International Journal of Pharmaceutics (v.405, #1-2)

Amelioration of dementia induced by Aβ 22-35 through rectal delivery of undecapeptide-hEGF to mouse brain by Bao-Quan Zhao; Yan-Ru Guo; Xu-Ling Li; Ting Zang; Heng-Yan Qu; Jian-Ping Zhou; Qian Li; Man-Ji Sun (1-8).
Distribution of P11-hEGF and hEGF after IV injection or per rectum in mice (n  = 6). P11-hEGF or hEGF (normalized to 40 μg hEGF) were IV injected to the mice. The animals were killed at various time intervals after injection, and the organs (brain, heart, liver, spleen, lung, kidney and stomach) were immediately removed. Tissue homogenates (1:10, w/v) were prepared and stored at −20 °C. The contents of EGF in various tissue homogenates were quantitatively determined by the radio-immunological method. (A and C) P 11-hEGF; (B and D) hEGF (Sigma).A group of growth factors have been shown to play important roles in amelioration of the malfunction of the neurodegenerative diseases. However, the proteins or polypeptides passing across the blood-brain barrier (BBB) to access the brain parenchyma are relatively few so that it hinders the therapies in clinic. Here a genetically reconstructed fusion peptide of human epidermal growth factor (hEGF) with an undecapeptide YGRKKRRQRRR (P11) was used to investigate the permeability between the cell membrane and the BBB via rectal administration. The efficiency to rescue the Aβ 22-35-induced dementia in mice was assessed after administration of P11-hEGF per rectal. Our results showed that P11-hEGF permeates across not only the 3T3 cell membrane in vitro, but also the endothelia of vessels after intravenous injection (IV), and the mucosa of the rectum after per rectal administration. Further results showed that the circulating P11-hEGF allowed penetrating through the blood-brain barrier and then getting into the brain manifesting biological responses. In the animal experiments, treatment with P11-hEGF not only ameliorated the dementia induced by Aβ 22-35 but also rescued the dementia of the aged mice, no matter how it was administrated (IV or per rectal). These results suggest that the rectal non-invasive delivery of the P11 polypeptide-conjugated growth factor is an efficient way for BBB transduction, thus raises the hope of real therapeutic progress against neurodegenerative diseases.
Keywords: Rectal delivery; P11-hEGF; Blood-brain barrier; Aβ 22-35 peptide; Dementia;

Plume temperature emitted from metered dose inhalers by G. Brambilla; T. Church; D. Lewis; B. Meakin (9-15).
The temperature of the drug cloud emitted from a pressurised metered dose inhaler (pMDI) may result in patient discomfort and inconsistent or non-existent dose delivery to the lungs. The effects of variations in formulation (drug, propellant, co-solvent content) and device hardware (metering volume, actuator orifice diameter, add-on devices) upon the temperature of pMDI plumes, expressed as replicate mean minimum values (MMPT), collected into a pharmacopoeial dose unit sampling apparatus (DUSA), have been investigated. Ten commercially available and two development products, including chlorofluorocarbon (CFC) suspensions and hydrofluoroalkane (HFA) solutions or suspensions, were examined together with a number of drug products in late stage development and a variety of HFA 134a placebo pMDIs. Plume temperatures were observed to be lowest in the proximity of the product's actuator mouthpiece where rapid flashing and evaporation of the formulation's propellant and volatile excipients cause cooling. The ability to control plume temperature by judicious choice of formulation co-solvent content, metering volume and the actuator orifice diameter is identified. An ethanol based HFA 134a formulation delivered through a fine orifice is inherently warmer than one with 100% HFA 134a vehicle delivered through a coarse actuator orifice. Of the 10 commercial products evaluated, MMPTs ranged from −54 to +4 °C and followed the formulation class rank order, HFA suspensions < CFC suspensions < HFA solutions. For all systems examined it was possible to raise pMDI plume temperature to that of the ambient surroundings by use of an add-on or integrated spacer device.
Keywords: Aerosol; Pressurised metered dose inhaler; Plume temperature; Hydrofluoroalkane; HFAs;

This study shows that the hydrodynamic conditions and level of planktonic bacteria in water are critical for the development of biofilm at very low TOC. The model investigated is sensitive for early detection of biofilm formation and can be used to establish an effective biofilm control strategy.Systems for storage and distribution of purified water at ambient temperature are highly susceptible to microbial contamination. The water flow, microbial content and chemical quality of the purified water in an industrial water system have been simulated in a biofilm annular reactor (BAR) to study the impact of different hydrodynamic conditions on biofilm development. Our results reveal the potential of stagnant purified water at total organic compounds (TOC) below 50 ppb to develop biofilm that allows detachment of planktonic bacteria and colonization of new surfaces within 24 h. However, under constant water flow over 7 days, the growth of initial biofilm was 40 times less, fewer bacteria were detached, and new surfaces were colonized to a lesser extent. Heterotrophic plate counts (HPCs) in biofilm were highly positively correlated with numbers of detached planktonic bacteria in effluent water. The study shows that the hydrodynamic conditions and level of planktonic HPC in water are critical for the development of biofilm at very low TOC. The results in the BAR agreed well with those from regular industrial microbial monitoring of purified water. To conclude, the BAR successfully simulates biofilm growth and can be used to establish an effective biofilm control strategy. However, the microbial quality of purified water in industrial system is a constant challenge; any increase of HPC in effluent water is a sign to take steps against excessive microbial growth.
Keywords: Biofilm; Purified water; Hydrodynamic conditions; Modelling; Quality assurance; Colonization; Laboratory study;

The pulmonary route of delivery offers a potential alternative to parenteral administration of peptides and proteins. Protection of protein structure is essential in both processing and storage of the final formulation. Sugars, such as trehalose and raffinose, have been employed to act as protein stabilisers. Optimisation of the aerodynamic characteristics of microparticles in dry powder inhaler formulations is critical to ensure optimum deposition of the formulation into the respiratory tract.In the present study we examine the adaptation to hydrophilic materials, specifically the disaccharide, trehalose and the trisaccharide, raffinose, of a previously reported spray drying process for producing nanoporous microparticles (NPMPs). We also investigate the feasibility of incorporating a model protein, lysozyme, into these sugar-based NPMPs.While spray drying raffinose or trehalose from aqueous solution or ethanol:water solutions resulted in non-porous microspheres, spray drying from a methanol:n-butyl acetate mixed solvent system resulted in microparticles which appeared to consist of an agglomeration of individual nanoparticles, i.e. nanoporous/nanoparticulate microparticles.NPMPs of trehalose and raffinose were amorphous, with glass transition temperatures (Tgs) that were sufficiently high (124 °C and ∼120 °C for trehalose and raffinose, respectively) to suggest good physical stability at room temperature and good potential to act as protein carriers and/or stabilisers.NPMPs demonstrated improved aerosolisation properties compared to spray dried non-porous particles. The successful incorporation of lysozyme into these NPMPs at a sugar to protein weight ratio of 1:4 demonstrated the potential of these systems to act as carriers for peptide or protein drugs which could be delivered via the pulmonary route.
Keywords: Nanoporous; Microparticles; Trehalose; Raffinose; Sugars; Proteins; Lysozyme; Pulmonary delivery; Amorphous; Spray drying; Stability;

Particle engineering of materials for oral inhalation by dry powder inhalers. II—Sodium cromoglicate by Lorraine M. Nolan; Jianhe Li; Lidia Tajber; Owen I. Corrigan; Anne Marie Healy (36-46).
Sodium cromoglicate is an antiasthmatic and antiallergenic drug used in inhalation therapy and commonly administered by a dry powder inhaler.In the present study we sought to examine the feasibility of producing nanoporous microparticles (NPMPs) of this hydrophilic material by adaptation of a spray drying process previously applied to hydrophobic drugs, and to examine the physicochemical and in vitro deposition properties of the spray dried particles in comparison to a commercial product. The storage stability of successfully prepared NPMPs was assessed under a number of conditions (4 °C with dessicant, 25 °C at 60% relative humidity and 25 °C with dessicant).Spray dried sodium cromoglicate was amorphous in nature. NPMPs of sodium cromoglicate displayed superior aerodynamic properties resulting in improved in vitro drug deposition, as assessed by Andersen Cascade Impactor and twin impinger studies, in comparison to the commercial product, Intal®. Deposition studies indicated that porosity and sphericity were important factors in improving deposition properties. The optimum solvent system for NPMP production was water:methanol:n-butyl acetate, as spherical NPMPs spray dried from this solvent system had a higher respirable fraction than non-spherical NPMPs of sodium cromoglicate (spray dried from methanol:n-butyl acetate), non-porous sodium cromoglicate (spray dried from water) and micronised sodium cromoglicate (Intal®).While particle morphology was altered by storage at high humidity (60% RH) and in vitro deposition performance deteriorated, it was possible to maintain NPMP morphology and aerosolisation performance by storing the powder with dessicant.
Keywords: Nanoporous; Microparticles; Sodium cromoglicate; Pulmonary delivery; Amorphous; Spray drying; Solid-state analysis; Stability;

Pictures of tablets made of native, 50% succinylated or 100% succinylated β-lg before and 1 after incubation in simulated gastric fluid with pepsin for 30, 60 or 120 min.The use of succinylated β-lactoglobulin as a novel functional tablet excipient for the protection of probiotic bacteria against the adverse gastric conditions and their delivery in the intestine was studied. Tablets were produced by direct compression of a dry mixture of Bifidobacterium longum HA-135 and the tested excipient. The results showed that tablets made of native β-lg did not ensure cell survival while grafting carboxylic acid groups on the protein revealed to be an innovative method to create a gastroresistant matrix that could allow the survival of up to 108  CFU and 107  CFU after 1 h and 2 h gastric incubation, respectively. When compared to other polymers, succinylated β-lg promoted the best survival both upon compression and after simulated gastric passage. The proportion of succinylated β-lg in the formulation could be lowered to 60% without modifying the protective ability of the matrix. Additionally, the tablets proved to be stable over a period of 3 months when refrigerated. Succinylated β-lg tablets are an interesting vehicle for the protection of acid-sensitive bacteria during transit in the upper gastro-intestinal tract.
Keywords: β-Lactoglobulin; Succinylation; Probiotic; Bifidobacteria; Tablet;

Solution interactions of diclofenac sodium and meclofenamic acid sodium with hydroxypropyl methylcellulose (HPMC) by Samuel R. Pygall; Peter C. Griffiths; Bettina Wolf; Peter Timmins; Colin D. Melia (55-62).
Many pharmaceutical agents require formulation in order to facilitate their efficacious delivery. However, the interaction between the active species and the formulation additives has the potential to significantly influence the pharmocokinetics of the active. In this study, the solution interactions between hydroxypropyl methylcellulose (HPMC) with two non-steroidal anti-inflammatories – the sodium salts of diclofenac and meclofenamate – were investigated using tensiometric, rheological, NMR, neutron scattering and turbidimetric techniques. The two drugs behaved very differently—meclofenamate addition to HPMC solutions led to substantial increases in viscosity, a depression of the gel point and a marked reduction in the self-diffusion coefficient of the drug, whereas diclofenac did not induce these changes. Collectively, these observations are evidence of meclofenamate forming self-assembled aggregates on the HPMC, a phenomenon not observed with diclofenac Na. Any process that leads to aggregation on a nonionic polymer will not be strongly favoured when the aggregating species is charged. Thus, it is hypothesised that the distinction between the two drugs arises as a consequence of the tautomerism present in meclofenamate that builds electron density on the carbonyl group that is further stabilised by hydrogen bonding to the HPMC. This mechanism is absent in the diclofenac case and thus no interaction is observed. These studies propose for the first time a molecular basis for the observed often-unexpected, concentration-dependant changes in HPMC solution properties when co-formulated with different NSAIDs, and underline the importance of characterising such fundamental interactions that have the potential to influence drug release in solid HPMC-based dosage forms.
Keywords: HPMC; Molecular interaction; PGSE-NMR; Drug; SANS;

The aim of this work was to develop an integrated process analytical technology (PAT) approach for a dynamic pharmaceutical co-precipitation process characterization and design space development. A dynamic co-precipitation process by gradually introducing water to the ternary system of naproxen–Eudragit L100–alcohol was monitored at real-time in situ via Lasentec FBRM and PVM. 3D map of count-time-chord length revealed three distinguishable process stages: incubation, transition, and steady-state. The effects of high risk process variables (slurry temperature, stirring rate, and water addition rate) on both derived co-precipitation process rates and final chord-length-distribution were evaluated systematically using a 33 full factorial design. Critical process variables were identified via ANOVA for both transition and steady state. General linear models (GLM) were then used for parameter estimation for each critical variable. Clear trends about effects of each critical variable during transition and steady state were found by GLM and were interpreted using fundamental process principles and Nyvlt's transfer model. Neural network models were able to link process variables with response variables at transition and steady state with R 2 of 0.88–0.98. PVM images evidenced nucleation and crystal growth. Contour plots illustrated design space via critical process variables’ ranges. It demonstrated the utility of integrated PAT approach for QbD development.
Keywords: Quality-by-Design (QbD); Process analytical technology (PAT); Co-precipitation; Real-time process monitoring; Critical process variable; Design space;

The purpose of the present study was to investigate the prediction accuracy of the fully mechanistic gastrointestinal unified theoretical (GUT) framework for in vivo oral absorption of low solubility drugs. Solubility in biorelevant media, molecular weight, log  P oct , pK a, Caco-2 permeability, dose and particle size were used as the input parameters. To neglect the effect of the low stomach pH on dissolution of a drug, the fraction of a dose absorbed (Fa%) of undissociable and free acids were used. In addition, Fa% of free base drugs with the high pH stomach was also included to increase the number of model drugs. In total twenty nine structurally diverse compounds were used as the model drugs. Fa% data at several doses and particle sizes in humans and dogs were collated from the literature (total 110 Fa% data). In approximately 80% cases, the prediction error was within 2 fold, suggesting that the GUT framework has practical predictability for drug discovery, but not for drug development. The GUT framework appropriately captured the dose and particle size dependency of Fa% as the particle drifting effect was taken into account. It should be noted that the present validation results cannot be applied for salt form cases and other special formulations such as solid dispersions and emulsion formulations.
Keywords: Solubility; Permeability; Dissolution; Bioavailability; Fraction of a dose absorbed; Simulation;

Assembled modules technology for site-specific prolonged delivery of norfloxacin by Paulo Renato Oliveira; Larissa Sakis Bernardi; Orazio Luca Strusi; Salvatore Mercuri; Marcos A. Segatto Silva; Paolo Colombo; Fabio Sonvico (90-96).
Floating modular drug delivery system for the release of norfloxacin in the upper GI tract.The aim of this research was to design and study norfloxacin (NFX) release in floating conditions from compressed hydrophilic matrices of hydroxypropylmethylcellulose (HPMC) or poly(ethylene oxide) (PEO). Module assembling technology for drug delivery system manufacturing was used. Two differently cylindrical base curved matrix/modules, identified as female and male, were assembled in void configuration by friction interlocking their concave bases obtaining a floating release system. Drug release and floatation behavior of this assembly was investigated.Due to the higher surface area exposed to the release medium, faster release was observed for individual modules compared to their assembled configuration, independently on the polymer used and concentration. The release curves analyzed using the Korsmeyer exponential equation and Peppas & Sahlin binomial equation showed that the drug release was controlled both by drug diffusion and polymer relaxation or erosion mechanisms. However, convective transport was predominant with PEO and at low content of polymers. NFX release from PEO polymeric matrix was more erosion dependent than HPMC. The assembled systems were able to float in vitro for up to 240 min, indicating that this drug delivery system of norfloxacin could provide gastro-retentive site-specific release for increasing norfloxacin bioavailability.
Keywords: Norfloxacin; Dome Matrix®; Release modules; Floating dosage form;

Propolis as potential cosmeceutical sunscreen agent for its combined photoprotective and antioxidant properties by Elena Gregoris; Sabrina Fabris; Mariangela Bertelle; Luigi Grassato; Roberto Stevanato (97-101).
Propolis, bee glue, and its main polyphenolic components show high antioxidant activity as found measuring their inhibitory action on lipid peroxidation of linoleic acid (LA) in sodium dodecyl sulfate (SDS) micelles. Furthermore, these substances evidence effectiveness as broad spectrum UVB and UVA photoprotection sunscreens, as it results by measurements of sun protection factor (SPF), the universal indicator related primarily to UVB radiations, and of the two parameters giving an indication of the UVA absorbance properties, i.e. UVA/UVB ratio and critical wavelength.The combination of these characteristics moves up propolis and its main polyphenolic components to the class of cosmeceuticals, as possible active ingredient of sunscreen commercial formulations for their protective and preventive properties.
Keywords: Propolis; Flavonoids; Lipid peroxidation; Broad spectrum sunscreen;

A controlled release resinate beads of betahistine diHCl (BHCl), a short half-life freely water soluble drug, was developed to allow once-daily administration to improve patient compliance and eliminate the risk of intolerance of the drug. BHCl–resin complex was subsequently coated with Eudragit® RS100. A 24 full factorial design was employed for optimization and to explore the effect of Eudragit® RS100 concentration in the coating solution (X 1), the coating percentage (X 2), the speed of rotation (X 3) and the concentration of plasticizer (PEG 400) (X 4) on the release rate of the drug from the microcapsules. The extent of coating (Y 1), and the percentage drug released at given times (Y 2, Y 3 and Y 4) were selected as dependent variables. The optimization process was performed for X 1, X 2, X 3 and X 4 using target ranges of these responses determined based on target release model deduced form zero-order dissolution profile of BHCl for once-daily administration. The levels of X 1, X 2, X 3 and X 4 of optimized BHCl microcapsules are 14.42%, 50.63%, 1495 rpm and 9.94%, respectively. The calculated value of f 2 for the optimized BHCl microcapsules filled into hard gelatin capsules was 67.03 indicating that the dissolution profiles of the optimized formulation is comparable to that of the target release model. It could be concluded that a promising once-daily extended-release microcapsules of the highly water soluble drug, BHCl, was successfully designed.
Keywords: Betahistine; Ménière's disease; Microcapsules; Ion exchange resin; Full factorial design; Optimization;

Method of modelling the compaction behaviour of cylindrical pharmaceutical tablets by Norhayati Ahmat; Hassan Ugail; Gabriela González Castro (113-121).
The mechanisms involved for compaction of pharmaceutical powders have become a crucial step in the development cycle for robust tablet design with required properties. Compressibility of pharmaceutical materials is measured by a force–displacement relationship which is commonly analysed using a well known method, the Heckel model. This model requires the true density and compacted powder mass value to determine the powder mean yield pressure. In this paper, we present a technique for shape modelling of pharmaceutical tablets based on the use of partial differential equations (PDEs). This work also presents an extended formulation of the PDE method to a higher dimensional space by increasing the number of parameters responsible for describing the surface in order to generate a solid tablet. Furthermore, the volume and the surface area of the parametric cylindrical tablet have been estimated numerically. Finally, the solution of the axisymmetric boundary value problem for a finite cylinder subject to a uniform axial load has been utilised in order to model the displacement components of a compressed PDE-based representation of a tablet. The Heckel plot obtained from the developed model shows that the model is capable of predicting the compaction behaviour of pharmaceutical materials since it fits the experimental data accurately.
Keywords: PDE method; Parametric surfaces; Axisymmetric deformation; Compression and compaction; Heckel model;

Impact of excipients on coating efficiency in dry powder coating by Martina Smikalla; Axel Mescher; Peter Walzel; Nora Anne Urbanetz (122-131).
The coating efficiency of dry powder coating can be increased by using excipients having a high spreadability resp. low contact angle on the polymer.Dry powder coating is a technique to coat substrates without the use of organic solvent or water. The polymer powder is directly applied to the cores to be coated. Liquid additives are often used to lower the glass transition temperature of the polymer and to enhance the adhesion of the powder to the cores. This leads to an increase in coating efficiency of the process.The impact of various liquid additives and their properties like spreading behavior, viscosity and plasticizing activity were investigated with respect to their influence on the coating efficiency of the process. Ethylcellulose and hydroxypropyl methylcellulose acetate succinate were used as coating polymers. Spreading behavior of the liquid additive on the polymer was the most influencing parameter and could be successfully predicted with contact angle measurements on polymer films. Calculations of works of adhesion and spreading coefficients also revealed to be promising predictive techniques for choosing suitable additives to improve process efficiency. Isopropyl myristate showed the best spreading behavior resulting in the highest coating efficiency.Based on these results, a formulation for ethylcellulose containing isopropyl myristate was developed and film formation was examined using dissolution testing and imaging techniques to evaluate the optimum curing conditions.
Keywords: Dry powder coating; Rotary fluid bed; Contact angle; Surface energy; Coating efficiency; Ethylcellulose; Sustained release;

Equilibrium drug solubility measurements in 96-well plates reveal similar drug solubilities in phosphate buffer pH 6.8 and human intestinal fluid by Tiina Heikkilä; Milja Karjalainen; Krista Ojala; Kirsi Partola; Frank Lammert; Patrick Augustijns; Arto Urtti; Marjo Yliperttula; Leena Peltonen; Jouni Hirvonen (132-136).
The 96-well plate equilibrium drug solubility results in human intestinal fluid and aqueous buffer at pH 6.8.This study was conducted to develop a high throughput screening (HTS) method for the assessment of equilibrium solubility of drugs. Solid-state compounds were precipitated from methanol in 96-well plates, in order to eliminate the effect of co-solvent. Solubility of twenty model drugs was analyzed in water and aqueous solutions (pH 1.2 and 6.8) in 96-well plates and in shake-flasks (UV detection). The results obtained with the 96-well plate method correlated well (R 2  = 0.93) between the shake-flask and 96-well plates over the wide concentration scale of 0.002–169.2 mg/ml. Thereafter, the solubility tests in 96-well plates were performed using fasted state human intestinal fluid (HIF) from duodenum of healthy volunteers. The values of solubility were similar in phosphate buffer solution (pH 6.8) and HIF over the solubility range of 102–105  μg/ml. The new 96-well plate method is useful for the screening of equilibrium drug solubility during the drug discovery process and it also allows the use of human intestinal fluid in solubility screening.
Keywords: Equilibrium drug solubility; High throughput screening; 96-well plate; Human intestinal fluid; UV-detection;

Enhanced efficacy of TD53, a novel algicidal agent, against the harmful algae via the liposomal delivery system by Hyo-Kyung Han; Yeon-Mi Kim; Soo-Jeong Lim; Soon-Seok Hong; Seul-Gi Jung; Hoon Cho; Wonjae Lee; EonSeon Jin (137-141).
The liposomal delivery system for TD53, a novel algicial drug appeared to be effective to enhance the efficacy of TD53 without any significant loss of selectivity to harmful algae.The present study aimed to design the liposomal delivery system for TD53, a novel algicial drug in order to improve the delivery properties of TD53 and evaluate its algicidal effects as well as selectivity against harmful and non-harmful algae. Liposomes of TD53 were prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by a lyophilization, resulting in relatively small size vesicles (234 ± 38 nm) and narrow size distribution (PI = 0.130 ± 0.027). The drug leakage from the liposome was negligible in the F/2 media (<2% during 96 h incubation). Subsequently algicidal activity of liposomal TD53 against harmful and nonharmful algae was evaluated at various concentrations. The IC50 values of TD53 in liposome against harmful algae such as Chattonella marina, Heterosigma akashiwo and Cocholodinium polykrikoides were 2.675, 2.029, and 0.480 μM, respectively, and were reduced by approximately 50% compared to those obtained from non-liposomal TD53. In contrast, the algicidal effect of liposomal TD53 was insignificant against non-harmful algae including Navicula pelliculosa, Nannochloropsis oculata and Phaeodactylum EPV. Those results suggested that liposomal delivery systems might be effective to enhance the efficacy of TD53 while maintaining the selectivity to harmful algal species.
Keywords: Harmful algae; Liposome; Algicidal activity; TD53; Delivery system;

Proniosomes as a carrier system for transdermal delivery of tenoxicam by H.O. Ammar; M. Ghorab; S.A. El-Nahhas; I.M. Higazy (142-152).
Effect of the transdermal tenoxicam proniosome gel formulation on inhibition of oedema in the hind paw of rats.Tenoxicam is a non steroidal anti-inflammatory drug (NSAID) widely used in the treatment of rheumatic diseases and characterized by its good efficacy and less side effects compared to other NSAIDs. Its oral administration is associated with severe side effects in the gastrointestinal tract. Transdermal drug delivery has been recognized as an alternative route to oral delivery. Proniosomes offer a versatile vesicle delivery concept with the potential for drug delivery via the transdermal route. In this study, different proniosomal gel bases were prepared, characterized by light microscopy, revealing vesicular structures, and assessed for their drug entrapment efficiency, stability, their effect on in vitro drug release and ex vivo drug permeation. The lecithin-free proniosomes prepared from Tween 20:cholesterol (9:1) proved to be stable with high entrapment and release efficiencies. The in vivo behaviour of this formula was studied on male rats and compared to that of the oral market product. The investigated tenoxicam loaded proniosomal formula proved to be non-irritant, with significantly higher anti-inflammatory and analgesic effects compared to that of the oral market tenoxicam tablets.
Keywords: Tenoxicam; Transdermal; Proniosomes; Anti-inflammatory;

Adsorption behavior of epirubicin hydrochloride on carboxylated carbon nanotubes by Zhe Chen; Dramou Pierre; Hua He; Shuhua Tan; Chuong Pham-Huy; Hao Hong; Jilong Huang (153-161).
Compared with MWNTs and SWNTs, c-MWNTs exhibit the highest adsorption efficiency, which could also stay stable with the increasing EPI concentration.The aim of this study was to understand the interaction between carboxylated carbon nanotubes (c-CNTs) and anticancer agents and evaluate the drug-loading ability of c-CNTs. We prepared carboxylated multi-walled carbon nanotubes (c-MWNTs) with nitric acid treatment, then evaluated the adsorption ability of c-MWNTs as adsorbents for loading of the anticancer drug, epirubicin hydrochloride (EPI), and investigated the adsorption behavior of EPI on c-MWNTs. Unmodified multi-walled carbon nanotubes (MWNTs) and single-walled carbon nanotubes (SWNTs) were included as comparative adsorbents. The results showed that carbon nanotubes were able to form supramolecular complexes with EPI via π–π stacking and possessed favorable loading properties as drug carriers. The Freundilich adsorption model was successfully employed to describe the adsorption process. Because of the high surface area and hydrogen bonding, c-MWNTs’ adsorption efficiency was the highest and the most stable and their drug-loading capacity was superior to that of MWNTs. With the increase of pH, the adsorption capacity of EPI on the c-MWNTs increased. Low-temperature facilitated the adsorption. More rapid EPI adsorption rate and higher drug-loading ability were observed from c-MWNTs with smaller diameter. Moreover, the adsorption kinetics of EPI on c-MWNTs could be well depicted by using the pseudo-second-order kinetic model.
Keywords: Carboxylated carbon nanotubes; Epirubicin hydrochloride; π–π stacking; Adsorption; Drug delivery;

The high content of surfactants is one of the major limits to microemulsions (MEs) use in pharmaceutical and cosmetic field. In this work MEs with low surfactant content were prepared by the phase inversion temperature (PIT) method using different oil phases and emulsifiers. The effects of vehicle composition on in vitro release and skin permeation of octylmethoxycinnamte (OMC), one of the most used UVB filter, was evaluated. These MEs showed droplet sizes in the range 32–77 nm and a single peak in size distribution. MEs prepared using the most lipophilic lipids (decyl oleate or cetyl stearyl isononanoate) showed the lowest stability. In vitro release and skin permeation profiles were affected by both lipophilicty and structure of the lipid used as internal phase and the formulation that released the lowest amount of OMC provided the lowest active compound skin permeation. It is noteworthy that no OMC release and skin permeation were observed using oleth-20/glyceryl oleate as emulsifiers. Furthermore, a skin permeation enhancement effect was observed depending on the vehicle components. The results of this work suggest that PIT MEs could provide controlled skin drug delivery by choosing proper associations of oil phase lipids and emulsifiers.
Keywords: Microemulsions; Phase inversion temperature; Oil phase lipophilicity; Non ionic surfactants; Octylmethoxycinnamate;

Particle formation of budesonide from alcohol-modified subcritical water solutions by Adam G. Carr; Raffaella Mammucari; Neil R. Foster (169-180).
Recently, subcritical water (SBCW: water that has been heated to a temperature between 100 °C and 200 °C at pressures of up to 70 bar) has been used to dissolve several hydrophobic pharmaceutical compounds (). Furthermore, a number of active pharmaceutical ingredients (APIs) have been rapidly precipitated from SBCW solutions (). It is possible to alter the precipitate morphology by altering the processing variables; including the SBCW-API solution injection temperature and adding impurities (such as pharmaceutical excipients, e.g. lactose) to the precipitation chamber.The work presented in this article demonstrates that the morphology of pharmaceutical particles can be tuned by adding organic solvents (ethanol and methanol) to the SBCW-API solutions. Particle morphology has also been tuned by adding different pharmaceutical excipients (polyethylene glycol 400 and lactose) to the precipitation chamber. Different morphologies of pharmaceutical particles were produced, ranging from nanospheres of 60 nm diameter to 5 μm plate particles. Budesonide was used as the model API in this study. Two experimental products were spray dried to form dry powder products. The aerodynamic particle size of the powder was established by running the powder through an Andersen Cascade Impactor. It has been shown that the drug particles produced from the SBCW micronization process, when coupled with a spray drying process, are suitable for delivery to the lungs.
Keywords: Subcritical water; Budesonide; Micronization; Spray drying; Inhalation;

Anionic ferrofluid was encapsulated in 200 nm-diameter liposomes. The process involved phase-reverse evaporation followed by sequential extrusion. Magnetoliposomes were characterized by transmission electron microscopy, Doppler laser electrophoresis, SQUID magnetometry, dynamic light scattering and iron content by atomic absorption spectrophotometry. The absence of hysteresis of the magnetic power of particles at room temperature is characteristic of a material with superparamagnetic properties. The encapsulation efficiency was determined for several iron/phospholipid ratios, and this parameter ranged from 0.016 to 0.024 mg iron per mmole of phospholipids, depending on the initial magnetite concentration. In comparison with magnetoliposomes that were obtained solely by extrusion, this method afforded significantly better encapsulation (P  = 0.0002). Magnetic particles were intravenously administered to healthy or inflammation-induced mice. After 1 h, the content of iron was determined in exudates, liver, spleen and plasma. Magnetoliposomes accumulated in the exudates collected from the inflammation site, which suggests that these particles could be loaded with the drugs needed to treat some inflammatory processes.
Keywords: Biomaterials; Inflammation; Liposomes; Magnetic properties; Magnetic materials;

Gene silencing mediated by RNA interference (RNAi) presents a promising strategy for gene therapy. The aim of this work is to evaluate a new gene delivery system for downregulation of survivin expression and enhanced chemosensitivity of MCF-7 cells to adriamycin (ADR). A new cationic poly(2-dimethylaminoethylamine/2-(2-aminoethyoxy)ethoxy)phosphazene (PDMAE) with multiple amino groups was synthesized through Michael addition for survivin shRNA (shSur) delivery in MCF-7 cells. PDMAE51/shSur complex nanoparticles with the size of 190 nm and zeta potential of +15 mV achieved maximal suppression of survivin, even superior to PEI25K or poly(2-(2-aminoethyoxy)ethoxy)phosphazene (PAEP) based complex nanoparticles. The significant downregulation of survivin expression was achieved by PDMAE51/shSur nanoparticles. The nuclear localization by confocal laser scanning microscopy (CLSM) and apparent apoptosis peak of cell cycle in MCF-7 cells were observed when transfected by PDMAE51/shSur nanoparticles The combined use of PDMAE51/shSur and ADR enhanced the sensitivity of MCF-7 cells to ADR at a larger extent than that of PEI or PAEP based complex nanoparticles. These results suggested that PDMAE51 could be potential as an efficient and safe gene carrier in RNAi therapeutics and tumor chemotherapy.
Keywords: RNAi; shRNA; Adriamycin; Survivin; Poly(2-dimethylaminoethylamine/2-(2-aminoethyoxy)ethoxy)phosphazene (PDMAE);

Evaluation of a multi-functional nanocarrier for targeted breast cancer iNOS gene therapy by Helen O. McCarthy; Alek V. Zholobenko; Yuhua Wang; Brenda Canine; Tracy Robson; David G. Hirst; Arash Hatefi (196-202).
The present study determines whether the novel designer biomimetic vector (DBV) can condense and deliver the cytotoxic iNOS gene to breast cancer cells to achieve a therapeutic effect. We have previously shown the benefits of iNOS for cancer gene therapy but the stumbling block to future development has been the delivery system.The DBV was expressed, purified and complexed with the iNOS gene. The particle size and charge were determined via dynamic light scattering techniques. The toxicity of the DBV/iNOS nanoparticles was quantified using the cell toxicity and clonogenic assays. Over expression of iNOS was confirmed via Western blotting and Griess test.The DBV delivery system fully condensed the iNOS gene with nanoparticles less than 100 nm. Transfection with the DBV/iNOS nanoparticles resulted in a maximum of 62% cell killing and less than 20% clonogenicity. INOS overexpression was confirmed and total nitrite levels were in the range of 18 μM.We report for the first time that the DBV can successfully deliver iNOS and achieve a therapeutic effect. There is significant cytotoxicity coupled with evidence of a bystander effect. We conclude that the success of the DBV fusion protein in the delivery of iNOS in vitro is worthy of future in vivo experiments.
Keywords: iNOS; Gene therapy; Designer biomimetic vector; Breast cancer;

Performance evaluation of PAMAM dendrimer based simvastatin formulations by Hitesh Kulhari; Deep Pooja; S.K. Prajapati; Abhay Singh Chauhan (203-209).
The basic purpose of this research was to evaluate the performance of three different G4 poly (amidoamine) (PAMAM) dendrimers to be used as drug carriers and to simultaneously develop the controlled release formulations of lipid lowering drug simvastatin.The purpose of this investigation was to evaluate the performance of poly (amidoamine) (PAMAM) dendrimers, with three different surface groups, to be used as drug carriers. Drug–dendrimers complexes were investigated for solubility studies, dissolution studies, in vitro drug release studies, and for stability studies. The solubility enhancement was found maximum with PEGylated dendrimers (33 times) followed by amine (23 times) and hydroxyl (17.5 times) dendrimers. The solubility profile of simvastatin–dendrimer complex showed a linear correlation (Higuchi AL-type diagram) between solubility and dendrimers concentration. The formation of the complexes between drug molecules and dendrimers were characterized by the FTIR spectra of these complexes, showing the appearance of the bond formed between the functional groups of the drug (OH and COOH) and dendrimers (NH2 and OH). The drug–dendrimer complexes displayed the controlled release action during in vitro release studies. Pure simvastatin (SMV) was released in 5 h whereas the PEGylated dendrimers–SMV complexes released the drug up to 5 days. The non-PEGylated formulations released the drug up to 24 h. Formulations with amine and PEGylated dendrimers were subjected to accelerated stability studies. Formulations with amine dendrimers were found to be most stable in dark, low temperature (0 °C) whereas the dark, RT was most suitable storage conditions for formulation with PEGylated dendrimers.
Keywords: Poly (amidoamine) dendrimers; Simvastatin; Surface groups; Complex;

Stability of liposomes containing bio-enhancers and tetraether lipids in simulated gastro-intestinal fluids by Johannes Parmentier; Matthias M.M. Becker; Udo Heintz; Gert Fricker (210-217).
Stable EPC/cholesterol based liposomes with bio-enhancers and tetraether lipids can be formed. They show a low release of FITC-dextran in pH 2 buffer, sodium taurocholate and pancreatin.The stability of egg phosphatidylcholine (EPC) and cholesterol (Chol) based liposomes and liposomes with the addition of the tetraether lipid glycerylcaldityl tetraether (GCTE) and the bio-enhancers cholylsarcosine, octadecanethiol and TPGS 1000 in Tris buffer pH 2, sodium taurocholate 10 mM and pancreatin was compared. At pH 2 all formulations released nearly 100% of the small hydrophilic fluorescent marker carboxyfluorescein (CF) within the first 10 min, whereas they were mostly stable in size as confirmed by dynamic light scattering (DLS) measurements. Also leakage of the macromolecule FITC-dextran 70 kDa over 60 min at pH 2 was at most 23.9%. After 20 min in 10 mM sodium taurocholate vesicles without GCTE showed a release of CF between 84.0% and 89.5%. In contrast, GCTE-stabilised formulations after 90 min in sodium taurocholate exhibited a CF release between 36.6% and 69.0% depending on the addition of bio-enhancers. Pancreatin had a minor influence on liposome stability in all assays. It is possible to form EPC/Chol vesicles containing different types of bio-enhancers and to stabilise them with GCTE against bile salts. This type of liposomes could be a versatile tool for the oral delivery of drug substances with poor stability in the GI tract and low permeability.
Keywords: Oral delivery; Liposomes; Enhancers; Tetraether lipids; Dynamic light scattering;

Simple and easy methods to prepare oral nanosuspension of a poorly water-soluble pharmaceutical candidate compound, called a candidate, have been developed to support the discovery and preclinical studies using animals. The different wet-milling processes in miniature, middle and large preparation scales have been established in order to cover the various types of studies with wide scale. The powder of phenytoin, a poorly water-soluble model drug candidate, was suspended in the aqueous medium, in which the appropriate dispersing agents were dissolved, and milled by agitating together with small hard beads made of zirconia. Three general-purpose equipments with stirring, oscillating and turbulent motions were applied instead of the specific milling machine with high power to avoid much investment at such early development stage. The operational condition and dispersing agents were optimized to obtain finer particles using the middle-scaled oscillating beads-milling apparatus in particular. It was found that the nanosuspension, which whole particle distribution was in the submicron range, was successfully produced within the running time around 10 min. By applying the newly developed dispersing medium, the nanoparticles with identical size distribution were also prepared using the stirring and turbulent methods on miniature and large scales, respectively; indicating only 50 mg to 30 g or more amount of candidate could be milled to nanosuspension using three equipments. The crystalline analysis indicated that the both crystal form and crystallinity of the original bulk drug completely remained after wet-milling process. The results demonstrated that the wet-milling methods developed in this research would be a fundamental technique to produce nanosuspension for poorly water-soluble and oral absorbable drug candidates.
Keywords: Wet-milling; Nanosuspension; Oscillating beads-mill; Oral formulation; Manufacturing scale; Discovery and preclinical researches;

Serial MRI study of the enhanced therapeutic effects of liposome-encapsulated citicoline in cerebral ischemia by Pedro Ramos-Cabrer; Jesús Agulla; Bárbara Argibay; María Pérez-Mato; José Castillo (228-233).
Liposome encapsulation of active principles enhances their bioavailability to the brain. We investigated whether encapsulation of citicoline in liposomes increases its therapeutic effects in ischemia, performing a longitudinal MRI study of lesion volumes and edema in an animal model of stroke. Nineteen rats were submitted to permanent occlusion of the middle cerebral artery and treated with: (1) saline, (2) intraperitoneal citicoline (500 mg/kg), (3) intravenous citicoline (48 mg/kg), and (4) intravenous liposome-encapsulated citicoline (48 mg/kg). Lesion volumes were measured by MRI at days 0, 1, 3 and 7 following surgery. Encapsulation in liposomes increased the therapeutic effects of citicoline, as reflected by a 32% reduction of the infarct sizes at day 7, in contrast with controls where infarct sizes at day 7 increased by 39%, respect to values at day 0. Intravenously injected citicoline reduced infarct sizes by 9% while intraperitoneal citicoline resulted in an increase of infarct sizes by 10%. A slight (not significant) reduction of edema formation was observed for animals treated with citicoline, in all of its delivery forms. Liposome-encapsulated citicoline causes a noticeable reduction in lesion volumes as compared to free citicoline (either i.p. or i.v.) at days 1, 3 and 7 following permanent stroke.
Keywords: Stroke; Citicoline; CDP-choline; Liposomes; MRI;