Ex-vivo Diffusion Research Articles

Effects of Early Alcohol Exposure on Functional Organization and Microstructure of a Visual-Tactile Integrative Circuit.

Children with fetal alcohol spectrum disorders (FASD) often have deficits associated with multisensory processing. Because ethanol (EtOH) disrupts activity-dependent neuronal plasticity, a process that is essential for refining connections during cortical development, we hypothesize that early alcohol exposure results in alterations in multisensory cortical networks, which could explain the multisensory processing deficits seen in FASD. Here, we use a gyrencephalic animal model to test the prediction that early alcohol exposure alters the functional connectivity and microstructural features of the rostral posterior parietal cortex (PPr), a visual-tactile integrative area. Ferrets were exposed to moderate doses of EtOH during the brain growth spurt period. Functional connectivity and microstructural features were assessed using resting-state functional magnetic resonance imaging and exvivo diffusion kurtosis imaging (DKI), respectively, when the animals reached juvenile age and adulthood, respectively. While the whole brain volume was smaller in alcohol-treated animals, the relative size of the frontal brain area was larger when compared to control animals. Altered functional connectivity was observed in alcohol-treated animals, where increased connectivity was observed between PPr and the region that provides its major visual inputs (the caudal portion of the parietal cortex), but not with the region that provides its major somatosensory inputs (tertiary somatosensory cortex). DKI revealed reduced microstructural tissue complexity in all investigated sensory areas of alcohol-treated animals. In this study, we observed alterations in cortical functional connectivity and microstructural integrity in a cortical area involved in multisensory processing in a ferret FASD model. These findings indicate an alteration in cortical networks that may be related to the multisensory processing deficiencies observed in FASD.

Differential microstructural alterations in rat cerebral cortex in a model of chronic mild stress depression.

Chronic mild stress leads to depression in many cases and is linked to several debilitating diseases including mental disorders. Recently, neuronal tracing techniques, stereology, and immunohistochemistry have revealed persistent and significant microstructural alterations in the hippocampus, hypothalamus, prefrontal cortex, and amygdala, which form an interconnected system known as the stress circuit. Most studies have focused only on this circuit, however, some studies indicate that manipulation of sensory and motor systems may impact genesis and therapy of mood disorders and therefore these areas should not be neglected in the study of brain microstructure alterations in response to stress and depression. For this reason, we explore the microstructural alterations in different cortical regions in a chronic mild stress model of depression. The study employs ex-vivo diffusion MRI (d-MRI) to assess cortical microstructure in stressed (anhedonic and resilient) and control animals. MRI is followed by immunohistochemistry to substantiate the d-MRI findings. We find significantly lower extracellular diffusivity in auditory cortex (AC) of stress groups and a significantly higher fractional anisotropy in the resilient group. Neurite density was not found to be significantly higher in any cortical ROIs in the stress group compared to control, although axonal density is higher in the stress groups. We also report significant thinning of motor cortex (MC) in both stress groups. This is in agreement with recent clinical and preclinical studies on depression and similar disorders where significant microstructural and metabolic alterations were found in AC and MC. Our findings provide further evidence that the AC and MC are sensitive towards stress exposure and may extend our understanding of the microstructural effects of stress beyond the stress circuit of the brain. Progress in this field may provide new avenues of research to help in diagnosis and treatment intervention for depression and related disorders.

Development and Antimicrobial Evaluation of Binary Ethosomal Topical Gel of Terbinafine Hydrochloride for the Treatment of Onychomycosis

The objective of this investigation was to prepare and evaluate the binary ethosomal gel containing terbinafine HCl (TH) to treat onychomycosis. It was reported that binary ethosomes possessed good permeation and stability characteristics than ethosomes. Binary ethosomes of TH were prepared by film hydration method. Prepared binary ethosomes were evaluated for optimized system. Further, ex-vivo skin permeation studies were conducted with rat skin. The formulation of binary ethosomes was converted to gel by using carbopol 934 and evaluated for rheological properties. Antifungal testing of gel was done by cup plate method, using Candida albicans and zones of inhibition of growth were measured. The size, ZP and EE of the prepared binary ethosomes ranged from 200-320nm, -20 to -30mV and 70-92% respectively. In-vitro and ex-vivo diffusion studies indicated that formulation BE4 with greater amount of binary alcoholic phase, showed faster release and increased flux over others. SEM studies revealed that binary ethosomes were in spherical shape. Based on size, EE, drug release profile and antifungal studies, the BE4 formulation was selected for gel preparation. The prepared 0.5% binary ethosomal BE4 gel showed good content uniformity, pH 5.5, no skin irritation, good consistency and better rheological behaviour. Further, antifungal studies of BE4 gel indicated that gel had lower antifungal activity than plain BE4 formulation, probably due to gelling effect in agar diffusion studies. In conclusion, we developed a pharmaceutical gel containing binary ethosomes of BE4 having TH to provide anti-fungal effects useful for the treatment of onychomycosis. 

FORMULATION AND OPTIMIZATION OF CELECOXIB NANOEMULGEL

Objective: The main objective of this experiment was to prepare and optimized celecoxib nanoemulgel. This formulation can be used for acuterheumatoid arthritis patients.Methods: Celecoxib is a poorly water soluble drug. We prepared celecoxib nanoemulgel to improve intrinsic solubility of celecoxib and enhancedeeper permeation throughout the skin. After several screening, the combination of acetonitrile, triacetin, campul 908P was considered for oil phase;acconon MC8-2EP as surfactant, and capmul MCM C-10 as a co-surfactant accordingly. As per Box-Behnken surface design model, optimization wasdone for all the 13 formulations.Results: Based on pseudo ternary plot, it was found that 4:1 Smix ratio was optimum and possessed maximum drug solubility. Further, screeningshown, 0.25-0.75% carbopol-940 can be a stable candidate for hydrogel preparation. Prepared nanoemulsions and hydrogels were admixed to preparenanoemulgel. Based on overlay plot, EG14* formulation was consider as optimum one, and various evaluation parameters were performed along withother formulations. Using Franz diffusion cell, in-vitro diffusion studies was performed. Almost all the formulations produces good qualitative drugrelease profile. The EG14* shown 95.50% drug release after 12th hrs with standard Higuchi plot (R2 value 0.9989). The optimum viscosity was foundto be 521±0.81 mPas at 100 rpm. The appearance of the formulations was milky, yellowish white with expectable pH ranged from 5.8 to 6.7. Theoptimized formulation has good spreadability coefficient, good ex-vivo diffusion enhancement factor (3.03) as compare to marketed gel. Mostly, ourformulations have less skin irritation and higher anti-inflammatory activity (92.56% of inhibition of paw edema for EG14*).Conclusion: From the thermodynamic studies, it was confirmed that EG14* maintained excellent stability profile in various heating-cooling cycle,centrifugation, and freeze-thaw cycle condition. Hence, it can be conclude that, our formulation, can be consider for pilot scale up.

Brain targeted delivery of mucoadhesive thermosensitive nasal gel of selegiline hydrochloride for treatment of Parkinson's disease

Selegiline hydrochloride (SL), is an anti-Parkinson’s agent, has low-oral bioavailability due to its high first pass metabolism and scarce oral absorption. In the present study, SL mucoadhesive nasal thermosensitive gel (SNT-gel) was prepared to enhance the bioavailability and subsequently, its concentration in the brain. The SNT-gel was prepared using Poloxamer 407-Chitosan combination and optimised formulation was further evaluated for physicochemical parameters. The comparative pharmacodynamic studies including behavioural studies, biochemical testing and histopathology of the brain was carried out in rats for SNT-gel, SL-nasal solution and SL Marketed Tablets. The optimised SNT-gel formulation (SNT-V) revealed sol-gel transition at 33–34°C. In-vitro diffusion study of SNT-V showed 102.37 ± 2.1% diffusion at 12 h which reduced to 89.64 ± 1.2% in Ex-vivo diffusion. Comparative results of behavioural studies indicated an improved score of photoactometer and reduced motor deficit (catalepsy score) in SNT-gel treatment group as compared with other groups. Similarly, a significant increase in brain dopamine, reduction in monoamine oxidase B level, increase in catalase activity and level of reduced glutathione upon treatment with SNT-gel indicated its effectiveness which was also supported by histopathology results. Therefore, nasal thermosensitive gel holds better potential for brain targeting in Parkinson’s disease over the conventional nasal or oral formulations.

Formulation and development of Silybin loaded solid lipid nanoparticle enriched gel for irritant contact dermatitis

The purpose of the present research was to develop Silybin loaded solid lipid nanoparticle gel (SIL-SLN gel) for irritant contact dermatitis (ICD). ICD is associated with reduced skin water content, emerging in dry skin condition and relapsing eczema. SIL is a naturally occurring flavones, which shows antioxidant activity and helps in the treatment of ICD. In this study, the SLN was prepared by the ultrasonic probe sonication method and further evaluated for particle size and entrapment efficiency. Results of optimized batch showed mean particle size 139 ± 0.35 nm and entrapment efficiency 90.97 ± 0.91%. Optimized batch was freeze dried and characterized by field emission scanning electron microscopy (FE-SEM), it shows particles are in nano range, with spherical morphology and smooth surface. Finally, the SLN was incorporated into gel for convenient topical application. The SIL-SLN-gel were evaluated for in-vitro skin occlusivity, skin irritation and ex-vivo diffusion and deposition study and further compared with SIL-plain gel. Efficacy of gel on dinitrochlorobenzene (DNCB) induced ICD mice were evaluated by skin water content, ear swelling and histopathology. Ex-vivo study of SIL-SLN gel exhibited prolonged drug release, whereas the skin irritation study shows no irritancy. In DNCB induced ICD mice SIL-SLN gel showed higher efficacy than SIL-plain gel.

Gellified Emulsion of Ofloxacin for Transdermal Drug Delivery System.

Purpose: Ofloxacin is a fluoroquinolone with broad-spectrum antibacterial action, used in treatment of systemic and local infections. Ofloxacin is BCS class II drug having low solubility, high permeability with short half-life. The present work was aimed to design, develop and optimize gellified emulsion of Ofloxacin to provide site targeted drug delivery. Transdermal drug delivery will enhance the bioavailability of the drug giving controlled drug release. Methods: Transdermal drug delivery system was designed with gelling agent (Carbopol 940 and HPMC K100M), oil phase (oleic acid) and emulsifying agent (Tween 80: Span 80). Effect of concentration of gelling agent on release of drug from transdermal delivery was studied by 32 factorial design. Emulgel was evaluated for physical appearance, pH, drug content, viscosity, spreadability, antimicrobial activity, in- vitro diffusion study and ex-vivo diffusion study. Results: FE-SEM study of the emulsion batch B5 has revealed formation of emulsion globules of approximately size 6-8 µm with -11.2 mV zeta potential showing good stability for the emulsion. Carbopol 940 had shown greater linear effect on drug release and viscosity of the formulations due to its high degree of gelling. In-vitro diffusion study through egg membrane had shown 88.58±1.82 % drug release for optimized batch F4. Ex-vivo diffusion study through goat skin indicated 76.68 ± 2.52% drug release. Conclusion: Controlled release Ofloxacin emulgel exhibiting good in-vitro and ex-vivo drug release proving good antimicrobial property was formulated.

Fabrication of topical metered dose film forming sprays for pain management

Topical film-forming metered dose spray formulations were designed for management of pain. Ropivacaine, a local anesthetic is explored for its topical efficacy in alleviating pain. Metered dose spray containers, organic solvents, film forming polymers and permeation enhancers were utilized to fabricate the Metered Dose topical spray. Factors like viscosity, spray pattern, spray angle, volume of actuation, droplet size distribution of the metered dose spray formulation and drying time, flexibility and wash-ability of the film formed after spraying were assessed. Permeation of the drug into the porcine skin was observed based on ex-vivo diffusion studies and confocal microscopy. The results indicated a high level of drug concentration in the skin layers. Anti-nociceptive efficacy of the formulations was assessed on Wistar rats by hot plate and tail flick tests, based on the response to pain perception. The results were comparable to the conventional lidocaine gel. Topical film forming sprays have the ability to provide an accurate, long lasting and patient compliant delivery of drugs on the skin as compared to conventional gels.

PREPARATION AND EVALUATION OF TRANSNASAL MICROEMULSION OF VIGABATRIN

Introduction: The objective of this study was to develop novel transnasal microemulsion containing Vigabatrin for treatment of epilepsy. Method: Oleic acid was selected as oil while Tween 80 and ethanol were selected as surfactant and co-surfactant respectively based on solubility results. Optimized ratio of Tween 80 and Ethanol was selected after developing pseudoternary phase diagrams for different ratio and microemulsions were prepared. The prepared microemulsions were evaluated for globule size, viscosity, pH, and % transmittance. Ex-vivo diffusion study for optimized microemulsion was performed through goat nasal mucosa where in diffusion flux and permeability coefficients were determined. Pharmacological performance was screened in rats by electrically induced seizures. Result: It was found that op mized microemulsion was stable and transparent. Pharmacological evaluation indicated significant reduction (p<0.001) of seizures in rats treated with optimized formula on in comparison to rats treated with oral Vigabatrin microemulsion and nasal Vigabatrin solu on which suggested Vigabatrin transnasal delivery system as an effective alternate therapy for treatment of epilepsy. Conclusion: Transnasal microemulsion of Vigabatrin was successfully formulated using Tween 80 as surfactant and ethanol as co-surfactant in the formula on to treat epilepsy. Key words: Epilepsy; Transnasal Microemulsion; Vigabatrin.

Development and Evaluation of Azelaic Acid Based Ethosomes for Topical Delivery for the Treatment of Acne

Abstract: Purpose: To design, develop and evaluate an Azelaic acid encapsulated Ethosomal formulation for acne. Methods: Encapsulated ethosomes were prepared by three methods viz. hot method, cold method and thin film hydration method. Central Composite Design was employed for optimisation of ethosomal formulations. Concentrations of phospholipid, cholesterol and ethanol were selected as independent variables and their effect on the dependent variables (Entrapment Efficiency and Drug Diffused) was studied. The optimised vesicular carriers were evaluated for vesicle size, entrapment efficiency, in-vitro and ex-vivo diffusion studies, anti-microbial activity, skin irritation studies and stability studies as per ICH guidelines. Ethosomal formulations with varying soya phosphatidylcholine, cholesterol and ethanol were prepared. Results: Vesicles were spherical, unilamellar with a smooth surface. The optimised formulation showed a vesicle size of 4.25 ± 1.35 μm and entrapment efficiency of 91.86 ± 2.25%. In-vitro and ex-vivo drug diffusion of the ethosomal gel was compared with a conventional gel and a marketed cream. The developed novel formulation exhibited enhanced anti-acne activity as compared to conventional gel and a marketed cream. Conclusion: We can conclude that the ethosomal formulation is an efficient vesicular carrier system for topical delivery. Key words: Ethosomes, Azelaic acid, Composite, Vesicles, Propionibacterium acne, Topical.

Intranasal delivery of venlafaxine loaded nanostructured lipid carrier: Risk assessment and QbD based optimization

The objective of the present investigation was to optimize and develop venlafaxine (VLF) loaded nanostructured lipid carrier (NLC) using QbD and risk assessment approach. Full factorial 32 design was applied using two independent variables viz., X1: Drug to total lipid ratio and X2: Surfactant concentration whereby their effect on dependent variables viz., Y1: Size, Y2: PDI and Y3: %EE was studied. Compritol 888 ATO and Capmul MCM EP showed highest solubility for VLF as solid and liquid lipid respectively which depicted superior miscibility at solid: liquid lipid ratio of 70:30% w/w. VLF NLC were formulated based on optimized formula obtained from design space which gave experimental values for CQA (Size: 77.08 ± 3.45 nm, PDI: 0.234 ± 0.062 and %EE: 81.33 ± 3.05). FTIR, DSC and XRD studies revealed formation of less ordered crystalline structure of lipid matrix favouring higher encapsulation of VLF. Ex-vivo diffusion study showed higher flux value with VLF NLC (14.2 ± 0.40 μg/cm2/h) in comparison to VLF solution (9.62 ± 0.59 μg/cm2/h) across goat nasal mucosa. VLF NLC did not show toxicity and nasal mucosa was intact indicating safety for intranasal administration. Overall, QbD approach sounds to be apt for the successful development of VLF NLC.

Evaluate the early changes of myocardial fibers in rhesus monkey during sub-acute stage of myocardial infarction using diffusion tensor magnetic resonance imaging

PurposeThe deterioration of cardiac mechanical function starts from the micro-alterations in the myocardial fibers after myocardial infarction (MI) due to the heart beats derived from the systole and diastole of the myocardial fibers. So, we want to evaluate quantitatively the early changes of myocardial fibers in rhesus monkey during sub-acute MI stage. Materials and methodsThree fixed hearts with infarction after left anterior descending coronary artery ligation for 7days and eight age-matched intact controls were scanned by ex-vivo diffusion tensor magnetic resonance imaging (DT-MRI) to measure apparent diffusion coefficient (ADC), fractional anisotropy (FA) and helix angle (HA). ResultsIn comparison with healthy controls, FA and transmural range of HA in MI regions showed a significant reduction whereas ADC showed a significant increment (p<0.01). The double-helix myocardial fibers shifted further to left-handed helix around the infarcted and adjacent myocardium but shifted further to right-handed helix in remote myocardium. ConclusionHA is sensitive to evaluate quantitatively the early changes of myocardial fibers in sub-acute MI rhesus monkeys. The myocardial fibers in normal monkeys are similar to those in normal humans, suggesting that early changes of myocardial fibers in sub-acute MI monkeys can contribute to more accurately understand those in patients suffering sub-acute MI.

Application of quality by design approach for intranasal delivery of rivastigmine loaded solid lipid nanoparticles: Effect on formulation and characterization parameters

In the present investigation, Quality by Design (QbD) approach was applied on the development and optimization of solid lipid nanoparticle (SLN) formulation of hydrophilic drug rivastigmine (RHT). RHT SLN were formulated by homogenization and ultrasonication method using Compritol 888 ATO, tween-80 and poloxamer-188 as lipid, surfactant and stabilizer respectively. The effect of independent variables (X1 – drug: lipid ratio, X2 – surfactant concentration and X3 – homogenization time) on quality attributes of SLN i.e. dependent variables (Y1 – size, Y2 – PDI and Y3 – %entrapment efficiency (%EE)) were investigated using 33 factorial design. Multiple linear regression analysis and ANOVA were employed to indentify and estimate the main effect, 2FI, quadratic and cubic effect. Optimized RHT SLN formula was derived from an overlay plot on which further effect of probe sonication was evaluated. Final RHT SLN showed narrow size distribution (PDI- 0.132±0.016) with particle size of 82.5±4.07nm and %EE of 66.84±2.49. DSC and XRD study showed incorporation of RHT into imperfect crystal lattice of Compritol 888 ATO. In comparison to RHT solution, RHT SLN showed higher in-vitro and ex-vivo diffusion. The diffusion followed Higuchi model indicating drug diffusion from the lipid matrix due to erosion. Histopathology study showed intact nasal mucosa with RHT SLN indicating safety of RHT SLN for intranasal administration.

Development and Statistical Optimisation of Buspirone Hydrochloride Buccoadhesive Films.

The aim of the present study was to prepare unidirectional buccal films of buspirone hydrochloride by solvent casting technique. Hydroxypropylmethylcellulose (HPMC K15M) and Eudragit RL-100 were used as polymers in different proportion. Polyethylene glycol 400 and sodium lauryl sulphate were used as plasticizer and permeation enhancer, respectively, in different concentration. In the formulation, total amount of polymer (X 1) and percentage of HPMC K15M (X 2) were kept as independent variables. Afterwards, statistically optimized process was carried out and two optimized formulations (OF1 and OF2) were developed. The observed results of optimized formulation were showed a greater degree of percentage of similarity with predicted values. The stability studies showed that there was no significant change found in physicochemical properties, in-vitro release, and ex-vivo diffusion studies.

Preparation and evaluation of transnasal microemulsion of carbamazepine

The objective of this study was to develop novel transanasl microemulsion containing carbamazepine for treatment of epilepsy. Oleic acid was selected as oil while Tween 80 and propylene glycol were selected as surfactant and cosurfactant respectively based on solubility results. Optimized ratio of Tween 80: propylene glycol was selected after developing pseudoternary phase diagrams for different ratio and microemulsions were prepared. The prepared microemulsions were evaluated for globule size, viscosity, pH, conductivity and % transmittance. Ex-vivo diffusion study for optimized microemulsion was performed through sheep nasal mucosa wherein diffusion flux and permeability coefficients were determined. Further pharmacodynamic performance was evaluated in rats by electrically induced seizures. It was found that optimized microemulsion was stable and transparent with average globule size of 190 nm and diffusion flux of 75.77 μg cm−2 min−1 and showed no toxicity during histopathological evaluation on sheep nasal mucosa. Pharmacodynamic evaluation also indicated lesser intensity of seizures in rats treated with optimized formulation in comparison to rats treated with oral carbamazepine microemulsion and nasal carbamazepine solution which suggested carbamazepine transnasal delivery system as an effective alternate therapy for treatment of epilepsy.