From co-delivery to synergistic anti-inflammatory effect: Studies on chitosan-stabilized Janus emulsions having chloroquine phosphate and flavopiridol in Complete Freund's Adjuvant induced arthritis rat model.

For the first time, the co-delivery of chloroquine phosphate and flavopiridol by intra-articular route was achieved to provide local joint targeting in Complete Freund's Adjuvant-induced arthritis rat model. The presence of paired-bean structure onto the dispersed oil droplets of o/w nanosized emulsions allows efficient entrapment of two drugs (85.86-96.22 %). The dual drug-loaded emulsions displayed a differential in vitro drug release behavior, near normal cell viability in MTT assay, better cell uptake (internalization) and better reducing effect of mean immunofluorescence intensity of inflammatory proteins such as NF-κB and iNOS at in vitro RAW264.7 macrophage cell line. The radiographical study, ELISA test, RT-PCR study and H & E staining also indicated a reduction in joint tissue swelling, IL-6 and TNF-α levels diminution, fold change diminution in the mRNA expressions for NF-κB, IL-1ß, IL-6 and PGE2 and maintenance of near normal histology at bone cartilage interface respectively. The results of metabolomic pathway analysis performed by LC-MS/MS method using the rat blood (plasma) collected from disease control and dual drug-loaded emulsions treatment groups revealed a new follow-up study to understand not only the disease progression but also the formulation therapeutic efficacy assessment.

Development, systematic optimisation and biofilm disruption activity of eugenol-based nanosized emulsions stabilised with Tween 80.

The aims of this study were to systematically optimise a formula for eugenol emulsions via face-centered central composite design and to assess the activity against two-different bacterial strains (Staphylococcus aureus and Propionibacterium acnes) present at planktonic and biofilm forms. The molecular interaction of excipients, mean particle size (MPS) including zeta potential (ZP), drug entrapment efficiency (DEE) and in vitro drug release of optimised emulsions was done using FT-IR, Malvern Zetasizer, ultracentrifugation technique and membrane-free dissolution model, respectively. The emulsions consisted of 151.3 ± 1.45 nm MPS, -21.3 ± 1.25 mV ZP and 93.98 ± 1.41% DEE values. On storage of emulsions at 25 °C for 3 months, the value of DEE was found to be 72.12 ± 2.82%. The Tween 80 emulsifier film coverage onto the dispersed eugenol droplets of emulsions delayed significantly the drug release (12%-19%) compared to the drug release occurred from pure eugenol. The treatment of planktonic S. aureus and P. acnes with diluted eugenol emulsions showed the minimum inhibitory concentration and minimum bactericidal concentration values at 1.25-2.5 mg/ml whereas it occurred at 10 mg/ml for pure eugenol. Treating the biofilms with eugenol emulsions (1-2 mg/ml) yielded 59-70% minimum biofilm eradication concentration but 10 mg/ml pure eugenol showed 60%. Hence, the eugenol emulsions displayed antibacterial activity and could be projected as an antibiofilm or biofilm disruption agent.

Analytical Quality by Design-Driven RP-HPLC Method Conditions to Concomitantly Determine Cinnarizine and Morin Hydrate in Combined Drug Solution and Dual Drug-Loaded Formulations.

BACKGROUND: The replacement of traditional oils with a camphor and menthol-based eutectic mixture is done to prepare oil-less emulsion-like dispersions for co-delivery of cinnarizine (CNZ) and morin hydrate (MH) for managing Meniére's disease (MD). Since two drugs are loaded into the dispersions, the development of a suitable reverse phase-high performance liquid chromatography (RP-HPLC) method for their simultaneous analysis becomes inevitable. OBJECTIVE: By applying the analytical quality by design (AQbD) approach, the RP-HPLC method conditions were optimized for the concomitant determination of two drugs. METHODS: The systematic AQbD started with identifying critical method attributes (CMA) through an Ishikawa fishbone diagram, risk estimation matrix, and risk priority number-based failure mode effect analysis followed by screening using fractional factorial design and optimization by face-centered central composite design. The concomitant determination of two drugs by the optimized RP-HPLC method condition was substantiated via specificity checking using combined drug solution, drug entrapment efficiency, and in vitro release of the two drugs from emulsion-like dispersions. RESULTS: The AQbD optimized RP-HPLC method conditions revealed the retention time for CNZ and MH at 5.017 and 5.323, respectively. The studied validation parameters were found within the ICH-prescribed limits. Exposing the individual drug solutions to acidic and basic hydrolytic conditions yielded extra chromatographic peaks for MH, probably due to the degradation of MH. The DEE % values of 87.40 ± 4.70 and 74.79 ± 2.94, respectively, were noticed for CNZ and MH in emulsion-like dispersions. More than 98% CNZ and MH release was occurred from emulsion-like dispersions within 30 min post-dissolution in artificial perilymph. CONCLUSIONS: Overall, the AQbD approach could be helpful for systematic optimization of RP-HPLC method conditions to estimate concomitantly other therapeutic moieties. HIGHLIGHTS: The proposed article shows the successful application of AQbD for the optimization of RP-HPLC method conditions to concomitantly estimate CNZ and MH in combined drug solution and dual-drug-loaded emulsion-like dispersions.

Studies on spray dried topical ophthalmic emulsions containing cyclosporin A (0.05% w/w): systematic optimization, in vitro preclinical toxicity and in vivo assessments.

Cyclosporin A (CsA, 0.05% w/w)-loaded positively charged emulsions were prepared based on castor oil, chitosan, poloxamer 188, glycerin and double-distilled water. To augment the shelf/storage-stability of original emulsions, the solid-dry powder for reconstitution was made by spray drying technique. The screening (Taguchi OA) and optimization (face-centered central composite) designs produced the optimized conditions for spray drying: 40 Nm3/h aspirator flow rate, 15 ml/min feed rate, 115 °C inlet temperature, 10% mannitol and 1.25% trehalose. The % drug entrapment efficiency values of original and reconstituted emulsions ranged from 73.20 ± 0.13 to 71.55 ± 1.25%. At 20 min post-dissolution, two times higher CsA release was seen from reconstituted emulsions than the original emulsions (85.78 ± 1.14 vs. 42.25 ± 1.84%) in simulated tear fluid. Using MTT assay, the reconstituted emulsions with or without CsA produced 94.512 ± 2.12 to 99.941 ± 1.89% cell viability values in HCE-2 cells. No appreciable change in capillary integrity was visualized in HET CAM following reconstituted emulsions treatment. At equivalent 15 µg drug, the in vitro protein denaturation assay showed augmented inhibition value (~ 85%) for tested CsA emulsions compared to diclofenac reference (68.30 ± 2.05) indicating enhanced anti-inflammatory activity. The CsA concentrations in multiple ocular matrices of rabbit eyes determined by the UPLC-MS/MS method attained the therapeutic drug level of 50-300 ng/ml even at 90 min post-topical instillation of both emulsions. Overall, the CsA emulsion eyedrops can be supplied as a spray dried storable intermediate product for reconstitution.

Analytical Quality-by-Design-Based Systematic Optimization of RP-HPLC Method Conditions to Analyze Simultaneously Chloroquine Phosphate and Flavopiridol in Stress-Induced Combined Drug Solution and Pharmaceutical Emulsions.

A design of experiments (DoE)-driven RP-HPLC method conditions was employed to analyze simultaneously chloroquine (CQ) phosphate and flavopiridol (FLAP) in emulsions and solution. After subjecting the various critical method attributes to preliminary risk assessment and screening by Pareto-chart-based fractional factorial design, the 17 runs were produced in Box-Behnken design for optimization. Analysis of variance, lack of fit, prediction equations, 3D response surface plots and contour plots were used to evaluate the critical analytical attributes such as retention time, tailing factor and theoretical plate count. The optimized RP-HPLC method conditions include 262 nm as detection wavelength, 37°C temperature for column, 20-µl injection volume, 1-ml/min flow rate and mobile phase mixture [70:30 ratio of 0.4% triethylamine in methanol&sodium phosphate buffer (11 mM, pH 3.0)]. The studied validation parameters were found within the ICH-prescribed limits. Exposing the combined drug solution at oxidative stress condition resulted to diminish the FLAP recovery value (53.39 ± 0.86) and arrival of an extra chromatographic peak. However, the % drug entrapment efficiency values of 96.22 ± 2.47 and 85.86 ± 3.66, respectively, were noticed for CQ phosphate and FLAP in emulsions. Thus, DoE-driven approach could be helpful for systematically optimizing RP-HPLC method conditions.

Dual Delivery of Cyclosporin A and Etodolac Using Polymeric Nanocapsules in a Rabbit Eye Model: Ocular Biodistribution and Pharmacokinetic Study.

Purpose: Commercially available eye drops are loaded only with a single drug. By using the polymeric nanocapsules, dual delivery of 0.05% w/w cyclosporin A (CsA) and 0.2% w/w etodolac (Edc) was achieved. An ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for determining simultaneously the biodistribution and pharmacokinetic profile of CsA and Edc in ocular tissues. Methods: After one single drop instillation of nanocapsules into healthy right eyes of rabbits, the eyeballs were enucleated at 5, 15, 30, 60, and 90 min time periods to separate the 5 different ocular tissues. A liquid/liquid extraction method was used for ocular sample extraction using darunavir as internal standard. Using 3 diverse conditions such as bench-top, autosampler, and freeze-thaw, the stability of the analytes at 3 quality control samples in ocular tissues was also checked. Results: Intra- and interday precisions for both CsA and Edc in multiple ocular tissues were <10.32%, and the accuracy was <11.98%. The % bias and % RSD values for CsA and Edc were found within the acceptable limit of ±15%. The highest Cmax values were attained in cornea for both the drugs at 60 min postinstillation time point. Despite molecular size and structural differences, both CsA and Edc after liberation from nanocapsule drops can permeate into the tissues of the anterior as well as posterior segments of the eye. Conclusion: The biodistribution and pharmacokinetic data might help and strengthen our understanding of synergetic anti-inflammatory activity of CsA and Edc from nanocapsules after its ocular topical application for managing keratoconjunctivitis sicca.

Application of Design of Experiments® Approach-Driven Artificial Intelligence and Machine Learning for Systematic Optimization of Reverse Phase High Performance Liquid Chromatography Method to Analyze Simultaneously Two Drugs (Cyclosporin A and Etodolac) in Solution, Human Plasma, Nanocapsules, and Emulsions.

The objectives of current investigation are (1) to find out wavelength of maximum absorbance (λmax) for combined cyclosporin A and etodolac solution followed by selection of mobile phase suitable for the RP-HPLC method, (2) to define analytical target profile and critical analytical attributes (CAAs) for the analytical quality by design, (3) to screen critical method parameters with the help of full factorial design followed by optimization with face-centered central composite design (CCD) approach-driven artificial neural network (ANN)-linked with the Levenberg-Marquardt (LM) algorithm for finding the RP-HPLC conditions, (4) to perform validation of analytical procedures (trueness, linearity, precision, robustness, specificity and sensitivity) using combined drug solution, and (5) to determine drug entrapment efficiency value in dual drug-loaded nanocapsules/emulsions, percentage recovery value in human plasma spiked with two drugs and solution state stability analysis at different stress conditions for substantiating the double-stage systematically optimized RP-HPLC method conditions. Through isobestic point and scouting step, 205 nm and ACN:H2O mixture (74:26) were selected respectively as the λmax and mobile phase. The ANN topology (3:10:4) indicating the input, hidden and output layers were generated by taking the 20 trials produced from the face-centered CCD model. The ANN-linked LM model produced minimal differences between predicted and observed values of output parameters (or CAAs), low mean squared error and higher correlation coefficient values in comparison to the respective values produced by face-centered CCD model. The optimized RP-HPLC method could be applied to analyze two drugs concurrently in different formulations, human plasma and solution state stability checking.

Orally Administered Drug Solubility-Enhancing Formulations: Lesson Learnt from Optimum Solubility-Permeability Balance.

Although oral drug delivery is considered as most acceptable route for administering the active pharmaceutical ingredients to patients of all age-groups with the exceptions of bed-ridden patients and infants, the extent and rate of drug reaching the systemic circulation (in other word, drug bioavailability) always depends on many factors such as drug solubility in gastrointestinal fluids and drug permeation into intraluminal epithelial membrane structure, absence (fasting state) and presence (fed state) of food materials in the gastrointestinal tract, and individual variations in gastric emptying time. Taking the most influential factors like drug solubility and its permeability into consideration, these two factors play a pivotal role and even act as the litmus test for the formulation scientists who involve in oral dosage form development. It is very clear that there should be an optimum solubility and permeability balance to be reachable for getting the desired drug bioavailability to exert the intended therapeutic activity. The objectives of current review are (1) to provide an overview of two-different categories of poorly water soluble API molecules, (2) to describe briefly three-different case studies taken from drug solubility-enhancing formulations dealing with interplay between solubility and permeability, and (3) to showcase selected examples of solubility-permeability interplay phenomena arising out from the various orally administrable dosage forms. The lessons learnt from the past and current literatures are certainly encouraging to go ahead for oral dosage form development but with the prior knowledge about the possible existence of solubility-permeability interplay/tradeoff phenomenon.

Protecting the Normal Physiological Functions of Articular and Periarticular Structures by Aurum Nanoparticle-Based Formulations: an Up-to-Date Insight.

Taking the articular and periarticular structures as a litmus test for gold-based nanoformulations, the potential of gold nanoparticles in protecting the normal physiological functions of these structures particularly in geriatric patients is one of the research areas of current interest. Aside from its use to make the traditional and fashionable ornaments for human usage, the gold metal is also known for its rich therapeutic activity. This is especially true when the gold is converted from its bulk form into nanosized form before its administering into the human body. Since it is the age of nanocomponents in medical and pharmaceutical research areas, this review is therefore mainly focused on nanoparticulate systems consisting of aurum. Accumulating research reports nevertheless show concrete evidence indicating the potential of gold-based nanoformulations to manage joint syndromes such as osteoarthritis and rheumatoid arthritis. This review embarks from preparation techniques and characterization methods to therapeutical application potentials of gold-based nanoformulations.

Systematic Optimization, In Vitro Drug Release, and Preliminary Nonclinical Toxicity Assessment of Nonphospholipid-Based Topical Ophthalmic Emulsions Containing 0.05 or 0.1% w/w Cyclosporin A for Dry-Eye Syndrome Management.

The objectives of the present investigations are (1) to envisage a risk assessment plan for nonphospholipid-based topical ophthalmic emulsions with the help of failure mode and effect analysis (FMEA), (2) to screen the risky formulation and process variables by the Taguchi design, (3) to optimize systematically an emulsion formula by face-centered central composite design (CCD), (4) to incorporate cyclosporin A (0.05 or 0.1% w/w) into the optimized emulsions and predict the in vitro drug release kinetic via a particle diffusion-controlled mathematical model equation, and (5) to assess the emulsion's toxicity using in vitro hemolysis study. Through the risk priority number (RPN) scores of FMEA, half-normal and Pareto charts of the Taguchi design, 3D-response surface graphs, and overlay plots of CCD, the emulsion formula was systematically optimized. Irrespective of the two different drug loadings into optimized emulsions, the drug entrapment efficiency values ranged from 73.20 ± 0.13 to 74.42 ± 0.15%. The film diffusion or ion-exchange process fails to interpret the in vitro drug release kinetic profile. A permissible percentage hemolysis value of above 10% but below 25% guidance was observed for emulsions with or without cyclosporin A. The systematically optimized phospholipidless ophthalmic emulsions could further be exploited commercially for managing dry-eye syndrome.

Studies on olive-and silicone-oils-based Janus macroemulsions containing ginger to manage primary dysmenorrheal pain.

Ginger (GIN) powder-loaded oil-in-water (o/w) macroemulsions were prepared based on olive-and silicone-oils. The dispersed oil droplets with paired-beans structure were evident and thus the final emulsion can be termed as Janus macroemulsions. The objectives of the present study are (1) to identify the marker compound present in GIN powder via HPLC analysis, (2) to process the GIN powder via anti-solvent precipitation technique, (3) to see the solubility of GIN powder in various single oils or oil combination, (4) to optimize the GIN-loaded o/w macroemulsions using the central composite design (CCD) with respect to mean particle size of dispersed oil droplets and highest percentage drug entrapment efficiency values (DEE) and (5) to evaluate the pain reducing activity of optimized GIN-loaded macroemulsion via in vivo primary dysmenorrhea (PD) mice model. Both predicted and obtained values of percentage DEE (76.29 Vs.76.09) and mean particle size (245.99 Vs. 272.51 µm) were almost the same indicating the CCD statistical design applicability. The optimized Janus macroemulsion was stable at 4 °C for over a period of 90 days. Using the PD mice model, the counting of writhing reaction produced by the tested GIN-loaded macroemulsions at low and high doses did not reveal significant difference in comparison to the positive control (aspirin treated). Only the high dose of GIN-loaded macroemulsion was able to restore the uterine tissue's normal histomorphological structure after the H & E staining. Nevertheless, the paired beans structure should be tested for entrapping the plant-derived drugs having dissimilar physicochemical characteristics but similar therapeutic activity.

Studies on Aspirin Crystals Generated by a Modified Vapor Diffusion Method.

The objectives of the current investigation were (1) to study the influence of selected two different non-solvents (diethylether and dichloromethane) on the drug crystal formation of a model drug, aspirin (ASP-I) by the modified vapor diffusion method and (2) to characterize and compare the generated crystals (ASP-II and ASP-III) using different analytical techniques with that of unprocessed ASP-I. When compared to the classical vapor diffusion method which consumes about 15 days to generate drug crystals, the modified method needs only 12 h to get the same. Fourier transform-infrared spectroscopy (FT-IR) reveals that the internal structures of ASP-II and ASP-III crystals were identical when compared with ASP-I. Although the drug crystals showed a close similarity in X-ray diffraction patterns, the difference in the relative intensities of some of the diffraction peaks (especially at 2θ values of around 7.7 and 15.5) could be attributed to the crystal habit or crystal size modification. Similarly, the differential scanning calorimetry (DSC) study speculates that only the crystal habit modifications might occur but without involving any change in internal structure of the generated drug polymorphic form I. This is further substantiated from the scanning electron microscopy (SEM) pictures that indicated the formation of platy shape for the ASP-II crystals and needle shape for the ASP-III crystals. In addition, the observed slow dissolution of ASP crystals should indicate polymorph form I formation. Thus, the modified vapor diffusion method could routinely be used to screen and legally secure all possible forms of other drug entities too.

In Vitro Anti-inflammatory and Antimicrobial Activities of Azithromycin After Loaded in Chitosan- and Tween 20-Based Oil-in-Water Macroemulsion for Acne Management.

The objectives of the current investigation are (1) to prepare and characterize (particle size, surface charge (potential zeta), surface morphology by transmission electron microscopy, drug content, and drug release) the azithromycin (AZM, 100 mg)-loaded oil-in-water (o/w) macroemulsion, (2) to assess the toxicity of macroemulsion with or without AZM using RBC lysis test in comparison with AZM in phosphate buffer solution of pH 7.4, (3) to compare the in vitro antimicrobial activity (in Escherichia coli using zone inhibition assay) of AZM-loaded macroemulsion with its aqueous solution, and (4) to assess the in vitro anti-inflammatory effect (using egg albumin denaturation bioassay) of the AZM-loaded macroemulsion in comparison with diclofenac sodium in phosphate buffer solution of pH 7.4. The AZM-loaded macroemulsion possessed the dispersed oil droplets with a mean diameter value of 52.40 ± 1.55 µm. A reversal in the zeta potential value from negative (-2.16 ± 0.75 mV) to positive (+6.52 ± 0.96 mV) was noticed when AZM was added into the macroemulsion. At a 1:5 dilution ratio, 2.06 ± 0.03 mg of drug was released from macroemulsion followed by 1.01 ± 0.01 and 0.25 ± 0.08 mg, respectively, for 1:10 and 1:40 dilution ratios. Antimicrobial activity maintenance and significant reduction of RBC lysis property were noticed for AZM after loaded in the macroemulsion. However, an increment in the absorbance values for emulsion-treated samples in comparison to the control samples was noticed in the anti-inflammatory test. This speculates the potential of the AZM-loaded emulsion to manage inflammatory conditions produced at Acne vulgaris.

Effect of production variables on the physicochemical characteristics of celecoxib-loaded stearic and alginic acids-based microparticles.

The objectives of the current investigation were (1) to prepare the microparticles based on stearic and alginic acids from an aqueous system by hot (melt) dispersion method, (2) to achieve a higher drug entrapment efficiency and process yield (%) by changing the production variables such as stirring speed, concentration of stabilizer in aqueous dispersion medium, volume of aqueous dispersion medium, and stirring time, and (3) to see whether or not a retardation in drug release profile was attained from the celecoxib-loaded stearic and alginic acids-based microparticles compared to that of the celecoxib alone. The addition of alginic acid into stearic acid produced spherical-shaped particles with an almost smooth surface. Higher drug entrapment efficiency and process yield (%) values were obtained when the microparticles were prepared at 1000 r/min using 0.1% w/v polyvinyl alcohol in 100 mL aqueous dispersion medium and 30 min stirring time. The in vitro dissolution study in 900 mL of 2% sodium lauryl sulfate (SLS) solution at 75 r/min, however, showed only around 10% retardation in drug release from microparticles compared to the drug release from pure celecoxib alone. This indicated that the gel-like network formed by the alginic acid around the microparticles could not prevent the drug leakage from the microparticles.

Microbial colonization of medical devices and novel preventive strategies.

Upon implantation or insertion into patient's body for exerting the intended purpose like salvage of normal functions of vital organs, the medical devices are unfortunately becoming the sites of competition between host cell integration and microbial adhesion. Moreover, since there is an increased use of implanted medical devices, the incidence of biofilm-and medical devices-related nosocomial infections is also increasing progressively. To control microbial colonization and subsequent biofilm formation of the medical devices, different approaches either to enhance the efficiency of certain antimicrobial agents or to disrupt the basic physiology of the pathogenic microorganisms including novel small molecules and antipathogenic drugs are being explored. In addition, the various lipid-and polymer-based drug delivery carriers are also investigated for applying antibiofilm coating of the medical devices especially over catheters. The main intention of this review is therefore to summarize the major and/breakthrough inventions disclosed in patent literature as well as in research papers related to microbial colonization of medical devices and novel preventive strategies. This review starts with an overview of the preventive strategies followed by a short description about the potential of different lipidic-and polymeric-drug delivery carriers in eradicating the biofilm-associated infections from the medical devices.