International Journal of Pharmaceutics (v.350, #1-2)

Ligand based dendritic systems for tumor targeting by Abhinav Agarwal; Surbhi Saraf; Abhay Asthana; Umesh Gupta; Virendra Gajbhiye; Narendra K. Jain (3-13).
Medications that can selectively target tumors at the same time avoid access of the drug to nontarget areas, employ utilization of homing devices termed as ligands, that can bind to specific epitopes expressed on the surface of the necrotic mass of cells. Molecular signatures for transferrin, Epidermal Growth Factor, Sialic Lewis and folic acid are expressed on the surface of these cells. Dendrimers are nanosized, non-immunogenic, and hyper-branched vehicles that can be efficiently tailored for spatial distribution of bioactives, thereby reducing untoward cytotoxicity on normal cells. These nanoparticulate drug delivery vehicles provide a unique platform that has precisely placed functional groups so that multiple copies of ligands can be attached to it and facilitate targeting to the tumor surface or neo-vascularizing vessels proliferating around these cells. The article reviews the scope of ligand based dendritic system as a prospective for delivery of anti-cancer drugs, via active targeting with interception of minimal side effects.
Keywords: Tumors; Site-specific delivery; Receptors; Ligands; Nanoparticulate carriers; Dendrimers;

How to achieve sustained and complete protein release from PLGA-based microparticles? by A. Giteau; M.C. Venier-Julienne; A. Aubert-Pouëssel; J.P. Benoit (14-26).
One of the most challenging tasks in the delivery of therapeutic proteins from PLGA-based microparticles is the sustained and complete release of the protein in its native form. The mechanisms responsible for incomplete protein release from these devices are numerous and complex; the beneficial effect of different formulations has often been evaluated in vitro. Strategies employed for overcoming protein destabilization during the release step are reviewed in this paper. Proteins have been protected in the deleterious environment by adding stabilizers to the formulation, or by modifying the protein or the polymer. Alternatively, some strategies have aimed at avoiding the formation of the destabilizing environment. As experimental conditions may influence the results from in vitro release studies, we initially report precautions to avoid adverse effects.
Keywords: Sustained release; Protein; Microspheres; Poly(lactic-co-glycolic acid) (PLGA); In vitro release; Release mechanism;

A chitosan hydrogel-based cancer drug delivery system exhibits synergistic antitumor effects by combining with a vaccinia viral vaccine by Hee Dong Han; Chung Kil Song; Yong Sung Park; Kyung Hee Noh; Jin Hee Kim; Taewon Hwang; Tae Woo Kim; Byung Cheol Shin (27-34).
Cancer treatment combining chemotherapy and immunotherapy has been vigorously exploited to further improve cancer therapeutic efficacy. This study investigated a new chemoimmunotherapy approach utilizing hydrogel as a local anti-cancer drug delivery system. Chitosan hydrogel containing doxorubicin (CH-DOX) and vaccinia virus vaccine expressing Sig/E7/LAMP-1 (Vac-Sig/E7/LAMP-1) were used as chemoimmunotherapeutic agents. It was found that intratumoral injection of CH-DOX effectively inhibited tumor growth itself and, in addition, exhibited a synergistic antitumor effect in combination with a vaccinia virus-based vaccine. This combination did not decrease but rather increased the number of tumor-specific CD8+ T cells primed by vaccinia virus-mediated vaccination; the resulting antitumor effects were further improved up to 60 days as compared with monotherapy after tumor challenge, and the survival of tumor-bearing mice was dramatically prolonged. This study is a pioneer report that demonstrates the use of a biodegradable hydrogel system as an anti-cancer drug delivery system for successful chemoimmunotherapy. It is hoped that, this study can provide a foundation for a rational approach to improve antitumor efficacy of chemoimmunotherapy.
Keywords: Hydrogel; Doxorubicin; Vaccinia vaccine; Chemoimmunotherapy;

Solid form selection of zwitterionic 5-HT4 receptor agonist by Takashi Kojima; Kiyohiko Sugano; Satomi Onoue; Noriaki Murase; Morimichi Sato; Yohei Kawabata; Takashi Mano (35-42).
From discovery synthesis of a zwitterionic pharmaceutical compound, 4-{[4-({[(3-isopropyl-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)carbonyl]amino}methyl)piperidin-1-yl]methyl}tetrahydro-2H-pyran-4-carboxylic acid (compound A), two anhydrous ZW-I and ZW-II and two hydrate forms ZW-III and ZW-IV were identified. Although stable form ZW-I was chemically stable at 70 °C/75% RH for 10 days, it was transformed to hydrate form ZW-IV under ambient conditions within a few days, taking up water from atmospheric moisture. In order to select a solid form for further investigation, solid-state characterization, salt screening on 96-well plate, stable polymorph and hydrate screening and physical stability were performed. Based on the results of the salt screening, besylate, camsylate, hemi-edisylate, hemifumarate, monosuccinate salts of compound A were prepared, and their polymorphism and chemical and physical stability were evaluated. From the viewpoint of stability and manufacturability, a stable form of besylate salt (BSA-I), which had two anhydrous forms BSA-I and BSA-II and hydrate form BSA-III, was selected as a solid form. BSA-I was quite stable at high relative humidity and provided significant improvement of physical stability compared with ZW-I.
Keywords: Solid form selection; Salt screening; Raman spectroscopy; Hydrate; Polymorph; Physical stability;

Effect of permeation enhancers and organic acids on the skin permeation of indapamide by Changshun Ren; Liang Fang; Ting Li; Manli Wang; Ligang Zhao; Zhonggui He (43-47).
The aim of present study was to investigate the transdermal properties of indapamide and to explore the efficacy of various permeation enhancers and organic acids with regard to the percutaneous absorption of indapamide. Permeation experiments were performed in vitro, using rat abdominal skin as a barrier. In the permeation studies, 2-chamber diffusion cells were used. The results obtained indicate that N-dodecylazepan-2-one, N-methyl-2-pyrrolidone, menthol and oleic acid had a strong enhancing effect on the permeation of indapamide and N-dodecylazepan-2-one exhibited the most potent enhancing effect. All eight of the organic acids chosen had a potent enhancing effect on the permeation of indapamide across rat abdominal skin. Among the organic acids examined, lactic acid had the greatest enhancing effect. The formation of an ion-pair between indapamide and organic acids may be responsible for the enhanced skin permeation of indapamide. Although the exact reason remains unknown, it is worth carrying out further investigations.
Keywords: Indapamide; Permeation enhancers; Organic acids; Percutaneous absorption; In vitro;

The thermal properties of two polymorphs (A and C) of a Merck development compound were studied using high-speed differential scanning calorimetry (Hyper-DSC™). The utility of this novel technique as a fast analytical tool for studying the polymorphic behaviour of metastable polymorphs has previously been demonstrated successfully for Carbamazepine. Accelerated heating rates can alter the kinetics of the melting transition of metastable polymorphs such that concurrent exothermic recrystallisation is inhibited. Here it is demonstrated that at heating rates of 400 °C/min concurrent recrystallisation of Polymorph A of the Merck development compound is inhibited allowing the enthalpy of fusion for the lower melting Polymorph C to be determined. The utility of the technique as a qualitative tool to detect the presence of polymorphic impurities was confirmed for levels much lower than 10% (w/w). However, seeding effects consistent with those reported previously for Carbamazepine were also observed for this structurally distinct molecule limiting the utility of the technique for accurate quantification of low levels of polymorphic impurities.
Keywords: High speed DSC; Hyper-DSC™; Polymorphism;

Clusterin antisense complexed with chitosan for controlled intratumoral delivery by Christopher M. Springate; John K. Jackson; Martin E. Gleave; Helen M. Burt (53-64).
The purpose of this work was to characterize an injectable, intratumoral, controlled release delivery system for clusterin antisense oligonucleotide (clusterin ASO) based on clusterin ASO complexed with chitosan microparticles (CC complexes) and blended with a biodegradable polymeric paste (CC in paste). The effect of clusterin ASO/chitosan ratio on the physicochemical properties of CC complexes and the influence of chitosan and polymeric paste on the in vitro release and stability of clusterin ASO were investigated. Chitosan had an intrinsic pK a of 6.2. Chitosan particles incubated at different pHs swelled to approximately 600% of their dry weight and had a mean diameter of approximately 200 μm. As the amount of chitosan in CC complexes was increased or as the pH was decreased, zeta potentials became increasingly less negatively charged and the amount of clusterin ASO complexed with chitosan increased. Clusterin ASO released into PBS or plasma in vitro from polymeric paste and CC in paste in a similar manner with a burst phase of release followed by a slow sustained release. The ratio of clusterin ASO to chitosan and incorporation into polymeric paste influenced the rate and extent of clusterin ASO release. Inclusion of clusterin ASO with or without chitosan in polymeric paste inhibited the in vitro degradation of clusterin ASO in plasma. Treatment of PC-3 cells in vitro with clusterin ASO alone or clusterin ASO released from the various formulations resulted in 52–62% inhibition of the expression of clusterin protein. Degradation studies showed that approximately 40% of the full-length clusterin ASO remained from both clusterin ASO alone and CC complex samples when incubated in 50% plasma in vitro for 4 days. In conclusion, the amount of clusterin ASO loaded into microparticulate chitosan was dependent on the amount of chitosan present and the pH of the environment and clusterin ASO released from the various formulations in a controlled manner and in a bioactive form.
Keywords: Oligonucleotide delivery; Controlled drug delivery; Chitosan; Polymeric paste; Clusterin antisense; Prostate cancer;

Comparison of the stability of split and intact gabapentin tablets by Donna A. Volpe; Abhay Gupta; Anthony B. Ciavarella; Patrick J. Faustino; Vilayat A. Sayeed; Mansoor A. Khan (65-69).
The purpose of this study was to determine the stability differences between split and intact gabapentin tablets. Gabapentin tablets from three different manufacturers (G1, G2 and G3) were tested for a period of 9 weeks under long-term (25 °C/60% RH) and intermediate stability (30 °C/60% RH) storage conditions after storage in closed amber pharmacy dispensing containers. Samples were analyzed for dissolution and potency using validated HPLC methods. Potency test also included the quantitation of gabapentin's main degradation product. Tablets from all manufacturers and at all time points had potency >90%. At 9 weeks, a statistically significant decrease (p  < 0.02) in gabapentin potency was observed for the whole and split G2 and G3 tablets under the intermediate storage conditions. At the end of 9 weeks, all samples also showed slightly higher levels of degradation product which was statistically significant (p  < 0.01) for samples stored under intermediate stability storage conditions and exceeded the proposed USP (PF, 2006) limit for the G3 split and intact tablets. No difference was observed between the potency and dissolution of the intact and the split tablets from the same manufacturer and the three products tested remained stable throughout the study period. The results suggest that splitting of gabapentin tablets did not affect the stability of these particular drug products tested as part of this study when stored under normal storage conditions for a period of up to 9 weeks. However, the results should not be extrapolated to other gabapentin drug products and to other tablet dosage forms.
Keywords: Split-tablet; Stability; Gabapentin;

A novel method to prepare oral formulations, normally suspended dosage form, for preclinical safety studies in animals has been developed using a rotation/revolution mixer. Small hard balls made of zirconia were added to the mixing process to evaluate effectiveness in making a high quality suspension. The driving with balls loaded in the cylindrical container (vessel) of the mixer was quite efficient in dispersing and milling the particles of the active pharmaceutical ingredient (API) in an aqueous medium. The API powder and a small amount of oral aqueous medium (vehicle) were successfully mixed by the spinning motion of the balls in the vessel as though the paste-like suspension was kneaded with a mortar and pestle. It was found that the milled suspension with the mean size of 10–20 μm could be prepared, in addition finer milling of less than 10 μm could be achieved by selecting the material of vessel. Optimum driving conditions including mixing time, size and quantity of balls, and the standard operational procedure was established using compounds varying in physicochemical properties. The particle size and quantitative analysis by HPLC showed that the resultant suspension was well-milled and highly homogeneous with the nearly intended concentration of API. The proposed method established by this experiment could be applied to the actual safety studies in the real preparation scale of oral suspension.
Keywords: Suspension; Preclinical safety; Rotation/revolution mixer; Zirconia balls; Milling; Oral formulation;

Ricin was encapsulated in various liposomes having neutral, negatively and positively charged and different density of DSPE-mPEG-2000 on the surface and cytotoxicity of ricin entrapped in these different charged liposomal formulations was studied in CHO pro cells and compared with free ricin with a view to develop an optimum delivery system for ricin in vivo. It was observed that the cytotoxicity of ricin entrapped in various charged liposomes was significantly dependent on the charge on the surface of liposomes. The maximum cytotoxicity of ricin was observed when it was delivered through negatively charged liposomes. Monensin enhances the cytotoxicity of ricin entrapped in various charged liposomes and the extent of enhancement of the cytotoxicity is significantly dependent on the charge on the surface of liposomes. Maximum potentiation (213.14-fold) of cytotoxicity of ricin was observed when it was delivered through positively charged liposomes followed by negatively charged (83.36-fold) and neutral (71.30-fold) liposomes, respectively. Studies on the kinetics of inhibition of protein synthesis by ricin entrapped in various charged liposomes revealed that lag period of inhibition of protein synthesis is significantly lengthened following delivery through various charged liposomes. However, in the presence of monensin, the lag period was reduced. There is a marginal variation in the cytotoxicity of ricin entrapped in various charged liposomes after incorporation of 5 mol% of DSPE-mPEG-2000 on the surface. However, there is a significant variation in the enhancing potency of monensin on the cytotoxicity of ricin entrapped in various charged liposomes in CHO pro cells following incorporation of 5 mol% DSPE-mPEG-2000 on the surface. Studies on the effect of variation of density of DSPE-mPEG-2000 on the surface of various charged liposomes on the enhancement of cytotoxicity of entrapped ricin by monensin in CHO pro cells showed that the enhancing potency of monensin on the cytotoxicity of ricin entrapped in various charged liposomes is significantly dependent on the density of DSPE-mPEG-2000 on their surface. It was also observed that the efficacies of monensin on the enhancement of cytotoxicity of ricin entrapped in various charged PEG-liposomes in CHO pro cells was highly related to their amount of cell-association. The present study has clearly shown that by suitable alteration of liposomal lipid composition, charge and density of hydrophilicity it would be possible to direct liposomal ricin to specific cells for their selective elimination in combination with monensin.
Keywords: Ammonium chloride; CHO cells; Golgi apparatus; Liposomal ricin; Monensin; Ricin cytotoxicity;

Thermal and alkaline stability of meropenem: Degradation products and cytotoxicity by Andreas Mendez; Pedro Chagastelles; Eduardo Palma; Nance Nardi; Elfrides Schapoval (95-102).
The stability of the broad-spectrum antibiotic meropenem was investigated in order to isolate and elucidate the mean degradation products involved in thermal and alkaline decomposition of meropenem in solution. The purification of thermal degradation product (45 °C) involved a combination of preparative chromatographic techniques. The degradates were characterized by NMR and ESI-MS. The thermal degradation product was a result of several chemical reactions, with modification of side chain and β-lactam ring, resulting in a pyrrolic derivative. Under alkaline conditions (NaOH 0.1 N), meropenem was converted totally to the corresponding β-lactam ring-opened derivative, in sodium salt state. The degraded samples of meropenem reconstituted solution, powder for injection and alkaline solution was also studied in order to determine the preliminary cytotoxicity in vitro against mononuclear cells. The results obtained indicated that samples could be toxic in high concentration (2.0 mg/mL) after 48 h of incubation. The present study confirms the lability of the drug in aqueous solution, specially when submitted to thermal and alkaline conditions. Thus, it is necessary attention during the handling and storage of this antibiotic.
Keywords: Meropenem; Thermal and alkaline degradation; Identification of degradation products; Cytotoxicity in vitro assay;

In this work, the interaction of the skin penetration enhancers dl-menthol, α-terpineol, 1,8-cineole and (+)-limonene with the uppermost skin layer, the stratum corneum and with multilamellar vesicles from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is investigated by electron paramagnetic resonance (EPR) spectroscopy of the small spin label 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO), which partitions the aqueous and hydrocarbon phases. The EPR spectrum allows for the determination of the actual partition coefficient and the rotational diffusion rates of the spin probe in the two environments. The enthalpy changes, ΔH°, to transfer the spin probe from the aqueous to the hydrocarbon phase, as well as the activation energies associated to its rotational motion, were considerably smaller for stratum corneum, indicating less pronounced thermal reorganization. For DPPC, the terpenes increased both the partition coefficient and the rotational diffusion rate of the spin label in the membrane, except in the liquid–crystalline phase, while these increases in stratum corneum were observed in the entire temperature range measured with the exception of the rotational motion parameter for dl-menthol and α-terpineol at temperatures below their melting point (32–41 °C). It is suggested that the terpenes effectively acting as spacers in the membrane fluidize the lipids and cause ruptures in the hydrogen-bonded network of the polar interface.
Keywords: Stratum corneum; EPR; Spin label; Lipid dynamics; Terpene;

Permeability of surface-modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers by Dipak S. Pisal; Venkata K. Yellepeddi; Ajay Kumar; Radhey S. Kaushik; Michael B. Hildreth; Xiangming Guan; Srinath Palakurthi (113-121).
Aim of this study was to prepare polyamine-conjugated PAMAM dendrimers and study their permeability across Caco-2 cell monolayers. Polyamines, namely, arginine and ornithine were conjugated to the amine terminals of the G4 PAMAM dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P app) for the PAMAM dendrimers increased by conjugating the dendrimers with both of the polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. The correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with both the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of the polyamine-conjugated dendrimers was evaluated in Caco-2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were slightly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer–polyamine conjugates as drug carriers to increase the oral absorption of drugs.
Keywords: PAMAM dendrimers; Polyamines; transepithelial electrical resistance (TEER); Caco-2 cells; Transepithelial transport; methylthiazoletetrazolium (MTT) assay;

Application of instrumental evaluation of color for the pre-formulation and formulation of rabeprazole by Yun-Seok Rhee; Chun-Woong Park; Yoon-Sub Shin; Sung-Hoon Kam; Kyu-Hyun Lee; Eun-Seok Park (122-129).
The aims of this study were to fast screen the compatibility of rabeprazole and excipients using a spectrocolorimeter and to examine the relationship between the color change value and drug contents/drug degradation products in solid dosage forms. The color change values of rabeprazole-excipient mixtures were measured using a spectrocolorimeter, with six tablet formulations compressed using a single-punch instrumental tablet press. The rabeprazole and degradation products contents in the tablets were analyzed using an HPLC method, with the color change values of the tablets measured using spectrocolorimetery for 4 weeks. These experiments indicated that the instrumental evaluation of color was a speedy, simple and useful tool in the determination of the interaction between the drug and excipients, as well as in the formulation of solid dosage forms. The relationships of the % reduced drug contents versus the color change value, and those of the % drug degradation products versus the color change value were exponentially increased in formulations containing zinc stearate. On stress testing, the color change value of rabeprazole was inconsistent with previous reports, as the degradation of rabeprazole can be greatly influenced by humidity as well as temperature. Consequently, these results highlight the potential of color formation in the application of pre-formulation and formulation of drugs.
Keywords: CIELAB; Discoloration; Pre-formulation; Formulation; Chemical stability; Rabeprazole;

Disodium norcantharidate loaded poly(ɛ-caprolactone) microspheres by Shaobing Wang; Shengrong Guo; Liang Cheng (130-137).
Poly(ɛ-caprolactone) (PCL) microspheres encapsulating disodium norcantharidate (DSNC), a drug in salt form and with high water solubility, were prepared by s/o/w solvent evaporation technique and characterized in terms of size, morphology, encapsulation efficiency and drug release. The viscosity of s/o dispersion was crucial to the successful encapsulation of DSNC. Scanning electron microscopy (SEM) studies showed that the drug-loaded microspheres had coarse surface and porous internal structure. The analysis of X-ray diffraction (XRD) indicated that there was no interaction between DSNC and PCL, but the degree of crystallinity of PCL decreased with the introduction of the drug. The drug release profiles indicated an initial burst release followed by a slow release, and a further investigation into the release mechanism implied that the release of DSNC from PCL microspheres was caused by a combination of diffusion and osmotic pressure.
Keywords: Poly(ɛ-caprolactone); Microspheres; Disodium norcantharidate; Water solubility; Solvent evaporation;

Anionic lipids like phosphatidylserine are known to significantly enhance electroporation mediated transepidermal transport of polar solutes of molecular weights up to 10 kDa. The underlying mechanism of the effect of anionic lipids on transdermal transport is not fully understood. The main barrier to transdermal transport lies within the intercellular lipid matrix (ILM) of the stratum corneum (SC) and our previous studies indicate that dimyristoyl phosphatidylserine (DMPS) can perturb the packing of this lipid matrix. Here we report on our investigation on water retention in the SC following electroporation in the presence and the absence of DMPS. The water content in the outer most layers of the SC of full thickness porcine skin was determined using ATR-FTIR-spectroscopy. The results show that in the presence of DMPS, the SC remains in a state of enhanced hydration for longer periods after electroporation. This increase in water retention in the SC by DMPS is likely to play an important role in trans-epidermal transport, since improved hydration of the skin barrier can be expected to increase the partitioning of polar solutes and possibly the permeability.
Keywords: Porcine skin; Stratum corneum; Hydration; Water content; Transdermal transport; Anionic lipids; Dimyristoyl phosphatidylserine; Electroporation; Infrared spectroscopy; ATR-FTIR;

The performance of microcrystalline cellulose (MCC) which had been modified by the inclusion of various levels of sodium carboxymethylcellulose (SCMC) in the wet cake prior to drying, in terms of their ability to form pellets by a standardised extrusion/spheronization process has been assessed. Initial screening of the ability of the modified MCCs to form pellets with an 80% level of lactose as a model drug identified two potential products containing 6 or 8% of SCMC (B 6 and B 8). These two products were compared with a standard grade of MCC (Avicel PH101) in terms of their ability to produce pellets with 80% of model drugs of low (ibuprofen), intermediate (lactose) and high (ascorbic acid) water solubility when subjected to a standardised extrusion/spheronization process. Also assessed was their ability to retain water with applied pressure using a pressure membrane technique and their ability to restrict water migration during extrusion with a ram extruder. The two new types of MCC (B 6 and B 8) were able to form good quality pellets with all three model drugs, whereas Avicel PH101 could not form pellets with this high level of ibuprofen. This improved performance was related to the ability of the new types of MCC to hold higher levels of water within their structure and restrict the migration of water in the wet mass when subjected to pressure applied during the process of preparing the pellets. There is evidence to show that the two new types of MCC can function over a wider range of water contents than Avicel PH101 and that they have an improved performance if the extrusion process is rapid and if, after incorporation of the water into the powder, the sample is stored for some time before extrusion.
Keywords: Extrusion/spheronization; High drug loading; Modified microcrystalline cellulose; Pellets;

Theoretical and experimental vibrational study of miconazole and its dimers with organic acids: Application to the IR characterization of its inclusion complexes with cyclodextrins by Valéry Barillaro; Georges Dive; Eric Ziémons; Pascal Bertholet; Brigitte Evrard; Luc Delattre; Geraldine Piel (155-165).
The geometry, frequency and intensity of the vibrational bands of miconazole were derived from the density functional theory (DFT) calculations with the hybrid functional B3LYP and the 6-31G(d) basis set. Starting from the fully AM1 optimized geometries of miconazole/βCD/acids complexes, the miconazole/acid dimers were reoptimized at the B3LYP/6-31G(d) level. Three acids were studied: maleic, fumaric and l-tartaric acids. To begin with the vibrational spectral data obtained from solid phase in mid FT-IR spectrum of miconazole and its dimers are assigned based on the results of the normal modes calculations. All the observed spectra and the calculated ones are found to be in good agreement. In a second step, theoretical results allowed the assignment of FT-IR spectrum for the miconazole/HPγCD inclusion complex produced by supercritical carbon dioxide treatment and confirmed the inclusion of miconazole. The experimental spectra for the miconazole/HPγCD/acids complexes prepared by supercritical carbon dioxide processing were also assigned using theoretical results. The results confirmed the presence of a genuine inclusion complex and also the interaction between miconazole and the acid.
Keywords: Miconazole; Molecular modeling; Density functional theory; Normal mode; Miconazole/acid dimer; Cyclodextrin inclusion complexes;

d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) was used as a novel additive to the poly(l-lactide) (PLLA) films for local drug delivery with paclitaxel as a prototype therapeutic agent. Paclitaxel-loaded PLLA/TPGS films were prepared by the solvent casting technique with dichloromethane as the solvent. Effects of TPGS component on the films’ physicomechanical properties and the drug release profile were investigated. It was found by field emission scanning microscopy (FESEM) that a biphasic honeycomb surface was formed for the PLLA/TPGS films, while the PLLA film exhibited a smooth and homogeneous surface. There was no significant effect of the drug loading on the morphological structure of the PLLA/TPGS films. Differential scanning calorimetry (DSC) demonstrated that the PLLA/TPGS films was a phase-separated system. Tensile testing showed that the flexibility of the PLLA/TPGS films was much higher than that of the PLLA film. The elongation at break for the PLLA/TPGS film of 5%, 10% and 15% TPGS content was 6.8, 8.9 and 19.4 times of that for the PLLA film, respectively. In vitro drug release studies found that incorporation of TPGS considerably facilitated paclitaxel release.
Keywords: Anticancer drugs; Antiproliferative drugs; Biodegradable films; Biodegradable polymers; Controlled release; Localized drug delivery;

Drying efficiency and particle movement in coating—Impact on particle agglomeration and yield by E.S.K. Tang; L. Wang; C.V. Liew; L.W. Chan; P.W.S. Heng (172-180).
The purpose of this study was to determine the influences of drying efficiency and particle movement on the degree of agglomeration and yield of pellets coated under different conditions. Thermodynamic conditions were varied using different inlet air temperatures and airflow rates, fluid dynamics were varied using different airflow patterns and air velocities, and two sizes of pellets were coated at different airflow rates and partition gaps. Agglomeration was minimized when all the moisture introduced into the system was removed by the drying air. Excessively dry conditions led to increased loss of yield due to spray-drying effect and attrition. Fluid dynamics were still important even with adequate drying, as the degree of agglomeration was relatively higher in the non-swirling airflow of Wurster coating than in the swirling airflow of precision coating. Increasing air velocities increased pellet velocities, resulting in lower degrees of agglomeration. Hence, agglomeration due to fluid dynamics was attributed to differences in pellet velocities, pellet proximity and pellet trajectories within the partition column. Smaller pellets agglomerated primarily from inadequate drying and not due to inadequate opportunities for particle movement. Larger pellets were more affected by the partition gap due to restriction of their movement through the partition gap. Hence, both thermodynamics and fluid dynamics were found to be important in minimizing agglomeration and ensuring quality coated products.
Keywords: Coating; Swirl; Drying; Airflow; Agglomeration;

Bioadhesive tablet formulations of ketoconazole for vaginal delivery were studied. Carbomer (Carbopol 974P, Carbopol 934P), hydroxypropylmethyl cellulose (HPMC) and hydroxypropyl cellulose (HPC) were used as candidate bioadhesive polymers. Effervescent was incorporated into the formulations as a disintegration agent. The swelling behavior and bioadhesive strength of the drug-free tablets were investigated. Carbopol 934P was selected as biopolymer in combination with HPMC or HPC at different ratios to develop five drug-loaded formulations. The swellings, tackiness and in vitro release were studied on the tablets. A good sustained effect and a moderate bioadhesion were obtained with the tablets. The formulation containing 100 mg of effervescent, with the Carbopol 934P:HPC ratio of 1:9, seemed to be the optimum one for the tablet. In vivo drug residence tests were carried out by administering the preferred formulation to female rats. The results showed that the drug remaining followed a one-order model. Even after 24 h of administration in vagina of rats, 17% of the original employed drug was retained on the vaginal tissue. Our study may provide a potential vaginal tablet formulation of ketoconazole against Candida albicans.
Keywords: Ketoconazole; Bioadhesion; Swelling; Vaginal tablets; Effervescent;

The aim of this study was to investigate the influence of sulfobutyl ether β-cyclodextrin (SBE7-β-CD; Captisol®) on the dissolution properties of a poorly water-soluble drug from extrudates prepared by hot-melt extrusion. Ketoprofen was employed as a model drug. Extrudates containing the parent β-cyclodextrin (β-CD) were also produced for comparative evaluation to assess the benefits of SBE7-β-CD. Hot-melt extrudates were produced at 100 °C, which was close to the melting point of ketoprofen. The physiochemical properties and the in vitro drug release properties of ketoprofen from extrudates were investigated and compared with samples prepared by physical mixing, co-grinding, freeze-drying and heat-treatment. The solubilizing effects and the interactions of ketoprofen with SBE7-β-CD and β-CD were investigated using phase solubility and NMR studies, respectively. The dissolution rate of ketoprofen from samples prepared by hot-melt extrusion with SBE7-β-CD was significantly faster than both the physical mixture and the hot-melt extrudates prepared with the parent β-CD. Moisture absorption studies revealed that the hygroscopic nature of SBE7-β-CD led to particle aggregation and a corresponding decrease in drug release rate for all samples. However, the samples prepared by melt extrusion were least affected by exposure to elevated humidity.
Keywords: Hot-melt extrusion; Sulfobutyl ether β-cyclodextrin; Captisol®; Poorly water-soluble drug; Ketoprofen;

Effect of pharmaceutical excipients on aqueous stability of rabeprazole sodium by Shan Ren; Mi-Jin Park; Hongkee Sah; Beom-Jin Lee (197-204).
The chemical stability of a proton-pump inhibitor, rabeprazole sodium, was evaluated in simulated intestinal fluid (pH 6.8) containing various ‘Generally Recognized As Safe (GRAS)’-listed excipients, including Brij® 58, Poloxamer 188, Cremophor RH40, Gelucire 44/14 and PEG 6000. After incubation at 37 and 60 °C, the amounts of rabeprazole and its degradation product, thioether-rabeprazole, were quantitated by HPLC analysis. The main degradation product was separated and characterized by LC/MS. The degradation of rabeprazole followed first-order kinetics. In the absence of any excipients, the rate constants (k) obtained at 37 and 60 °C were 0.75 and 2.78 h−1, respectively. In contrast, the addition of excipients improved its stability. Among several excipients tested in this study, Brij® 58 displayed the greatest stabilizing effect. For instance, at 37 and 60 °C, Brij® 58 reduced the k values to 0.22 and 0.53 h−1, respectively. The stabilizing mechanisms of these hydrophilic polymeric excipients with optimal HLB values could be partially explained in terms of their solubilizing efficiency and micellar formation for thioether-rabeprazole. In conclusion, rabeprazole formulations that contain suitable excipients would improve its stability in the intestinal tract, thereby maximizing bioavailability.
Keywords: Aqueous stability; Pharmaceutical excipients; Rabeprazole; Thioether-rabeprazole; Stabilizing mechanism;

A colonic-release delivery system containing naproxen inclusion complex with 2-hydroxypropyl-β-cyclodextrin (2-HPβCD) was originally proposed. The core tablets consisting of the naproxen inclusion complex and disintegrants (Ac-Di-Sol®, Primojel®, Avicel® or Polyplasdone®) were formed by direct compression, and then coated with the polymers, either pH-dependent Eudragit® S100 and/or pH-independent Eudragit® RS100 with plasticizers like dibutyl sebacate (DBS) and aluminum tristearate (AT). The in vitro release characteristics were evaluated in simulated gastric fluid for 2 h and then subsequently in simulated intestinal fluid for 12 h. The potential histological changes were also evaluated after direct dosing of suspensions of naproxen alone and powdered mixtures of inclusion complex-loaded tablet into rat intestinal segments. No distinct colonic release was observed when disintegrants were excluded in the single-layered coated tablets regardless of coated structures, giving a zero-order fashion over 12 h. The coated tablet with double-layered structures of Eudragit® S100 and Eudragit® RS100 was not also applicable. In contrast, colonic release was achieved when the core tablet containing inclusion complex and disintegrant was coated with only Eudragit® S100 in a single-layered structure. The colonic-release tablet was resistant in gastric fluid for 2 h and for 2–4 h in intestinal fluid, followed by rapid release of the drug after a total of 4–6 h of lag time depending on the type of disintegrants. The lag time was advanced in case of DBS while delayed in case of AT. On histological examination, the inclusion complex-loaded suspension caused less intestinal tissue damage than naproxen alone. Based on these findings, the colonic-release tablet with enteric coatings which contains inclusion complex and disintegrants could be useful to deliver drugs like naproxen to the lower small intestine and upper colon with increased dissolution and reduced intestinal tissue damage.
Keywords: Naproxen; Inclusion complex; Colonic release; Different coated structures; Reduced intestinal tissue damage;

Within the past decade, more than half of the drug candidates generated are poorly water soluble and therefore overcoming the low aqueous solubility of drug candidates becomes critical for product development. Vitamin E TPGS (VeTPGS), a non-ionic surfactant, has been used in both liquid and solid dosage forms to solubilize compounds and improve their bioavailability. To prepare solid dosage forms using VeTPGS, VeTPGS is often mixed with other excipients, mostly polymers. However, there is still a lack of understanding of miscibility between VeTPGS and polymers from a thermodynamic point of view. In this paper, the miscibility of VeTPGS with polymers has been studied in the light of the Flory–Huggins (F–H) theory with an objective to understand the effect of dispersion forces (solubility parameter) and nondispersive interactions on the miscibility between VeTPGS and polymers. A series of polymers with similar solubility parameters and structure similarity were selected. Binary blends of polymers and VeTPGS were prepared using a vapor evaporation technique followed by XRPD, DSC, and SEM characterization. Results suggest that the miscibility between VeTPGS and PMMA is very likely due to a specific interaction between the hydrophobic portion of VeTPGS (Vitamin E) and PMMA.
Keywords: Vitamin E TPGS; Solid dispersion; Flory–Huggins theory; F–H interaction parameter; Miscibility and modified F–H theory;

Beneficial properties for insulin absorption using superporous hydrogel containing interpenetrating polymer network as oral delivery vehicles by Lichen Yin; JieYing Ding; Likun Fei; Miao He; Fuying Cui; Cui Tang; Chunhua Yin (220-229).
In this investigation, superporous hydrogels containing poly (acrylic acid-co-acrylamide)/O-carboxymethyl chitosan (O-CMC) full-interpenetrating polymer networks (SPH-IPNs) were evaluated for their potentials in effective insulin absorption via the oral route. Insulin release from the SPH-IPNs exhibited sensitivity towards pH and ionic strength. After drug loading and release, the circular dichroism (CD) spectra revealed that conformation of insulin had no significant alteration and bioactivity of insulin was well preserved according to hypoglycaemic effect in mice. Through their abilities to bind Ca2+ and to entrap the enzymes, SPH-IPNs could partly inactivate trypsin and α-chymotrypsin, and SPH-IPN with higher O-CMC/monomer ratio appeared more potent. Swollen SPH-IPNs could attach mechanically and muco-adhere to the intestinal wall, thus achieving improved retentive properties compared to commonly used muco-adhesive excipient Carbopol® 934. Transport of insulin across rat intestine and colon ex vivo was enhanced around two- to three-fold after application of the SPH-IPN. Insulin-loaded SPH-IPN showed significant hypoglycaemic effects following oral administration to healthy rats, achieving a 4.1% pharmacological availability compared to subcutaneous insulin injection. These pronounced properties demonstrated that the SPH-IPN would be a promising peroral carrier for insulin and other peptide drugs.
Keywords: Insulin; Superporous hydrogels containing full-interpenetrating polymer networks; Enzymatic inhibition; Transport enhancement; Muco-adhesion; Oral administration;

Hepatobiliary excretion and enterohepatic circulation of colchicine in rats by Yu-Jen Chen; Shiou-Mei Huang; Chia-Yuan Liu; Pen-Ho Yeh; Tung-Hu Tsai (230-239).
This study investigated the pharmacokinetics of unbound colchicine in rat blood, liver and bile, and its interaction with cyclosporin A (CsA; P-glycoprotein inhibitor) and proadifen (non-specific cytochrome P450 inhibitor) by using a microdialysis and liquid chromatographic system. The pharmacokinetics of colchicine in rat blood showed elimination in a nonlinear manner within the dosage ranges of 1–10 mg/kg. Twenty minutes after administration, colchicine reached maximum concentration in the liver and bile. The liver-to-blood distribution ratios (AUCliver/AUCblood) were 1.8 ± 0.6, 1.0 ± 0.2 and 0.8 ± 0.1, and the bile-to-blood distribution ratios (AUCbile/AUCblood) were 121.6 ± 24.7, 102.2 ± 13.4 and 116.5 ± 18.4 at dosages of 1, 3 and 10 mg/kg, respectively. The high hepatobiliary excretion of colchicine may lead to increased toxicity in normal tissues and indicates that colchicine undergoes hepatobiliary excretion against the concentration gradient from bile-to-blood. The area under the curse (AUC) of colchicine in the liver increased in the proadifen-treated groups, suggesting that metabolism of colchicine may involve cytochrome P450. CsA pretreatment caused an increase in the AUC of colchicine in the blood, a decreased AUC in the bile, and a profound decline in the bile-to-blood distribution ratio. Furthermore, the acute diarrhea and body weight loss caused by colchicine were delayed by pretreatment with CsA. These results indicate that the hepatobiliary excretion of colchicine was regulated by P-glycoprotein (P-gp) and the related acute diarrhea could be modulated by CsA. By using a paired rats model, the enterohepatic circulation of colchicine was also observed.
Keywords: Colchicine; Cytochrome P450; Enterohepatic circulation; Hepatobiliary excretion; P-glycoprotein;

Pharmacokinetics and metabolism of lithospermic acid by LC/MS/MS in rats by Li Wang; Qiang Zhang; Xiaochuan Li; Youli Lu; Zhimou Xue; Lijiang Xuan; Yiping Wang (240-246).
The pharmacokinetics and metabolism of lithospermic acid (LA), a component isolated from Salvia miltiorrhiza, and its two O-methylated metabolites (3′-monomethyl- and 3′,3″-dimethyl-lithospermic acid), were analyzed by a rapid and specific isocratic liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Rat serum samples collected after intravenous and oral administration were analyzed for obtaining pharmacokinetic data of LA. Two O-methylated metabolites, namely one 3′-monomethyl- and one 3′,3″-dimethyl-lithospermic acid were detected in rat serum and bile samples after intravenous and oral administration of LA, respectively. An oral bioavailability of 1.15% was found, with the AUC0–t values of 301.89 and 3.46 mg h/L for intravenous and oral administration, respectively. The total recovery from bile was 75.36% (0.46% for LA, 17.23% for M1, and 57.67% for M2) after intravenous administration, and 4.26% (0.00% for LA, 0.10% for M1, and 4.16% for M2) after oral administration. These results indicate that methylation is the main metabolic pathway of LA, and that LA is excreted into rat bile and finally into feces.
Keywords: Lithospermic acid; Pharmacokinetics; Metabolism; Rat; Bile; LC/MS/MS;

Pluronic F127-g-poly(acrylic acid) copolymers as in situ gelling vehicle for ophthalmic drug delivery system by Wen-Di Ma; Hui Xu; Chao Wang; Shu-Fang Nie; Wei-San Pan (247-256).
To prolong the precorneal resident time and improve ocular bioavailability of the drug, Pluronic F127-g-poly(acrylic acid) copolymers were studied as in situ gelling vehicle for ophthalmic drug delivery system. The rheological properties and in vitro drug release of Pluronic-g-PAA copolymer gels were investigated. The rheogram and in vitro drug release studies indicated that the drug release rates decreased as acrylic acid/Pluronic molar ratio and copolymer solution concentration increased. But the drug concentration had no obvious effect on drug release. The release rates of the drug from such copolymer gels were mainly dependent on the gel dissolution. In vivo resident experiments showed the drug resident time and the total resident amount in rabbit's conjunctiveal sac increased by 5.0 and 2.6 folds for in situ gel, compared with eye drops. The decreased loss angle at body temperature and prolonged precorneal resident time also indicated that the copolymer gels had bioadhesive properties. These in vivo experimental results, along with the rheological properties and in vitro drug release studies, demonstrated that in situ gels containing Pluronic-g-PAA copolymer may significantly prolong the drug resident time and thus improve bioavailability. Pluronic-g-PAA copolymer can be a promising in situ gelling vehicle for ophthalmic drug delivery system.
Keywords: Ophthalmic drug delivery system; Temperature-responsive in situ gel; Rheology; Pluronic F127-g-poly(acrylic acid) copolymers; Bioadhesive properties;

Preparation of chitosan–gelatin scaffold containing tetrandrine-loaded nano-aggregates and its controlled release behavior by An Xiaoyan; Yang Jun; Wang Min; Zhang Haiyue; Chang Li; Yao Kangde; Yao Fanglian (257-264).
A well-timed delivery of bioactive macromolecules from the porous scaffolds is very important in tissue engineering. Tetrandrine (Ted) is one of a large number of known plant derived bisbenzylisoquinoline alkaloids and is obtained from the roots of Stephania tetrandria. Ted can be used as a modifier to poly(l-lactic acid) scaffolds to promote chondrocyte differentiation and secrete type II collagen. But the effect of Ted on chondrocyte's behavior strongly depends on the concentration of Ted in the culture media. Here amphiphilic diblock copolymer (PLAE) composed of l-lactide and methoxy poly(ethylene glycol) (MePEG) was prepared and the Ted loaded copolymeric nanospheres were obtained by self-emulsification and then solvent evaporation. The mean sizes of core/shell type PLAE nanospheres and Ted-loaded nanospheres are about 60 and 100 nm, respectively. Chitosan–gelatin (Cs–Gel) porous scaffolds loaded with PLAE–Ted nanospheres were fabricated through freeze drying. Ted release behaviors from PLAE–Ted nanospheres and porous scaffolds were investigated. The result shows that the Ted-loaded nanospheres could be embedded within Cs–Gel scaffolds and no initial burst release could be observed in the release patterns. Here a room can be provided for the scaffolds to sustained release bioactive component in tissue engineering.
Keywords: PLA/MePEG diblock copolymer; Nanosphere; Scaffolds; Drug release; Tetrandrine;

Effect of cyclosporin A on the brain regional distribution of doxorubicin in rats by Yen-Ju Hsieh; Chih-Hsien Chang; Shu-Pei Huang; Chia-Wen Lin; Meng-Nan Wang; Yu-Tse Wu; Yu-Jen Chen; Tung-Hu Tsai (265-271).
Doxorubicin (DOX) is an anthracycline antibiotic that possesses broad-spectrum antineoplastic activity, and is one of the most important anticancer agents. The purpose of this study was to investigate the effects of cyclosporine A (CsA) on the brain regional distribution of DOX and its liposome DOX formulation (Lipo-Dox). Liquid chromatography with tandem mass spectrometry (LC–MS/MS) was used to measure DOX in rat plasma and in various brain regions (cerebral cortex, hippocampus, striatum, midbrain, cerebellum, and the rest of brain). Good linearity was achieved over the 5–5000 ng/mL range, with coefficients of correlation greater than 0.995. The limit of quantification for doxorubicin was 5 ng/mL. This study was divided into the following four groups: DOX alone, DOX + CsA, Lipo-Dox alone and Lipo-Dox + CsA. After administering DOX (5 mg/kg, i.v.) alone and DOX + CsA (10 mg/kg, i.v.), it was undetectable in various brain regions. When the same dose of Lipo-Dox (5 mg/kg, i.v.) and Lipo-Dox + CsA (10 mg/kg, i.v.) were given individually, the plasma level and the brain regional level of DOX were much greater than those of DOX given alone. These results indicate that Lipo-Dox prolongs the DOX level in plasma and enhances brain distribution of DOX. The disposition of DOX might be regulated by P-glycoprotein.
Keywords: Brain regional distribution; Doxorubicin; P-glycoprotein; Pharmacokinetics;

High throughput methods to characterize protein permeation and release by Martinus A.H. Capelle; Robert Gurny; Tudor Arvinte (272-278).
Spectroscopic methods have been developed to study protein permeation and release kinetics in multi-well plates. The permeation of bovine serum albumin (BSA) through a membrane, which separated a 96-well plate in two compartments, was characterized. A change in fluorescence intensity was measured corresponding to the permeation of BSA from one compartment to another. The permeation of BSA was influenced by the pore size and pore density size of the membrane. The multi-well plates were also used to study the release of a protein drug, hirudin, from an agar hydrogel. A hirudin formulation was mixed at 60 °C with liquid agar and the mixture turned to a gel by cooling at room temperature. The gel entrapping hirudin was formed inside the wells of a 96-well plate. On top of the 100 μl agar–hirudin gel a volume of 200 μl of 10 mM phosphate buffer pH 7.4, 140 mM NaCl was added. The release kinetics of hirudin from the gel were measured following the changes in the hirudin intrinsic tyrosine fluorescence. The release of hirudin over 12 h was measured at three positions: at the bottom of the agar gel, at the interface of the gel with the solution, and in the middle of the receiver solution. The data presented in this paper indicate that high throughput methods can be applied in the characterization of protein drug release from drug delivery systems using small sample volumes.
Keywords: Protein formulation; Drug release; Permeability; Hirudin; High throughput screening;

Enhancement of dissolution rate and bioavailability of aceclofenac: A chitosan-based solvent change approach by Srinivas Mutalik; Parambil Anju; Krishnan Manoj; Achutha Nayak Usha (279-290).
In this study the significant effect of chitosan on improving the dissolution rate and bioavailability of aceclofenac has been demonstrated by simple solvent change method. Chitosan was precipitated on aceclofenac crystals using sodium citrate as the salting out agent. The pure drug and the prepared co-crystals with different concentrations of chitosan (0.05–0.6%) were characterized in terms of solubility, drug content, particle size, thermal behaviour (differential scanning calorimetry, DSC), X-ray diffraction (XRD), morphology (scanning electron microscopy, SEM), in vitro drug release and stability studies. The in vivo performance was assessed by preclinical pharmacodynamic (analgesic and anti-inflammatory activity) and pharmacokinetic studies. The particle size of the prepared co-crystals was drastically reduced during the formulation process. The DSC showed a decrease in the melting enthalpy indicating disorder in the crystalline content. The XRD also revealed a characteristic decrease in crystallinity. The dissolution studies demonstrated a marked increase in the dissolution rate in comparison with pure drug. The considerable improvement in the dissolution rate of aceclofenac from optimized crystal formulation was attributed to the wetting effect of chitosan, decreased drug crystallinity, altered surface morphology and micronization. The optimized co-crystals exhibited excellent stability on storage at accelerated conditions. The in vivo studies revealed that the optimized crystal formulation provided a rapid pharmacological response in mice and rats besides exhibiting improved pharmacokinetic parameters in rats.
Keywords: Aceclofenac; Chitosan; Crystals; Sodium citrate; Dissolution; Pharmacokinetics; Pharmacodynamics;

The aim of this research is to investigate the effects of sodium lauryl sulfate (SLS) on ionotropically cross-linked alginate beads. Different levels of SLS were mixed with sodium alginate and chlorpheniramine maleate (as loaded model drug). The resulting viscous solutions were dropped onto aqueous solutions of zinc or calcium ions for ionotropic curing. The generated beads were assessed by their drug releasing profiles, infrared and differential scanning colorimetery (DSC) traits.SLS was found to exert profound concentration-dependent impacts on the characteristics of zinc-crosslinked alginate beads such that moderate modifications in the levels of SLS switched drug release from enteric coating-like behavior to a biphasic release modifiable to sustained-release by the addition of minute amounts of xanthan gum.Calcium cross-linking failed to reproduce the same behavior, probably due to the mainly ionic nature of calcium–carboxylate bonds compared to the coordinate character of their zinc–carboxylate counterparts. Apparently, moderate levels of SLS repel water penetration into the beads, and therefore minimize chlorpheniramine release. However, higher SLS levels seem to discourage polymeric cross-linking and therefore allow biphasic drug release.
Keywords: Sodium alginate; Beads; Ionotropic cross-linking; Calcium; Zinc; Chlorpheniramine; SLS; Release profiles; Infrared; DSC;

Floating dosage forms to prolong gastro-retention—The characterisation of calcium alginate beads by Frances Stops; John T. Fell; John H. Collett; Luigi G. Martini (301-311).
Floating calcium alginate beads, designed to improve drug bioavailability from oral preparations compared with that from many commercially available and modified release products, have been investigated as a possible gastro-retentive dosage form. A model drug, riboflavin, was also incorporated into the formula.The aims of the current work were (a) to obtain information regarding the structure, floating ability and changes that occurred when the dosage form was placed in aqueous media, (b) to investigate riboflavin release from the calcium alginate beads in physiologically relevant media prior to in vivo investigations.Physical properties of the calcium alginate beads were investigated. Using SEM and ESEM, externally the calcium alginate beads were spherical in shape, and internally, air filled cavities were present thereby enabling floatation of the beads. The calcium alginate beads remained buoyant for times in excess of 13 h, and the density of the calcium alginate beads was <1.000 g cm−3. Riboflavin release from the calcium alginate beads showed that riboflavin release was slow in acidic media, whilst in more alkali media, riboflavin release was more rapid.The characterisation studies showed that the calcium alginate beads could be considered as a potential gastro-retentive dosage form.
Keywords: Floating dosage forms; Riboflavin; Citric acid; Gastro-retentive dosage form; In vitro release; Dissolution release profile; Zero order;

Tyrosine-derived nanospheres for enhanced topical skin penetration by L. Sheihet; P. Chandra; P. Batheja; D. Devore; J. Kohn; B. Michniak (312-319).
The objective of this study was to investigate the passive skin penetration of lipophilic model agents encapsulated within tyrosine-derived nanospheres. The nanospheres were formed by the self-assembly of a biodegradable, non-cytotoxic ABA triblock copolymer. The A-blocks were poly(ethylene glycol) and the hydrophobic B-blocks were oligomers of suberic acid and desaminotyrosyl-tyrosine alkyl esters. These nanospheres had an average hydrodynamic diameter of about 50 nm and formed strong complexes with fluorescent dyes, 5-dodecanoylaminofluorescein (DAF, Log D = 7.54) and Nile Red (NR, Log D = 3.10). These dyes have been used here as models for lipophilic drugs. The distribution of topically applied nanosphere-dye formulations was studied in human cadaver skin using cryosectioning and fluorescence microscopy. Permeation analysis (quantified fluorescence) over a 24 h period revealed that the nanospheres delivered nine times more NR to the lower dermis than a control formulation using propylene glycol. For DAF, the nanosphere formulation was 2.5 times more effective than the propylene glycol based control formulation. We conclude that tyrosine-derived nanospheres facilitate the transport of lipophilic substances to deeper layers of the skin, and hence may be useful in topical delivery applications.
Keywords: Biodegradable nanospheres; Topical delivery; Skin penetration; Lipophilic; Fluorescence;

PLGA nanoparticles simultaneously loaded with vincristine sulfate and verapamil hydrochloride: Systematic study of particle size and drug entrapment efficiency by Xiangrong Song; Yu Zhao; Wenbin Wu; Yueqi Bi; Zheng Cai; Qiuhong Chen; Yuanbo Li; Shixiang Hou (320-329).
PLGA nanoparticles simultaneously loaded with vincristine sulfate (VCR) and verapamil hydrochloride (VRP) were prepared via combining O/W emulsion solvent evaporation and salting-out method. Ten independent processing parameters and two materials characteristics were assessed systematically to enhance the incorporation of the two hydrophilic low molecular weight drugs into PLGA nanoparticles and minimize nanoparticles size. Approaches investigated for the enhancement of drug entrapment efficiencies and the minimization of particle size included the influence of the molecular weight (MW) of PLGA and the lactide to glycolide (L:G) ratio of PLGA, PLGA concentration, the degrees of hydrolyzation and polymerization of PVA, PVA concentration, initial VCR and VRP content, acetone to dichloromethane volume ratio, aqueous phase pH, salt concentration of aqueous phase, aqueous to organic phase volume ratio, sonication time, sonication energy and removal rate of organic solvents. The nanoparticles produced by optimal formulation were submicron size (111.4 ± 2.35 nm, n  = 3) and of low polydispersity (0.062 ± 0.023, n  = 3). Nanoparticles observed by transmission electron microscopy (TEM) showed extremely spherical shape. The entrapment efficiencies determined with high performance liquid chromatogram (HPLC) by ultracentrifuge method were 55.35 ± 4.22% for VCR and 69.47 ± 5.34% for VRP, respectively (n  = 3).
Keywords: Vincristine sulfate; Verapamil hydrochloride; PLGA; Nanoparticles; O/W; Salting-out;

Self-assembled drug delivery systems by Yiguang Jin; Rui Xin; Ping Ai; Dawei Chen (330-337).
Self-assembled drug delivery systems (SADDS) are defined as the self-aggregates of amphiphilic prodrugs. Prodrug, molecular self-assembly and nanotechnology are involved in SADDS manufacturing. But the knowledge of the self-assembly of amphiphilic prodrugs and the formation rules of SADDS is very limited. In this paper, five cholesteryl derivatives of antiviral nucleoside analogues were synthesized, involving antiviral acyclovir, didanosine and zidovudine, and the different acyl linkers, succinyl, adipoyl and phosphoryl. The derivatives are typical amphiphiles with nucleosides as polar heads and long-chained lipids as hydrophobic tails. The derivatives showed the similar soluble behavior, and the solubility highly depended on the types of solvents. Two forces, hydrogen bonding and hydrophobic interaction in alcohol solutions could improve the derivatives dissolving. However, the molecular self-assembly of derivatives could prefer to happen in the noncompetitive solvents including chloroform and tetrahydrofuran (THF) based on the intermolecular hydrogen bonding between nucleobase moieties, which could greatly increase their solubility. The derivatives formed nanosized vesicles based on hydrophobic interaction after injecting their THF solutions into water. The volume ratios of polar heads and hydrophobic tails of amphiphiles could determine the vesicle size, and the amphiphiles with large ratios would prefer to form small vesicles. The self-assembled vesicles would likely become SADDS.
Keywords: Antiviral; Cholesteryl; Molecular self-assembly; Nucleosides; Prodrugs; Vesicles;

Preparation and characterization of intravenously injectable nimodipine nanosuspension by Ruolan Xiong; Weigen Lu; Jun Li; Peiquan Wang; Rong Xu; Tingting Chen (338-343).
The purpose of this study was to develop an alternative, improved and better tolerated injectable nimodipine nanosuspension compared with commercially available ethanol solution. In this study, nimodipine nanosuspension was prepared by high-pressure homogenization (HPH). The effects of the production parameters such as pressure, cycle numbers and crushing principles on the mean particle size, 99% diameter and polydispersity of the nanosuspension were investigated. Characterization of the product was performed by scanning electron microscope (SEM) and differential scanning calorimeter (DSC). The safety of the nimodipine nanosuspension was discussed with special attention to contamination by microparticles and the increase in saturation solubility C s. Irritability study in rabbits showed that this formulation provided less local irritation and phlebitis risks than the commercial ethanol product, which represented a promising new drug formulation for intravenous therapy of subarachnoid hemorrhage (SAH)-related vasospasm.
Keywords: Nimodipine; Nanosuspension; High-pressure homogenization; Crystal form; Vascular irritation;

Design, synthesis and in vitro evaluation of a novel “stealth” polymeric gene vector by He Guosen; Feng Min; Luo Xin; Subbu Venkatraman (344-350).
We report on the synthesis of a novel gene carrier that has low interaction with serum components, as well as low cytotoxicity. Cationic copolymers composing branched poly(ethylenimine) (PEI) grafted with hydrophilic poly(ethylene glycol) (PEG) and poly(l-lactic acid) (PLLA) or small-molecule oleoyl were synthesized and evaluated as novel gene carriers in this study. The copolymers were complexed with plasmid DNA and the resulting polyplexes were approximately 140 nm in diameter and had a positive surface potential (ζ  = +13.8 mV) at the N/P ratio of 10/1. The experiments showed that copolymers with the oleoyl moiety were superior to the other two copolymers (with PLLA), in terms of in vitro gene transfection efficiency. Safety studies using MTT assay indicated much lower cytotoxicity of the oleoyl polyplexes than the pDNA/PEI complexes. The intracellular behavior of the polyplexes was monitored by confocal laser scanning microscopy, and it was found that the polyplexes were internalized into HeLa cells very effectively. At the same time, the plasmid DNA carried by the oleoyl-containing copolymers was found to localize in the nucleus of the recipient cells. One experiment comparing serum-free and serum-containing media indicated that the oleoyl polyplexes may be able to evade the reticulo-endothelial system (RES) better than the PEI–pDNA complex.
Keywords: Gene vector; Cationic copolymer; Intracellular distribution; Polyplex;

Microemulsions containing lecithin and sugar-based surfactants: Nanoparticle templates for delivery of proteins and peptides by Anja Graf; Elisabeth Ablinger; Silvia Peters; Andreas Zimmer; Sarah Hook; Thomas Rades (351-360).
Two pseudo-ternary systems comprising isopropyl myristate, soybean lecithin, water, ethanol and either decyl glucoside (DG) or capryl-caprylyl glucoside (CCG) as surfactant were investigated for their potential to form microemulsion templates to produce nanoparticles as drug delivery vehicles for proteins and peptides. All microemulsion and nanoparticle compounds used were pharmaceutically acceptable and biocompatible. Phase diagrams were established and characterized using polarizing light microscopy, viscosity, conductivity, electron microscopy, differential scanning calorimetry and self-diffusion NMR. An area in the phase diagrams containing optically isotropic, monophasic systems was designated as the microemulsion region and systems therein identified as solution-type microemulsions. Poly(alkylcyanoacrylate) nanoparticles prepared by interfacial polymerisation from selected microemulsions ranged from 145 to 660 nm in size with a unimodal size distribution depending on the type of monomer (ethyl (2) or butyl (2) cyanoacrylate) and microemulsion template. Generally larger nanoparticles were formed by butyl (2) cyanoacrylate. Insulin was added as a model protein and did not alter the physicochemical behaviour of the microemulsions or the morphology of the nanoparticles. However, insulin-loaded nanoparticles in the CCG containing system decreased in size when using butyl (2) cyanoacrylate. This study shows that microemulsions containing sugar-based surfactants are suitable formulation templates for the formation of nanoparticles to deliver peptides.
Keywords: Microemulsions; Nanoparticles; Alkylcyanoacrylate; Self-diffusion NMR; Protein delivery; Insulin;

In this study, a well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles was stabilized by chitosan (CS) and o-carboxymethylchitosan (OCMCS), respectively. The resulting magnetic Fe3O4 nanoparticles were characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscope (TEM), zeta-potential measurement and vibrating sample magnetometry (VSM). TEM results demonstrated a spherical or ellipsoidal morphology with an average diameter of 14–20 nm. The adsorbed layer of CS and OCMCS on the magnetite surface was confirmed by FTIR. XRD illustrated that the resulting magnetic nanoparticles have a spinel structure and lastly VSM results showed the modified magnetic Fe3O4 nanoparticles were superparamagnetic. The adsorption mechanism of CS and OCMCS onto the surface of Fe3O4 nanoparticles is believed to be the electrostatic and coordination interactions, respectively. The mechanisms of both CS and OCMCS stabilizing the suspension of Fe3O4 nanoparticles were supposed electrostatic repulsion. These well-dispersed superparamagnetic Fe3O4 nanoparticles stabilized by the biocompatible CS or OCMCS dispersant should have potential applications in biotechnology fields.
Keywords: Superparamagnetic; Fe3O4; Nanoparticles; Well-dispersed; Chitosan; o-Carboxymethylchitosan;

Measurement of drug agglomerates in powder blending simulation samples by near infrared chemical imaging by Weiyong Li; Abraham Woldu; Richard Kelly; Jim McCool; Rick Bruce; Henrik Rasmussen; John Cunningham; Denita Winstead (369-373).
This research note describes a powder blending simulation study conducted using 20-mL scintillation vials and a bench-top rotating mixer on a scale of 2 g for each sample. In order to investigate the impact of mean particle size and size distribution on blending behavior of an active pharmaceutical ingredient (API), the drug substance was separated into sieve fractions using the US standard sieves of 60, 80, 100, 200, and 325 mesh. Each of the fractions was mixed with two excipients (hydroxypropyl methylcellulose and microcrystalline cellulose) for up to 20 min. Then the blending samples were analyzed by a near infrared chemical imaging (NIR-CI) system. The NIR-CI system was able to measure API particles/domains (agglomerates) at 0.001 mm2 and above within a 11.2 mm × 9.0 mm field of view. It was found that blends prepared with larger API particles (60–200 mesh) contain agglomerated API domains ≥0.1 mm2. The blends prepared with finer API particles (≤325 mesh) show the characteristics of a randomized mixing. This simple and effective method can be used for evaluation of blending behavior for APIs in formulation development.
Keywords: Near infrared spectroscopy; Chemical imaging; Blend uniformity; Simulation; Randomized mixing;

Noticeboard (374).