International Journal of Pharmaceutics (v.377, #1-2)

Formulation and in vitro assessment of minoxidil niosomes for enhanced skin delivery by Prabagar Balakrishnan; Srinivasan Shanmugam; Won Seok Lee; Won Mo Lee; Jong Oh Kim; Dong Hoon Oh; Dae-Duk Kim; Jung Sun Kim; Bong Kyu Yoo; Han-Gon Choi; Jong Soo Woo; Chul Soon Yong (1-8).
Niosomes have been reported as a possible approach to improve the low skin penetration and bioavailability characteristics shown by conventional topical vehicle for minoxidil. Niosomes formed from polyoxyethylene alkyl ethers (Brij™) or sorbitan monoesters (Span™) with cholesterol molar ratios of 0, 1 and 1.5 were prepared with varying drug amount 20–50 mg using thin film-hydration method. The prepared systems were characterized for entrapment efficiency, particle size, zeta potential and stability. Skin permeation studies were performed using static vertical diffusion Franz cells and hairless mouse skin treated with either niosomes, control minoxidil solution (propylene glycol–water–ethanol at 20:30:50, v/v/v) or a leading topical minoxidil commercial formulation (Minoxyl). The results showed that the type of surfactant, cholesterol and incorporated amount of drug altered the entrapment efficiency of niosomes. Higher entrapment efficiency was obtained with the niosomes prepared from Span 60 and cholesterol at 1:1 molar ratio using 25 mg drug. Niosomal formulations have shown a fairly high retention of minoxidil inside the vesicles (80%) at refrigerated temperature up to a period of 3 months. It was observed that both dialyzed and non-dialyzed niosomal formulations (1.03 ± 0.18 to 19.41 ± 4.04%) enhanced the percentage of dose accumulated in the skin compared to commercial and control formulations (0.11 ± 0.03 to 0.48 ± 0.17%) except dialyzed Span 60 niosomes. The greatest skin accumulation was always obtained with non-dialyzed vesicular formulations. Our results suggest that these niosomal formulations could constitute a promising approach for the topical delivery of minoxidil in hair loss treatment.
Keywords: Niosomes; Minoxidil; Stability; Penetration; Skin accumulation; Dermal delivery;

Granule size control and targeting in pulsed spray fluid bed granulation by Henrik Ehlers; Anchang Liu; Heikki Räikkönen; Juha Hatara; Osmo Antikainen; Sari Airaksinen; Jyrki Heinämäki; Honxiang Lou; Jouko Yliruusi (9-15).
The primary aim of the study was to investigate the effects of pulsed liquid feed on granule size. The secondary aim was to increase knowledge of this technique in granule size targeting. Pulsed liquid feed refers to the pump changing between on- and off-positions in sequences, called duty cycles. One duty cycle consists of one on- and off-period. The study was performed with a laboratory-scale top-spray fluid bed granulator with duty cycle length and atomization pressure as studied variables. The liquid feed rate, amount and inlet air temperature were constant. The granules were small, indicating that the powder has only undergone ordered mixing, nucleation and early growth. The effect of atomizing pressure on granule size depends on inlet air relative humidity, with premature binder evaporation as a reason. The duty cycle length was of critical importance to the end product attributes, by defining the extent of intermittent drying and rewetting. By varying only the duty cycle length, it was possible to control granule nucleation and growth, with a wider granule size target range in increased relative humidity. The present study confirms that pulsed liquid feed in fluid bed granulation is a useful tool in end product particle size targeting.
Keywords: Fluid bed granulation; Granule size; Inlet air relative humidity; Atomization pressure; Pulsed spray; Process control;

Layered growth with bottom-spray granulation for spray deposition of drug by Dawn Z.L. Er; Celine V. Liew; Paul W.S. Heng (16-24).
The gap in scientific knowledge on bottom-spray fluidized bed granulation has emphasized the need for more studies in this area. This paper comparatively studied the applicability of a modified bottom-spray process and the conventional top-spray process for the spray deposition of a micronized drug during granulation. The differences in circulation pattern, mode of growth and resultant granule properties between the two processes were highlighted. The more ordered and consistent circulation pattern of particles in a bottom-spray fluidized bed was observed to give rise to layered granule growth. This resulted in better drug content uniformity among the granule batches and within a granule batch. The processes’ sensitivities to wetting and feed material characteristics were also compared and found to differ markedly. Less robustness to differing process conditions was observed for the top-spray process. The resultant bottom-spray granules formed were observed to be less porous, more spherical and had good flow properties. The bottom-spray technique can thus be potentially applied for the spray deposition of drug during granulation and was observed to be a good alternative to the conventional technique for preparing granules.
Keywords: Fluidized bed; Wurster process; Bottom-spray granulation; Layered growth; Drug distribution; Circulation pattern;

Comparison of solid dispersions produced by supercritical antisolvent and spray-freezing technologies by Elisabeth Badens; Viktor Majerik; Géza Horváth; László Szokonya; Nathalie Bosc; Eric Teillaud; Gérard Charbit (25-34).
Oxeglitazar is a new orally administered poorly water soluble active substance used in the treatment of type II diabetes. The objective of this work was to improve its dissolution kinetics using supercritical antisolvent (SAS) and spray-freezing (SF) techniques. Oxeglitazar was formulated with various excipients, including: Poloxamer 188 and 407, polyethylene glycol (PEG) 8000 and polyvinilpyrrolidone (PVP) K17 in a 1:1 weight ratio. In the SAS technology, pharmaceutical ingredients were dissolved in an appropriate solvent, and the feed solution was dispersed through a capillary nozzle in supercritical CO2 (SC CO2). Dichloromethane (DCM), chloroform (CHCl3), and a binary co-solvent system of chloroform–ethanol (EtOH/CHCl3 50:50, v/v%) were tested. In the SF process, tert-butanol (tBuOH) was used as solvent. The feed solution was injected into liquid nitrogen through a capillary nozzle located above the surface of the boiling nitrogen. Frozen particles were collected and freeze-dried for 30 h. Formulations were compared in terms of particle morphology, particle size, flow properties, crystallinity, polymorphic purity, residual solvent content, precipitation yield, drug content, specific surface area and dissolution kinetics. SAS and SF processed formulations exhibited enhanced dissolution rates. Within 5 min, the amount of dissolved drug varied from 31.6 to 64.3% for SAS and from 77.9 to 96.9% for freeze-dried formulations while only 30.5% was dissolved from raw drug. Apart from oxeglitazar/PVP K17, SAS prepared solid dispersions were characterized by high crystallinity and acicular shape. Freeze-dried formulations consisted of porous spherical particles with high amorphous content (94.2–100%).
Keywords: Oxeglitazar; Supercritical antisolvent; Spray-freezing; Poorly water soluble drug; Solid dispersion; Bioavailability;

The mechanical property anisotropy of compacts made from four commercially available pharmaceutical excipient powders (microcrystalline cellulose, lactose monohydrate, ascorbic acid, and aspartame) was evaluated. The speed of pressure (longitudinal) waves in the uni-axially compressed cubic compacts of each excipient in the three principle directions was determined using a contact ultrasonic method. Average Young's moduli of each compact in the axial (x) and radial (y and z) directions were characterized. The contact ultrasonic measurements revealed that average Young's modulus values vary with different testing orientations which indicate Young's modulus anisotropy in the compacts. The extent of Young's modulus anisotropy was quantified by using a dimensionless ratio and was found to be significantly different for each material (microcrystalline cellulose > lactose > aspartame > ascorbic acid). It is also observed that using the presented contact method, compacts at high solid fraction (0.857–0.859) could be differentiated than those at the solid fraction of 0.85 in their groups. The presented contact ultrasonic method is an attractive tool since it has the advantages of being sensitive to solid fraction ratio, non-destructive, requiring small amount of material and rapid. It is noteworthy that, since the approach provides insight into the performance of common pharmaceutical materials and fosters increased process knowledge, it can be applied to broaden the understanding of the effect of the mechanical properties on the performance (e.g., disintegration profiles) of solid oral dosage forms.
Keywords: Tablet; Anisotropy; Mechanical property; Young's modulus; Excipients; Contact ultrasonic measurement;

Numerical calibration of the Andersen cascade impactor using a single jet model by Yuriy Gulak; Eric Jayjock; Fernando Muzzio; Andrea Bauer; Paul McGlynn (45-51).
A single jet model is described and used to analyze the performance of an 8-stage Andersen cascade impactor (ACI). The two-dimensional axisymmetric jet flow field was calculated numerically by solving the Navier–Stokes equations and particle trajectories were then analyzed to obtain the collection efficiency curves at different flow rates. The resulting efficiency curves and corresponding cutoff diameters were compared and found to be in good agreement with the available experimental data. We also examined the effect of gravity on impactor performance and discuss the limitations of the single jet model.
Keywords: Andersen cascade impactor (ACI); Single jet model; d 50 ; Cutoff diameter;

The efficacy of an antitumor nucleoside, 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine (3′-ethynylcytidine, ECyd), was analyzed in vitro and in vivo. The in vivo antitumor effect of ECyd encapsulated into long-circulating liposomes was also examined. Based on pharmacokinetic (PK) and pharmacodynamic (PD) analyses, a model that quantitatively explains the in vivo effects of ECyd was proposed, using the concept of minimum effective concentration. The model suggests that ECyd followed a time-dependent mechanism of action in vivo, and that availability of ECyd in tumor tissue was highly important. To improve the availability of ECyd, its phospholipid derivatives were synthesized and encapsulated into long-circulating liposomes, which increased the antitumor effect. These results indicate that it is very important to design carriers of antitumor drugs based on PK–PD modeling.
Keywords: 3′-Ethynylcytidine; Antitumor effect; PK–PD modeling; Phospholipid derivatives; Liposome; Minimum effective concentration;

The surfactant assisted polymerization of 1-vinyl-2-pyrrolidone in supercritical carbon dioxide in the presence of Piroxicam, selected as a model of a low aqueous solubility drug, was studied in order to prepare in a single step a polymeric composite to enhance the rate of dissolution of the pharmaceutical compound. Reactive entrapping was carried out at 65 °C in the P range 21–38 MPa. Under proper operative conditions we obtained the composite under the form of sub-micron spherical particles with relatively narrow particle size distribution. Drug loadings higher than 12% (w/w) were obtained and XRD and Raman spectroscopy suggest that the anti-inflammatory agent is dispersed in the matrix with a non-crystalline structure. The dissolution rate of the drug from the composites was significantly faster both than that of the pure compound and of its physical mixture with the polymer. Collected results suggest that the proposed one-pot process can be used to prepare polymer based composites to increase bioavailability of low solubility drugs without utilization of toxic solvents and under mild temperature conditions.
Keywords: Supercritical fluid; Drug release kinetics; Solid dispersion; Dissolution enhancement; Polymer microspheres;

The effect of drug concentration on solvent activity in silicone membranes by P. Santos; M. Machado; A.C. Watkinson; J. Hadgraft; M.E. Lane (70-75).
The effects of supersaturated formulations on drug permeation through artificial and biological membranes have been reported by a number of research groups. However, little information is known about solvent permeation from these supersaturated formulations, and in particular the effect of high drug concentrations and degree of saturation (DS) on solvent activity. The aim of this study was to determine the effect of the DS of a model drug, oxybutynin, on solvent and drug permeation. Supersaturated residues of oxybutynin in propylene glycol (PG) or (octyl salicylate) OSAL were prepared by the solvent evaporation method. In both formulations a high percentage (25%, v/v) of solvent was used in order to avoid solvent depletion. Permeation of PG and OSAL through silicone was monitored by GC and HPLC, respectively. All OSAL formulations permeated to a higher extent than PG formulations. A decrease in OSAL permeation with 5 DS formulations was observed in comparison with 1 DS or 2 DS formulations, indicating a decrease in solvent activity with drug concentration. In addition, the drug transport from the 5 DS formulation of OSAL was higher than the 1 and 2 DS formulations but lower than predicted. Based on both solvent and drug permeation, this suggests that the low drug permeation observed with 5 DS resulted from a decrease in solvent thermodynamic activity rather than a decrease in solute activity as a result of drug crystallisation. Using PG formulations, the PG permeation remained unaffected with the DS of the formulation, up to 5 DS.
Keywords: Supersaturation; Oxybutynin; Propylene glycol; Octyl salicylate; Silicone; Permeation;

Chitosan–phospholipid blend for sustained and localized delivery of docetaxel to the peritoneal cavity by Payam Zahedi; Raquel De Souza; Micheline Piquette-Miller; Christine Allen (76-84).
Localized and sustained delivery of chemotherapeutics presents a “magic bullet” effect by providing high drug concentrations at the target site, extended drug exposure and reduced systemic toxicity. In the present study, an injectable chitosan–phospholipid (PoLigel) blend is put forth as a strategy to achieve sustained and localized delivery of docetaxel (DTX) following intraperitoneal (IP) administration. The stability of the blend was confirmed in vitro, by turbidity measurements and attributed to specific molecular interactions and the organization of the materials within the blend, as evidenced by FTIR analysis and confocal laser scanning microscopy, respectively. The chitosan and phospholipid were found to colocalize in regions surrounding a mean object area of 11.2 μm2 with colocalization coefficients of 43% and 46% for the chitosan and phospholipid, respectively. The PoLigel blend afforded sustained drug release as seen both in vitro (2.4 ± 0.7% DTX per day) and in vivo (4.4 ± 0.7% DTX per day). Constant concentrations of DTX were observed over a 2-week period in plasma and relevant peritoneal tissues, with no signs of toxicity or inflammation, following IP administration of the blend in healthy CD-1 mice. At DTX doses of 28.8 and 19.2 mg/kg, the blend showed significant tumor inhibition of 87.3 ± 9.3% and 74.1 ± 25.9%, respectively, in a murine xenograft model of human ovarian adenocarcinoma. This localized delivery system has shown excellent potential for sustained IP treatment of cancers, such as ovarian, that reside in the peritoneal cavity.
Keywords: Chitosan; Docetaxel; Injectable; Intraperitoneal; Localized sustained delivery; Phospholipid;

The transnasal delivery of 5-fluorouracil to the rat brain is enhanced by acetazolamide (the inhibitor of the secretion of cerebrospinal fluid) by Tomotaka Shingaki; Ismael J. Hidalgo; Tomoyuki Furubayashi; Hidemasa Katsumi; Toshiyasu Sakane; Akira Yamamoto; Shinji Yamashita (85-91).
The purpose of the research is to evaluate the effect of acetazolamide (AZA), an inhibitor of the secretion of cerebrospinal fluid (CSF), on the direct drug transport from the nasal cavity to the CSF and the brain uptake of a model drug, 5-fluorouracil (5FU). 5FU was infused intravenously or perfused nasally in the presence and absence of intravenously administered AZA. Concentrations of 5FU in plasma, CSF and the cerebral cortex were measured. The AUC and the concentration of 5FU in the brain were used to calculate the apparent brain uptake clearance (CLup) of 5FU, which is an index of drug delivery to the brain under the two experimental conditions. Intravenous AZA markedly increased the concentration of 5FU in the CSF and brain following the nasal perfusion of 5FU, although the plasma concentrations of 5FU were similar with intravenous infusion and nasal perfusions of 5FU. CLup of 5FU after the nasal perfusion with AZA was significantly increased by 104% and 46% as compared to intravenous infusion and nasal perfusion without AZA, respectively. AZA enhanced the 5FU delivery to the brain through a nose-to-brain pathway by increasing the concentration of the nasally applied drug in the CSF.
Keywords: Nasal absorption; Cerebrospinal fluid; Brain delivery; Brain uptake clearance; Acetazolamide; 5-Fluorouracil;

Effect of cationic liposomes/DNA charge ratio on gene expression and antibody response of a candidate DNA vaccine against Maedi Visna virus by A.M. Henriques; C. Madeira; M. Fevereiro; D.M.F. Prazeres; M.R. Aires-Barros; G.A. Monteiro (92-98).
Maedi Visna virus (MVV) is an ovine lentivirus with high prevalence all over the world. Since conventional vaccines had failed in protecting animals against the infection, the development of a DNA vaccine can be an alternative. The candidate vaccine was constructed by cloning the sequence encoding MVV p25 protein and was tested both in vitro and in vivo experiments associated with cationic liposomes. The lipoplexes (plasmid DNA–liposome complexes) with charge ratios ranging from 0 to 18 were prepared in physiological saline solution and characterized at a physical-chemistry level. Agarose gel electrophoresis was used as a first approach to evaluate qualitatively the amount of unbounded DNA by the liposomes. Dynamic light scattering measurements revealed that under the studied conditions lipoplexes with theoretical charge ratios (+/−) from 3 to 6 are unstable and prone to aggregation displaying sizes higher than 1 μm. At lower and higher charge ratios lipoplex size range from 200 to 500 nm. Using a Foster Resonance Energy Transfer methodology previously reported by us, complexation efficiency of the same complexes was related to in vitro and in vivo results.Higher transfection efficiencies were obtained in vitro with lipoplexes with charge ratio (+/−) = 10, where 97% of the DNA were protected by the liposomes. However, the subcutaneous immunization of mice induced higher antibody titers with lipoplexes at charge ratio (+/−) = 1, in which only 23% DNA is protected by the liposomes. Moreover, use of cationic liposomes has shown an increased antibody response when compared with a naked DNA immunization.
Keywords: Maedi Visna virus; DNA vaccine; Lipoplex characterization;

Safety, tolerability and pharmacokinetics of phenoprolamine hydrochloride floating sustained-release tablets in healthy Chinese subjects by Libo Zhao; Xiaoyan Yang; Rong Xu; Jianhong Wu; Shifen Gu; Li Zhang; Peili Gong; Hui Chen; Fandian Zeng (99-104).
The present study was designed to assess the safety, tolerability and pharmacokinetics of phenoprolamine hydrochloride floating sustained tablets (PHFST) in healthy Chinese subjects. 116 volunteers were randomized into single- or multiple-dose groups for oral administration 30–240 mg of PHFST once or 60–120 mg twice daily. Safety and tolerability were appraised by monitoring adverse events and laboratory parameters. Pharmacokinetics was assessed by determining the plasma concentrations of phenoprolamine hydrochloride with a validated HPLC method. In single-dose studies, no severe adverse events were observed in volunteers, and all adverse events were mild; the percentages of treatment-emergent events judged to be possibly related to the drug were 3/6 in the 240 mg dose group, 1/6 in the 180–210 mg dose groups, and none in the 30–150 mg dose groups; system exposure (AUC, C max) increased with respect to dose at 30–120 mg, whereas AUC raised disproportionately with dose escalating from 120 to 240 mg; the absorption of phenoprolamine hydrochloride was unaffected by food. In multiple studies, no safety concerns were revealed up to 7 days; steady-state plasma concentration was achieved after approximately 4–5 days of repeated twice-daily dosing. PHFST is safe and well tolerated in healthy Chinese subjects. The mean C max of PHFST is proportional to dose, but not the AUC. Oral dosing regimen selected for subsequent Phase II/III clinical trials was 60 mg of PHFST, b.i.d., and dose up to 120 mg, b.i.d. – may be used to achieve better antihypertensive effect.
Keywords: Safety; Tolerability; Pharmacokinetics; Phenoprolamine hydrochloride floating sustained tablets; Healthy Chinese subjects;

Investigation of the use of solution NMR spectroscopy to determine the effect of organic solvents on chemical shift changes in bases on addition of acids is reported. This information can be useful in the evaluation of solvents and counterion selection for salt formation. 1H and 15N chemical shift changes in three bases (pyrazine, phthalazine, and pyridine) on the addition of acids (1:1 ratio) were determined in various solvents. The effect of acid strength on chemical shift changes was examined. 1H and 15N chemical shift changes indicated protonation (salt formation). The media used affected the observed chemical shift changes. In D2O the data followed the ΔpK a (base–acid) general rule, that the pK a value of the acids should be 2 units lower than the pK a of the base to ensure proton transfer. Protonation, as measured by chemical shift changes using solution NMR spectroscopy, provided novel insight on potential salt formation in different media. Solution NMR spectroscopy appears to be a useful tool to evaluate counter ion and solvent selection for salt formation reaction.
Keywords: NMR; Chemical shift changes; Protonation; pK a; Salt screening; Solvent effect;

Poly(ɛ-caprolactone) (PCL) implants containing praziquantel (PZQ), a broad-spectrum antiparasite drug, are fabricated by injection molding and characterized in terms of content uniformity, morphology, drug physical state and stability. In vitro drug release from the implants is also studied. It is found that drug is dispersed uniformly in all implants and keeps stable over 365 days at 4 °C/60% RH. X-ray diffraction analysis reveals that PZQ exists primarily in its crystalline state in implants with high drug contents (50% and 25%). All implants exhibit similar release behaviors and about 70% of the drug is released after 365 days. The cross-sections of all implants present two distinct zones (i.e. peripheral white zone and inner pink zone) and the boundary between the two zones changes as time progresses. Drug content in the white zone is very low (less than 1%), but drug content in the pink zone is almost the same as the predefined value. Porous structures in the white zone but dense structures in the pink zone are observed by SEM. Obvious PCL degradation occurs till up to 365 days. These results show that the release process of PZQ is a gradual diffusion from the exterior to the interior of the implants.
Keywords: Poly(ɛ-caprolactone); Praziquantel; Implant; Injection molding; In vitro release;

FG90 chitosan as a new polymer for metronidazole mucoadhesive tablets for vaginal administration by Luana Perioli; Valeria Ambrogi; Cinzia Pagano; Stefania Scuota; Carlo Rossi (120-127).
Topical administration of the antibacterial metronidazole (MET) represents the most common therapy in the treatment of bacterial vaginosis (BV). The formulations generally available for BV therapy are creams, gels, vaginal lavages and vaginal suppositories. In this study, a new dosage form, containing MET, was developed with the aim to realize vaginal mucoadhesive tablets by including bioadhesive polymers as chitosan (FG90C), polyvinylpyrrolidone (PVPK90) and polycarbophil (PCPAA1), blended in different ratios. All formulations were characterized by studies of DSC, friability, hardness, hydration, mucoadhesion, in vitro release and antibacterial activity.All polymer mixtures employed were used to prepare tablets with the compactness and hardness so as allow the application on vaginal mucosa.FG90C performances improved in particular when mixed to PVPK90 (1:1 ratio). This kind of delivery system is suitable for formulating MET for topical application representing a good alternative to traditional dosage forms for vaginal topical administration.
Keywords: Vaginosis; FG90 chitosan; Metronidazole; Tablets; Mucoadhesion; Bacterial inhibition;

Double-layer weekly sustained release transdermal patch containing gestodene and ethinylestradiol by Yanli Gao; Jinying Liang; Jianping Liu; Yan Xiao (128-134).
The combination therapy of gestodene (GEST) and ethinylestradiol (EE) has shown advanced contraception effect and lower side effect. The present study was designed to develop a weekly sustained release matrix type transdermal patch containing GEST and EE using blends of different polymeric combinations. The multiple-layer technique was adopted in order to maintain a steady permeation flux for 7 days. The effects of polymer types, polymer ratios, permeation enhancers, drug loadings and drug ratios in different layers on the skin permeations of the drugs were evaluated using excised mice skin. Polariscope examination was carried out to observe the drug distribution behavior. The formulation with the mixture of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) (7:1) was found to provide the regular release and propylene glycol (PG) could enhance the permeation fluxes of drugs. Double-layer transdermal drug delivery system (TDDS) could sustain the steady permeation flux of drugs for 7 days when the ratio of drug in drug release layer and drug reservoir layer was 1:4 with the identical total drug amount. The in vitro transdermal permeation fluxes were 0.377 μg/cm2/h and 0.092 μg/cm2/h, for GEST and EE respectively. The uniformity of dosage units test showed that the distribution of drugs in the matrix was homogeneous, which was further demonstrated by the polariscope result. The developed transdermal delivery system containing GEST and EE could be a promising non-oral contraceptive method.
Keywords: Transdermal; Gestodene; Ethinylestradiol; 7-day patch; Contraception;

A microparticle preparation method based on an emulsion of ethanol in liquid paraffin stabilised using sorbitan sesquioleate which produces enteric microparticles of excellent morphology, size and pH-sensitive drug release was assessed for its robustness to changes in formulation and processing parameters. Prednisolone and methacrylic acid and methyl methacrylate copolymer (Eudragit S) were the drug and polymer of choice. Emulsion solvent evaporation procedures are notoriously sensitive to changes in methodology and so emulsion stirring speed, drug loading, polymer concentration and surfactant (emulsifier) concentration were varied; microparticle size, encapsulation efficiency, yield and in vitro dissolution behaviour were assessed. The yield and encapsulation efficiency remained high under all stirring speeds, drug loadings and polymer concentrations. This suggests that the process is flexible and efficiency can be maintained. Surfactant concentration was an important parameter; above an optimum concentration resulted in poorly formed particles. All processing parameters affected particle size but this did not alter the acid resistance of the microparticles. At high pH values the smaller microparticles had the most rapid drug release. In conclusion, the microparticle preparation method was resistant to many changes in processing, although surfactant concentration was critical. Manipulation of particle size can be used to modify the drug release profiles without adversely affecting the gastro-resistant properties of these pH-responsive microparticles.
Keywords: Emulsion solvent evaporation; Microencapsulation; Microspheres; Eudragit S; Arlacel 83; Enteric polymers; Modified release; Colonic delivery;

The universality of low-energy nano-emulsification by Nicolas Anton; Thierry F. Vandamme (142-147).
Extensive studies have been done on nano-emulsions and emulsification methods to provide nanometric-scaled templates for the formulation of nanoparticles. The so-called “low-energy” methods are of particular interest as they prevent the potential degradation of fragile encapsulated molecules. This work deals with new concepts in nano-emulsification using low-energy methods. Three-model ternary systems, water/nonionic surfactant/oil, were studied and compared. Nano-emulsions were generated using both spontaneous emulsification and the PIT method, so as to study the links between these two nano-emulsification methods. The influence of the composition and formulation variables on the nano-emulsion properties and emulsification procedures were thus investigated. This study pioneers the concept of the universality of low-energy nano-emulsification, proving that all these low-energy methods (i.e. spontaneous emulsification and the phase inversion temperature (PIT) method) are governed by a single unique mechanism. It thus provides a better understanding of low-energy nano-emulsification processes and notably the PIT method, useful in the fields of nanoparticle and nano-pharmaceutic formulations. These results are fundamental in establishing experimental procedures for the incorporation of drugs in nano-emulsions.
Keywords: Nano-emulsion; Emulsification; Low-energy; Spontaneous emulsification; Phase inversion temperature method;

Nanodispersions of taxifolin: Impact of solid-state properties on dissolution behavior by Alexander N. Shikov; Olga N. Pozharitskaya; Inna Miroshnyk; Sabiruddin Mirza; Irina N. Urakova; Samuli Hirsjärvi; Valery G. Makarov; Jyrki Heinämäki; Jouko Yliruusi; Raimo Hiltunen (148-152).
Nanosizing is an advanced formulation approach to address the issues of poor aqueous solubility of active pharmaceutical ingredients. Here we present a procedure to prepare a nanoparticulate formulation with the objective to enhance dissolution kinetics of taxifolin dihydrate, a naturally occurring flavonoid with antioxidant, anti-inflammatory, and hepatoprotective activities. Polyvinylpirrolidone was selected as a carrier and the solid nanodispersions of varying compositions were prepared by a co-precipitation technique followed by lyophilization. The formulation technology reported herein resulted in aggregate-free, spherical particles with the mean size of about 150 nm, as observed by scanning electron microscopy and measured by photon correlation spectroscopy. Furthermore, the co-precipitation process caused taxifolin dihydrate to convert into an amorphous form as verified by X-ray powder diffraction, differential scanning calorimetry, hot stage microscopy and Raman spectroscopy. Finally, in vitro dissolution behavior of the nanodispersion of taxifolin was shown to be superior to that of either pure drug or a drug–polymer physical mixture, reaching 90% of taxifolin released after 30 min. Such enhanced drug release kinetics from the nanodispersion was attributed to both the reduced particle size and the loss of crystallinity.
Keywords: Nanoparticles; Taxifolin; Lyophilization; Nanodispersion; Solid-state properties; Drug release kinetics;

Evaluation of dissolution kinetics of hydrophilic polymers by use of acoustic spectroscopy by Giulia Bonacucina; Marco Cespi; Giovanni F. Palmieri (153-158).
This paper seeks to demonstrate the feasibility of using a novel analytical technique, acoustic spectroscopy, to characterize the dissolution kinetics of hydrophilic polymers, in particular, three different model polysaccharides: lambda carrageenan, gellan gum, and xanthan gum.The influence of particle size and of analysis temperature (25 or 45 °C) was evaluated through the evolution over time of the microrheological acoustic parameters G′ and G″. This new method was then compared with classical rheological analysis. To better compare acoustic spectroscopy and rheological analysis, the initial dissolution rate from the slope of the first part of the G′ or viscosity versus time curves was calculated.Both analytical techniques gave the same rank order of kinetics for the powder types and fractions examined; differences in absolute values were due to the fact that the two methods measured different parameters and had different stirring efficiencies.The rheological data obtained with both methods of analysis and their modelling confirmed that acoustic spectroscopy is an effective tool for monitoring and quantifying dissolution kinetics and hence affords a powerful technique for following over time a great number of physical changes occurring in a specific system.
Keywords: Ultrasound; Acoustic spectroscopy; Rheology; Hydrocolloids; Dissolution kinetics;

Anti-inflammatory and anti-oxidant activity of anionic dendrimer–N-acetyl cysteine conjugates in activated microglial cells by Bing Wang; Raghavendra S. Navath; Roberto Romero; Sujatha Kannan; Rangaramanujam Kannan (159-168).
Dendrimers are emerging as potential intracellular drug delivery vehicles. Understanding and improving the cellular efficacy of dendrimer–drug conjugates, can lead to significant in vivo benefits. This study explores efficacy of anionic polyamidoamine (PAMAM–COOH) dendrimer–N-acetyl cysteine (NAC) conjugates for applications in neuroinflammation. The anti-oxidative and anti-inflammatory effects of PAMAM–(COOH)46–(NAC)18 conjugate is evaluated on microglial cells in vitro. Cell entry and localization of PAMAM–(COOH)62–(FITC)2 conjugate in BV-2 microglial cells were assessed using flow cytometry and confocal microscopy. ELISA assays were used to evaluate markers of oxidative stress (ROS, NO) and inflammation (TNF-α) after stimulation of microglial cells with lipopolysaccharides (LPS), following treatment with increasing doses of free N-acetyl-l-cysteine (NAC) or PAMAM–(COOH)46–(NAC)18 conjugate containing an equivalent molar concentration of NAC. Flow cytometry and confocal microscopy demonstrated the PAMAM–(COOH)62–(FITC)2 conjugate entered BV-2 cells rapidly with significant increase in fluorescence within 15 min and localized mostly in the cytoplasm. PAMAM–(COOH)46–(NAC)18 conjugate was non-toxic, and significantly reduced ROS, NO and TNF-α release by activated microglial cells after 24 h and 72 h stimulation of LPS following 3 h pre-treatment when compared to the same concentration of free NAC (P  < 0.05 or P  < 0.01). Anionic PAMAM dendrimer–NAC conjugate was synthesized with a glutathione sensitive linker for intracellular release. The non-toxic conjugate is a more effective anti-oxidant and anti-inflammatory agent when compared to free NAC in vitro. The conjugate showed significant efficacy even at the lowest dose (0.5 mM NAC), where the activity was comparable or better than that of free drug at 8 mM (16× higher dosage). The improved efficacy of the conjugate, when combined with the intrinsic neuroinflammation-targeting ability of the PAMAM dendrimers, may provide new opportunities for in vivo applications.
Keywords: Dendrimers; PAMAM; Drug delivery; Neuroinflammation; N-acetyl cysteine;

Influence of nanocarrier type and size on skin delivery of hydrophilic agents by Sarah Küchler; Mona Abdel-Mottaleb; Alf Lamprecht; Michal R. Radowski; Rainer Haag; Monika Schäfer-Korting (169-172).
The nanoparticulate carrier systems solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters gained interest for the topical treatment of skin diseases as they facilitate the skin penetration of loaded lipophilic drugs. Here, we studied if these carrier systems are also suitable drug delivery systems for more hydrophilic agents using the dye rhodamin B as model compound. Furthermore, the influence of the particle size on the skin penetration was investigated. Loading rhodamin B onto SLN (250–340 nm) and CMS nanotransporters (20–30 nm), the dye amount increased significantly in viable epidermis and dermis as compared to a conventional cream. CMS nanotransporters were most efficient. Creating nanoparticles of 50–200 nm demonstrated only marginal size effect for the skin penetration. Therefore, the superiority of the CMS nanotransporters seems to be attributed to the character of the nanoparticles and not to its smaller size.
Keywords: Dendritic core-multishell nanotransporters; Solid lipid nanoparticles; Skin penetration; Size effect; Rhodamin B;

Insulin-micro- and nanoparticles for pulmonary delivery by Claudia Klingler; Bernd W. Müller; Hartwig Steckel (173-179).
The pulmonary application of insulin via oral inhalation turned out to be a promising option due to the large surface area and good vascularisation the lung is offering for the systemic delivery of peptides and proteins. To have a systemic effect, inhaled particles need to attain the alveoli and should therefore have a mass median diameter of less than 2 μm. To achieve such a particle size for dry powders spray drying of drug solutions is a common method. In this study, a nano-precipitation of the drug prior to spray drying was carried out using the solvent change method. The produced powders were compared to powder produced out of a solution and to the marketed product Exubera®. The Aerolizer® device was used representing a simple capsule-based dry powder inhaler.It could be shown that the insulin yield of the precipitation process highly depends on the used pH-value and the amount of non-solvent. Also the particle size after spray drying decreases with increasing amount of non-solvent. Aerodynamic assessment of insulin powders showed that the precipitated insulin particles behave superior to powders spray dried from solution with respect to particles smaller than 2 μm. The deposition pattern of the originator powder delivered with the Exubera® device showed significantly lower fine particle fractions and higher residues in comparison to the Aerolizer® device.In summary, precipitated insulin particles combined with the delivery from a standard capsule-based inhaler were found to be at least as effective in vitro as the marketed Exubera® product. With an optimised powder having an increased particle fraction smaller than 2 μm more insulin may reach the deeper lung. Therefore, a lower dose could be used for an effective diabetic therapy.
Keywords: Insulin; DPI; Precipitation; Solvent change; Pulmonary drug delivery; Nanoparticles;

Freeze-dried nifedipine-lipid nanoparticles with long-term nano-dispersion stability after reconstitution by Hiroyuki Ohshima; Atsuo Miyagishima; Takurou Kurita; Yuji Makino; Yasunori Iwao; Takashi Sonobe; Shigeru Itai (180-184).
Nifedipine (NI) is a poorly water-soluble drug and its oral bioavailability is very low. To improve the water solubility, NI-lipid nanoparticle suspensions were prepared by a combination of co-grinding by a roll mill and high-pressure homogenization without any organic solvent. The mean particle size and zeta potential of the NI-lipid nanoparticle suspensions were about 52.6 nm and −61.8 mV, respectively, and each parameter remained extremely constant during a period of 4 months under 6 °C and dark conditions, suggesting that the negative charge of the phospholipid, dipalmitoyl phosphatidylglycerol, is very effective in preventing coagulation of the particles. In order to assure the nano-order particle size of the suspensions in view of long-term stability, a freeze-drying technique was applied to the NI-lipid nanoparticle suspensions. The mean particle size of freeze-dried NI-lipid nanoparticles after reconstitution was significantly increased in comparison to that of the preparations before freeze-drying. It was found, however, that the addition of sugars (glucose, fructose, maltose or sucrose) to the suspensions before freeze-drying inhibited the aggregation of nanoparticles, suggesting that the long-term stability storage of freeze-dried NI-lipid nanoparticles after reconstitution would be overcome. In addition, freeze-dried nanoparticles with 100 mg sugar (glucose, fructose, maltose or sucrose) showed excellent solubility (>80%), whereas without sugar, as a control, showed low solubility (<20%). It was found that negatively charged phospholipids and sugars prevent coagulation of NI nanoparticle suspensions, and reproduce the nanoparticle dispersion after reconstitution; and remarkably increase the apparent solubility of nifedipine.
Keywords: Nanoparticles; Long-term nano-dispersion stability; Freeze-dry; Sugar; Water solubility;

Quality by design approach to understand the process of nanosuspension preparation by Sudhir Verma; Yan Lan; Rajeev Gokhale; Diane J. Burgess (185-198).
Quality by design (QbD) principles were explored to maximize the understanding of the unit operation of microfluidization, for the preparation of nanosuspensions using indomethacin as a model drug. The effects of key formulation and process variables (drug concentration, stabilizer type, stabilizer concentration, temperature, milling time and microfluidization pressure) were investigated by executing a 2(5−1) factorial design. Particle size, zeta potential and the physical form of the drug constituted the critical quality attributes (CQAs). Multiple linear regression analysis and ANOVA were employed to identify and estimate the effect of important parameters, establish their relationship with CQAs, create design space and model the process of microfluidization for predictive purposes. In order of importance, milling time, microfluidization pressure, stabilizer type, temperature and stabilizer concentration were identified as critical parameters affecting the formation and stability of nanosuspensions. Interaction between homogenization pressure, temperature and milling time also significantly affected the nanosuspension particle size. No correlation was found between the zeta potential and the storage stability. No change in the physical form of indomethacin was observed on storage for 28 days at 4 °C and 25 °C. This research highlights the level of understanding that can be accomplished through a well designed study based on the philosophy of QbD.
Keywords: Quality by design; Nanosuspensions; Milling; Optimization; Screening; Factorial design;

Long-circulation of hemoglobin-loaded polymeric nanoparticles as oxygen carriers with modulated surface charges by Feng Xu; Yuan Yuan; Xiaoqian Shan; Changsheng Liu; Xinyi Tao; Yan Sheng; Huanjun Zhou (199-206).
The aim of this study was to investigate the effects of the surface charges on the in vitro macrophage cellular uptake and in vivo blood clearance and biodistribution of the hemoglobin-loaded polymeric nanoparticles (HbPNPs). The surface charges of the HbPNPs fabricated from mPEG–PLA–mPEG were modulated with cationized cetyltrimethylammonium bromide (CTAB) and anionized sodium dodecyl sulphate (SDS), respectively. In vitro macrophage cellular uptake and in vivo biodistribution of the coumarin 6-labeled HbPNPs with different electric charges were investigated, and the half-lives in the circulation were pharmacokinetically analyzed. The particle sizes of the HbPNPs were all below 200 nm with a narrow size distribution and high encapsulation efficiency (>84%). And the ζ-potentials of the untreated, cationized and anionized HbPNPs in phosphate buffered sodium chloride solution (PBS) were −12.3, +3.28 and −25.4 mV, respectively. The HbPNPs did not occur significant aggregation or sedimentation, even after 5 days. Compared with the untreated HbPNPs, 1-fold decrease/increase of the uptake percentage associated with the cationized/anionized HbPNPs was observed. In vivo experiment demonstrated that the calculated half-life of the cationized HbPNPs was 10.991 h, 8-fold longer than that of the untreated HbPNPs (1.198 h). But the anionized HbPNPs displayed opposite effect. Furthermore, the cationized HbPNPs mainly accumulated in the liver, lung and spleen after 48 h injection. MTT results showed that the HbPNPs with different surface charges all exhibited slight toxicity. These results demonstrated that the CTAB-modulated HbPNPs with low positive charge and suitable size have a promising potential as a long-circulating oxygen carrier system with desirable biocompatibility and biofunctionality.
Keywords: Hb-loaded polymeric nanoparticles; Blood long-circulation; ζ-Potential; Biodistribution;

Q10-loaded NLC versus nanoemulsions: Stability, rheology and in vitro skin permeation by Varaporn B. Junyaprasert; Veerawat Teeranachaideekul; Eliana B. Souto; Prapaporn Boonme; Rainer H. Müller (207-214).
In this study, nanoemulsions (NE) of medium chain triacylglycerols (MCT) and nanostructured lipid carriers (NLC) of cetyl palmiate/MCT were produced to load coenzyme Q10 (Q10) and characterized for their stability before and after incorporation into xanthan gum hydrogels. After storage at 4, 25 and 40 °C, the particles remained in the nanosize range for 12 months, with zeta potential higher than |40 mV|. Similar results were found in xanthan gum-based hydrogels containing NE or NLC. The crystallinity index of Q10-loaded NLC increased after being incorporated into hydrogels. The Q10 entrapped in NLC and NE remained higher than 90% at all temperatures for 12 months but dramatically decreased when exposed to light. From the rheological studies, both NLC and NE dispersions possessed pseudoplastic flow having more liquid characteristics, whereas NLC and NE hydrogels exhibited plastic flow with thixothopy, showing more elastic rather than viscous properties. The occurrence of a spatial arrangement of lipid molecules was observed in the matrix of NLC when entrapped into hydrogels. From in vitro permeation studies, it could be stated that the amount of Q10 released and occlusiveness were major keys to promote the deep penetration of Q10 into the skin.
Keywords: Nanostructured lipid carriers; NLC; Q10; Chemical stability; In vitro skin permeation;

Noticeboard (216).