European Journal of Pharmaceutics and Biopharmaceutics (v.72, #3)

APV Diary (S1).

Biopharmaceuticals based on recombinant proteins have started to go off-patent, opening the way for other manufacturers to place follow-on products to the market. Meanwhile it has been recognized by all stakeholders that there are fundamental differences between conventional small-molecule based drugs and biopharmaceuticals. This has led to the adoption of distinct legal and regulatory frameworks for biosimilars (follow-on products to biopharmaceuticals) in various parts of the world. This review gives an overview on the scientific basis for the approval requirements, the regulatory and market status, open issues, and the strategic perspective.
Keywords: Biosimilars; Follow-on biologicals (FOBs); Subsequent-entry biologicals; Protein structure; Protein manufacturing; Comparability; INN;

The aim of this study was to prepare bioadhesive sulfacetamide sodium (SA) microspheres to increase their residence time on the ocular surface and to enhance their treatment efficacy on ocular keratitis. Microspheres were fabricated by spray drying method using mixture of polymers such as pectin, polycarbophil and hydroxypropylmethyl cellulose (HPMC) at different ratios. The particle size and distribution, morphological characteristics, thermal behavior, encapsulation efficiency, mucoadhesion and in vitro drug release studies on formulations have been investigated. After optimisation studies, SA-loaded polycarbophil microsphere formulation with polymer:drug ratio of 2:1 was found to be the most suitable for ocular application and used in in vivo studies. In vivo studies were carried out on New Zealand male rabbit eyes with keratitis caused by Pseudomonas aeruginosa and Staphylococcus aureus. Sterile microsphere suspension in light mineral oil was applied to infected eyes twice a day. Plain SA suspension was used as a positive control. On 3rd and 6th days of the antimicrobial therapy, the eyes were examined in respect to clinical signs of infection (blepharitis, conjunctivitis, iritis, corneal oedema and corneal infiltrates) which are the main symptoms of bacterial keratitis and then cornea samples were counted microbiologically. The rabbit eyes treated with microspheres demonstrated significantly lower clinical scores than those treated with SA alone. A significant decrease in the number of viable bacteria in eyes treated with microspheres was observed in both infection models when compared to those treated with SA alone. In conclusion, in vitro and in vivo studies showed that SA-loaded microspheres were proven to be highly effective in the treatment of ocular keratitis.
Keywords: Sulfacetamide sodium; Microsphere; Mucoadhesion; Texture analyzer; Cornea; Bacterial keratitis; Pseudomonas aeruginosa; Staphylococcus aureus;

Silica nanoparticles as hepatotoxicants by Hikaru Nishimori; Masuo Kondoh; Katsuhiro Isoda; Shin-ichi Tsunoda; Yasuo Tsutsumi; Kiyohito Yagi (496-501).
Nano-size materials are increasingly used in cosmetics, diagnosis, imaging and drug delivery, but the toxicity of the nano-size materials has never been fully investigated. Here, we investigated the relationship between particle size and toxicity using silica particles with diameters of 70, 300 and 1000 nm (SP70, SP300, and SP1000) as a model material. To evaluate acute toxicity, we first performed histological analysis of liver, spleen, kidney and lung by intravenous administration of silica particles. SP70-induced liver injury at 30 mg/kg body weight, while SP300 or 1000 had no effect even at 100 mg/kg. Administration of SP70 dose-dependently increased serum markers of liver injury, serum aminotransferase and inflammatory cytokines. Repeated administration of SP70 twice a week for 4 weeks, even at 10 mg/kg, caused hepatic fibrosis. Taken together, nano-size materials may be hepatotoxic, and these findings will be useful for future development in nanotechnology-based drug delivery system.
Keywords: Silica particle; Nano-size particle; Liver injury;

Development of a long-acting injectable formulation with nanoparticles of rilpivirine (TMC278) for HIV treatment by Lieven Baert; Gerben van ‘t Klooster; Willy Dries; Marc François; Alfons Wouters; Esther Basstanie; Koen Iterbeke; Fred Stappers; Paul Stevens; Laurent Schueller; Pieter Van Remoortere; Guenter Kraus; Piet Wigerinck; Jan Rosier (502-508).
Long-acting parenteral formulations of antiretrovirals could facilitate maintenance and prophylactic treatment in HIV. Using the poorly water- and oil-soluble non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC278 (rilpivirine) as base or hydrochloride (HCl), nanosuspensions were prepared by wet milling (Elan NanoCrystal® technology) in an aqueous carrier. Laser diffraction showed that the average particles size were (1) close to the targeted size proportionality (200–400–800 nm), with increasing distributions the larger the average particle size, and (2) were stable over 6 months. Following single-dose administration, the plasma concentration profiles showed sustained release of TMC278 over 3 months in dogs and 3 weeks in mice. On comparison of intramuscular and subcutaneous injection of 5 mg/kg (200 nm) in dogs, the subcutaneous route resulted in the most stable plasma levels (constant at 25 ng/mL for 20 days, after which levels declined slowly to 1–3 ng/mL at 3 months); 200 nm nanosuspensions achieved higher and less variable plasma concentration profiles than 400 and 800 nm nanosuspensions. In mice, the pharmacokinetic profiles after a single 20 mg/kg dose (200 nm) were similar with two different surfactants used (poloxamer 338, or d-alpha-tocopheryl polyethylene glycol 1000 succinate). In conclusion, this study provides proof-of-concept that 200-nm sized TMC278 nanosuspensions may act as long-acting injectable.
Keywords: Rilpivirine; Long-acting; Injectable; HIV; Pharmacokinetics;

The use of Eudragit® RS 100/cyclodextrin nanoparticles for the transmucosal administration of glutathione by Angela Lopedota; Adriana Trapani; Annalisa Cutrignelli; Laura Chiarantini; Elena Pantucci; Rosa Curci; Elisabetta Manuali; Giuseppe Trapani (509-520).
The aim of this work was to develop and characterize new nanoparticle systems based on Eudragit RS 100 and cyclodextrins (CDs) for the transmucosal administration of glutathione (GSH). For this purpose, nanoparticles (NPs) with the mucoadhesive properties of Eudragit RS 100 and the penetration enhancing and peptide protective properties of CDs were prepared and evaluated. The quasi-emulsion solvent diffusion technique was used to prepare the NPs with natural and chemically modified (HP-β-CD and Me-β-CD) CDs. The NPs prepared showed homogeneous size distribution, mean diameters between 99 and 156 nm, a positive net charge and spherical morphology. Solid state FT-IR, thermal analysis (DSC), and X-ray diffraction studies suggest that the nanoencapsulation process produces a marked decrease in crystallinity of GSH. The encapsulation efficiency of the peptide was found to be between 14.8% and 24%. The results indicate that mean diameters, surface charges and drug-loaded NPs were not markedly affected by the CD, whereas the presence of the latter influences drug release and to some extent peptide stability and absorption. Finally, it has been shown that CD/Eudragit RS 100 NPs may be used for transmucosal absorption of GSH without any cytotoxicity using the epithelial human HaCaT and murine monocyte macrophage RAW264.7 cell lines.
Keywords: GSH; Eudragit RS 100; Cyclodextrins; Nanoparticles; Transmucosal administration;

A dynamic topical hydrofluoroalkane foam to induce nanoparticle modification and drug release in situ by Yanjun Zhao; Mojgan Moddaresi; Stuart A. Jones; Marc B. Brown (521-528).
Topical nanoparticles are usually applied using semi-solid formulations, but the delivery process is often inefficient due to the poor drug release from the particles. The aim of this study was to investigate the capability of a dynamic foam to break open nanoparticles upon application to the skin and enhance drug delivery efficiency. Vitamin E acetate (VEAc) was selected as a model drug and loaded into lipid nanoparticles (50–60 nm) prepared by phase inversion. The highest drug loading was 18.9 ± 1.2 mg/ml and the corresponding encapsulation efficiency was 81.5 ± 4.1%. Dynamic foams were generated by emulsifying VEAc-loaded nanoparticle suspensions with hydrofluoroalkane using pluronic L62D. An in vitro permeation study demonstrated that VEAc did not release from the nanoparticles when administered as an aqueous suspension, but attained a flux of 18.0 ± 2.1 (μg cm−2  h−1) when applied using the foam. Drug release from the foam was shown to be a consequence of nanoparticle modification after dose administration and this led to the foam delivering 0.7 ± 0.3% VEAc into the stratum corneum (SC) when applied to human skin.
Keywords: Foam; Vehicle; Vitamin E acetate; Nanoparticle; Penetration;

Dexamethasone-containing biodegradable superparamagnetic microparticles for intra-articular administration: Physicochemical and magnetic properties, in vitro and in vivo drug release by Nicoleta Butoescu; Olivier Jordan; Pierre Burdet; Pierre Stadelmann; Alke Petri-Fink; Heinrich Hofmann; Eric Doelker (529-538).
Compared with traditional drug solutions or suspensions, polymeric microparticles represent a valuable means to achieve controlled and prolonged drug delivery into joints, but still suffer from the drawback of limited retention duration in the articular cavity. In this study, our aim was to prepare and characterize magnetic biodegradable microparticles containing dexamethasone acetate (DXM) for intra-articular administration. The superparamagnetic properties, which result from the encapsulation of superparamagnetic iron oxide nanoparticles (SPIONs), allow for microparticle retention with an external magnetic field, thus possibly reducing their clearance from the joint. Two molecular weights of poly(lactic-co-glycolic acid) (PLGA) were used, 12 and 19 kDa. The prepared batches were similar in size (around 10 μm), inner morphology, surface morphology, charge (neutral) and superparamagnetic behaviour. The SPION distribution in the microparticles assessed by TEM indicates a homogeneous distribution and the absence of aggregation, an important factor for preserving superparamagnetic properties. DXM release profiles were shown to be quite similar in vitro (ca. 6 days) and in vivo, using a mouse dorsal air pouch model (ca. 5 days).
Keywords: PLGA microparticles; Dexamethasone acetate; Superparamagnetic iron oxide nanoparticles (SPIONs); Microscopy; SQUID; EELS; Drug release; Dorsal air pouch model; Arthritis; Intra-articular;

Enhanced oral bioavailability of dexibuprofen by a novel solid Self-emulsifying drug delivery system (SEDDS) by Prabagar Balakrishnan; Beom-Jin Lee; Dong Hoon Oh; Jong Oh Kim; Myung Ja Hong; Jun-Pil Jee; Jung Ae Kim; Bong Kyu Yoo; Jong Soo Woo; Chul Soon Yong; Han-Gon Choi (539-545).
The main objective of this study was to prepare a solid form of lipid-based self-emulsifying drug delivery system (SEDDS) by spray drying liquid SEDDS with an inert solid carrier Aerosil 200 to improve the oral bioavailability of poorly water-soluble drug dexibuprofen. The liquid SEDDS was a system that consisted of dexibuprofen, Labrasol, Capryol 90 and Labrafil M 1944 CS. The particle size analysis revealed no difference in the z-average particle diameter of the reconstituted emulsion between liquid and solid SEDDS. The solid SEDDS was characterized by SEM, DSC and XRD studies. In vivo results of solid SEDDS and dexibuprofen powder in rats at the dose of 10 mg/kg showed that the initial plasma concentrations of drug in solid SEDDS were significantly higher than those of dexibuprofen powder (P  < 0.05). The solid SEDDS gave significantly higher AUC and Cmax than did dexibuprofen powder (P  < 0.05). In particular, the AUC of solid SEDDS was about twofold higher than that of dexibuprofen powder. Our results suggested that this solid SEDDS could be used as an effective oral solid dosage form to improve the bioavailability of poorly water-soluble drug dexibuprofen.
Keywords: Poorly water-soluble drugs; Self-emulsifying systems; Spray drying; Solid dosage form; Bioavailability;

Subconjunctivally injected, liposome-encapsulated streptokinase enhances the absorption rate of subconjunctival hemorrhages in rabbits by Seung-Hee Baek; Sung Jin Park; Su-Eon Jin; Jin-Ki Kim; Chong-Kook Kim; Jeong-Min Hwang (546-551).
Liposome-encapsulated streptokinase (SK) was prepared with distearoylphosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000). In vitro release assay demonstrated over 81% of SK was released from liposomes at 48 h, and the effect of its subconjunctival injection on the absorption rate of induced subconjunctival hemorrhage (SH) in rabbits was evaluated. After 8 h of SH induction, eyes were randomly assigned to one of four subconjunctival injection groups (10 eyes each): group A: the free form of SK (1000 IU/mL); group B: liposome-encapsulated SK (1000 IU/mL); group C: 0.1 mL of liposomes; and group D: no injection. SHs were photographed at 8, 24, 48, 72, and 120 h after SH induction and their sizes were compared. Size decrease of the SH was faster in groups A and B than in groups C and D. Group B displayed significantly different absorption rates than group A at 24 and 48 h and with groups C and D at 24, 48, and 72 h, with the shortest mean elapsed time among all groups. The ocular absorption of SK was lower after the injection of the liposome-encapsulated SK than the free form. These results demonstrated that subconjunctival injection of liposome-encapsulated SK enhances the rate of SH absorption, especially in the early phases.
Keywords: Streptokinase; PEG-containing liposome; Liposome-encapsulated streptokinase; Subconjunctival hemorrhage; Subconjunctival injection;

Dermal and transdermal targeting of dihydroavenanthramide D using enhancer molecules and novel microemulsions by Sandra Heuschkel; Johannes Wohlrab; Reinhard H.H. Neubert (552-560).
Specific accumulation of drugs in certain skin layers or in the blood circulation is the aim of (trans-)dermal targeting. As demonstrated previously, high dermal concentrations of the model drug dihydroavenanthramide D can be reached by the addition of 1,2-alkanediols as penetration enhancer to a conventional o/w cream. The focus of the present study is on an increased permeation by the choice of a modern colloidal drug carrier. Microemulsions based on a vegetable protein surfactant and 1,2-alkanediols as co-surfactant were developed. The respective pseudoternary phase diagrams revealed an increasing area of the optical isotropic phase with increasing chain length of the glycol (C3–C4–C5). Pentylene glycol-containing systems were characterized by electrical conductivity and differential scanning calorimetry indicating the presence of water-continuous microemulsions. Two selected formulations containing pentylene glycol and propylene glycol, respectively, were further investigated by TEM, conductivity, viscosity, and temperature stability. In the subsequently performed Franz type diffusion studies using full thickness human skin dihydroavenanthramide D was applied as model drug. Both formulations showed sufficient penetration into viable skin layers and particularly high permeation rates. Compared to the previously investigated glycol-containing cream, the microemulsions revealed a smaller fraction of the model drug within viable epidermis and dermis, but a strongly increased amount in the acceptor solution. Therefore, the formulations might find different application areas depending on needs concerning localization, beginning and duration of the drug effect.
Keywords: Microemulsion; Protein-based surfactant; Penetration enhancer; Pentylene glycol; Penetration; Physicochemical characterization; DSC; Electrical conductivity; Dihydroavenanthramide D;

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates by Melanie Greindl; Florian Föger; Juliane Hombach; Andreas Bernkop-Schnürch (561-566).
Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA250-Cys, displaying 580 μmol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA250 and 0.5% (w/v) PAA250-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA250 or 4% (w/v) thiolated PAA250 were orally given to rats. The PAA250-Cys solution increased the area under the plasma concentration–time curve (AUC0-12) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA250. This in vivo study revealed that PAA250-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.
Keywords: Poly(acrylic acid); Thiomers; MRP2; MRP2 inhibition; Sulforhodamine;

Influence of polymer adjuvants on the ultrasound-mediated transfection of cells in culture by Sophie Mehier-Humbert; Thierry Bettinger; Feng Yan; Richard Guy (567-573).
The purpose of this study was to further understand the mechanisms involved in ultrasound-mediated delivery of DNA (sonoporation); in particular, to understand how a plasmid should be formulated with an ultrasound contrast agent (UCA). Different polymer adjuvant–UCA combinations were formulated, and their impact on in vitro DNA transfection, was determined, under various experimental conditions. When present in the medium surrounding a cell suspension, and in the presence of a plasmid encoding for the green fluorescent protein (GFP), expression following sonoporation was increased by more than 1.5-fold compared to that achieved in control experiments (without the adjuvants). The effects of the adjuvants were not influenced by the nature of the UCA, nor by that of the transfected cells; in contrast, the adjuvant concentrations, their physico-chemical properties, and the manner in which they were used, did have an impact on transfection. Close association of the adjuvants to the UCA inhibited their action, suggesting that these substances must have access to the cell membrane to be effective. Indeed, Pluronic® F127 appeared to improve the efficacy of transfection (percentage of GFP-positive cells and cell viability), via fluidization of the cell membrane, perhaps facilitating thereby the formation of transient pores and their re-sealing. The mechanism of action of polyethylene glycols, on the other hand, remains unclear.
Keywords: Sonoporation; Ultrasound contrast agents; Polymer adjuvants; Membrane fluidization; Transfection; Cell viability;

In this study, the changes in the physico-chemical properties of different high amylose maize starches, i.e., Hylon® VII, Hylon® V and IM-DS acetate starch, were studied prior and after heat treatment used in the preparation of film coatings (WO 2008/012573 A1).Characterisation of the unprocessed maize starches was carried out with regard to the outer particle morphology, particle size distribution, specific surface area, moisture content, apparent particle density, swelling, polarised light microscopy, Fourier Transform Infrared (FT-IR), X-ray powder diffraction and modulated Differential Scanning Calorimetry (mDSC). Pure amylopectin and low amylopectin samples (LAPS) were also used to aid the interpretation of the results. The effect of heat processing was evaluated in terms of degree of crystallinity, FT-IR and mDSC. Enzymatic digestibility of both processed and unprocessed maize starches was estimated qualitatively using various α-amylases resembling those present under in vivo conditions.A significant decrease in the degree of crystallinity of the dried samples after processing was observed, in particular for amylopectin. Only LAPS and Hylon® VII samples showed differences in their thermal behaviour upon heat treatment, thus suggesting that a minimum amount of amylose is required for an effect to be detectable. High amylose starches maintained a well-ordered arrangement of their macromolecular chains, as was seen by X-ray and FT-IR studies. This effect could be explained by a formation of retrograded forms of the starches. The retrograded starches were found to be less digestible by various types of amylase, in particular those found in the upper intestines, indicating that the formation of a butanol complex as claimed elsewhere is not essential in the preparation of colon delivery devices.
Keywords: High amylose starches; Enzymatic digestion; Fourier Transform Infrared; Heat treatment; Modulated Differential Scanning Calorimetry; X-ray diffraction;

This work reports an investigation into free-film properties of a high amylose maize starch-based film coating that has been used in the preparation of formulations for drug delivery to the colon (WO 2008/012573 A1) and relates these properties to in vitro drug release from pellets.Maize starch/ethylcellulose free films were prepared and characterised by scanning electron microscopy (SEM), light microscopy, modulated differential scanning calorimetry (mDSC), Fourier-transform infrared (FT-IR), X-ray and % swelling in aqueous fluids with pH conditions similar to the stomach and small intestine. 5-ASA release from film-coated pellets was tested in enzyme free simulated gastric fluid and phosphate buffer pH 7.2. Selected formulations were further assessed in simulated gastric and intestinal fluids containing pepsin and pancreatin, respectively.The free films prepared were smooth and homogeneous in their appearance. The two polymers are immiscible, and neither mDSC nor FT-IR could detect interactions between them. Films made from high amylose starches were found to have a considerably lower swelling ability than high amylopectin-based films, and they suppressed drug release in the enzyme free media successfully.5-ASA release from pellets coated with mixtures of high amylose starches (Hylon® VII, Hylon® V or LAPS) and Surelease® in a ratio of 1 to 2 w/w was found to be minimal in simulated gastric and intestinal fluids. This suggests that these mixed films provide starch domains that are resistant to the enzymes present in the upper GI tract and thus can potentially be used in the preparation of colon-specific delivery devices. Starches with a minimum amylose content of 56% such as the starches used in this study (Hylon® VII and Hylon® V) are preferred, and although pure amylose can also be used this is not essential.
Keywords: Film coating; Fourier-transform infrared; High-amylose maize starch; In vitro drug release; Modulated differential scanning calorimetry; Swelling; X-ray;

Electroosmotic flow as a result of buccal iontophoresis – Buccal mucosa properties by Aleksandra Moscicka-Studzinska; Ewa Kijeńska; Tomasz Ciach (595-599).
The objective of this study was to investigate and to better understand the properties of buccal mucosa as a semipermeable membrane and a portal for drug administration by iontophoretic and electroosmotic means. In vitro experiments showed that buccal mucosa at the pH of about 7.4 behaved as a cation-exchange membrane and non-linear resistor. It had lower resistance and was more permeable for water than a skin. The electroosmotic volume flow through mucosa depended on current density, mucosa resistance and electrolyte concentration. Sodium dodecyl sulfate (in concentration range 0.001–0.005 mol L−1) and urea (in concentration range 0.42–1.67 mol L−1) did not promote a water transfer through buccal mucosa, however, both substances enhanced flow through the skin.
Keywords: Electroosmosis; Electroosmotic flow; Iontophoresis; Buccal mucosa; Skin;

Comparison of two in vitro models for the analysis of follicular penetration and its prevention by barrier emulsions by Jürgen Lademann; Alexa Patzelt; Heike Richter; Sabine Schanzer; Wolfram Sterry; Alexander Filbry; Kerstin Bohnsack; Frank Rippke; Martina Meinke (600-604).
The penetration of topically applied substances in and through the human skin is of special interest for the development and optimization of topically applied drugs and cosmetic products. In the present study, the efficacy of barrier emulsions in the prevention of the penetration of pollen allergens into the hair follicles was investigated. Because of the sensitising potential of the used pollen allergens, the study was carried out under in vitro conditions. Therefore, excised human skin and porcine ear skin were used as tissue models. Applying laser-scanning microscopy and fluorescent-labeled grass pollen allergens, we found that the preventive efficacy of the barrier emulsions could be significantly better investigated on porcine ear skin than on excised human skin. This might be due to the contraction of the elastic fibres around the hair follicles in excised human skin after its removal. In contrast to the excised human skin, the porcine ear skin remains on the cartilage during the experiment. Therefore, contraction of the tissue can be avoided. The results give further indication that in vitro studies based on membranes of excised skin are not suitable for the investigation of the follicular penetration pathway of topically applied substances.
Keywords: Follicular penetration; Skin models; Pollen allergens; Barrier formulations; Laser scanning microscopy;

Purpose: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein BCRP (ABCG2). Methods: BCRP expression was verified by immunostaining and Western blots in intact porcine brain capillaries, isolated endothelial cells (PBCECs) and in MDCKII-cells over-expressing human wild-type BCRP (MDCKII-hBCRP). Transport of fluorescent mitoxantrone across cells was determined to assess a preferred transport direction. Sensitivity of cultured cells versus mitoxantrone in the absence and in the presence of transport modulators was examined at increasing concentrations of the cytostatic using the AlamarBlue™ assay. In addition, cells were incubated with mitoxantrone in the absence and presence of increasing concentrations of different compounds with the potential to interact with BCRP. Intracellular fluorescence accumulation was measured using a flow cytometer. Results: Isolated capillaries as well as 7-day old PBCECs showed expression of BCRP. Cell sensitivity to mitoxantrone significantly increased in the presence of the BCRP inhibitors KO143 and GF120918. Transport of mitoxantrone across PBCEC monolayers was directed with P app (apical to basolateral) 5.6 × 10−6  cm s−1 and with P app (basolateral to apical) 2.8 × 10−5  cm s−1. FACS analysis revealed a different extent of fluorescence accumulation dependent on the kind and concentration of BCRP modulating compounds. Conclusions: The mitoxantrone-based assay can be used as a rapid FACS screening system to assess drug interactions with BCRP at the blood–brain barrier and therefore represents a useful tool in drug profiling.
Keywords: BCRP; ABCG2; Fluorescence assay; ABC-protein; Mitoxantrone;

Improved bioavailability of darunavir by use of κ-carrageenan versus microcrystalline cellulose as pelletisation aid by Markus Thommes; Lieven Baert; Gerben van ’t Klooster; Marian Geldof; Laurent Schueller; Jan Rosier; Peter Kleinebudde (614-620).
The aim of this study was to produce pellet formulations containing a high drug load (80%) of the poorly soluble HIV-protease inhibitor darunavir, using wet extrusion/spheronisation with κ-carrageenan or microcrystalline cellulose (MCC) as pelletisation aid. Drug release was assessed in vitro by a standardized paddle-dissolution test and in vivo by a single-dose pharmacokinetic study in dogs. Mean dissolution time (MDT) was 78.2 ± 3.5 h from MCC pellets (1301 ± 301 μm) and 6.1 ± 0.7 min from κ-carrageenan pellets (966 ± 136 μm). In contrast to κ-carrageenan pellets, MCC pellets did not disintegrate and showed a diffusion-controlled drug release. In line with the in vitro findings, the darunavir peak plasma levels and exposure after the administration of a 300 mg dose were more than 60-fold higher when formulated with κ-carrageenan pellets when compared with MCC pellets, and 10-fold higher after co-administration with 10 mg/kg of ritonavir. The relative bioavailability of darunavir versus the reference tablet (F rel) was 155% with κ-carrageenan pellets and 2% with MCC pellets without ritonavir, while 78% and 9%, respectively, in presence of ritonavir. In conclusion, when compared with MCC pellets, the bioavailability of darunavir was substantially improved in κ-carrageenan pellets, likely due to their better disintegration behavior.
Keywords: Bioavailability; Darunavir; TMC114; Carrageenan; Microcrystalline cellulose; Pellets; Extrusion; Spheronisation;

Compaction properties of isomalt by Gerad K. Bolhuis; Jeffrey J.P. Engelhart; Anko C. Eissens (621-625).
Although other polyols have been described extensively as filler-binders in direct compaction of tablets, the polyol isomalt is rather unknown as pharmaceutical excipient, in spite of its description in all the main pharmacopoeias. In this paper the compaction properties of different types of ispomalt were studied. The types used were the standard product sieved isomalt, milled isomalt and two types of agglomerated isomalt with a different ratio between 6-O-α-d-glucopyranosyl-d-sorbitol (GPS) and 1-O-α-d-glucopyranosyl-d-mannitol dihydrate (GPM). Powder flow properties, specific surface area and densities of the different types were investigated. Compactibility was investigated by compression of the tablets on a compaction simulator, simulating the compression on high-speed tabletting machines. Lubricant sensitivity was measured by compressing unlubricated tablets and tablets lubricated with 1% magnesium stearate on an instrumented hydraulic press. Sieved isomalt had excellent flow properties but the compactibility was found to be poor whereas the lubricant sensitivity was high. Milling resulted in both a strong increase in compactibility as an effect of the higher surface area for bonding and a decrease in lubricant sensitivity as an effect of the higher surface area to be coated with magnesium stearate. However, the flow properties of milled isomalt were too bad for use as filler-binder in direct compaction. Just as could be expected, agglomeration of milled isomalt by fluid bed agglomeration improved flowability. The good compaction properties and the low lubricant sensitivity were maintained. This effect is caused by an early fragmentation of the agglomerated material during the compaction process, producing clean, lubricant-free particles and a high surface for bonding. The different GPS/GPM ratios of the agglomerated isomalt types studied had no significant effect on the compaction properties.
Keywords: Tablets; Direct compaction; Filler-binders; Polyols; Isomalt;

Histological analysis of 70-nm silica particles-induced chronic toxicity in mice by Hikaru Nishimori; Masuo Kondoh; Katsuhiro Isoda; Shin-ichi Tsunoda; Yasuo Tsutsumi; Kiyohito Yagi (626-629).
Nano-sized silica is a promising material for disease diagnosis, cosmetics and drugs. For the successful application of nano-sized material in bioscience, evaluation of nano-sized material toxicity is important. We previously found that nano-sized silica particles with a diameter of 70 nm showed acute liver failure in mice. Here, we performed histological analysis of major organs such as the liver, spleen, lung, kidney, brain and heart in mice, chronically injected with 70-nm silica particles for 4 weeks. Histological analysis revealed hepatic microgranulation and splenic megakaryocyte accumulation in these 70-nm silica particles treated mice, while the kidney, lung, brain and heart remained unaffected. Thus, liver and spleen appear to be the major target organs for toxicity by the chronic administration of the 70-nm silica particles.
Keywords: Nanotoxicology; Silica particle; Histological analysis; Liver; Spleen;

The finding that imatinib enhances the drug transport from bloodstream to neoplastic cells suggested a possible role of this drug as an adjuvant to the chemotherapeutics given in the treatment of solid malignancies. The present experiments aimed to verify whether imatinib can selectively increase the penetration of a doxorubicin–lactosaminated human albumin conjugate (L-HSA-DOXO) in chemically induced rat hepatocellular carcinomas (HCCs). We observed that imatinib increased the uptake of L-HSA-DOXO by HCCs but at the same time caused a similar enhanced penetration of the conjugate in liver and bone marrow. To our knowledge, this is the first demonstration that the enhancing effect of imatinib on interstitial drug transport is not restricted to the tumors, but can be also displayed in normal tissues. This observation casts some doubts about the possibility that the value of anticancer agents with toxic side effects on liver and bone marrow can be improved by imatinib.
Keywords: Imatinib; Drug delivery; Interstitial fluid pressure; Hepatocellular carcinoma; HCC-targeted doxorubicin;