International Journal of Pharmaceutics (v.438, #1-2)

Pigment epithelium-derived factor gene loaded in cRGD–PEG–PEI suppresses colorectal cancer growth by targeting endothelial cells by Lei Li; Jun Yang; Wei-Wei Wang; Ya-Chao Yao; Shu-Huan Fang; Zhi-Yu Dai; Hong-Hai Hong; Xia Yang; Xin-Tao Shuai; Guo-Quan Gao (1-10).
Pigment epithelium-derived factor (PEDF) recombinant protein has been investigated in many kinds of solid tumors due to its potent antiangiogenic activity. However, the complexity of protein purification, instability of recombinant protein and requirement of repeated injections are obstacles for the recombinant PEDF therapy for solid tumors. We successfully synthesized polyethyleneglycol–polyetherimide (PEG–PEI) and cRGD–PEG–PEI which was coupled with a cyclic RGD peptide, a special ligand for integrin αvβ3 receptor, as the vehicle for PEDF gene therapy in this study. In vitro, the competitive binding assay showed that cRGD contributed to the enhanced gene transfection efficiency of PEG–PEI in human umbilical vein endothelial cells (HUVECs). PEDF gene delivered by cRGD–PEG–PEI apparently suppressed growth of tumor with a 67.4% reduction and decreased microvessel density in nude mice bearing SW620 human colorectal xenografts. Accordingly, SW620 tumors from cRGD–PEG–PEI/PEDF–pcDNA3.1 (+)-treated mice expressed more PEDF than that of the control groups. Our study demonstrated that cRGD–PEG–PEI transported the PEDF gene into endothelia cells more efficiently than PEG–PEI, resulting in more effective inhibitory effects on tumor growth by anti-angiogenesis. Therefore, for the first time, we have explored an effective non-viral vehicle for PEDF gene therapy by targeting endothelial cells.
Keywords: PEDF; PEG–PEI; cRGD; HUVECs; Gene therapy;

Preparation and characterization of fusion processed solid dispersions containing a viscous thermally labile polymeric carrier by Justin R. Hughey; Justin M. Keen; Dave A. Miller; Chris Brough; James W. McGinity (11-19).
The primary aim of the present study was to investigate the ability of hydroxypropyl and methoxyl substituted cellulose ethers to stabilize supersaturated concentrations of itraconazole (ITZ), a poorly water-soluble weak base, after an acid-to-neutral pH transition. A secondary aim of the study was to evaluate the effect of fusion processes on polymer stability and molecular weight. Polymer screening studies showed that stabilization of ITZ supersaturation was related to the molecular weight of the polymer and levels of hydroxypropyl and methoxyl substitution. METHOCEL™ E50LV (E50LV), which is characterized as having a high melt viscosity, was selected for solid dispersion formulation studies. Hot-melt extrusion processing of E50LV based compositions resulted in high torque loads, low material throughput and polymer degradation. KinetiSol® Dispersing, a novel fusion based processing technique, was evaluated as a method to prepare the solid dispersions with reduced levels of polymer degradation. An experimental design revealed that polymer molecular weight was sensitive to shearing forces and high temperatures. However, optimal processing conditions resulted in significantly reduced E50LV degradation relative to HME processing. The technique was effectively utilized to prepare homogenous solid solutions of E50LV and ITZ, characterized as having a single glass transition temperature over a wide range of drug loadings. All prepared compositions provided for a high degree of ITZ supersaturation stabilization.
Keywords: Poorly water soluble; Polymer stability; Hydroxypropyl methylcellulose; Hot melt extrusion; KinetiSol Dispersing;

Twin screw granulation: Steps in granule growth by Ranjit M. Dhenge; James J. Cartwright; Michael J. Hounslow; Agba D. Salman (20-32).
The present work focuses on the study of the progression of granules in different compartments along the length of screws in a twin screw granulator (TSG). The effects of varying powder feed rate; liquid to solid ratio and viscosity of granulation liquid on properties of granules was studied. The bigger granules produced at the start of the process were found to change in terms of size, shape and strength along the screw length at all the conditions investigated. The granules became more spherical and their strength increased along the screw length. Tracer granules were also introduced in order to understand the role of kneading and conveying elements in the TSG. The kneading elements promoted consolidation and breakage while the conveying elements led to coalescence, breakage and some consolidation. The results presented here help to provide a qualitative and quantitative understanding of the twin screw granulation process.
Keywords: Twin screw; Wet granulation; Viscosity; Growth; Mechanism;

Tablet coating thicknesses were estimated using several techniques such as weight gain and scanning electron microscopy (SEM), in comparison with acoustic microscopy and diffuse reflectance spectroscopy. Acoustic microscopy, used for the first time in such an application, is based on the physical phenomenon of ultrasound propagation through the materials and the echoes generated by their interfaces. Based on the time of flights (TOFs) of the echoes from the coating surface and the tablet, it is possible to calculate the coating thickness. In order to evaluate the accuracy and robustness of these methods, drug tablets were coated with Kollicoat® SR polymer for several times, so that to prepare tablets with different coating thicknesses. Tablets with 3, 6 and 9 wt% coating material have been prepared and based on SEM micrographs it was found that the tablet coating thickness is 71.99 ± 1.2 μm, 92.5 ± 1.7 μm and 132.3 ± 2.1 μm, respectively (SEM analysis). The tablet coating thicknesses measured with acoustic microscopy and infrared diffuse reflectance spectroscopy, were in agreement with those obtained using SEM. This verifies that both techniques can be successfully applied for real time and non-destructive thickness measurements of tablet coating. Furthermore, both techniques, compared with SEM and weight gained measurements, are fast and fully automated.
Keywords: Tablet coating thicknesses; Acoustic microscopy; Infrared diffuse reflectance spectroscopy; Real time analysis;

Sustained in vitro release and cell uptake of doxorubicin adsorbed onto gold nanoparticles and covered by a polyelectrolyte complex layer by L. Minati; V. Antonini; S. Torrengo; M. Dalla Serra; M. Boustta; X. Leclercq; C. Migliaresi; M. Vert; G. Speranza (45-52).
Gold nanoparticles functionalized with doxorubicin and stabilized with multilayers of degradable polyelectrolyte were allowed to age in aqueous medium in vitro in order to show the possibility of drug release in cellular environment. The chemico-physical characteristics of the nanoparticles are reported. The observed release of doxorubicin (DOX) was pH-dependent, and it increased in acidic environment. Cell uptake of nanoparticles and drug release were monitored by laser scanning confocal microscopy. Data showed that drug-bearing nanoparticles delivered DOX into the nuclei of A549 cells, leading to pronounced cytotoxic effects to this lung tumor cells. Our results suggest that gold nanoparticles conjugated with doxorubicin could be used as a pH-triggered drug releasing carrier for tumor drug delivery.
Keywords: Gold colloids; Doxorubicin; Polyelectrolyte coating; Drug delivery;

Development of novel microprecipitated bulk powder (MBP) technology for manufacturing stable amorphous formulations of poorly soluble drugs by Navnit Shah; Harpreet Sandhu; Wantanee Phuapradit; Rodolfo Pinal; Raman Iyer; Antonio Albano; Ashish Chatterji; Shalini Anand; Duk Soon Choi; Kin Tang; Hung Tian; Hitesh Chokshi; Dharmendra Singhal; Waseem Malick (53-60).
A novel method was developed to manufacture amorphous formulations of poorly soluble compounds that cannot be processed with existing methods such as spray drying and melt extrusion. The manufacturing process and the characterization of the resulting amorphous dispersion are presented via examples of two research compounds. The novel process is utilized N,N-dimethylacetamide (DMA) to dissolve the drug and the selected ionic polymer. This solution is then co-precipitated into aqueous medium. The solvent is extracted out by washing and the co-precipitated material is isolated by filtration followed by drying. The dried material is referred to as microprecipitated bulk powder (MBP). The amorphous form prepared using this method not only provides excellent in vitro and in vivo performance but also showed excellent stability. The stabilization of amorphous dispersion is attributed to the high T g, ionic nature of the polymer that help to stabilize the amorphous form by possible ionic interactions, and/or due to the insolubility of polymer in water. In addition to being an alternate technology for amorphous formulation of difficult compounds, MBP technology provides advantages with respect to stability, density and downstream processing.
Keywords: Amorphous; Solid dispersion; Solid solution; MBP; Co-precipitation;

A proposed pathway for the formation of degradation products of trandolapril.Various regulatory authorities like International Conference on Harmonization (ICH), US Food and Drug Administration, Canadian Drug and Health Agency are emphasizing on the purity requirements and the identification of impurities in active pharmaceutical drugs. Qualification of the impurities is the process of acquiring and evaluating data that establishes biological safety of an individual impurity; thus, revealing the need and scope of impurity profiling of drugs in pharmaceutical research.As no stability-indicating method is available for identification of degradation products of trandolapril, a new angiotensin converting enzyme inhibitor (ACEI), under stress testing, the development of an accurate method is needed for quantification and qualification of degradation products. Ultra high performance liquid chromatography (UPLC) coupled to electrospray tandem mass spectrometry was used for the rapid and simultaneous analysis of trandolapril and its degradation products. Chromatographic separation was achieved in less than 4 min, with improved peak resolution and sensitivity. Thanks to this method, the kinetics of trandolapril degradation under various operating conditions and the characterization of the structure of the by-products formed during stress testing have been determined. Thereafter, a mechanism of trandolapril degradation in acid and neutral conditions, including all the identified products, was then proposed.
Keywords: Trandolapril; ICH guideline (Q1A R2); Method development; UPLC–MS/MS; Degradation products; Degradation pathway;

Interaction and destabilization of a monoclonal antibody and albumin to surfaces of varying functionality and hydrophobicity by Ruairidh G. Couston; Dimitrios A. Lamprou; Shahid Uddin; Christopher F. van der Walle (71-80).
Protein products come into contact with many surfaces of differing wettability during bioprocessing, formulation and delivery, but corresponding data for the adsorbed conformations and the associated force of adhesion (F ad) is sparse. Here we have generated a series of hydrophilic and hydrophobic surfaces through silanization of silica with various terminal groups, characterizing the surface energies and droplet contact angles. F ad measured by atomic force microscopy for oriented monolayers of a human monoclonal antibody (mAb-1) clearly distinguished hydrophobic surfaces (low F ad values) from hydrophilic surfaces (high F ad values). High F ad for a methoxy capped polyethylene glycol (1000 MW) surface supports the interaction of mAb-1 with buried ethylene oxide groups, consistent with mAb-1 compression into a distorted brush border. Solid state circular dichroism showed that mAb-1 (β-sheet) or albumin (α-helical) adsorbed to bare silica beads largely retained their secondary structures. However, the extent of structural loss upon protein adsorption to functionalized silica beads could not be simply correlated to hydrophilic/hydrophobic surface interaction as seen for the F ad measurements. For example, of the hydrophilic surfaces mAb-1 unfolded notably more when adsorbed to the aminopropyl surface, and of the hydrophobic surfaces both mAb-1 and albumin retained most secondary structure when adsorbed to the perfluorooctyl surface (consistent with the lipophobic perfluorinated moiety limiting exposure of the protein hydrophobic core). The data show that F ad values are not necessarily predictive of the subsequent extent of structural relaxation, and that significant structural loss is evident for proteins adsorbed to both hydrophilic and hydrophobic surfaces.
Keywords: Monoclonal antibody; Adhesion; Adsorption; Silanization; Solid/liquid interface;

Development of oral taste masked diclofenac formulations using a taste sensing system by Marie Guhmann; Maren Preis; Frédéric Gerber; Norbert Pöllinger; Jörg Breitkreutz; Werner Weitschies (81-90).
To achieve patient compliance, organoleptic aspects of drug substances play a key role in the development of oral pharmaceutical preparations. In this study, the ability of an electronic tongue to help in selecting a drug candidate and rationalize the development of oral taste masked formulations was evaluated. As drug, diclofenac, in the form of acid, sodium salt and potassium salt, was used. The taste sensing system Insent TS-5000Z was capable of differentiating diclofenac acid from its salts; and also distinguishing sodium and potassium salts eliciting similar taste modalities. Differences between qualities and intensities of sensor responses were mainly attributed to the different cations. Fewer taste and aftertaste stimuli were recorded for diclofenac acid. Based on this screening, the acid form was selected to formulate different diclofenac oral taste masked preparations further evaluated using electronic tongue data by comparing formulation prototypes against corresponding placebos. Output information demonstrated the ability of the taste sensors to detect and discriminate different formulation concepts and taste masking strategies. Comparative dissolution studies showed insufficient discrimination of formulation prototypes. Supported by these results, the electronic tongue proved to be a valuable additional tool for assessing and predicting the taste of active pharmaceutical ingredients in the early development stage.
Keywords: Electronic taste sensing system; Formulation development; Diclofenac; Orodispersible tablets; ODT; Taste masking;

Competitive molecular interaction among paeonol-loaded liposomes: Differential scanning calorimetry and synchrotron X-ray diffraction studies by Rui-guang Wu; Jun-dong Dai; Fu-gen Wu; Xiao-hua Zhang; Wei-feng Li; Yu-rong Wang (91-97).
Thermotropic phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes containing 5 mol% cholesterol, or 5 mol% stigmasterol, or 5 mol% paeonol have been investigated by differential scanning calorimetry (DSC) and synchrotron X-ray diffraction (XRD) techniques, to investigate the competitive molecular interaction among paeonol-loaded liposomes. The results show that both sterol and paeonol can incorporate into hydrophobic region and interact with acyl chains of DPPC. Both 5 mol% sterols and 5 mol% paeonol can promote the formation of rippled gel phase of DPPC liposomes at room temperature. 5 mol% paeonol can induce the occurrence of phase separation in DPPC liposomes, but 5 mol% cholesterol or 5 mol% stigmasterol cannot induce this phenomenon. Both the repeat distance and the correlation length of paeonol-poor domain are larger than those of coexisted paeonol-rich domain. Both calorimetric data and SAXS patterns show that sterols have more favorable, stabilizing interactions with DPPC than paeonol, implying that high concentrations of sterols will have a negative effect on the loading of paeonol. In addition, calorimetric data show that cholesterol have a little more favorable, stabilizing interactions with DPPC than stigmasterol. The results of this study will play an important role in optimizing the formulation of paeonol-loaded liposomes.
Keywords: Paeonol; Liposomes; Sterols; Competitive molecular interaction; Differential scanning calorimetry; Synchrotron X-ray diffraction;

Double emulsion has been used most often in formulation of hydrophilic drugs by nanoparticles of biodegradable polymers, which has disadvantages such as low drug loading and low drug encapsulation efficiency due to the drug loss in the process. The drug release may be too fast for sustained chemotherapy. We developed in this research a d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) prodrug micelle system with cisplatin as a model hydrophilic drug. We demonstrated that such a system can successfully deliver the model hydrophilic drug with a low critical micelle concentration (CMC) of only 5.01 mg/L, a high drug load of 4.95% (w/w) and a pH-responsive drug release kinetics and higher cellular uptake in comparison with the original drug and the TPGS-cisplatin prodrug itself. The cell viability experiment showed great enhancement of the cisplatin chemotherapy, which is demonstrated by the IC50 value reduced from 3.95, 0.98, 0.19 for cisplatin to 1.36, 0.51, 0.08 μg/mL for the TPGS prodrug micelle formulation after 24, 48, 72 h culture with the HepG2 hepatocarcinoma cells, respectively. Furthermore, such a TPGS prodrug micellar formulation showed significant neuroprotective effects for the cisplatin chemotherapy, which is demonstrated by the greatly increased IC50 value for the SH-SY5Y neuroblast-like cells in comparison between cisplatin and the TPGS prodrug micelle formulation. The TPGS prodrug micelles can also be generalized to become a new strategy for codelivery of hydrophilic and hydrophobic drugs and/or imaging agents.
Keywords: Anti-cancer drugs; Biodegradable polymers; Cancer nanotechnology; Drug formulation; Nanomedicine;

Impact of gold nanoparticle coating on redox homeostasis by J. Tournebize; A. Boudier; O. Joubert; H. Eidi; G. Bartosz; P. Maincent; P. Leroy; A. Sapin-Minet (107-116).
Gold nanoparticles (AuNP) hold great potential for biomedical applications. This study was aimed at examination of the effect of AuNP coating on the redox status of their environment. Two kinds of AuNP were tested, similar by shape and size, but with different surface coatings: either stabilized with citrate or functionalized with dihydrolipoic acid (Au@DHLA NP). Interestingly, whereas citrate-stabilized AuNP interact in vitro with reduced glutathione (GSH) and S-nitrosoglutathione, Au@DHLA NP do not interfere with both biomolecules. Albumin exhibits higher affinity toward citrate-stabilized AuNP than Au@DHLA NP, increasing their hydrodynamic diameter (8.0- and 1.3-fold, respectively). Furthermore, the AuNP coating affects also their internalization by macrophages (which was two fold higher for citrate-stabilized AuNP), following an exposure to a subtoxic NP concentration (10 nM, 80% viability). Citrate-stabilized AuNP were found to decrease the intracellular GSH level (ca. 20%), with no increase in reactive oxygen species production. Furthermore, these AuNP did not induce apoptosis (as shown by caspase-3 activity and nfkb2 transcription factor), and also did not activate gene expression related to oxidative stress (ncf1) and inflammatory response (tnfα). The present data highlight that the functionalization of AuNP with DHLA decreases their reactivity with biomolecules and cells, resulting in a promising medical platform.
Keywords: Gold nanoparticles; Redox status; Reduced glutathione; S-nitrosoglutathione; Macrophages; Cell uptake;

Electrospun aceclofenac and pantoprazole loaded zein/eudragit nanofibers that individually controls the release of two drugs and reduces the gastritis induced by aceclofenac (NSAIDs).Electrospun composite zein/eudragit nanofibers were developed with an aim to deliver two different classes of drugs simultaneously that would restrict/compensate the adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs). Co-administration of proton pump inhibitors is beneficial for patients consuming NSAIDs for treating chronic ailments like arthritis. In this study, aceclofenac/pantoprazole loaded zein/eudragit S 100 nanofibers were developed using a single nozzle electrospinning process. The morphological analysis revealed the uniform and smooth surface of the drug loaded nanofibers. The physico-thermal characterization of nanofibers depicted the molecular integration of the drugs with the polymers and also confirmed that the drugs were evenly distributed in the nanofibers in an amorphous state. In vitro release studies ensure the efficiency of the developed fibers in sustaining the release of both the drugs up to 8 h. In vivo animal experiments further confirmed that the co-administration of pantoprazole along with aceclofenac reduced the gastro-intestinal toxicity induced by NSAIDs. The histological evaluation revealed the preserved mucosal architecture of rat gastric tissue treated with drug loaded composite nanofibers. Thus, dual drug delivery system comprising polymers with different release characteristics has been successfully developed and further, oral delivery of aceclofenac with reduced side effects was achieved.
Keywords: Composite nanofibers; Drug delivery; Zein; Eudragit S 100; Aceclofenac; Pantoprazole; Gastric toxicity;

Sirolimus solid self-microemulsifying pellets: Formulation development, characterization and bioavailability evaluation by Xiongwei Hu; Chen Lin; Dingxiong Chen; Jing Zhang; Zhihong Liu; Wei Wu; Hongtao Song (123-133).
The prepared solid sirolimus self-microemulsifying pellets had a good appearance and in vitro redispersibility. No crystalline sirolimus was observed in the pellets and the oral bioavailability in beagle dogs was significantly improved relative to the commercial sirolimus tablets.To enhance the dissolution and oral absorption of water insoluble drug sirolimus (SRL), self-microemulsifying pellets of SRL were developed and evaluated. Solubility test, self-emulsifying grading test, ternary phase diagrams and central composite design were adopted to screen and optimize the composition of liquid SRL-SMEDDS. The selected liquid SRL-SMEDDS formulations were prepared into pellets by extrusion–spheronization method and the optimal formulation of 1 mg SRL-SMEDDS pellets capsule (1.0, 22.4, 38.4, 19.2, 121.6, 30.4 and 8.0 mg of SRL, Labrafil M1944CS, Cremophor EL, Transcutol P, MCC, Lactose and CMS-Na, respectively) was finally determinated by the feasibility of the preparing process and redispersibility. The optimal SRL-SMEDDS pellets showed a significant quicker redispersion rate than the dissolution rate of commercial SRL tablets Rapamune® in water. The droplet size and polydispersity index of the reconstituted microemulsion was almost unchanged after solidification, and pellet size and friability were all qualified. Visual observation and scanning electron microscopic analysis confirmed good appearance of the solid pellets. DSC, XRPD, and IR analysis confirmed that there was no crystalline sirolimus in the pellets. Pharmacokinetic study in beagle dogs showed the oral relative bioavailability of SRL-SMEDDS pellets to the commercial SRL tablets Rapamune® was about 136.9%. In conclusion, the solid SMEDDS pellets might be an encouraging strategy to improve the oral absorption of SRL and the extrusion–spheronization method was a feasible technology for the solidification of liquid SMEDDS.
Keywords: Sirolimus; Self-microemulsifying drug delivery system; Solidification; Pellets; Redispersibility; Oral bioavailiability;

Blend uniformity analysis of pharmaceutical products by Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) by Dara Fitzpatrick; Eoin Scanlon; Jacob Krüse; Bastiaan Vos; Rachel Evans-Hurson; Eileen Fitzpatrick; Seán McSweeney (134-139).
Intra and inter-batch comparison of a powder blend.Blend uniformity analysis (BUA) is a routine and highly regulated aspect of pharmaceutical production. In most instances, it involves quantitative determination of individual components of a blend in order to ascertain the mixture ratio. This approach often entails the use of costly and sophisticated instrumentation and complex statistical methods.In this study, a new and simple qualitative blend confirmatory test is introduced based on a well known acoustic phenomenon. Several over the counter (OTC) product powder blends are analysed and it is shown that each product has a unique and highly reproducible acoustic signature. The acoustic frequency responses generated during the dissolution of the product are measured and recorded in real time. It is shown that intra-batch and inter-batch variation for each product is either insignificant or non-existent when measured in triplicate. This study demonstrates that Broadband Acoustic Resonance Dissolution Spectroscopy or BARDS can be used successfully to determine inter-batch variability, stability and uniformity of powder blends. This is just one application of a wide range of BARDS applications which are more cost effective and time efficient than current methods.
Keywords: Blend uniformity; BARDS; Intra-batch variation; Dissolution;

Flux of ionic dyes across microneedle-treated skin: Effect of molecular characteristics by Yasmine A. Gomaa; Martin J. Garland; Fiona J. McInnes; Ryan F. Donnelly; Labiba K. El-Khordagui; Clive G. Wilson (140-149).
Drug flux across microneedle (MN)-treated skin is influenced by the characteristics of the MN array, formed microconduits and physicochemical properties of the drug molecules in addition to the overall diffusional resistance of microconduits and viable tissue. Relative implication of these factors has not been fully explored. In the present study, the in vitro permeation of a series of six structurally related ionic xanthene dyes with different molecular weights (MW) and chemical substituents, across polymer MN-pretreated porcine skin was investigated in relation of their molecular characteristics. Dyes equilibrium solubility, partition coefficient in both n-octanol or porcine skin/aqueous system, and dissociation constants were determined. Results indicated that for rhodamine dyes, skin permeation of the zwitterionic form which predominates at physiological pH, was significantly reduced by an increase in MW, the skin thickness and by the presence of the chemically reactive isothiocyanate substituent. These factors were generally shown to override the aqueous solubility, an important determinant of drug diffusion in an aqueous milieu. The data obtained provided more insight into the mechanism of drug permeation across MN-treated skin, which is of importance to both the design of MN-based transdermal drug delivery systems and of relevance to skin permeation research.
Keywords: Microneedles; Molecular weight; Solubility; Porcine skin; Isothiocyanate; Partition coefficient;

Polymer coating of carrier excipients modify aerosol performance of adhered drugs used in dry powder inhalation therapy by Daniela Traini; Santo Scalia; Handoko Adi; Elisabetta Marangoni; Paul M. Young (150-159).
The potential of excipient coating to enhance aerosol performance of micronized drugs in carrier excipient–drug blends, used in dry powder inhalers, was investigated. Both EC (ethyl cellulose) and PVP (polyvinylpyrrolidone) were used as coating agents. Carriers were prepared via sieve fractioning followed by spray drying, with and without polymer additive. Each uncoated and coated carrier salbutamol sulphate (SS) blended systems were evaluated for particle size, morphology, drug carrier adhesion and aerosolisation performance, after blending and storage for 24 h. All carrier-based systems prepared had similar particle sizes and morphologies. The surface chemistries of the carriers were significantly different, as was drug-carrier adhesion and aerosolisation performance. Particle adhesion between SS and aerosol performance (fine particle fraction; FPF) followed the rank: PVP coated > un-coated > EC coated lactose. This rank order could be attributed to the surface energy measured by contact goniometry and related to the chemistry of lactose and each polymer. Storage did not significantly affect aerosol performance, however a rank increase in mean FPF value was observed for uncoated and EC coated lactose. Finally, the net electrostatic charge across the aerosol cloud indicated that the EC coated lactose transferred less charge to SS particles. The performance of each carrier system could be attributed to the carrier surface chemistry and, in general, by careful selection of the coating polymer, drug-carrier adhesion, electrostatic charge and aerosol performance could be controlled
Keywords: Dry powder inhaler (DPI); Lactose; Salbutamol sulphate; Polyvinylpyrrolidone; Ethyl cellulose; Coating;

Protein spheres prepared by drop jet freeze drying by Sören N. Eggerstedt; Mathias Dietzel; Martin Sommerfeld; Richard Süverkrüp; Alf Lamprecht (160-166).
In spray freeze drying (SFD) solutions are frozen by spraying into a very cold environment and subsequently dried by sublimation. In contrast to conventional freeze drying, spray freeze drying has the possibility to produce flowable lyophilizates which offers a variety of new pharmaceutical applications. Here, a drop jet nozzle is proposed as liquid dispenser that is able to produce droplets with a very narrow size distribution compared to standard methods. The drop jet nozzle is mounted in a spray tower designed to prevent direct contact of the product with the freezing medium. Various formulations have been tested containing lysozyme as model protein and stabilizers such as bovine serum albumin, polyvinylpyrrolidone or dextran in various concentrations and mannitol. Excellent free flowing and nearly monodispersed, porous particles are produced where particle properties can be controlled by formulation and process conditions. The particle diameter varied between 231 ± 3 μm and 310 ± 10 μm depending on the formulation composition. The lysozyme activity was >94 ± 5% for all formulations exhibiting a full preservation of enzyme activity. This new method is very promising for the production of nearly monodisperse particulate lyophilizates in various therapeutic applications.
Keywords: Spray freeze drying; Protein formulations; Lyophilization; Porous particles;

Quality by design: Impact of formulation variables and their interactions on quality attributes of a lyophilized monoclonal antibody by David Awotwe-Otoo; Cyrus Agarabi; Geoffrey K. Wu; Elizabeth Casey; Erik Read; Scott Lute; Kurt A. Brorson; Mansoor A. Khan; Rakhi B. Shah (167-175).
The purpose of this study was to use QbD approaches to evaluate the effect of several variables and their interactions on quality of a challenging model murine IgG3κ monoclonal antibody (mAb), and then to obtain an optimized formulation with predefined quality target product profile.This antibody was chosen because it has a propensity to precipitate and thus represents a challenge condition for formulation development. Preliminary experiments were conducted to rule out incompatible buffer systems for the mAb product quality. A fractional factorial experimental design was then applied to screen the effects of buffer type, pH and excipients such as sucrose, sodium chloride (NaCl), lactic acid and Polysorbate 20 on glass transition temperature ( T ′ g ), monoclonal antibody concentration (A 280), presence of aggregation, unfolding transition temperature (T m) of the lyophilized product, and particle size of the reconstituted product. A Box–Behnken experimental design was subsequently applied to study the main, interaction, and quadratic effects of these variables on the responses.Pareto ranking analyses showed that the three most important factors affecting the selected responses for this particular antibody were pH, NaCl, and Polysorbate 20. The presence of curvature in the variables’ effects on responses indicated interactions. Based on the constraints set on the responses, a design space was identified for this mAb and confirmed with experiments at three different levels of the variables within the design space. The model indicated a combination of high pH (8) and NaCl (50 mM) levels, and a low Polysorbate 20 (0.008 mM) level at which an optimal formulation of the mAb could be achieved. Moisture contents and other analytical procedures such as size exclusion chromatography, protein A analysis and SDS-PAGE of the pre-lyophilized and final reconstituted lyophilized products indicated an intact protein structure with minimal aggregation after formulation and lyophilization.In conclusion, experimental design approach was effective in identifying optimal concentrations of excipients and pH for this challenging monoclonal antibody formulation.
Keywords: Lyophilization; Monoclonal antibody; IgG3κ; Quality by design; Formulation; Design of experiments;

We previously developed a PEG-coated cationic liposome that enabled dual targeting delivery of oxaliplatin (l-OHP) to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation consequently elicited potent antitumor efficacy in a murine solid tumor model after 3 sequential injections. However, the probable mechanism(s) for this enhanced antitumor activity has not been fully elucidated. In the present study, therefore, the changes in tumor microenvironment induced by sequential administration of liposomal l-OHP were investigated, with emphasis on its impact to the intratumoral localization of the subsequently injected dose. In addition, the potential for anti-PEG IgM production upon repeated administration of liposomal l-OHP-containing PEGylated lipid was clearly revealed. Two sequential injections of liposomal l-OHP induced superior apoptotic activity in tumor tissue and thus resulted in broader intratumor distribution of the subsequent test dose of PEG-coated cationic liposomes, compared with a single injection of liposomal l-OHP. In addition, it was confirmed that repeated administration of liposomal l-OHP did not induce a significant anti-PEG IgM response, indicating that l-OHP encapsulated in PEG-coated liposomes was efficient in abrogating the ABC phenomenon. These results suggest that sequential treatment strategies with liposomal cytotoxic agents might be superior to mono-treatment strategies in achieving alterations in the tumor microenvironment and maintaining/restoring the pharmacokinetics of the formulation, and, therefore, would result in substantial therapeutic efficacy.
Keywords: ABC phenomenon; Antitumor activity; Apoptosis; Oxaliplatin; PEG-coated cationic liposomes; Sequential administration;

AFM study of hydrophilicity on acetaminophen crystals by Kalyana C. Pingali; Troy Shinbrot; Alberto Cuitino; Fernando J. Muzzio; Eric Garfunkel; Yevgeny Lifshitz; Adrian B. Mann (184-190).
Pharmaceutical powder processing is notoriously subject to unpredictable jamming, sticking and charging disturbances. To unveil the material science underlying these effects, we use atomic force microscopy (AFM) on a common pharmaceutical, acetaminophen (APAP). Specifically, we study surface adhesion and morphology as a function of relative humidity (RH) for monoclinic acetaminophen, using both plain AFM tips and tips functionalized to be hydrophobic or hydrophilic. Results indicate that the (0 0 1) crystal face exhibits significantly higher adhesion (surface potential) than the other crystal faces. For all the faces clear peaks in adhesion occur at 50–60% RH when they are examined using hydrophilic tips. The surface morphology of some facets showed a strong dependence on RH while others showed little or no significant change. In particular, the morphology of the (1 −1 0) faces developed large terraces at high humidity, possibly due to deliquescence followed by recrystallization. These results confirm the hypothesis that different crystal facets exhibit distinct surface potentials and morphology that change with environmental exposure. The work suggests that future studies of powder behaviors would benefit from a more detailed modeling of crystal surface contact mechanics.
Keywords: Organic crystals; Hydrophobic; Hydrophilic; Humidity; AFM;

Effect of peptides and their introduction methods on target gene transfer of gene vector based on disulfide-containing polyethyleneimine by Hui-Yuan Wang; Yun-Xia Sun; Ji-Zhe Deng; Juan Yang; Ren-Xi Zhuo; Xian-Zheng Zhang (191-201).
To evaluate the effect of different peptides as well as their introduction methods on target gene transfer of gene vectors based on disulfide-containing polyethyleneimine (SS-PEI), a series of peptides including N3-GRGDSF, GRGDSF, and EEEEEEEEGRGDSF (E8GRGDSF) were prepared. N3-GRGDSF was conjugated to SS-PEI by click chemistry and SS-PEI-GRGDSF was obtained. GRGDSF was non-covalently introduced into SS-PEI/DNA mainly through hydrogen bonding to obtain SS-PEI/DNA/GRGDSF complexes, whereas E8GRGDSF was further non-covalently introduced to SS-PEI/DNA through electrostatic force to obtain SS-PEI/DNA/E8GRGDSF complexes. Transfection efficiency of all complexes with peptides was lower than that of SS-PEI/DNA in COS-7 cells due to the fact that nonspecific endocytosis was prohibited after peptide introduction. Whereas in HeLa cells, transfection activity of SS-PEI-GRGDSF/DNA and SS-PEI/DNA/E8GRGDSF at certain w/w ratios was higher than that of SS-PEI/DNA. But the transfection efficiency of SS-PEI/DNA/E8GRGDSF at peptide/DNA w/w ratios higher than 30 dropped due to targeted binding interactions between surplus E8GRGDSF and the integrins in HeLa cells, which would prohibit specific endocytosis of E8GRGDSF in complexes. Transfection activity of SS-PEI/DNA/GRGDSF was lower than or comparable to that of SS-PEI/DNA because of loose complexes constructed by hydrogen bonding between GRGDSF and SS-PEI/DNA.
Keywords: Inclusion complexes; RGD peptide; Targeted gene delivery;

Comparison of the cutaneous iontophoretic delivery of rasagiline and selegiline across porcine and human skin in vitro by Dhaval R. Kalaria; Pratik Patel; Vandana Patravale; Yogeshvar N. Kalia (202-208).
The objective was to investigate the anodal iontophoresis of the MAO-B inhibitors rasagiline (RAS) and selegiline (SEL) across porcine and human skin in vitro. Passive delivery of RAS and SEL from aqueous solution was minimal; however, increasing current density from 0.1 to 0.3 and 0.5 mA/cm2 produced a linear increase in steady-state iontophoretic flux (J ss,RAS  = 49.1i d  + 27.9 (r 2  = 0.96) and J ss,SEL  = 27.8i d  + 25.8 (r 2  = 0.98)). In the absence of background electrolyte, a four-fold change in donor concentration (10, 20 and 40 mM) did not produce a statistically significant increase in cumulative permeation of either drug after iontophoresis at 0.5 mA/cm2 for 7 h. Co-iontophoresis of acetaminophen confirmed that electromigration was the dominant transport mechanism for both drugs (∼90%). Total iontophoretic delivery of RAS and SEL across porcine and human skin in vitro was statistically equivalent (RAS: 1512.7 ± 163.7 and 1523.6 ± 195.9 μg/cm2, respectively, and SEL: 1268.7 ± 231.2 and 1298.3 ± 253.3 μg/cm2, respectively). Transport efficiencies for RAS and SEL were good (ranged from 6.81 to 8.50 and 2.86 to 3.61%, respectively). Furthermore, the delivery efficiency, i.e., the fraction of the drug in the formulation that was delivered was very high (>56% at 0.5 mA/cm2). Cumulative permeation of RAS and SEL from carbopol gels, potential drug reservoirs for iontophoretic systems, was 891.5 ± 148.3 and 626.6 ± 162.4 μg/cm2, respectively; this was less than from solution and was tentatively attributed to either different partitioning or slower drug diffusion in the gel matrix. The results demonstrated that therapeutic amounts of rasagiline and selegiline could be easily delivered by transdermal iontophoresis with simple gel patches of modest surface area.
Keywords: Parkinson's disease; MAO-B inhibitors; Iontophoresis; Transdermal drug delivery;

Cyclodextrin as membrane protectant in spray-drying and freeze-drying of PEGylated liposomes by Jolanda M. van den Hoven; Josbert M. Metselaar; Gert Storm; Jos H. Beijnen; Bastiaan Nuijen (209-216).
In this study it was investigated whether hydroxypropyl-β-cyclodextrin (HPβCD) is able to stabilize the liposomal membranes during drying of long circulating polyethylene glycol (PEG) coated liposomes, as compared to the disaccharides trehalose and sucrose. PEGylated liposomes loaded with prednisolone disodium phosphate (PLP) were dried by spray-drying or freeze-drying. The dried powders were tested on their residual moisture content, glass transition temperature and amorphous character. Upon reconstitution the liposomal size, size distribution and drug retention were determined and the results were compared to the characteristics of the formulation solution before drying. In contrast to the disaccharides, HPβCD stabilizes the liposomal membranes of the PEGylated liposomes during the drying process of both spray drying and freeze-drying when present in a lipid:carbohydrate ratio of 1:6 (w/w). The resulting powder can be stored at room temperature. No changes in size and size distribution were seen upon reconstitution of the HPβCD containing formulations. Drying resulted in a minimal leaking of PLP from the liposomes. Its relatively high T ′ g and T g of HPβCD, as compared to the disaccharides, make HPβCD an excellent membrane protectant for dry PEGylated liposomal formulations.
Keywords: PEGylated liposomes; Freeze-drying; Spray-drying; Lyoprotection; Hydroxypropyl-β-cyclodextrin; Disaccharides;

Preactivated thiomers: Permeation enhancing properties by Xueqing Wang; Javed Iqbal; Deni Rahmat; Andreas Bernkop-Schnürch (217-224).
The study was aimed to prepare a series of poly(acrylic acid)-cysteine-2-mercaptonicotinic acid conjugates (preactivated thiomers) and to evaluate the influence of molecular mass or degree of preactivation with 2-mercaptonicotinic acid (2MNA) on their permeation enhancing properties. Preactivated thiomers with different molecular mass and different degree of preactivation were synthesized and categorized on the basis of their molecular mass and degree of preactivation as PAA100-Cys-2MNA (h), PAA250-Cys-2MNA (h), PAA450-Cys-2MNA (h), PAA450-Cys-2MNA (m) and PAA450-Cys-2MNA (l). In vitro permeation studies, the permeation enhancement ability for preactivated thiomers was ranked as PAA450-Cys-2MNA (h) > PAA250-Cys-2MNA (h) > PAA100-Cys-2MNA (h) on both Caco-2 cell monolayers and rat intestinal mucosa. Comparing the influence of degree of preactivation with 2MNA on permeation enhancement, the following order PAA450-Cys-2MNA (h) > PAA450-Cys-2MNA (m) ≈ PAA450-Cys-2MNA (l) on Caco-2 cell monolayers and PAA450-Cys-2MNA (m) > PAA450-Cys-2MNA (h) > PAA450-Cys-2MNA (l) on intestinal mucosa was observed. The P app of sodium fluorescein was 5.08-fold improved on Caco-2 cell monolayers for PAA450-Cys-2MNA (h) and 2.46-fold improved on intestinal mucosa for PAA450-Cys-2MNA (m), respectively, in comparison to sodium fluorescein in buffer only. These results indicated that preactivated thiomers could be considered as a promising macromolecular permeation enhancing polymer for non-invasive drug administration.
Keywords: Thiomers; Poly(acrylic acid)-cysteine; Preactivated thiomers; Permeation enhancer;

View cell showing PEG 1000 (T m  ∼ 37.5 °C) and palmitic acid (T m  ∼ 62.6 °C) under scCO2 at 35 °C and 140 bar. PEG 1000 has liquefied (T m depression) and shows flakes of solid palmitic acid dispersed within it.The thermodynamic behaviour of selected polymeric components for preparation of controlled release microparticles using supercritical carbon dioxide (scCO2) processing was investigated. The polymeric materials selected were egg lecithin (a model for the lung surfactant phospholipid), poly(ethyleneglycol) (PEG) of different molecular weights, fatty acids (C18, C16, and C14), and physical blends of PEGs and fatty acids. In addition a range of PEG-stearates was also assessed. Analysis of thermodynamic behaviour was performed by differential scanning calorimetry (DSC) and by assessment of their interaction with scCO2 in a high-pressure variable volume view cell. The key criterion was to demonstrate a strong interaction with scCO2 and to show liquefaction of the polymeric material at acceptable processing temperatures and pressures. Positive results should then indicate the suitability of these materials for processing by the Particle from Gas Saturated Solutions (PGSS) technique using scCO2 to create microparticles for pulmonary administration. It was found that the materials tested interacted with scCO2 and showed a sufficient lowering of their melting temperature (T m) to make them suitable for use in the PGSS microparticle production rig. Fatty acids of low T m were shown to act as a plasticising agent and to lower the T m of PEG further during interaction with scCO2.
Keywords: Supercritical carbon dioxide; Fatty acids; PEG; PGSS; Polymer blends; High pressure variable volume view cell;

Electrospun drug-loaded core–sheath PVP/zein nanofibers for biphasic drug release by Yong-Nan Jiang; Hong-Ying Mo; Deng-Guang Yu (232-239).
Core–sheath nanofibers prepared using coaxial electrospinning were investigated for providing biphasic drug release profiles. With ketoprofen (KET) as the model drug, polyvinylpyrrolidone and zein as the sheath polymer and core matrix, respectively, the coaxial process could be carried out smoothly and continuously without any clogging of the spinneret. Scanning electron microscopy and transmission electron microscopy observations demonstrated that the nanofibers were linear with homogeneous structure and had a clear core–sheath structure with an average diameter of 730 ± 190 nm, in which the sheath had a thickness of ca. 90 nm. Differential scanning calorimetric and X-ray diffraction analyses verified that all the components in the core–sheath nanofibers were present in an amorphous state. Attenuated total reflectance Fourier transform infrared spectra demonstrated both the sheath and core matrix had good compatibility with KET owing to hydrogen bonding. In vitro dissolution tests showed that the nanofibers could provide an immediate release of 42.3% of the contained KET, followed by a sustained release over 10 h of the remaining drug. The present study exhibited a simple and useful approach to systematically design and fabricate nanostructures using coaxial electrospinning for providing biphasic drug release profiles.
Keywords: Biphasic release; Coaxial electrospinning; Nanofibers; Core–sheath structure; Zein;

Preparation and evaluation of liposome-encapsulated codrug LMX by Yan Zhong; Jing Wang; Yao Wang; Bin Wu (240-248).
A novel codrug (LMX) consisting of Lamivudine (LMV) and Ursolic acid (UA) was synthesized, and a liposomal formulation of LMX was designed and optimized to facilitate its in vivo administration.A novel codrug (LMX) consisting of Lamivudine and Ursolic acid has been shown to possess the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury in vivo. Because of the limited water solubility of LMX, our aims were to design and optimize a liposomal formulation that could facilitate its in vivo administration, and to estimate the potential of LMX-loaded liposomes as oral or intravenous delivery system. In this work, LMX-loaded liposomes were prepared by the thin film hydration method coupled with sonication. LMX-loaded liposomes showed spherical morphology under transmission electron microscope (TEM) analysis. The mean particle size of liposomes was about 210 nm, and the drug entrapment efficiency was more than 90%. Stability data indicated that lyophilized liposomes were stable for at least 6 months at 4 °C. In vitro drug release profile of LMX-loaded liposomes showed a sustained release profile of LMX and an initial mild burst was observed. The relative bioavailability of LMX-loaded liposomes was 1074.8% compared with LMX suspension after oral administration, and 135.2% relative to 50% alcohol solution after intravenous (i.v.) administration. These results indicated that LMX-loaded liposomes were valued to develop as a practical preparation for oral or i.v. administration.
Keywords: Liposomes; Codrug; LMX; Bioavailability; Lamivudine, Ursolic acid;

Solid-in-oil dispersion: A novel core technology for drug delivery systems by Yoshiro Tahara; Noriho Kamiya; Masahiro Goto (249-257).
Drug delivery systems using a solid-in-oil (S/O) dispersion as a core technology have advanced significantly over the past ten years. A novel, effective and practical preparation method for a S/O dispersion was originally established in 1997 as a tool for enzymatic catalysis in organic media. This oil-based dispersion containing proteins in non-aqueous media had great potential for applications to other research with one of the most successful being its adaptation as a drug delivery system. The history and features of novel processes for preparing S/O dispersions are presented in this article. In addition, recent research into the use of S/O dispersions for innovative oral and skin drug delivery systems is discussed.
Keywords: Emulsion; Enzymatic catalysis; Oral drug delivery; Skin drug delivery; Solid-in-oil dispersion;

Quaternized chitosan–organic rectorite intercalated composites based nanoparticles for protein controlled release by Ruifen Xu; Shangjing Xin; Xue Zhou; Wei Li; Feng Cao; Xuyang Feng; Hongbing Deng (258-265).
Organic rectorite (OREC) was added in the quaternized chitosan (QC)/alginate (ALG) nanoparticles using an ionic gelation method to fabricate a controllable release system for proteins for the first time. The morphology of nanoparticles, the intercalated structure of OREC, bovine serum albumin encapsulation efficiency and in vitro release properties were investigated. Fourier transform infrared spectra, energy dispersive X-ray, X-ray photoelectron spectroscopy, small angle X-ray diffraction and size distribution analysis were performed to characterize the composite nanoparticles. With the addition of OREC, the encapsulation efficiency and the loading capacity of nanoparticles had increased from 21.2% to 44.9% and from 13.7% to 25.0%, respectively. In addition, the rapid initial release was inhibited successfully from 20.15% to 11.07% in stimulated gastric fluid and from 14.69% to 4.52% in stimulated intestinal fluid. The results verified that the addition of OREC could make these nanoparticles effective carriers to encapsulate drug and slow the drug controlled release of nanoparticles.
Keywords: Quaternized chitosan; Organic rectorite; Intercalation; Nanoparticles; Drug delivery;

Controlled release of rasagiline mesylate promotes neuroprotection in a rotenone-induced advanced model of Parkinson's disease by M. Fernández; E. Barcia; A. Fernández-Carballido; L. Garcia; K. Slowing; S. Negro (266-278).
FE-SEM microphotographs of RM-loaded PLGA microspheres, in vitro release profiles of RM from PLGA microspheres, confocal images of mitochondrial staining (red fluorescence) of SKN-AS control cells, cells treated with H2O2 (1 mM) and co-treated with RM (87.6 μM). Catalepsy (bar and grid) and akinesia results obtained in the rotenone-induced rat model of PD. Group G1: control group; G2: rotenone-treated control group; and rotenone-treated animals also receiving: G3: PLGA-blank microspheres; G4: RM in saline (1 mg/kg/day for 45 days, starting on day 15th of the study) and, G5: RM-loaded PLGA microspheres (amount of microspheres equivalent to 15 mg/kg RM injected on days 15 and 30 of the study). RM: rasagiline mesylate.Microencapsulation of rasagiline mesylate (RM) into PLGA microspheres was performed by method A (O/W emulsion) and method B (W/O/W double emulsion). The best formulation regarding process yield, encapsulation efficiency and in vitro drug release was that prepared with method A, which exhibited constant drug release for two weeks (K 0  = 62.3 μg/day/20 mg microspheres). Exposure of SKN-AS cells to peroxide-induced oxidative stress (1 mM) resulted in cell apoptosis which was significantly reduced by RM (40.7–102.5 μM) as determined by cell viability, ROS production and DNA fragmentation. Daily doses of rotenone (2 mg/kg) given i.p. to rats for 45 days induced neuronal and behavioral changes similar to those occurring in PD. Once an advanced stage of PD was achieved, animals received RM in saline (1 mg/kg/day) or encapsulated within PLGA microspheres (amount of microspheres equivalent to 15 mg/kg RM given on days 15 and 30). After 45 days RM showed a robust effect on all analytical outcomes evaluated with non-statistically significant differences found between its administration in solution or within microparticles however; with this controlled release system administration of RM could be performed every two weeks thereby making this new therapeutic system an interesting approach for the treatment of PD.
Keywords: Rasagiline mesylate; Neuroprotection; SKN-AS; Microspheres; Rotenone; Advanced Parkinson's disease;

Evaluation of thermoresponsive properties and biocompatibility of polybenzofulvene aggregates for leuprolide delivery by Mariano Licciardi; Giovanni Amato; Andrea Cappelli; Marco Paolino; Germano Giuliani; Beatrice Belmonte; Carla Guarnotta; Giovanna Pitarresi; Gaetano Giammona (279-286).
In this study, a polybenzofulvene derivative named poly-6-MOEG-9-BF3k, was evaluated as polymeric material for the production of injectable thermoresponsive nano-aggregates able to load low molecular weight peptidic drug, like the anticancer leuprolide. Thermoresponsive behavior of poly-6-MOEG-9-BF3k was studied in aqueous media by evaluating scattering intensity variations by means of DLS in function of temperature. Zeta potential measurements and SEM observations were also carried out. Moreover, critical aggregation temperature of the poly-6-MOEG-9-BF3k polymer was evaluated by pyrene fluorescence analysis.Then, the ability of prepared thermoresponsive aggregates to protect this model oligopeptide drug and regulate its release rate in function of external temperature was evaluated in vitro.Finally, biocompatibility of poly-6-MOEG-9-BF3k aggregates was tested in vitro on a healthy cell line (human bronchial epithelial cell; 16-HBE) and in vivo on rat animal model upon subcutaneous administration.
Keywords: Polybenzofulvene; Thermoresponsive; Nano-aggregates; Leuprolide;

In this study, aripiprazole (APZ), a weak alkaline drug with pH-dependent solubility, was selected as model drug to examine the feasibility of preparing nanosuspensions using nanoprecipitation/homogenization technique based on acid–base neutralization. The related substances in nanosuspensions prepared under optimal conditions were slightly increased as compared with APZ raw material. The resultant APZ nanosuspensions showed a mean particle size of 350 nm with polydispersion index (PI) value of 0.20. Good physical stability was kept for over 40 days. SEM observation showed the morphology of oval crystals with rough surface. Nanosuspensions significantly increased the solubility as well as the dissolution of APZ due to the decreased particle size. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of APZ in nanosuspensions. APZ nanosuspensions got maximum absorption rate and extent comparing with APZ commercial tablet and suspensions with relative bioavailability of 123.43 ± 12.98% and 171.41 ± 14.62%, respectively. This technique has the potential to prepare nanosuspensions of insoluble drugs with pH-dependent solubility.
Keywords: Nanosuspensions; Acid–base neutralization; Aripiprazole; Solubility; Dissolution; Oral bioavailability;

Ability of fibrinogen γ-derived dodecapeptides with different sequences to bind to rat platelets by Koji Tokutomi; Toshiaki Tagawa; Maki Korenaga; Masatoshi Chiba; Tomohiro Asai; Naohide Watanabe; Shinji Takeoka; Makoto Handa; Yasuo Ikeda; Naoto Oku (296-301).
No significant difference in the inhibitory effect on the aggregation of rat platelets was observed between human-H12 (HHLGGAKQAGDV) and rat-H12 (HHMGGSKQVGDM), even though 4 out of the 12 amino acid residues are different.A dodecapeptide (γ400–411) derived from a fibrinogen γ-chain carboxyl-terminal sequence recognizes specifically the active form of GPIIb/IIIa on the surface of activated platelets. For the purpose of efficient hemostasis, we previously developed ADP-encapsulated liposomes modified with human-dodecapeptide (HHLGGAKQAGDV, human-H12). On the other hand, the amino-acid sequence of H12 from rats is HHMGGSKQVGDM, having only 67% homology to that from humans. Here, we investigated the ability of rat-H12 in comparison with human-H12 to bind to platelets. Firstly, rat platelets were activated with phorbol-12-myristate-13-acetate (PMA), and the activation was confirmed by flow cytometry. Next, we evaluated the dissociation constant (K d ) of human-H12 and rat-H12 for dissociation from rat platelets by using FACS. As a result, the K d of human-H12 and rat-H12 with respect to rat platelets was 2.78 ± 0.21 and 2.91 ± 0.22 μM, respectively. Furthermore, H12 from both species inhibited quite similarly the aggregation of rat platelets in platelet-rich plasma (PRP). These results suggest that H12 from different species with different amino acid sequences interacts similarly with GPIIb/IIIa on platelets.
Keywords: Hemostasis; Dodecapeptide; Fibrinogen; GPIIb/IIIa; Human; Rat;

The aim of this study was to characterize the protein release from PLGA-based implants prepared by hot-melt extrusion with special emphasis on identifying reasons for incomplete release. Biodegradable PLGA-implants loaded with BSA were prepared with a syringe-die extrusion device. A burst-free release was achieved up to 25% BSA loading by milling the protein prior to extrusion. The release was incomplete at 70% at loadings below the percolation threshold of the protein; higher protein loadings increased the release to 97%. However, an insoluble implant mass remained for over 180 days, which was attributed to the acylation of BSA by PLGA oligomers via a thioester bond. The incomplete protein release due to the formation of this covalent bond was overcome when increasing the porosity of the implant, which effectively reduced the contact between BSA and PLGA oligomers. Accordingly, melt-extrusion facilitated incorporating high loadings of BSA into burst-free biodegradable implants. Additionally, it enhanced complete protein release by a process- or formulation controlled increase of the implant porosity.
Keywords: Biodegradable implant; Hot melt extrusion; Poly(lactide-co-glycolide); Protein formulation; Incomplete release;

A novel manufacturing strategy based on continuous processing integrated with online monitoring tools coupled with efficient automatic feedback control system is highly desired for efficient Quality by Design (QbD) based manufacturing of the next generation of pharmaceutical products with optimal consumption of time, space and resources. In this manuscript, an efficient plant-wide control strategy for an integrated continuous pharmaceutical tablet manufacturing process via roller compaction has been designed in silico. The designed control system consists of five cascade control loops and three single control loops resulting in 42 controller tuning parameters. An effective controller parameter tuning strategy involving an ITAE method coupled with an optimization strategy has been proposed and the designed control system has been implemented in a first principle model-based flowsheet that was simulated in gPROMS (Process System Enterprise). The advanced techniques (e.g. anti-windup) have been employed to improve the performance of the control system. The ability of the control system to reject the unknown disturbances as well as to track the set point has been analyzed. Results demonstrated enhanced performance of critical quality attributes (CQAs) under closed-loop control compared to open-loop operation thus illustrating the potential of closed-loop feedback control in improving pharmaceutical manufacturing operations.
Keywords: Process control; Pharmaceutical; Roller compactor; Continuous processing; Simulation;

Coformer selection based on degradation pathway of drugs: A case study of adefovir dipivoxil–saccharin and adefovir dipivoxil–nicotinamide cocrystals by Yuan Gao; Jing Gao; Ziling Liu; Hongliang Kan; Hui Zu; Wanjin Sun; Jianjun Zhang; Shuai Qian (327-335).
Adefovir dipivoxil (AD) is a bis(pivaloyloxymethyl) prodrug of adefovir with chemical stability problem. It undergoes two degradation pathways including hydrolysis and dimerization during storage. Pharmaceutical cocrystallization exhibits a promising approach to enhance aqueous solubility as well as physicochemical stability. In this study we attempted to prepare and investigate the physiochemical properties of AD cocrystals, which were formed with two coformers having different acidity and alkalinity (weakly acidic saccharin (SAC) and weakly basic nicotinamide (NCT)). The presence of different coformer molecules along with AD resulted in altered physicochemical properties. AD–SAC cocrystal showed great improvement in solubility and chemical stability, while AD–NCT did not. Several potential factors giving rise to different solid-state properties were summarized. Different coformers resulted in different cocrystal formation, packing style and hydrogen bond formation. This study could provide the coformer selection strategy based on degradation pathways for some unstable drugs in pharmaceutical cocrystal design.
Keywords: Adefovir dipivoxil; Cocrystal; Saccharin; Nicotinamide; Degradation pathway;