at the mucosal surface (208.4 µg; 20.8%) and (ii) through mucosa (664.8 µg; 66.5%). Histopathological examinations showed no significant adverse effects confirming that the barrier function of nasal mucosa remains unaffected even after treatment with venlafaxine in situ gel. Permeation through sheep nasal mucosa using in situ gel demonstrated a harmless nasal delivery of venlafaxine, providing new dimension to the treatment of chronic depression.

Gastroretentive delivery of rifampicin: in vitro mucoadhesion and in vivo gamma scintigraphy.

Rifampicin, a first line anti-tubercular drug, has maximum solubility and permeability in the stomach. An oral multi-particulate formulation with site specific sustained delivery of rifampicin was developed. This oral gastroretentive rifampicin formulation consisted of rifampicin pellets for immediate release as the loading dose and a bio/mucoadhesive rifampicin tablet for extended release. Immediate release pellets of rifampicin were prepared by extrusion-spheronization process and were evaluated for physico-mechanical properties: usable yield, size, shape, abrasion resistance, mechanical crushing force, residual moisture and drug release. For the mucoadhesive rifampicin formulation, statistical experimental strategy was utilized to simultaneously optimize the effect of two independent variables namely amount of Carbopol and MCC. The two dependent responses selected were, work of adhesion; estimated using Texture Analyzer and T(50%); determined from dissolution studies. Graphical and mathematical analysis of the results allowed the identification and quantification of the formulation variables influencing the selected responses. To study the gastrointestinal transit of the optimized gastroretentive formulation, the in vivo gamma scintigraphy was carried out in six healthy human volunteers, after radiolabeling the formulation with (99m)Tc. The transit profiles demonstrated that the dosage form was retained in the stomach for more than 320 min. The human data validates the design concept and signifies the potential of the developed system for stomach targeted delivery of rifampicin for improved bioavailability.

Multivariate optimization of formulation and process variables influencing physico-mechanical characteristics of site-specific release isoniazid pellets.

In the present study, isoniazid was formulated as site-specific release pellets with high drug loading (65%, w/w) using extrusion-spheronization followed by aqueous coating of Sureteric (35% weight gain). A statistical experimental strategy was developed to optimize simultaneously the effect of the two formulation variables and one process variable on the critical physico-mechanical properties of the core pellets of isoniazid. Amount of granulating fluid and amount of binder were selected as formulation variables and spheronization speed as a process variable. A 2(3) full factorial experimental design was employed for the present study. Pellets were characterized for physico-mechanical properties viz. usable yield, pellet size, pellips, porosity, abrasion resistance, mechanical crushing force, residual moisture and dissolution efficiency. Graphical and mathematical analysis of the results allowed the identification and quantification of the formulation and process variables active on the selected responses. A polynomial equation fitted to the data was used to predict the responses in the optimal region. The optimum formulation and process parameters were found to be 44.24% (w/w) of granulating fluid, 2.13% (w/w) of binder and spheronization speed of 1000rpm. Optimized formulation showed usable yield 84.95%, particle size 1021.32microm, pellips 0.945, porosity 46.11%, and abrasion resistance 0.485%. However, mechanical crushing force, residual moisture and dissolution efficiency were not significantly affected by the selected independent variables. These results demonstrate the importance of, amount of water, binder and spheronization speed, on physico-mechanical characteristics of the isoniazid core pellets with high drug loading.

Dissolution test for site-specific release isoniazid pellets in USP apparatus 3 (reciprocating cylinder): optimization using response surface methodology.

The present work aims to predict drug release from novel site-specific release isoniazid pellets, in USP dissolution test apparatus 3, using the response surface methodology (RSM). Site-specific release isoniazid pellets were prepared by extrusion-spheronization followed by aqueous coating of Acryl-EZE. RSM was employed for designing of the experiment, generation of mathematical models and optimization study. A 3(2) full factorial design was used to study the effect of two factors (at three levels), namely volume of dissolution medium (150, 200, 250 ml) and reciprocation rate (5, 15, 25 dips per min). Amount of drug released in 0.1N hydrochloric acid at 2h and in pH 6.8 phosphate buffer at 45 min were selected as responses. Results revealed that both, the volume of medium and reciprocation rate, are significant factors affecting isoniazid release. A second order polynomial equation fitted to the data was used to predict the responses in the optimal region. The optimized conditions resulted in dissolution data that were close to the predicted values. The proposed mathematical model is found to be robust and accurate for optimization of dissolution test conditions for site-specific release isoniazid pellets.