International Journal of Pharmaceutics (v.444, #1-2)

Characteristics of sequential targeting of brain glioma for transferrin-modified cisplatin liposome by Qing Lv; Li-Min Li; Min Han; Xin-Jiang Tang; Jin-Na Yao; Xiao-Ying Ying; Fan-Zhu Li; Jian-Qing Gao (1-9).
Methods on how to improve the sequential targeting of glioma subsequent to passing of drug through the blood–brain barrier (BBB) have been occasionally reported. However, the characteristics involved are poorly understood. In the present study, cisplatin (Cis) liposome (lipo) was modified with transferrin (Tf) to investigate the characteristics of potential sequential targeting to glioma. In bEnd3/C6 co-culture BBB models, higher transport efficiency across the BBB and cytotoxicity in basal C6 cells induced by Cis-lipo(Tf) than Cis-lipo and Cis-solution, suggest its sequential targeting effect. Interestingly, similar liposomal morphology as that of donor compartment was first demonstrated in the receptor solution of BBB models. Meanwhile, a greater acquisition in the lysosome of bEnd3, distributed sequentially into the nucleus of C6 cells were found for the Cis-lipo(Tf). Pre-incubation of chlorpromazine and Tf inhibited this process, indicating that a clathrin-dependent endocytosis is involved in the transport of Cis-lipo(Tf) across the BBB.
Keywords: Sequential targeting; Brain-targeted delivery; Transferrin; Cisplatin; Glioma;

In vivo/in vitro pharmacokinetic and pharmacodynamic study of spray-dried poly-(dl-lactic-co-glycolic) acid nanoparticles encapsulating rifampicin and isoniazid by L.L.I.J. Booysen; L. Kalombo; E. Brooks; R. Hansen; J. Gilliland; V. Gruppo; P. Lungenhofer; B. Semete-Makokotlela; H.S. Swai; A.F. Kotze; A. Lenaerts; L.H. du Plessis (10-17).
Poly-(dl-lactic-co-glycolic) acid (PLGA) nanoparticles were prepared by a double emulsion solvent evaporation spray-drying technique and coated with polyethylene glycol (PEG 1% v/v). The PLGA nanoparticles had a small size (229 ± 7.6 to 382 ± 23.9 nm), uniform size distribution and positive zeta potential (+12.45 ± 4.53 mV). In vitro/in vivo assays were performed to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) performance of these nanoparticles following nanoencapsulation of the anti-tuberculosis drugs rifampicin (RIF) and isoniazid (INH). The results demonstrated the potential for the reduction in protein binding of these drugs by protection in the polymer core. Furthermore, in vitro efficacy was demonstrated using Mycobacterium tuberculosis (M. tb.) (strain H37Rv). Sustained drug release over seven days were observed for these drugs following once-off oral administration in mice with subsequent drug distribution of up to 10 days in the liver and lungs for RIF and INH, respectively. It was concluded by these studies combined with our previous reports that spray-dried PLGA nanoparticles demonstrate potential for the improvement of tuberculosis chemotherapy by nanoencapsulation of anti-tuberculosis drugs.
Keywords: PLGA nanoparticles; In vitro; In vivo; Pharmacokinetic; Pharmacodynamic; Rifampicin; Isoniazid; PEG coated;

Improved dissolution rate and oral bioavailability of lovastatin in red yeast rice products by Chia-Hao Chen; Jyh-Chin Yang; Yow-Shieng Uang; Chun-Jung Lin (18-24).
Lovastatin, categorized as a class II compound according to the Biopharmaceutics Classification System, is mainly responsible for the blood cholesterol lowering effect of red yeast rice (RYR). The aim of this study was to compare the dissolution rate, physical state, and oral bioavailability of lovastatin in three RYR products (LipoCol Forte, Cholestin, or Xuezhikang) to those of two lovastatin tablets (Mevacor or Lovasta). The results showed that the dissolution rate of lovastatin in various dissolution media in the registered RYR products was faster and higher than that of lovastatin in lovastatin tablets. Powder X-ray diffraction and differential scanning calorimetry patterns showed that the crystallinity of lovastatin was reduced in RYR products. In human studies, the AUC and C max values for both lovastatin and its active metabolite, lovastatin acid, were significantly higher in volunteers receiving LipoCol Forte capsules or powder than in those receiving lovastatin tablets or powder. In addition, shorter and less variable T max values were observed in volunteers taking LipoCol Forte than in those taking lovastatin tablets. These findings suggest that the oral bioavailability of lovastatin is significantly improved in RYR products as a result of a higher dissolution rate and reduced crystallinity.
Keywords: Lovastatin; Bioavailability; Dissolution; Crystallinity; Red yeast rice;

General procedure to aid the development of continuous pharmaceutical processes using multivariate statistical modeling – An industrial case study by Emanuele Tomba; Marialuisa De Martin; Pierantonio Facco; John Robertson; Simeone Zomer; Fabrizio Bezzo; Massimiliano Barolo (25-39).
Streamlining the manufacturing process has been recognized as a key issue to reduce production costs and improve safety in pharmaceutical manufacturing. Although data available from earlier developmental stages are often sparse and unstructured, they can be very useful to improve the understanding about the process under development. In this paper, a general procedure is proposed for the application of latent variable statistical methods to support the development of new continuous processes in the presence of limited experimental data.The proposed procedure is tested on an industrial case study concerning the development of a continuous line for the manufacturing of paracetamol tablets. The main driving forces acting on the process are identified and ranked according to their importance in explaining the variability in the available data. This improves the understanding about the process by elucidating how different active pharmaceutical ingredient pretreatments, different formulation modes and different settings on the processing units affect the overall operation as well as the properties of the intermediate and final products. The results can be used as a starting point to perform a comprehensive and science-based quality risk assessment that help to define a robust control strategy, possibly enhanced with the integration of a design space for the continuous process at a later stage.
Keywords: Latent variable methods; Pharmaceutical development; Continuous processing; Design space; Quality by design; Quality risk assessment; Control strategy;

Dissolution testing of amorphous solid dispersions by K. Kogermann; A. Penkina; K. Predbannikova; K. Jeeger; P. Veski; J. Rantanen; K. Naelapää (40-46).
Dissolution of co-milled amorphous solid dispersions of piroxicam (PRX): dissolution of PRX; recrystallization of amorphous PRX (aPRX) to PRX monohydrate, swelling of polymer (polyvinyl pyrrolidone,PVP) dissolution of polymer (PVP).The main purpose of this study was to investigate the effect of different polymers, with varying physicochemical properties and molecular weight on the stability and dissolution of co-milled amorphous solid dispersions (ASDs) of piroxicam (PRX). The stability of amorphous PRX (aPRX) in ASDs was significantly improved by the polymers. In-line Raman spectroscopy revealed that solvent mediated solid state changes occurred in biorelevant medium, however differences between ASDs were found. Thus, the dissolution behavior of ASDs of PRX and the respective polymer during conventional large volume (900 ml) and a commercial small volume (20 ml) dissolution testing was evaluated. The results of these studies indicated that the molecular weight of the polymer (PVP90 vs PVP25) is influencing the solubility of PRX from ASD. Interestingly the effect of molecular weight of the polymer was different than reported previously in the literature for the similar ASDs prepared by spray drying. Furthermore, the dose related bioavailability was determined by investigating the experimental saturation concentrations for different doses. These studies confirmed the findings of the dissolution studies. The differences are presumably caused by the formation of physically different diffusion layers around the ASD particles.
Keywords: Amorphous solid dispersion; Solubility; Polymer; Molecular weight of polymer; Dissolution; Co-milling;

Tamoxifen-loaded lecithin organogel (LO) for topical application: Development, optimization and characterization by Amit Bhatia; Bhupinder Singh; Kaisar Raza; Sheetu Wadhwa; Om Prakash Katare (47-59).
Lecithin organogels (LOs) are semi-solid systems with immobilized organic liquid phase in 3-D network of self-assembled gelators. This paper attempts to study the various attributes of LOs, starting from selection of materials, optimization of influential components to LO specific characterization. After screening of various components (type of gelators, organic and aqueous phase) and construction of phase diagrams, a D-optimal mixture design was employed for the systematic optimization of the LO composition. The response surface plots were constructed for various response variables, viz. viscosity, gel strength, spreadability and consistency index. The optimized LO composition was searched employing overlay plots. Subsequent validation of the optimization study employing check-point formulations, located using grid search, indicated high degree of prognostic ability of the experimental design. The optimized formulation was characterized for morphology, drug content, rheology, spreadability, pH, phase transition temperatures, and physical and chemical stability. The outcomes of the study were interesting showing high dependence of LO attributes on the type and amount of phospholipid, Poloxamer™, auxillary gelators and organic solvent. The optimized LO was found to be quite stable, easily applicable and biocompatible. The findings of the study can be utilized for the development of LO systems of other drugs for the safer and effective topical delivery.
Keywords: Response surface methodology; Gels; Poloxamer; Drug delivery; Formulation by design; Design of experiments;

Bicellar systems as new delivery strategy for topical application of flufenamic acid by L. Rubio; C. Alonso; G. Rodríguez; M. Cócera; C. López-Iglesias; L. Coderch; A. De la Maza; J.L. Parra; O. López (60-69).
In this work, bicellar systems, bilayered disc-shaped nanoaggregates formed in water by phospholipids, are proposed as a novel strategy for delivery of the anti-inflammatory flufenamic acid (FFA) to the skin. A comparative percutaneous penetration study of this drug in bicellar systems and other vehicles was conducted. The effects induced on the skin by the application of FFA in the different vehicles were analyzed by attenuated total reflectance-fourier transform infrared (ATR-FTIR). Additionally, using the microscopic technique freeze-substitution transmission electron microscopy (FSTEM) and X-ray scattering technique using synchrotron radiation (SAXS-SR), we studied the possible microstructural and organizational changes that were induced in the stratum corneum (SC) lipids and the collagen of the skin by the application of FFA bicellar systems. Bicellar systems exhibited a retarder effect on the percutaneous absorption of FFA with respect to the other vehicles without promoting disruption in the SC barrier function of the skin. Given that skin disruption is one of the main effects caused by inflammation, prevention of disruption and repair of the skin microstructure should be prioritized in anti-inflammatory formulations.
Keywords: Bicellar systems; Flufenamic acid; DSC; ATR-FTIR; SAXS-SR; FS-TEM; In vitro percutaneous absorption; Drug delivery;

Vitamin B12 loaded polycaprolactone nanofibers: A novel transdermal route for the water soluble energy supplement delivery by Kalaipriya Madhaiyan; Radhakrishnan Sridhar; Subramanian Sundarrajan; Jayarama Reddy Venugopal; Seeram Ramakrishna (70-76).
Biocompatible PCL polymer nanofiber mediated sustained release of hydrophilic drug and applicability as transdermal delivery system is attempted. This new attempt to investigate water soluble vitamin delivery with hydrophobic polymer nanofiber sustained the release of the vitamin and the method is suited for the transdermal patch applications. The drug loaded fibers were characterized with SEM for morphology, porometer for pore size measurements, mechanical strength calculation and FT-IR for drug load characterization. The contact angle measurement showed surface wettability and controlled release of drug was quantified with UV absorption measurements. To further enhance the release of vitamin, the polymer fiber was plasma treated at different time intervals and made hydrophilic gradually. Since the increased surface area and drug encapsulation in nano-reservoirs can able to release drug in small quantities and in a sustained manner we attempted the release of the energy supplement with nanofibrous delivery mode.
Keywords: Vitamin B12; Nanofiber; Transdermal patch; Sustained release;

Biocompatible and biodegradable doxorubicin loaded nanoparticles with targeting ability were prepared from BSA–dextran–folic acid conjugate via a pH adjustment and heating process. The BSA–dextran–folic acid conjugate was produced by an esterification reaction between folic acid and dextran and then the Maillard reaction between the modified dextran and BSA. The nanoparticles have a size about 90 nm and excellent dispersibility at pH 7.4 aqueous solution. The doxorubicin loading efficiency and loading amount of the nanoparticles are larger than 90% and 14%, respectively. The antitumor activity and toxicity of the nanoparticles were evaluated through murine ascites hepatoma H22 tumor-bearing mice. The nanoparticles allow the administration of the doxorubicin with higher dose. At doxorubicin dose of 10 mg/kg, the nanoparticles can achieve 88.9% of the tumor inhibition rate that is the same as the free doxorubicin at the dose of 5 mg/kg. Importantly, the nanoparticles can decrease the toxicity of doxorubicin that results in a significant increase of the average life time in comparison with the free doxorubicin as well as the nanoparticles without folic acid.
Keywords: Antitumor activity; BSA; Drug delivery; Folic acid; Nanoparticles;

Biocompatibility of boron nitride nanotubes: An up-date of in vivo toxicological investigation by Gianni Ciofani; Serena Danti; Simone Nitti; Barbara Mazzolai; Virgilio Mattoli; Mario Giorgi (85-88).
Boron nitride nanotubes (BNNTs) represent an innovative and extremely intriguing class of nanomaterials, and preliminar but encouraging studies about their applications in biomedicine have emerged in the latest years. As a consequence, a systematic investigation of their biosafety has become of fundamental importance in the biomedical research. Extending our previous pilot in vivo study, here we present biocompatibility data of BNNTs injected in rabbits at a dose up to 10 mg/kg. No significant adverse effects were found up to 7 days since their administration, and no impairments in blood, liver and kidney functionality were highlighted. Moreover, a terminal half-life circulation of about 90 min was found. All the collected data are very promising, suggesting the optimal biocompatibility of BNNTs, and thus enabling their exploitation in nanomedicine as nanotransducers and nanocarriers.
Keywords: Boron nitride nanotubes; In vivo testing; Toxicology; Pharmacokinetics;

Water vapour permeability (WVP) and water absorption capacity (WAC) influence physicochemical properties and wearability of transdermal patches considerably. For determination of WVP, a modified transepidermal water loss (TEWL) measurement was developed. These measurements continuously measure WVP of transdermal patches in vitro along with time required to reach steady state, and its magnitude according to the type of polymer used. Additionally, WAC of the patches was examined and related to WVP. According to literature in the field of WVP determination, usually selected points are taken from the evaporation time curve and averaged over a given time span without knowing whether steady state has already been reached or not. The latter causes errors upon averaging. The advantage of the in vitro TEWL measurement presented includes reproducibly adjustable conditions for every time span desired, thus providing information on the kinetics of the experiment and avoiding biased results from averaging. Knowing the shape of the evaporation time curve and thus the kinetics of the experiment allows for focusing on the relevant part of the measurement, i.e. the determination of the steady state level and the time to reach it.Four different polymers (P1–P4) based on sugar-modified polyacrylates were investigated with regard to WVP and WAC of the matrices prepared thereof along with the influence of drug loading and the incorporation of a variety of additives commonly used for transdermal patches. A clear correlation between WVP and the hydrophilicity in terms of the number of free hydroxyl groups of the polymer was elaborated. Additives of higher hydrophilicity compared to that of the polymer itself led to higher WVPs and vice versa. The combination of the model drug lidocaine in its free base form together with the additive succinic acid (Suc) resulted in ionization of the drug and thus in substantially increased WVPs. Addition of α-tocopherol acetate (Toc) into P3 and P4 and Suc into the drug-free matrix of P3 decreased WVP probably by affecting the structure of the polymer network. The same effects were found for WAC upon incorporation of succinic acid into drug-loaded matrices of P3 and P4 (drug-loaded patches of P1 and P2 were not tested) but not for additives which were likely to modify the polymer network structure.
Keywords: TEWL; Water vapour permeability; Transdermal patch;

A thermosensitive morphine-containing hydrogel for the treatment of large-scale skin wounds by Sarah Heilmann; Sarah Küchler; Christian Wischke; Andreas Lendlein; Christoph Stein; Monika Schäfer-Korting (96-102).
(Per)-cutaneous morphine absorption using the finite dose approach over a period of 6 h.Topically applied opioids are an option to induce efficient analgesia in patients with severe skin wounds. For ongoing pain reduction, the vehicle should provide sustained drug release in order to increase the intervals during the regular wound dressing changes. In addition, the formulation should not impair wound healing. Hydrogels provide a moist wound environment, which is known to facilitate the healing process.Investigating poloxamer hydrogels as a carrier system for morphine in terms of release behavior and (per-)cutaneous absorption, poloxamer 407 25 wt.% hydrogel sustained morphine release up to 24 h. The drug release rate decreased with increasing concentration of the gel forming triblock copolymer. Poloxamer 407 25 wt.% hydrogel retarded morphine uptake into reconstructed human skin and percutaneous drug absorption compared to a hydroxyethyl cellulose reference gel.The results of our in vitro study indicate that the thermosensitive poloxamer 407 25 wt.% hydrogel is an appropriate carrier system for the topical application of morphine with regard to sustained drug release and ongoing analgesia.
Keywords: Morphine; Thermosensitive hydrogel; Sustained release; Percutaneous absorption; Reconstructed human skin;

Immunological effects of microneedle-mediated insulin delivery: Preliminary rat studies by Yuqin Qiu; Yunhua Gao; Suohui Zhang; Lei Guo; Jianmin Chen; Bai Xu (103-105).
Antibody levels in female rats immunized with insulin by microneedles or subcutaneous injection.The objective of the present study is to compare the immunogenicity of insulin by microneedle-mediated intradermal delivery or subcutaneous injection. Female and male rats were treated with insulin by either microneedle-mediated intradermal delivery or subcutaneous injection twice a week for 4 weeks. A control group without insulin administration was also studied. Human anti-insulin antibody (AIA) levels were measured by radioimmunoassay (RIA) before starting insulin treatment and after that within 20 weeks. The male rats did not induce positive AIA levels, and there was no significant difference between all male groups. In contrast, in female rats the AIA levels were significantly higher in microneedle group compared with injection group, and lasted from 6 to 20 weeks after starting insulin treatment. The increased immunogenicity of insulin delivery by microneedle might be due to the large number of immune cells in skin.
Keywords: Insulin; Microneedles; Immunogenicity; Intradermal delivery;

A new paradigm in dermatopharmacokinetics – Confocal Raman spectroscopy by R. Mateus; H. Abdalghafor; G. Oliveira; J. Hadgraft; M.E. Lane (106-108).
Confocal Raman spectroscopy (CRS) is a novel approach which has been used to determine stratum corneum thickness and to profile endogenous skin components. However, the utility of this technique for probing drug disposition in vivo has not been explored to date. In this paper we report the use of CRS to investigate the fate of ibuprofen after application from simple formulations which were previously investigated using the tape-stripping approach. Ibuprofen was prepared in propylene glycol (PG) and propylene glycol/water (PG/H2O) solutions. The formulations were then applied to the volar aspect of the forearm of human volunteers. The results confirmed that ibuprofen distribution profiles in the stratum corneum were comparable to previously published data from tape stripping experiments. We propose CRS as a non-invasive method for dermatopharmacokinetic evaluation of topical pharmaceutical formulations.
Keywords: Confocal Raman spectroscopy; Dermatopharmacokinetics; Ibuprofen; Propylene glycol; In vivo;

Self-assembled polymeric nanoparticles for percutaneous co-delivery of hydrocortisone/hydroxytyrosol: An ex vivo and in vivo study using an NC/Nga mouse model by Zahid Hussain; Haliza Katas; Mohd Cairul Iqbal Mohd Amin; Endang Kumolosasi; Fhataheya Buang; Shariza Sahudin (109-119).
In this study, hydroxytyrosol (HT; a potent antioxidant) was co-administered with hydrocortisone (HC) to mitigate the systemic adverse effects of the latter and to provide additional anti-inflammatory and antioxidant benefits in the treatment of atopic dermatitis (AD). The co-loaded nanoparticles (NPs) prepared had shown different particle sizes, zeta potentials, loading efficiencies, and morphology, when the pH of the chitosan solution was increased from 3.0 to 7.0. Ex vivo permeation data showed that the co-loaded NPs formulation significantly reduced the corresponding flux (17.04 μg/cm2/h) and permeation coefficient (3.4 × 10−3  cm/h) of HC across full-thickness NC/Nga mouse skin. In addition, the NPs formulation showed higher epidermal (1560 ± 31 μg/g of skin) and dermal (880 ± 28 μg/g of skin) accumulation of HC than did a commercial HC formulation. Moreover, an in vivo study using an NC/Nga mouse model revealed that compared to the other treatment groups, the group treated with the NPs formulation efficiently controlled transepidermal water loss (13 ± 2 g/m2/h), intensity of erythema (207 ± 12), and dermatitis index (mild). In conclusion, NPs co-loaded with HC/HT is proposed as a promising system for the percutaneous co-delivery of anti-inflammatory and antioxidative agents in the treatment of AD.
Keywords: Allergic contact dermatitis; Corticosteroid; Polyphenol; Topical co-delivery; Polymeric nanoparticles;

Drug-inorganic-polymer nanohybrid for transdermal delivery by Myung Hun Kim; Dae-Hwan Park; Jae-Hun Yang; Young Bin Choy; Jin-Ho Choy (120-127).
For transdermal drug delivery, we prepared a drug-inorganic nanohybrid (FB-LDH) by intercalating a transdermal model drug, flurbiprofen (FB), into the layered double hydroxides (LDHs) via coprecipitation reaction. The X-ray diffraction patterns and FT-IR spectra of the FB-LDH indicated that the FB molecules were successfully intercalated via electrostatic interaction within the LDH lattices. The in vitro drug release revealed that the Eudragit® S-100 in release media could facilitate the drug out-diffusion by effectively replacing the intercalated drug and also enlarging the lattice spacing of the FB-LDH. In this work, a hydrophobic gel suspension of the FB-LDH was suggested as a transdermal controlled delivery formulation, where the suspensions were mixed with varying amounts of Eudragit® S-100 aqueous solution. The Frantz diffusion cell experiments using mouse full-skins showed that a lag time and steady-state flux of the drug could be controlled from 12.8 h and 3.28 μg cm−2  h−1 to less than 1 h and 14.57 μg cm−2  h−1, respectively, by increasing the mass fraction of Eudragit® S-100 solution in gel suspensions from 0% to 20% (w/w), respectively. Therefore, we conclude gel formulation of the FB-LDH have a potential for transdermal controlled drug delivery.
Keywords: Controlled delivery; Drug-inorganic nanohybrid; Formulation; Layered double hydroxide; The Frantz diffusion cell experiments; Transdermal delivery;

Stability and physicochemical characterization of novel milk-based oral formulations by J. Kytariolos; G. Charkoftaki; J.R. Smith; G. Voyiatzis; A. Chrissanthopoulos; S.N. Yannopoulos; D.G. Fatouros; P. Macheras (128-138).
Stokes-side Raman spectra of milk powder, crystalline meloxicam and meloxicam/milk dried mixtures.The purpose of this work was to assess the colloidal stability of novel milk-based formulations.Milk-based formulations were prepared in situ by adding into milk alkaline- or ethanolic-drug solutions containing an array of drugs namely; ketoprofen, tolfenamic acid, meloxicam, tenoxicam and nimesulide, mefenamic acid, cyclosporine A, danazol and clopidogrel besylate. The produced formulations were characterized by means of dynamic lightscattering, ζ-potential studies, atomic force microscopy, fluorescence spectroscopy, Raman spectroscopy complemented with ab initio calculations and stability studies.The presence of the drugs did not induce significant changes in most cases to the particle size and ζ-potential values of the emulsions pointing to the colloidal stability of these formulations. Raman spectroscopy studies revealed interactions of the drugs and the milk at the intermolecular level. Complementary analysis with ab initio calculations confirmed the experimental observations obtained by Raman spectroscopy. Finally the produced drug containing alkaline/ethanolic solutions exhibited stability over a period of up to 12 months.The current data demonstrate that milk is a promising drug carrier.
Keywords: Milk-based formulations; Stability studies; NSAIDs; Cyclosporine; Danazol;

Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation by Christina Avanti; Nur Alia Oktaviani; Wouter L.J. Hinrichs; Henderik W. Frijlink; Frans A.A. Mulder (139-145).
Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under tropical conditions is problematic. In a previous study, we have found that the stability of oxytocin in aspartate buffered formulation is improved by the addition of divalent metal ions (unpublished results). The stabilizing effect of Zn2+ was by far superior compared to that of Mg2+. In addition, it was found that stabilization correlated well with the ability of the divalent metal ions to interact with oxytocin in aspartate buffer. Furthermore, LC–MS (MS) measurements indicated that the combination of aspartate buffer and Zn2+ in particular suppressed intermolecular degradation reactions near the Cys1,6 disulfide bridge. These results lead to the hypothesis that in aspartate buffer, Zn2+ changes the conformation of oxytocin in such a way that the Cys1,6 disulfide bridge is shielded from its environment thereby suppressing intermolecular reactions involving this region of the molecule. To verify this hypothesis, we investigate here the conformation of oxytocin in aspartate buffer in the presence of Mg2+ or Zn2+, using 2D NOESY, TOCSY, 1H–13C HSQC and 1H–15N HSQC NMR spectroscopy. Almost all 1H, 13C and 15N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for 13C or 15N enrichment. 1H–13C and 1H–15N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D2O, with the largest chemical shift changes observed for Cys1. Zn2+ causes more extensive changes in oxytocin in aqueous solution than Mg2+. Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys1, allowing the interactions with Zn2+ to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.
Keywords: Oxytocin; Conformation; Aspartate buffer; Aqueous solution; Divalent metal ions; NMR;

Liposomal encapsulation of the natural flavonoid fisetin improves bioavailability and antitumor efficacy by Johanne Seguin; Laura Brullé; Renaud Boyer; Yen Mei Lu; Miriam Ramos Romano; Yasmine S. Touil; Daniel Scherman; Michel Bessodes; Nathalie Mignet; Guy G. Chabot (146-154).
Liposomal formulation of fisetin improves its bioavailability (A) and antitumor efficacy in mice (B). Lewis lung carcinoma bearing mice treated with empty liposomes (solid squares), empty liposomes and cyclophosphamide at 30 mg/kg (solid triangles), and liposomal fisetin at 35 mg/kg in combination with cyclophosphamide at 30 mg/kg (empty triangles).The natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) has shown antiangiogenic and anticancer properties. Because of fisetin limited water solubility, we designed a liposomal formulation and evaluated its biological properties in vitro and in Lewis lung carcinoma (LLC) bearing mice.A liposomal formulation was developed with DOPC and DODA-PEG2000, possessing a diameter in the nanometer range (173.5 ± 2.4 nm), a high homogeneity (polydispersity index 0.181 ± 0.016) and high fisetin encapsulation (58%). Liposomal fisetin incubated with LLC cells were internalized, induced a typical fisetin morphological effect and increased the sub-G1 cell distribution. In vivo, liposomal fisetin allowed a 47-fold increase in relative bioavailability compared to free fisetin. The effect of liposomal fisetin on LLC tumor growth in mice at low dose (21 mg/kg) allowed a higher tumor growth delay (3.3 days) compared to free fisetin at the same dose (1.6 day). Optimization of liposomal fisetin therapy was attempted by co-treatment with cyclophosphamide which led to a significant improvement in tumor growth delay (7.2 days) compared to cyclophosphamide with control liposomes (4.2 days).In conclusion, fisetin liposomes markedly improved fisetin bioavailability and anticancer efficacy in mice and this formulation could facilitate the administration of this flavonoid in the clinical setting.
Keywords: Fisetin; Liposomes; Pharmacokinetics; Lewis lung carcinoma; Cyclophosphamide; Anticancer effect;

Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine by Janne Raula; Martina Hanzlíková; Antti Rahikkala; Juho Hautala; Esko I. Kauppinen; Arto Urtti; Marjo Yliperttula (155-161).
Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125 nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA.
Keywords: Solid state nanoparticles; Plasmid DNA; Non-viral gene delivery; Leucine coating; Aerosol flow reactor;

Porous starch based self-assembled nano-delivery system improves the oral absorption of lipophilic drug by Zhiwen Zhang; Jian Huang; Shijun Jiang; Zeying Liu; Wangwen Gu; Haijun Yu; Yaping Li (162-168).
A new porous starch based self-assembled nano-delivery system improves the oral absorption of lipophilic drugs in rats.The therapeutic efficacy of lipophilic drugs is commonly restricted by the low systemic exposure after oral administration. In this work, a new delivery system combining the advantages of porous starch and self-assembled nanocarrier was designed to improve the oral absorption of lipophilic drugs. The lipophilic probucol loaded porous starch based self-assembled nano-delivery (PSN) system was developed and characterized. The probucol loaded nanocarrier (PLN) could be formed by self-assembly when PSN was dispersed into the gastrointestinal (GI) fluids. PLN was nanometer-sized particles with narrow size distribution and exhibited good stability in GI fluids. The aqueous solubility of probucol was increased over 50,000-fold by PSN delivery system and the cumulative release of lipophilic probucol was increased over 80% in GI fluids. The distribution of probucol in duodenum, jejunum and ileum was respectively improved 7.17, 15.99 and 33.61-fold by PSN. In particular, the oral bioavailability of probucol from PSN was greatly improved about 9.96-fold than that from free drug suspension and 3.71-fold higher than that from the directed adsorbed probucol loaded porous starch system, which effectively confirmed the high potential of the strategy in enhancing the oral absorption of lipophilic drugs.
Keywords: Porous starch; Self-assembly; Nanoparticles; Oral absorption; Probucol;

High prevalence of pulmonary diseases in childhood requires inhalable medication even for young children. Little is known about the efficiency of aerosol therapy especially in preschool children. One factor which limits the lung dose is the upper airway geometry. Based on clinical data a recently developed idealized pediatric upper airway model (children 4–5 years) was used to investigate the performance of two dry powder inhalers (Easyhaler and Novolizer).In vitro investigations were first examined using steady flow rates and an inhalation volume of 1 L. Chosen flow rates were 28, 41 and 60 L/min (Easyhaler) and 45, 60 and 75 L/min (Novolizer). Afterwards inhalation profiles simulated by an electronic lung were included.The investigations showed high amounts of drug particles (up to 80%) which were deposited in the upper airway model. The pulmonary deposition in vitro using the Easyhaler was about 28% (28–60 L/min) and 22% (inhalation profile). Using the Novolizer in vitro pulmonary doses of 8–12% (45–75 L/min) and about 5% (inhalation profile) were observed.The idealized model shows good performance reproducibility of dry powder inhalers. We have shown that age-dependent models might be appropriate tools for formulation and device development in pediatric age groups.
Keywords: Pediatric upper airway model; Idealized throat model; Children; Dry powder inhaler; Easyhaler; Novolizer;

Cyclodextrin-crosslinked poly(acrylic acid): Synthesis, physicochemical characterization and controlled release of diflunisal and fluconazole from hydrogels by Marguerite J. Kutyła; Lynette K. Lambert; Nigel M. Davies; Ross P. McGeary; P. Nicholas Shaw; Benjamin P. Ross (175-184).
The aim of this work was to develop mucoadhesive hydrogels with variable drug delivery properties by crosslinking poly(acrylic acid) (PAA) with cyclodextrins (CDs). CD-PAA polymers with high CD content and good inter-batch reproducibility were synthesized by activating PAA with SOCl2, then reacting PAA chloride with CD in the presence of 4-dimethylaminopyridine at 50 °C. Manipulation of the synthesis conditions affected the physicochemical character of the CD-PAA polymers and hydrogels in terms of CD content, the average number of ester bonds to an individual CD, viscosity, and the association and release of model drugs. Inclusion complexation of diflunisal (DIF) and fluconazole (FLZ) with CD-PAA hydrogels was assessed by 19F NMR spectroscopy and association constants (K a s) for DIF were in the range 220–486 M−1 with βCD-PAA and 1327–6055 M−1 with hydroxypropyl-βCD-PAA. For FLZ the K a range was 34–171 M−1 with hydroxypropyl-βCD-PAA. The hydrogels were found to release both drugs by means of Fickian diffusion as the predominant mechanism. A slight trend toward negative correlation was found between the K a and Higuchi k H values for DIF. These results highlight the potential of CD-PAA hydrogels to control the release of model drugs through inclusion complexation.
Keywords: Hydroxypropyl-β-cyclodextrin; β-Cyclodextrin; Poly(acrylic acid); Hydrogel; Association constant; Drug delivery;

This work evaluates the influence of FcγR on the pharmacokinetics and pharmacodynamics of a rat anti-integrin-αIIb IgG1 monoclonal antibody, MWReg30, in mice. The pharmacokinetics and pharmacodynamics of MWReg30 were investigated in C57BL/6 control mice, FcγRI/RIII knockout mice, and FcγRIIb knockout mice, following intravenous doses of 0.04–0.4 mg/kg. MWReg30 treatment resulted in a dose-dependent induction of thrombocytopenia in all strains, but sensitivity to MWReg30 was increased in FcγRIIb knockout mice and decreased in FcγRI/RIII knockout mice, relative to results found in control mice. Expressed as a percentage of pre-treatment platelet counts, nadir platelet counts were 28.6 ± 5.0, 88.7 ± 16.6 and 25.3 ± 6.1% at 0.05 mg/kg, 28.4 ± 13.7, 56.7 ± 5.1, and 20.6 ± 9.5% at 0.2 mg/kg, and 24.9 ± 7.2, 38.7 ± 7.5, and 7.4 ± 2.2% at 0.4 mg/kg in control, FcγRI/RIII(−/−) and FcγRIIb(−/−) mice. However, knocking out FcγR did not affect MWReg30 pharmacokinetics. Plasma areas under the concentration vs. time curves (AUC0–10 days) ±SD for MWReg30 were: 5.24 ± 0.68, 5.51 ± 0.24, and 5.39 ± 1.05 nM × d at 0.04 mg/kg, and 12.7 ± 0.5, 13.6 ± 1.1, and 14.5 ± 2.0 nM × d at 0.1 mg/kg in control, FcγRI/RIII(−/−) and FcγRIIb(−/−) mice. The findings further emphasize the role of activating vs. inhibitory FcγR in processing immune complexes (i.e., MWReg30-platelets), while also providing an example where monoclonal antibody pharmacokinetics are not substantially influenced by FcγR expression.
Keywords: Anti-platelet antibody; IgG; Fcγ-receptors; Pharmacokinetics; Pharmacodynamics; Tissue disposition;

Air-jet and vibrating-mesh nebulization of niosomes generated using a particulate-based proniosome technology by Abdelbary Elhissi; Kanar Hidayat; David A. Phoenix; Enosh Mwesigwa; StJohn Crean; Waqar Ahmed; Ahmed Faheem; Kevin M.G. Taylor (193-199).
The aerosol properties of niosomes were studied using Aeroneb Pro and Omron MicroAir vibrating-mesh nebulizers and Pari LC Sprint air-jet nebulizer. Proniosomes were prepared by coating sucrose particles with Span 60 (sorbitan monostearate), cholesterol and beclometasone dipropionate (BDP) (1:1:0.1). Nano-sized niosomes were produced by manual shaking of the proniosomes in deionized water followed by sonication (median size 236 nm). The entrapment of BDP in proniosome-derived niosomes was higher than that in conventional thin film-made niosomes, being 36.4% and 27.5% respectively. All nebulizers generated aerosols with very high drug output, which was 83.6% using the Aeroneb Pro, 85.5% using the Pari and 72.4% using the Omron. The median droplet size was 3.32 μm, 3.06 μm and 4.86 μm for the Aeroneb Pro, Pari and Omron nebulizers respectively and the “fine particle fraction” (FPF) of BDP was respectively 68.7%, 76.2% and 42.1%. The predicted extrathoracic deposition, based on size distribution of nebulized droplets was negligible for all devices, suggesting all of them are potentially suitable for pulmonary delivery of niosomes. The predicted drug deposition in the alveolar region was low using the Omron (3.2%), but greater using the Aeroneb Pro (17.4%) and the Pari (20.5%). Overall, noisome-BDP aerosols with high drug output and FPF can be generated from proniosomes and delivered using vibrating-mesh or air-jet nebulizers.
Keywords: Aerosol; Beclometasone dipropionate; Liposome; Nebulizer; Niosome; Proniosome;

Corrigendum to “Pulmonary delivery of dry powders to rats tolerability limits of an intra-tracheal administration model” [Int. J. Pharm. 434 (2012) 481–487] by A. Guillon; J. Montharu; L. Vecellio; V. Schubnel; G. Roseaud; J. Guillemain; P. Diot; M. de Monte (200).

Corrigendum to “Multi-equilibrium system based on sertraline and β-cyclodextrin supramolecular complex in aqueous solution” [Int. J. Pharmaceut. 421 (2011) 24–33] by Joel J. Passos; Frederico B. De Sousa; Ivana S. Lula; Elison A. Barreto; Juliana F. Lopes; Wagner B. De Almeida; Rubén D. Sinisterra (201).