European Journal of Pharmaceutics and Biopharmaceutics (v.75, #2)

Announcement (I-II).

APV Diary (S1).

Editorial by Robert Gurny; Irene Kanter-Schlifke (79).

The block copolymers PEG2000-b-PLA2200, PEG2000-b-PCL2600 and PEG5000-b-PCL5000 have been currently identified as optimal solubilizing agents for Sagopilone, a poorly water-soluble anticancer drug. In the present study, the stability, formulation feasibility and in vitro as well as in vivo toxicity were evaluated.Dispersion media, storage conditions, and dilutions were varied for stability assessment. The critical micelle concentration (CMC) was determined using a fluorescent probe technique. Lyophilizates and polymeric films were investigated as formulation options. Furthermore, the toxicity was studied in vitro and in vivo using HeLa/MaTu cells and a nude mouse model, respectively.A drug–polymer ratio as low as 1:20 (w/w) was sufficient to solubilize Sagopilone effectively and to obtain stable dispersions (24 h: drug content ⩾95%). Although the micelles exhibited a similar thermodynamic stability (CMC: 10−7–10−6  M), PEG-b-PCL micelles were kinetically more stable than PEG2000-b-PLA2200 (24 h at 37 °C: drug content ⩾90% compared to 30%, respectively). Lyophilization of PEG-b-PCL micelles and storage stability of solid drug-loaded PEG2000-b-PLA2200 films (3 m, 6 °C: drug content of (95.6 ± 1.4)%) were demonstrated for the first time. The high antiproliferative activity has been maintained in vitro (IC50<1 nM). Carrier-associated side effects have not been observed in vivo and the maximum tolerated dose of micellar Sagopilone was determined to be 6 mg/kg.The results of this study indicate that polymeric micelles, especially PEG-b-PCL micelles, offer excellent potential for further preclinical and clinical cancer studies using Sagopilone.
Keywords: Block copolymer micelles; Epothilone; Solubilization; Lyophilization; Polymeric films; Cytotoxicity; In vivo toxicity;

Development of a polymeric micellar formulation for valspodar and assessment of its pharmacokinetics in rat by Ziyad Binkhathlan; Dalia A. Hamdy; Dion R. Brocks; Afsaneh Lavasanifar (90-95).
The aim of this study was to assess the potential of polymeric micelles to solubilize valspodar and modify its pharmacokinetics following intravenous and oral administration in rat. Drug-loaded methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) micellar solutions were prepared and administered either intravenously or orally to healthy Sprague–Dawley rats. Plasma pharmacokinetic parameters of valspodar in its polymeric micellar formulation were compared to its clinical formulation, which uses Cremophor EL and ethanol as solubilizing agents. High loading level was achieved for valspodar in PEO-b-PCL leading to an aqueous solubility of 2.8 mg/mL. Following i.v. administration (5 mg/kg), valspodar in the PEO-b-PCL micelles provided significantly higher (∼77%) plasma AUC compared to the Cremophor EL formulation. The PEO-b-PCL micelles also significantly decreased the volume of distribution (Vdss) and clearance (CL) of valspodar by nearly 49% and 34%, respectively. After oral administration (10 mg/kg), the average C max were similar for both formulations and were both reached at ∼2 h. The plasma unbound fraction of valspodar in the polymeric micellar formulation was significantly lower than control (8.27% versus 14.85%). Our results show that PEO-b-PCL micelles can efficiently solubilize valspodar and favorably modify its pharmacokinetic profile in rat after i.v. administration by decreasing the CL and Vd.
Keywords: Valspodar; PSC 833; Oral; Pharmacokinetics; Protein binding; Polymeric micelles;

The aim of the present study is to evaluate the effect of polyethylene glycol (PEG) chain organization on various physicochemical aspects of drug delivery from poly(d, l-lactide) (PLA) based nanoparticles (NPs). To reach that goal, two different pegylated polymers of poly(d, l-lactide) (PLA) were synthesized. Polymers used in this study are grafted ones in which PEG was grafted on PLA backbone at 7% (mol/mol of lactic acid monomer), PEG7%-g-PLA, and multiblock copolymer of both PLA and PEG, (PLA–PEG–PLA)n with nearly similar PEG insertion ratio and the same PEG chain length. Blank and ibuprofen-loaded NPs were prepared from both polymers and their properties were compared to PLA homopolymer NPs as a control. Encapsulation efficiency of ibuprofen was found to be ∼25% for (PLA–PEG–PLA)n NPs and ∼80% for PEG7%-g-PLA NPs. (PLA–PEG–PLA)n NPs either blank or loaded showed larger hydrodynamic diameter (∼200 nm) than PEG7%-g-PLA NPs (∼135 nm). A significant difference was observed in the amount of PVA associated with the surface of both NPs where 3.6% and 0.4% (wt/wt) were found on the surface of PEG7%-g-PLA and (PLA–PEG–PLA)n NPs, respectively. No observed difference in zeta potential values for both NPs formulations was found. DSC showed the existence of the drug in a crystalline state inside NPs matrix irrespective of the type of polymer used with either shifting or/ and broadening of the drug melting endotherm. Both AFM phase imaging and XPS studies revealed the possibility of existence of more PEG chains at the surface of grafted polymer NPs than (PLA–PEG–PLA)n during NPs formation. The in vitro release behavior showed that (PLA–PEG–PLA)n NPs exhibited faster release rates than PEG7%-g-PLA NPs. The physicochemical differences obtained between both polymers were probably due to different chain organization during NPs formulation. Such pegylated NPs made from these two different polymers might find many applications, being able to convert poorly soluble, poorly absorbed substances into promising drugs, improving their therapeutic performance, and helping them reach adequately their target area. Our results suggest that the properties of pegylated PLA-based NPs can be tuned by proper selection of both polymer composition and polymer architecture.
Keywords: Poly(d, l-lactide); Polymer architecture; Graft polymers; Multiblock; Chain organization; X-ray photoelectron spectroscopy (XPS);

A toxicological evaluation of inhaled solid lipid nanoparticles used as a potential drug delivery system for the lung by M. Nassimi; C. Schleh; H.D. Lauenstein; R. Hussein; H.G. Hoymann; W. Koch; G. Pohlmann; N. Krug; K. Sewald; S. Rittinghausen; A. Braun; C. Müller-Goymann (107-116).
Inhalation is a non-invasive approach for both local and systemic drug delivery. This study aimed to define the therapeutic window for solid lipid nanoparticles (SLNs) as a drug delivery system by inhalation from a toxicological point of view.To estimate the toxic dose of SLNs in vitro, A549 cells and murine precision-cut lung slices (PCLS) were exposed to increasing concentrations of SLNs. The cytotoxic effect of SLNs on A549 cells was evaluated by MTT and NRU assays. Viability of lung tissue was determined with WST assay and by life/dead staining using calcein AM/EthD-1 for confocal microscopy (CLSM) followed by quantitative analysis with IMARIS. Inflammation was assessed by measuring chemokine KC and TNF-α levels. The in vivo effects were determined in a 16-day repeated-dose inhalation toxicity study using female BALB/c mice, which were daily exposed to different concentrations of SLN30 aerosols (1–200 μg deposit dose). Local inflammatory effects in the respiratory tract were evaluated by determination of total protein content, LDH, chemokine KC, IL-6, and differential cell counts, performed on days 4, 8, 12, and 16 in bronchoalveolar lavage fluid. Additionally, a histopathological evaluation of toxicologically relevant organs was accomplished.The in vitro and ex vivo dose finding experiments showed toxic effects beginning at concentrations of about 500 μg/ml. Therefore, we used 1–200 μg deposit doses/animal for the in vivo experiments. Even after 16 days of challenge with a 200-μg deposit dose, SLNs induced no significant signs of inflammation. We observed no consistent increase in LDH release, protein levels, or other signs of inflammation such as chemokine KC, IL-6, or neutrophilia. In contrast, the particle control (carbon black) caused inflammatory and cytotoxic effects at corresponding concentrations.These results confirm that repeated inhalation exposure to SLN30 at concentrations lower than a 200-μg deposit dose is safe in a murine inhalation model.
Keywords: Solid lipid nanoparticle; Precision-cut lung slices; A549; BALB/c mice; Pro-inflammatory cytokines; Drug delivery; Cytotoxicity; Inflammation;

Influence of the introduction of a solubility enhancer on the formulation of lipidic nanoparticles with improved drug loading rates by A. Malzert-Fréon; G. Saint-Lorant; D. Hennequin; P. Gauduchon; L. Poulain; S. Rault (117-127).
The objective of the present paper is to develop lipidic nanoparticles (NP) able to encapsulate drugs presenting limited solubility in both water and lipids, with high loading rates, and without using organic solvents. In this goal, a solubility enhancer, a macrogolglyceride (Labrasol®), was incorporated in a formulation process based on a low-energy phase inversion temperature method. From electrical conductivity through the temperature scans, it appears that presence of Labrasol® does not prevent the phase inversion, and it takes part in the microemulsion structuring, probably of bicontinuous type. After screening pseudo-ternary diagrams, the feasibility of NP was established. From results of a partial least square analysis, it appears that these NP present a core–shell structure where Labrasol® is well encapsulated and contributes to the formation of the oily liquid core of the NP. The diameter of the NP, assessed by dynamic light scattering, remains kinetically stable. These NP, smaller than 200 nm, spherical in shape as attested by cryo-transmission electron micrographs, are able to encapsulate a tripentone, a new anticancer agent, with drug loading rates up to 6.5% (w/w). So highly drug-loaded lipidic nanocarriers were developed without using the slightest organic solvent trace, and making it easily possible dose adjustment.
Keywords: Nanoparticles; Labrasol; Phase inversion; Partial least square; Tripentone;

Effect of various additives and polymers on lysozyme release from PLGA microspheres prepared by an s/o/w emulsion technique by A. Paillard-Giteau; V.T. Tran; O. Thomas; X. Garric; J. Coudane; S. Marchal; I. Chourpa; J.P. Benoît; C.N. Montero-Menei; M.C. Venier-Julienne (128-136).
Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a two-dimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA–PEG–PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.
Keywords: Sustained release; Protein; Microspheres; Poly(lactic-co-glycolic acid) (PLGA); Poloxamer; Lysozyme;

Tumor targeting of functionalized lipid nanoparticles: Assessment by in vivo fluorescence imaging by Mathieu Goutayer; Sandrine Dufort; Véronique Josserand; Audrey Royère; Emilie Heinrich; Françoise Vinet; Jérôme Bibette; Jean-Luc Coll; Isabelle Texier (137-147).
Lipid nanoparticles (LNP) coated by a poly(oxyethylene) polymer have been manufactured from low cost and human use-approved materials, by an easy, robust, and up-scalable process. The incorporation in the formulation of maleimide-grafted surfactants allows the functionalization of the lipid cargos by targeting ligands such as the cRGD peptide binding to αvβ3 integrin, a well-known angiogenesis biomarker. LNP are able to encapsulate efficiently lipophilic molecules such as a fluorescent dye, allowing their in vivo tracking using fluorescence imaging. In vitro study on HEK293(β3) cells over-expressing the αvβ3 integrins demonstrates the functionalization, specific targeting, and internalization of cRGD-functionalized LNP in comparison with LNP-cRAD or LNP-OH used as negative controls. Following their intravenous injection in Nude mice, LNP-cRGD can accumulate actively in slow-growing HEK293(β3) cancer xenografts, leading to tumor over skin fluorescence ratio of 1.53 ± 0.07 (n  = 3) 24 h after injection. In another fast-growing tumor model (TS/A-pc), tumor over skin fluorescence ratio is improved (2.60 ± 0.48, n  = 3), but specificity between the different LNP functionalizations is no more observed. The different results obtained for the two tumor models are discussed in terms of active cRGD targeting and/or passive nanoparticle accumulation due to the Enhanced Permeability and Retention effect.
Keywords: Lipid nanoparticles; cRGD functionalization; Fluorescence imaging; Cellular targeting; In vivo targeting; Tumor nanoparticle uptake;

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes were prepared by high-pressure homogeniser and coated with two cationic polymers, chitosan (CS) and for the first time Eudragit EPO (EU), respectively. Compared to the control liposomes, the polymeric liposomes showed greater physicochemical stability in terms of mean particle size and zeta potential at room temperature. In the present study, aciclovir and minoxidil have been used as hydrophilic and hydrophobic candidates. In the presence of the drugs, the polymeric liposomes still showed constant particle size and zeta potential. Influences of polymers and model drugs on thermotropic phase transition of DPPC liposomes were studied by micro-differential scanning calorimetry (microDSC). The influences on configuration of DPPC liposomes were investigated by Fourier transform infrared spectroscopy (FTIR). According to DSC results, cationic polymers had a stabilising effect, whereas aciclovir and minoxidil changed the physical properties of the DPPC bilayers by influencing the main phase transition temperature and erasing the pre-transition. The investigation of C=O stretching bands of DPPC at 1736 cm−1 in FTIR spectra showed that aciclovir has strong hydrogen bonding with C=O groups of DPPC, whereas carbonyl groups were free in minoxidil presence. Moreover, the coating of liposomes with CS or EU led to higher skin diffusion for both drugs. This could be explained as an effect of positively charged liposomes to interact stronger with skin negatively charged surface and their possible interactions with structures below the stratum corneum.
Keywords: Liposomes; Skin permeation; DPPC; Chitosan;

Improved membrane transport of astaxanthine by liposomal encapsulation by Chiung-Huei Peng; Chi-Huang Chang; Robert Y. Peng; Charng-Cherng Chyau (154-161).
Astaxanthine (3,3′-dihydroxy-β,β′-caroten-4,4′-dione) (AST), a red-colored carotenoid pigment, possesses extremely powerful antioxidative activity. However, its drawbacks reside in poor solubility in aqueous system, resulting in extremely low bioavailability. To ameliorate such defects, we prepared AST encapsulated within liposomes (AST-L) and tested with Hep3B and HepG2 cell lines. AST-L had size 251 ± 23 nm with AST content 89.0 ± 8.6 mg/g. AST-L apparently showed improved stability and transportability. The overall transport time was 7.55 h and 6.00 h for free AST and AST-L, respectively. AST-L more effectively activated antioxidant enzymes like superoxide dismutase, catalase and glutathione S-transferase than free AST. Hep3B consumed AST more rapidly than HepG2 cell lines. Moreover, AST-L when combined with gamma radiation (10 Gy) therapy potentially triggered subG1 arrest in Hep3B and HepG2 cell lines in a dose-responsive manner (p  < 0.05). To conclude, the poor bioavailability of AST can be improved by liposomal encapsulation, which can be a good adjuvant remedy in gamma radiotherapy.
Keywords: Cell cycle arrest; Antioxidative enzymes; Hepatoma cells; Nanoencapsulation; Blood–Brain Barrier; Radiation therapy;

Dermal targeting using colloidal carrier systems with linoleic acid by Alexandra S.B. Goebel; Ulrich Knie; Christoph Abels; Johannes Wohlrab; Reinhard H.H. Neubert (162-172).
In the basic therapy of chronic skin diseases characterized by xerosis, the local treatment is an essential strategy to reach ideal therapeutic effects. Suitable active ingredients for this aim are fatty acids, in particular linoleic acid, which is an essential component for the organization and perpetuation of the skin barrier. In the present work, the development of a well-tolerated colloidal carrier system (microemulsion) containing linoleic acid as active ingredient is described. A comprehensive physiochemical characterization of the novel microemulsion system was performed using different techniques. The potential of the developed microemulsion system compared to a cream as suitable carrier for the dermal delivery of linoleic acid was determined. Penetration studies showed higher linoleic acids concentrations after administration of the colloidal carrier system in all skin layers independent of the time of incubation. Up to 23% of applied dose reached the skin from the colloidal carrier system whereas at most 8% of the active ingredient could be detected after applying the cream. Particularly, the percentage of the linoleic acids penetrated through the microemulsion in the stratum corneum and the viable epidermis differed significantly (p  < 0.01) when compared to that through a standard cream. Furthermore, linoleic acids accumulated in the epidermis at longer incubation times. Using the microemulsion, the penetration of linoleic acids was enhanced significantly (p  < 0.01). Hence, the microemulsion might be an innovative vehicle for the delivery of linoleic acids to the epidermis improving its use as their barrier regeneration and providing possible anti-inflammatory effects.
Keywords: Colloidal carrier system; Microemulsion; Skin; Penetration; Linoleic acid; Xerosis;

Transdermal iontophoresis of dexamethasone sodium phosphate in vitro and in vivo: Effect of experimental parameters and skin type on drug stability and transport kinetics by J. Cázares-Delgadillo; C. Balaguer-Fernández; A. Calatayud-Pascual; A. Ganem-Rondero; D. Quintanar-Guerrero; A.C. López-Castellano; V. Merino; Y.N. Kalia (173-178).
The aim of this study was to investigate the cathodal iontophoresis of dexamethasone sodium phosphate (DEX-P) in vitro and in vivo and to determine the feasibility of delivering therapeutic amounts of the drug for the treatment of chemotherapy-induced emesis. Stability studies, performed to investigate the susceptibility of the phosphate ester linkage to hydrolysis, confirmed that conversion of DEX-P to dexamethasone (DEX) upon exposure to samples of human, porcine and rat dermis for 7 h was limited (82.2 ± 0.4%, 72.5 ± 4.8% and 78.6 ± 6.0% remained intact) and did not point to any major inter-species differences. Iontophoretic transport of DEX-P across dermatomed porcine skin (0.75 mm thickness) was studied in vitro as a function of concentration (10, 20, 40 mM) and current density (0.1, 0.3, 0.5 mA cm−2) using flow-through diffusion cells. Increasing concentration of DEX-P from 10 to 40 mM resulted in a ∼4-fold increase in cumulative permeation (35.65 ± 23.20 and 137.90 ± 53.90 μg cm−2, respectively). Good linearity was also observed between DEX-P flux and the applied current density (id ; 0.1, 0.3, 0.5 mA cm−2; JDEX (μg cm2  h−1) = 237.98 id  − 21.32, r 2  = 0.96). Moreover, separation of the DEX-P formulation from the cathode compartment by means of a salt bridge – hence removing competition from Cl ions generated at the cathode – produced a 2-fold increase in steady-state iontophoretic flux (40 mM, 0.3 mA cm−2; 20.98 ± 7.96 and 41.82 ± 11.98 μg cm−2  h−1, respectively).Pharmacokinetic parameters were determined in Wistar rats (40 mM DEX-P; 0.5 mA cm−2 for 5 h with Ag/AgCl electrodes and salt bridges). Results showed that DEX-P was almost completely converted to DEX in the bloodstream, and significant DEX levels were achieved rapidly. The flux across rat skin in vivo (1.66 ± 0.20 μg cm−2  min−1), calculated from the input rate, was not statistically different from the flux obtained in vitro across dermatomed porcine skin (1.79 ± 0.49 μg cm−2  min−1). The results suggest that DEX-P delivery rates would be sufficient for the management of chemotherapy-induced emesis.
Keywords: Dexamethasone sodium phosphate; Iontophoresis; Transdermal; Pharmacokinetics; Antiemetics;

Controlled release of metronidazole benzoate from poly ε-caprolactone electrospun nanofibers for periodontal diseases by Maedeh Zamani; Mohammad Morshed; Jaleh Varshosaz; Marziyeh Jannesari (179-185).
Poly ε-caprolactone (PCL) nanofibers containing metronidazole benzoate (MET) were successfully electrospun and evaluated for periodontal diseases. Solutions of 10.5% w/v PCL and 5–15% w/w MET in mixtures of dichloromethane (DCM)/N,N-dimethylformamide (DMF) with ratios of 90:10, 80:20 and 70:30 v/v were prepared, and the nanofibers were produced by electrospinning technique. Scanning electron microscopy (SEM) was used to investigate the morphology and average diameter of the electrospun nanofibers. DSC results indicated a molecular dispersion of MET in the PCL nanofibers and showed a decrease in crystallinity of PCL nanofibers by adding MET. Results showed that an increase in the DCM:DMF ratio led to a decrease in the solution conductivity and an increase in the solution viscosity as well as in the nanofibers diameter. Also increasing metronidazole benzoate concentration caused an increase in the solution conductivity and a decrease in the solution viscosity as well as in the nanofibers diameter. In vitro drug release studies in phosphate buffer solution (pH 7.4) showed that the drug release rate was affected by the solvents ratio and the drug concentration. Moreover, the burst release was low, and sustained drug release was prolonged to at least 19 days.
Keywords: Nanofibers; Electrospinning; Drug delivery; Periodontal diseases; Metronidazole benzoate; Poly ε-caprolactone;

A poloxamer/chitosan in situ forming gel with prolonged retention time for ocular delivery by Taís Gratieri; Guilherme Martins Gelfuso; Eduardo Melani Rocha; Victor Hugo Sarmento; Osvaldo de Freitas; Renata Fonseca Vianna Lopez (186-193).
The aim of the present work was to obtain an ophthalmic delivery system with improved mechanical and mucoadhesive properties that could provide prolonged retention time for the treatment of ocular diseases. For this, an in situ forming gel comprised of the combination of a thermosetting polymer, poly (ethylene oxide)–poly (propylene oxide)–poly (ethylene oxide) (PEO–PPO–PEO, poloxamer), with a mucoadhesive agent (chitosan) was developed. Different polymer ratios were evaluated by oscillatory rheology, texture and mucoadhesive profiles. Scintigraphy studies in humans were conduced to verify the retention time of the formulations developed. The results showed that chitosan improves the mechanical strength and texture properties of poloxamer formulations and also confers mucoadhesive properties in a concentration-dependent manner. After a 10-min instillation of the poloxamer/chitosan 16:1 formulation in human eyes, 50–60% of the gel was still in contact with the cornea surface, which represents a fourfold increased retention in comparison with a conventional solution. Therefore, the developed formulation presented adequate mechanical and sensorial properties and remained in contact with the eye surface for a prolonged time. In conclusion, the in situ forming gel comprised of poloxamer/chitosan is a promising tool for the topical treatment of ocular diseases.
Keywords: Thermosetting; Poloxamer; Chitosan; Oscillatory Rheology; Mucoadhesion; Human scintigraphy;

Previously, a quaternary ammonium (N+)–chitosan (Ch) conjugate (N+(60)–Ch) characterized by short pendant chains, made of 1.7 ± 0.1 adjacent diethyl-dimethylene-ammonium groups, substituted onto the primary amino group of the chitosan repeating units (degree of substitution, 59.2 ± 4.5%) was used to synthesize a multifunctional non-cytotoxic thiomer (N+(60)–Ch–SH(5)), carrying 4.5 ± 0.7% thiol-bearing 3-mercaptopropionamide besides quaternary ammonium groups. The present work was aimed at evaluating the potential of N+(60)–Ch–SH(5) and N+(60)–Ch as bioactive excipients for dexamethasone (DMS) eyedrops. The DMS permeability across excised rabbit cornea was enhanced over the control value by the thiomer and the parent polymer to about the same extent (3.8 vs. 4.1 times). The mean precorneal retention time and AUC in the aqueous of DMS instilled in rabbit eyes via eyedrops were enhanced by the thiomer (MRT = 77.96 ± 3.57 min, AUC = 33.19 ± 6.96 μg ml−1  min) more than the parent polymer (MRT = 65.74 ± 4.91 min, AUC = 21.48 ± 3.81 μg ml−1  min) over the control (MRT = 5.07 ± 0.25 min, AUC = 6.25 ± 0.65 μg ml−1  min). The quaternary ammonium ions were responsible for both permeabilization of corneal epithelium and polymer adhesion to precorneal mucus, while the thiols increased the latter. This synergistic action is the basis of the higher thiomer bioactivity in vivo. A good ocular tolerability of the chitosan derivatives resulted from in vivo experiments.
Keywords: Thiolated quaternary ammonium chitosan; Thiomer; Corneal permeability enhancement; Precorneal retention enhancement; Intraocular absorption enhancement; Dexamethasone;

In situ measurement of spectral changes in the anterior eye following application of ultraviolet-absorbing compounds by W.K. Kek; J. Miller; E. Rawson-Lax; C.G. Wilson; D. Uttamchandani (200-205).
The ocular structures are very sensitive to damage from ultraviolet (UV) radiation, exposure is linked to corneal and conjunctival damage, cataract formation and may also be implicated in the aetiology of age-related macular degeneration. These structures are usually protected by wearing suitable eyeglasses and goggles. An alternative to conventional eyeglasses/goggles is the concept of “liquid sunglasses” which involve the topical application of eye drops that are designed to block harmful UV radiation reaching the sensitive ocular surfaces. The evaluation of such compounds directly applied to the eye surface requires in situ measurements to compare the efficacy of different formulations. A novel ocular spectrometer system has been used to evaluate changes in the transmission of ultraviolet (UV) radiation through the anterior eye following topical application of candidate UV-absorbing formulations. The key feature of the system is the ability to propagate a beam of light tangentially through the anterior eye using a compact, hand-held lens assembly incorporating UV-transmitting optical fibres. A range of formulations containing UV-absorbing compounds were topically applied to ex vivo rabbit eyes. Significant increases in the absorption of the UV spectrum were detected in seven of the eight formulations studied, demonstrating the potential of this measurement technique in the evaluation of formulations developed as potential topical ocular sunscreens.
Keywords: Ocular sunscreen; Spectroscopy; Optical fibre; Ocular spectrometer;

Degradation of parabens by Pseudomonas beteli and Burkholderia latens by Aeshna Amin; Sateesh Chauhan; Manish Dare; Arvind Kumar Bansal (206-212).
p-Hydroxybenzoic acid esters (parabens) are commonly used antimicrobial preservatives in pharmaceutical formulations. Two microorganisms, isolated from non-sterile methyl paraben (MP) and propyl paraben (PP) solutions, were found to degrade the respective parabens. Identification by 16S rRNA partial gene sequencing revealed them to be Pseudomonas beteli and Burkholderia latens, respectively. The present work describes a previously unreported interaction of the parabens with P. beteli and B. latens. Degradation of MP at various concentrations by P. beteli, followed a logarithmic pattern, while that of PP by B. latens was found to be linear. It was subsequently observed that P. beteli could degrade only MP, while B. latens could degrade both the parabens. Absence of HPLC chromatogram peaks of expected degradation products indicated that the parabens were used up as a carbon source. The behaviour of pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger) of the pharmacopoeial preservative effectiveness test (PET), towards MP, showed that none had the ability to degrade the paraben. It was concluded that, for a paraben-preserved multi-dose ophthalmic formulation, the sole use of the four pathogens that are recommended by the pharmacopoeia for PET can falsely indicate the formulation to be effective against ‘in-use’ contamination.
Keywords: Methyl paraben; Propyl paraben; Microbial degradation; Antimicrobial preservative; Preservative effectiveness test;

Claudin-4-targeting of diphtheria toxin fragment A using a C-terminal fragment of Clostridium perfringens enterotoxin by Hideki Kakutani; Masuo Kondoh; Rie Saeki; Makiko Fujii; Yoshiteru Watanabe; Hiroyuki Mizuguchi; Kiyohito Yagi (213-217).
Claudin (CL)-4, a tight junction protein, is overexpressed in some human neoplasias, including ovarian, breast, pancreatic and prostate cancers. The targeting of CL-4 is a novel strategy for tumor therapy. We previously found that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to CL-4. In the present study, we genetically prepared a novel CL-4-targeting molecule (DTA–C-CPE) by fusion of C-CPE and diphtheria toxin fragment A (DTA). Although DTA is not toxic to CL-4-expressing L cells, even at 20 μg/ml, DTA–C-CPE is toxic to CL-4-expressing L cells at 1 μg/ml. DTA–C-CPE-induced cytotoxicity was attenuated by pretreatment of the cells with C-CPE but not bovine serum albumin, indicating that DTA–C-CPE may bind to CL-4-expressing L cells through its C-CPE domain. To evaluate the specificity of DTA–C-CPE, we examined its cytotoxic effects in L cells that express CL-1, -2, -4 or -5. We found that DTA–C-CPE was toxic to only CL-4-expressing L cells. Thus, C-CPE may be a promising ligand for the development of cancer-targeting systems.
Keywords: Claudin; Targeting; Cancer; Diphtheria toxin; Clostridium perfringens enterotoxin;

We previously developed chitosan (CS)-modified poly (d,l-lactide-co-glycolide) (PLGA) nanospheres (NS) by an emulsion solvent diffusion method as a gene delivery system. In this study, PLGA NS were modified using polysorbate 80 (P80) to improve their cellular uptake. We investigated the cellular uptake, intracellular distribution, and transfection efficiency of P80-modified PLGA NS (P80-PLGA NS) for a plasmid DNA delivery system in A549 cells. The cellular uptake and transfection efficiency of P80-PLGA NS were greater than CS-modified PLGA NS (CS-PLGA NS). The uptake of unmodified NS and CS-PLGA NS was mediated, predominantly, by clathrin-mediated endocytosis. In contrast, a specific endocytic pathway could not be determined for the cellular uptake of P80-PLGA NS. The intracellular distributions of PLGA NS depended on their surface properties. P80-PLGA NS were not cytotoxic for A549 cells. Thus, P80-PLGA NS could be used as an effective gene delivery system; the surface properties of PLGA NS are key parameters for optimal intracellular uptake and distribution.
Keywords: Poly (d,l-lactide-co-glycolide); Nanosphere; Polysorbate 80; Surface modification; Gene delivery system;

Transdermal fentanyl matrix patches Matrifen® and Durogesic® DTrans® are bioequivalent by Hans G. Kress; Hildegard Boss; Thomas Delvin; Gezim Lahu; Søren Lophaven; Michael Marx; Sophie Skorjanec; Thomas Wagner (225-231).
The pharmacokinetic profiles of the two commercially available transdermal fentanyl patches Matrifen® (100 μg/h) and Durogesic® DTrans® (100 μg/h), used to manage severe chronic pain, were compared regarding their systemic exposure, rate of absorption, and safety.Transdermal matrix fentanyl patches [Matrifen® or Durogesic® DTrans® (100 μg/h)] were applied for 72 h to 30 healthy male subjects in a randomized, four-period (two replicated treatment sequences), crossover study; 28 subjects completed the study. The pharmacokinetic parameters of fentanyl were determined for 144 h after application using plasma samples. Safety of the patches (adverse events) and performance (adhesion, skin irritation, residual fentanyl content in the patch) were evaluated.The plasma concentration–time curves of Matrifen® (Test) and Durogesic® DTrans® (Reference) were similar. The geometric least square means of the Test/Reference ratio (90% confidence intervals [CI]) were within the range of 80–125%, demonstrating bioequivalence of Matrifen® and Durogesic® DTrans®: AUC0-tlast 92.5 (CI 88.7–96.4), AUC0-inf 91.7 (CI 88.0–95.7), and C max 98.3 (CI 92.9–104.1). After 72 h application, Matrifen® had a more efficient utilization of fentanyl (mean ± SD 82.3 ± 9.43%) than Durogesic® DTrans® (52.3 ± 12.8%), with substantially lower residual fentanyl in patch after use. The pharmacokinetic parameters showed lower intra- and inter-subject variability for Matrifen® than for Durogesic® DTrans® patch.Despite different technologies, the transdermal fentanyl patches Matrifen® and Durogesic® DTrans® are bioequivalent. Compared with Durogesic® DTrans®, the Matrifen® patch had lower initial and lower residual fentanyl content, as well as lower intra- and inter-subject variability, allowing reproducible drug delivery and reliable analgesia.
Keywords: Bioequivalence; Fentanyl; Healthy subjects; Pharmacokinetics; Phase I; Pain; Opioid; Safety; Matrifen®; Durogesic® DTrans®;

In vivo evaluation of two new sustained release formulations elaborated by one-step melt granulation: Level A in vitro–in vivo correlation by Lourdes Ochoa; Manoli Igartua; Rosa M. Hernández; Maria Ángeles Solinís; Alicia R. Gascón; Jose Luis Pedraz (232-237).
The objective of this study was to evaluate in vivo two sustained release formulations elaborated by a one-step melt granulation method using theophylline as model drug. Both formulations presented differences in the in vitro release profile due to the hydrophilic or lipophilic nature of the binder employed (PEG 6000 or glycerol monostearate). The formulations were administered to Beagle dogs, and plasma levels were compared. Both formulations provided a sustained plasma concentration profile after oral administration to dogs. Significant differences (p  < 0.05) in the plasma concentration–time curves between the two formulations were found, with higher C max (6.05 ± 2.00 vs. 2.55 ± 0.82 μg/mL), higher AUC0–∞ (70.24 ± 16.10 vs. 33.00 ± 8.96 h μg/mL) and delayed T max (6.00 ± 2.12 vs. 3.17 ± 0.98 h) for the formulation containing PEG 6000. Absolute bioavailability of theophylline was 96% and 46% for the formulations containing PEG 6000 and glycerol monostearate, respectively. These results are consistent with those obtained in vitro, with slower release rate of theophylline from tablets elaborated with glycerol monostearate than that obtained with tablets elaborated with PEG 6000. Moreover, the formulation containing PEG 6000 provided a plasma concentration–time profile similar to that obtained with the marketed formulation Theo-Dur®. A very good Level A IVIVC was observed between dissolution and absorption profiles of the drug from both test formulations. Our results showed that one-step melt granulation in a high shear mixer allows for an easy modulation of the release profile and, consequently, of the plasma level profile of the drug by selecting the type of binder used.
Keywords: Melt granulation; Sustained release; IVIVC; Theophylline; Binder;

Colonoscopic method for estimating the colonic absorption of extended-release dosage forms in dogs by Shinichiro Tajiri; Taro Kanamaru; Kazuhiro Yoshida; Yasue Hosoi; Sachiko Fukui; Tsutomu Konno; Shuichi Yada; Hiroaki Nakagami (238-244).
The purpose of this study was to develop a new method using beagle dogs in order to evaluate the colonic absorption properties of oral extended-release (ER) solid dosage forms. The established method is not only noninvasive and inexpensive but full-sized ER dosage forms are also directly administered to the colons of conscious dogs through the anus with an endoscope and modified bioptome. In the method, it was possible to administer the ER dosage forms into the ascending colon of dogs within 30 s–1 min. The colonic absorption of Voltaren-XR (Diclofenac sodium), Glucophage-XR (metformin), Pacif (morphine hydrochloride), Herbesser-R (diltiazem hydrochloride) and Plendil (felodipine), which are currently on the market, were investigated by this method. The relative bioavailabilities of these ER dosage forms to oral drug solution were 100.3%, 42.5%, 60.6%, 46.3% and 29.8%, respectively. Some of these results reflected the human colonic absorption profiles reported in the literature. This newly developed method could provide researchers with an alternative way to predict the human colon absorption performance of oral ER delivery systems.
Keywords: Extended-release dosage forms; Colonic absorption; Dogs; Endoscope; Bioptome;

Biopolymers such as pectin, alginate, and chitosan have a great potential in colon drug delivery. The aim of this study was to produce pellets with calcium and chitosan in the core and then by an interfacial complexation reaction coat the cores with pectin or alginate in combination with calcium or chitosan. Pellets with calcium in the core acted as a reference. The drug release was investigated in environments mimicking the stomach and the small intestine.The morphology of the coatings indicated a more wrinkled and irregular structure for coatings composed of pectin or alginate in combination with chitosan compared to the coat consisting of alginate in combination with calcium. The results from the drug release experiments showed that all the investigated coatings, especially with alginate, slowed down the drug release compared to the uncoated cores. The release from the chitosan-containing pellets was higher than the reference. The swelling studies revealed a high degree of swelling of the core consisting of chitosan. This probably explains the higher drug release from the coated chitosan pellets.
Keywords: Pectin; Alginate; Chitosan; Interfacial complexation reaction; Colon drug delivery;

Despite recent advances in the formulation of lyophilised rapid disintegrating tablets (RDTs), the inclusion of matrix supporting/disintegration enhancing agents has been limited to the use of saccharides and polyols. In this study, the feasibility of using amino acids as matrix forming agents in lyophilised RDTs was investigated. Twelve amino acids were chosen (alanine, arginine, threonine, glycine, cysteine, serine, histidine, lysine, valine, asparagine, glutamine and proline), and the suitability for freeze drying, mechanical properties and disintegration time after inclusion of the amino acids at varied concentration were studied. In addition, the porosity of the RDTs and wettability profile of the amino acids were investigated to understand the mechanisms of disintegration. The results suggest the suitability of these amino acids for the lyophilisation regime, as they displayed satisfactory safety margin between the glass transition and shelf temperature (−40 °C), except proline-based formulations. Moreover, the crystallisation behavior of alanine, glycine, cysteine and serine at high concentration increased the stability of the formulation. The characterisation of the RDTs suggests that high concentration of the amino acids is required to enhance the mechanical properties, whereas only optimum concentrations promote the disintegration. Moreover, wetting time of the amino acid and porosity of the tablet are the two factors that control the disintegration of RDTs.
Keywords: Amino acids; Matrix forming agents; Rapid disintegrating tablets; Lyophilisation; Wettability;

Synchrotron X-ray microtomographic study of tablet swelling by P.R. Laity; R.E. Cameron (263-276).
Tablet swelling behaviour was investigated by following the movements of embedded glass microsphere tracers, using X-ray microtomography (XμT) with intense illumination from a synchrotron. Specimens were prepared using combinations of hydroxypropyl-methyl-cellulose (HPMC) and microcrystalline cellulose (MCC) or pre-gelatinised starch (PGS), three materials commonly used as excipients for compacted tablets. The results revealed significant differences in swelling behaviour due to excipient type and compaction conditions. In particular, a sudden change was observed from gel-forming behaviour of formulations containing PGS or high HPMC content, to more rapid expansion and disintegration for formulations above 70% MCC. Although some radial expansion was observable with the higher PGS formulations and during later stages of swelling, axial expansion (i.e. the reverse of the compaction process) appeared to dominate in most cases. This was most pronounced for the 10/90 HPMC/MCC specimens, which rapidly increased in thickness, while the diameter remained almost unchanged. The expansion appeared to be initiated by hydration and may be due to the relaxation of residual compaction stress. This occurred within ‘expansion zones’, which initially appeared as thin bands close to the compacted (upper and lower) faces, but gradually advanced towards the centre and spread around the sides of the tablets. These zones exhibited lower X-ray absorbance, probably because they contained significant amounts of bubbles, which were formed by air released from the swelling excipients. Although, in most cases, these bubbles were too small to be resolved (<60 μm), larger bubbles (diameter up to 1 mm) were clearly evident in the rapidly swelling 10/90 HPMC/MCC specimens. It is suggested that the presence of these bubbles may affect subsequent water ingress, by increasing the tortuosity and occluding part of the gel, which may affect the apparent diffusion kinetics (i.e. Fickian or Case II). These observations also suggested that axial expansion, initiated by water ingress, may be an important mechanism during tablet swelling.
Keywords: X-ray microtomography (XμT); Tablet; Swelling; Hydroxypropyl-methyl-cellulose (HPMC); Pre-gelatinised starch (PGS); Microcrystalline cellulose (MCC); Gel forming;

A new method for modifying atomic force microscope (AFM) probe with polymer was proposed to estimate the interaction between Chitosan (CS)-coated poly (lactic-co-glycolic acid) (PLGA) polymer and mucin film. In this method, the mixture of polymers and its suitable volatile dissolvent were deposited on the AFM probe tip through a micropipette controlled by micromanipulator. After being dried, the polymer film forms on the probe tip. Several evaluation experiments show that using the new method, the AFM probe tip can be smoothly and uniformly coated with PLGA and CS with approximately same curvature radius as that of probe tip less than 500 nm. As a preliminary application of the proposed method, the interaction force between PLGA/CS and mucin film in air was investigated. It was revealed that when a PLGA probe is retracting from the mucin film, a repulsive force appeared; however, after the PLGA probe was further overcoated with CS, the force became attractive if the amount of CS was enough, such as at CS concentrations of 0.2% W/V. The observed force can be interpreted by the surface hydrophilic/hydrophobic characteristics of PLGA/CS and mucin film.
Keywords: Chitosan; PLGA; Mucin film; Atomic force microscopy; Repulsive force; Attractive force; Probe modification;

To establish a fluorescence-based assay for drug interactions with the ABC-export-protein MRP2 (ABCC2).Apical membrane vesicles were isolated by differential centrifugation from polarized MDCKII cells and MDCKII cells transfected with human MRP2. Vesicle fractions were characterized by electron microscopy, determination of the marker enzyme alkaline phosphatase and Western blot analysis of MRP2. Vesicle orientation was determined by measurement of 5′-nucleotidase activity in the absence and in the presence of detergents. To assess MRP2 activity, the uptake of the fluorescent MRP2-substrate 5-(6)-carboxy-2′,7′-dichlorofluorescein (CDF) was determined in the absence and in the presence of other compounds potentially interacting with MRP2.Apical membrane vesicles could be isolated from cells in considerable purity as indicated by electron microscopy, enrichment of alkaline phosphatase and high enrichment of MRP2 in vesicles of MDCKII-MRP2 cells. About half of the vesicles showed “inside-out” orientation. CDF was taken up into the membrane vesicles in a time- and concentration-dependent manner following a Michaelis–Menten type of kinetics with a K M of 39 μM and a V max of 465.3 fmol/(mg protein × min). Thereby, uptake into vesicles from transfected cells was significantly higher than uptake into vesicles from control cells. Presence of known MRP2-substrates/inhibitors in the incubation medium decreased CDF uptake into the vesicles in a concentration-dependent manner, whereas nonsubstrates/inhibitors had no effect.This CDF-based uptake assay can be used as a rapid and sensitive screening system to assess drug interactions with human MRP2 and therefore represents a useful tool in compound profiling.
Keywords: MRP2; ABCC2; Fluorescence assay; ABC protein; Carboxydichlorofluorescein (CDF);

Active packaging for topical cosmetic/drug products: A hot-melt extruded preservative delivery device by L. Zema; M.E. Sangalli; A. Maroni; A. Foppoli; A. Bettero; A. Gazzaniga (291-296).
A delivery device intended for the prolonged release of antimicrobial agents, able to enhance the stability profile of liquid/semi-solid cosmetic/pharmaceutical products for topical application, was proposed in the present study. With the aid of a simulation program based on compartment models, the relevant kinetic and formulation parameters were defined using dehydroacetic acid sodium salt (DHA.Na, Prevan®) as the model preservative. Indeed, the overall DHA.Na degradation rate is increased in the presence of formaldehyde releasers that are often employed as co-preservatives. Inert matrices (3 g weight and 18 mm diameter) based on high-density polyethylene (HDPE), possibly consistent with the design of an active packaging meant for preservative delivery, were prepared by hot-melt extrusion. Units with satisfactory physical-technological properties could be obtained up to 50% w/w loads of antimicrobial agent. In an attempt to modify the relevant Fickian release profiles by varying the area exposed to the medium, matrix systems coated with an impermeable film except for one base (CMs) or for the inner surface of a central drilled hole (PCMs) were investigated. On the basis of the n exponent of power equation and the outcome of linear fitting, PCMs were proven able to yield the zero-order release behaviour needed to ensure constant DHA.Na levels over a predetermined time period, as indicated by the simulation process.
Keywords: Active packaging; Preservative; Simulation program; Inert matrices for prolonged release; Hot-melt extrusion;

Helium leak testing of packages for oral drug products by Jürgen Kossinna; Andreas Meyer (297-303).
This article presents an overview on what helium leak testing is and how it can be implemented as a test method to determine the tightness of packages for oral drug products in a quality control or development laboratory. Whereas earlier publications on helium leak testing mainly focused on testing vials and the correlation between helium leak rates and microbial ingress, this paper provides various examples how helium leak testing contributes to assure the integrity of containers for oral formulations. The results clearly show whether optimal tightness is achieved or an improvement in materials or the closing/sealing conditions should be envisaged. Helium leak testing using a flexible test chamber and a mass spectrometer as detector is of advantage in particular for flexible packages containing moisture and oxygen sensitive products, where a dye ingress test is not applicable and other detectors or methods might not be sensitive enough or give qualitative results only.
Keywords: Helium leak testing; Flexible test chamber; Container closure integrity; Tightness; Stick packs; Sachets;