European Journal of Pharmaceutics and Biopharmaceutics (v.60, #3)

APV Diary (S1-S2).

The Summary of Product Characteristics (SPC) approved by the European Medicines Agency (EMEA) and the Package Insert (PI) approved by the Food and Drug Administration (FDA) were examined for 32 biopharmaceutical products. The aim was to identify differences in the product information since such information may have an impact on the planning of global clinical development programmes. The EU SPC contained more detailed instructions to the prescriber, including the positioning of the product with regard to the stage of the disease and to other therapies. The approach to safety information, notably to contraindications and warnings was more conservative in the EU SPC. The conservative approach in the EU may reflect the central position of the SPC in risk management of new pharmaceuticals. A typical feature of the US PI was the detailed description of the efficacy and safety result of the pivotal clinical trials.
Keywords: Biopharmaceuticals; Regulation; EMEA; FDA; Marketing authorisation; Product information; Labelling;

Targeting delivery of oligonucleotide and plasmid DNA to hepatocyte via galactosylated chitosan vector by Shuying Gao; Jiangning Chen; Lei Dong; Zhi Ding; Yong-hua Yang; Junfeng Zhang (327-334).
Delivery of oligonucleotide to specific cells and maintenance of its biological function are important for nucleic acid therapy. The objective of this paper is to demonstrate that galactosylated low molecular weight chitosan (gal-LMWC) is a safe and effective vector of antisense oligonucleotide (ASO) and plasmid DNA for the hepatocyte targeting delivery. Gal-LMWC has been successfully prepared and MTT cytotoxic assay shows that cytotoxicity of gal-LMWC is lower than that of high molecular weight chitosan (HMWC) and low molecular weight chitosan (LMWC) in HepG2 cells. Using a complex coacervation process, gal-LMWC can form stable nano-complexes with plasmid DNA or with ASO by the electrostatic interaction. The morphometrics, particle size, and the zeta potential of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes are very similar. The transfection efficiency by using gal-LMWC vector is significantly higher than that of naked DNA or naked ASO in HepG2 cells. Transfection efficiency of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes depends on the molar ratio of the positive chitosan amino group and the negative DNA phosphate group (N/P ratio) strongly. Inhibition experiments confirm that the enhanced transfection efficiency is due to the ASGR mediated endocytosis of the gal-LMWC/ASO complexes or gal-LMWC/DNA complexes. These results suggest that gal-LMWC can be used in gene therapy to improve the transfection efficiency in vitro and in vivo.
Keywords: Galactosylated low molecular weight chitosan; Plasmid DNA; Antisense oligonucleotide; Hepatocyte targeting; Transfection efficiency; Non-viral vector;

Physical characterisation of formulations for the development of two stable freeze-dried proteins during both dried and liquid storage by Stéphanie Passot; Fernanda Fonseca; Muriel Alarcon-Lorca; Dominique Rolland; Michèle Marin (335-348).
The development of stable freeze-dried proteins requires maintaining the physical and biological integrity of the protein as well as increasing the efficiency of the manufacturing process. Our objective was to study the effects of various excipients on both the physical characterisation and the dried and liquid stability of two proteins. Thermo-physical properties of 13 formulations were determined using both differential scanning calorimetry and freeze–drying microscopy. The antigenic activity was evaluated immediately after freeze–drying and after subsequent storage in both dried and liquid state. From the comparison between glass transition (Tg) and collapse (T coll) temperatures, we concluded that the collapse temperature was a more relevant parameter than Tg for freeze–drying cycle development and optimisation. One crystalline formulation composed of 4% mannitol and 1% of sucrose protected efficiently both proteins during subsequent storage in dried state (6 months at 25 °C) and in liquid state (3 months at 4 °C after rehydration). However, the freeze–drying behaviour of this crystalline formulation remained difficult to predict and control. On the other hand, two amorphous formulations composed of 4% of maltodextrin and 0.02% of Tween 80, or 5% of BSA preserved antigenic activity during storage in dried state. The glassy character of these formulations as well as their high collapse temperature values (−9 and −12 °C, respectively) should allow simplification and shortening of freeze–drying process.
Keywords: Protein formulation; Storage stability; Crystalline and amorphous bulking agents; Differential scanning calorimetry; Freeze–drying microscopy; Glass transition temperature; Collapse temperature; Freeze–drying;

Development of an enteric-coated formulation containing freeze-dried, viable recombinant Lactococcus lactis for the ileal mucosal delivery of human interleukin-10 by Nathalie Huyghebaert; An Vermeire; Sabine Neirynck; Lothar Steidler; Erik Remaut; Jean Paul Remon (349-359).
Recombinant hIL-10 producing Lactococcus lactis (Thy12) looks a promising intestinal mucosal delivery system for treatment of Crohn's disease [L. Steidler, W. Hans, L. Schotte, S. Neirynck, F. Obermeirer, W. Falk, W. Fiers, E. Remaut, Treatment of murine colitis by L. lactis secreting interleukin-10, Science 289 (2000) 1352–1355. L. Steidler, S. Neirynck, N. Huyghebaert, V. Snoeck, A. Vermeire, B.M. Goddeeris, E. Cox, J.P. Remon, and E. Remaut, Biological containment of genetically modified L. lactis for intestinal delivery of human interleukin-10, Nat. Biotechnol. 21 (7) (2003) 785–789]. As the hIL-10 production is strictly related to Thy12's viability and gastric fluid negatively influences this viability, an enteric-coated formulation had to be developed with maintenance of its viability after production and storage. L. lactis MG1363, used for optimization, was grown until stationary phase in milk (glucose/casiton supplemented) and freeze-dried. This resulted in a viability of about 60%. Storage at different conditions showed that viability remained highest at 8 °C/N2 atmosphere (32.5% of initial remained viable after 6 months). To increase the concentration of bacteria in the freeze-dried powder, they were concentrated by centrifugation. L. lactis tolerated this procedure. However, the concentration factor was limited to 10. Freeze-dried Thy12 was filled in ready-to-use enteric-coated capsules. Despite the good enteric properties of the capsules, viability of Thy12 dropped to about 43 and 28% after gastric fluid stage, depending on the enteric polymer used. Freeze-dried Thy12 filled in ready-to-use enteric-coated capsules, packed in Alu sachets (sealed at 20% RH) maintained 6.1 and 44.3% of initial viability after storage for 1 year at 8 and −20 °C, respectively, as well as its hIL-10 producing capacity.
Keywords: Freeze-drying; Recombinant Lactococcus lactis; Viability; Enteric-coated capsules;

Solid lipid nanoparticles (SLN) were produced using a full range of poloxamer polymers and poloxamine 908 for stabilization. The protein adsorption pattern acquired on the surface of these particles after intravenous injection is the key factor determining the organ distribution. Two-dimensional polyacrylamide gel electrophoresis (2-DE) was employed for determination of particle interactions with human plasma proteins. The objective of this study was to investigate changes in the plasma protein adsorption patterns in the course of variation of the polymers stabilizing the SLN. Considerable differences in the protein adsorption with regard to preferential adsorbed proteins were detected for the different stabilizers. Possible correlations between the polyethylene oxide (PEO) chain length and the adsorption of various proteins (first of all apolipoproteins) are shown and discussed. Besides the study of protein adsorption patterns, the total protein mass adsorbed to the SLN was also evaluated using the bicinchoninic acid (BCA)-protein assay. The knowledge concerning the interactions of proteins and nanoparticles can be used for a rational development of particulate drug carriers. Based on the findings presented in this paper, we anticipate that the in vivo well-tolerable SLN are a promising site-specific drug delivery system for intravenous injection.
Keywords: Solid lipid nanoparticles; Poloxamer; Poloxamine; Protein adsorption patterns; Two-dimensional polyacrylamide gel electrophoresis; Site-specific drug delivery;

Silk fibroin as a novel coating material for controlled release of theophylline by Oguz Bayraktar; Özge Malay; Yarkın Özgarip; Ayşegül Batıgün (373-381).
The aim of this study was to explore potential use of the silk fibroin (SF) as an aqueous coating material for theophylline tablets. We have examined the film forming and coating properties of heat-treated fibroin, SF solution having different amounts of polyethylene glycol (PEG) and 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) cross-linked SF. Heat-treated SF material possessed a brittle structure, which resulted in poor film forming and coating properties. The optimum PEG amount in SF solution was determined as 17% (by weight) for an acceptable film forming and zero order release profile. EDC cross-linked SF has shown a very good film forming and coating property with a potential for sustaining the drug release from coated theophylline tablets. Dissolution data for coated theophylline tablets were analyzed using Ritger and Peppas equation to describe the mechanism of drug release. Drug release from the EDC coated tablets followed zero-order kinetics. Release rate constants were found to be 0.26, 0.19, 0.16% min−1 for single-coated, double coated, and triple coated tablets, respectively. These results clearly demonstrated that silk fibroin has high utility as a novel aqueous coating material for controlled release products.
Keywords: Silk fibroin; Theophylline; Aqueous coating; Controlled release; Cross-linking;

Poly(ethylene-co-methyl acrylate) (EMA) membranes with different amounts of methyl acrylate (MA) content were studied in terms of the thermal and mechanical properties, swelling and drug permeation. The increase in MA content in the copolymer significantly increased the percentage of elongation and decreased the tensile strength and modulus of elasticity of the membranes. The degree of swelling of the EMA membranes increased with the ethanol composition and MA content. The contact angle of a sessile drop (10 μL of ethanol/water solution) decreased with an increase in the ethanol fraction suggesting that the membrane wettibility increased with the ethanol content. The flux of diltiazem hydrochloride increased from 0.012 to 0.018 mg cm−2  h−1 with an increase in the MA content from 16.5 to 29.0%. By increasing the ethanol fraction from 0.4 to 1.0, the flux of diltiazem hydrochloride into the membranes with 29.0% MA, increased from 2.56 (±0.09)×10−3 to 18.38 (±0.62)×10−3  mg cm−2  h−1. The permeability coefficient increased from 5.85×10−6 to 3.53×10−4  cm h−1 with an increase in the ethanol fraction. The flux can also be correlated with the drug solubility in the membrane and ethanol. For example, the solubilities of diltiazem hydrochloride, paracetamol and ibuprofen were 0.64, 6.68 and 504.48 mg cm−3 in the membrane, respectively. Under the same conditions, the flux for the above mentioned drugs was 0.08 (±0.01), 0.53 (±0.01) and 45.11 (±2.00) mg cm−2  h−1.
Keywords: Poly(ethylene-co-methyl acrylate); Membranes; Diltiazem hydrochloride; Ibuprofen; Paracetamol; Permeability coefficient;

Molecular weights of poly(butyl cyanoacrylate) nanoparticles determined by mass spectrometry and size exclusion chromatography by Alexander Bootz; Thomas Russ; Friedhelm Gores; Michael Karas; Jörg Kreuter (391-399).
Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) were employed to elucidate the chemical composition, mean number average molecular weight (M n), mean weight average molecular weight (M w), and polydispersity (PD) of poly(butyl cyanoacrylate) (PBCA) manufactured by emulsion polymerisation. Both methods gave similar results for M n, but substantial differences were observed for M w and PD, with MALDI producing consistently lower values which could not be improved by off-line coupling of SEC and MALDI. MALDI gave a more detailed view on the chemical composition of the cyanoacrylate and revealed the presence of two additional polymer series with different end groups besides the expected PBCA series, which showed different retention in SEC. Their formation is explained by the secession/addition of formaldehyde from/to the regular polymer via (reverse) Knoevenagel reaction. In additional experiments, the influence of different pH on PBCA-NP during polymerisation was examined by comparison of polymerisation yield and particle diameter to their chemical composition as revealed by the MALDI spectra. The most uniform nanoparticles, with the highest polymerisation yield, narrowest particle size, and mass distribution were produced at pH 1.
Keywords: Nanoparticles; Polyalkylcyanoacrylate; PBCA; Molecular weight; Gel permeation chromatography; Size exclusion chromatography; Matrix-assisted laser desorption ionization time of flight mass spectrometry;

Two-month ciprofloxacin implants for multibacterial bone infections by C. Castro; C. Évora; M. Baro; I. Soriano; E. Sánchez (401-406).
A ciprofloxacin implant formulation composed of 12% hydroxyapatite, 36% tricalcium phosphate, 12% poly(DL-lactide) (PLA) and 40% ciprofloxacin was characterized in vivo for use in treatment of multibacterial bone infection. After the implant was inserted in the femur of rabbits, approximately 90% of the total ciprofloxacin was released within 8 weeks, maintaining therapeutic levels in the femur and tibia. Throughout the femoral cortex and marrow these remained higher than the minimum inhibitory concentrations (MIC) against the most common pathogens causing osteomyelitis. Levels in tibia cortex were also above MIC for 6 weeks. The implant was characterized in terms of polymer degradation and morphological and crystallographic changes. X-ray analyses confirmed the osteoconductivity and biocompatibility of these materials. The sequential changes in the femur were those of a normal surgical trauma reaction followed by a repair process. All the results confirmed that ciprofloxacin release is limited by its low solubility, and that implant erosion and bone ingrowth into the implants enhance the antibiotic release.
Keywords: Ciprofloxacin implants; Bone infection; Biocompatibility; Calcium phosphates; Poly(lactic acid);

Use of CP/MAS solid-state NMR for the characterization of solvate molecules within estradiol crystal forms by Jeong-Sook Park; Hye Won Kang; Sung Jean Park; Chong-Kook Kim (407-412).
To investigate the solvate molecules within estradiol crystal forms, four crystal forms of estradiol (EM, ET, EP and EC) were obtained by recrystallization from various organic solvents such as methanol, ethanol, isopropanol and acetone, and their physicochemical properties were characterized using XRD, TGA, DSC and solid-state NMR. The XRD patterns showed that the estradiol crystal forms were composed of high crystallinity and their degrees of crystallinity were nearly identical. The solvent phases and solvate molecules within the estradiol crystal forms were detected by thermal analysis and the solvate molecular structures were identified by further observation using solid-state NMR. Estradiol typically tends to crystallize in form of its hemihydrate by the solvate molecules from the mother solvents used for recrystallization. The residual solvent might be incorporated into the estradiol molecules to change the local chemical environment and cause the observed 13C CP/MAS NMR spectral changes. These results suggest that the thermal analysis and CP/MAS solid-state NMR spectroscopy could be useful at the dried solid state to characterize the solvate molecule within the recrystallized estradiols.
Keywords: Estradiol; Crystal form; Solvate molecule; CP/MAS solid-state NMR;

Simulation of fasting gastric conditions and its importance for the in vivo dissolution of lipophilic compounds by Maria Vertzoni; Jennifer Dressman; James Butler; John Hempenstall; Christos Reppas (413-417).
In this study, the importance of accurate simulation of fasting gastric environment for the assessment of the absorption process of two model lipophilic compounds, GR253035X (weak base) and atovaquone (non-ionizable), was assessed. Dissolution profiles were constructed in previously proposed simulated gastric fluids and in a new medium that comprises only of components that have been recovered from the fasting stomach. Dissolution data obtained in a more physiologically relevant medium led to better correlation between the simulated and actual intralumenal dissolution vs. time profiles for GR253035X. In contrast, accurate simulation of gastric environment did not affect the simulated plasma profile of atovaquone. Accurate simulation of the fasting gastric contents may be crucial for the assessment of the absorption profile of lipophilic weak bases.
Keywords: Dissolution; Fasted state; Gastric composition; GR253035X; Atovaquone; Weak base;

The purpose of this study was to evaluate the local tolerance of vaginal gels (three gels containing dapivirine, the placebo gel, and Conceptrol®) with the Slug Mucosal Irritation test and to compare the results with those of the rabbit vaginal irritation test. The irritation potential on the slug mucosa was assessed by the mucus production caused by a repeated treatment for 5 successive days. Additionally, membrane damage was estimated by the protein and enzyme release. By means of a classification prediction model the formulations were classified into four irritation classes. The effect of a 10-day intravaginal application of the gels on the rabbit vaginal and cervical mucosa was evaluated by means of macroscopic and microscopic examination. The placebo and dapivirine gels induced no irritation of the slug mucosa (low mucus production and protein release, no enzyme release) and no vaginal or cervical irritation in rabbits. Conceptrol® caused severe irritation of the slug mucosa (increased mucus production, protein release, and enzyme release) and irritation of the rabbit vagina and cervix. The results obtained with the Slug Mucosal Irritation test were comparable to those of the rabbit vaginal irritation test.
Keywords: Local tolerance; Vaginal formulation; Mucosal irritation; Alternative test;

The limited permeation of 5-aminolevulinic acid (ALA) through excised human stratum corneum could be improved by using 5-aminolevulinic acid-n-butyl ester (ABE). Furthermore drug permeation could be increased by choice of a permeation enhancing formulation. In this study, permeation of ALA and ABE was investigated from various formulations. In addition, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD) experiments were performed in order to reveal an interaction between the tested formulations and stratum corneum lipid structure. Drug incorporation into Dolgit® Mikrogel showed the highest increase in permeability with both ALA and ABE. Especially, ABE together with Dolgit® Mikrogel was the most promising combination. Further permeation studies with poloxamer based ABE formulations, partially enriched with ibuprofen acid and medium chained triglycerides showed that both compounds promote permeation. The permeation coefficients of either drug from Excipial® Creme and Basiscreme DAC were found to be very similar. These results were in accordance with those of DSC and WAXD experiments. Interaction between formulation and stratum corneum lipid structure resulting in an increased drug permeation only occurred after pretreatment with formulations enriched with ibuprofen acid. After pretreatment with Excipial® Creme, Basiscreme DAC or Excipial® Fettcreme stratum corneum structure and subsequently permeability remained unchanged. Nevertheless permeation of ALA from Excipial® Fettcreme is slower than from the tested hydrophilic formulations and therefore believed to be influenced by the affinity of ALA to the vehicle and stratum corneum.
Keywords: 5-Aminolevulinic acid; 5-Aminolevulinic acid-n-butyl ester; Stratum corneum; Permeation studies; Permeation enhancement; DSC experiments; WAXD experiments;

Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin by Regilene Steluti; Fernanda Scarmato De Rosa; John Collett; Antônio Cláudio Tedesco; Maria Vitória Lopes Badra Bentley (439-444).
Photodynamic therapy (PDT), a potential therapy for cancer treatment, utilizes exogenously applied or endogenously formed photosensitizers, further activated by light in an appropriate wavelength and dose to induce cell death through free radical formation. 5-Aminolevulinic acid (5-ALA) is a pro-drug which can be converted to the effective photosensitizer, protoporphyrin IX (PpIX). However, the use of 5-ALA in PDT is limited by the low penetration capacity of this highly hydrophilic molecule into appropriate skin layers. In the present study, we propose to increase 5-ALA penetration by using formulations containing glycerol monooleate (GMO), an interesting and useful component of pharmaceutical formulations. Propylene glycol solutions containing different concentrations of GMO significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin, after the use of topical 5-ALA formulations containing GMO in a concentration-dependent manner. The results show that skin 5-ALA penetration and PpIX accumulation, important factors for the success of topical 5-ALA-PDT in skin cancer, are optimized by GMO/propylene glycol formulations.
Keywords: 5-Aminolevulinic acid; Monoolein; Photodynamic therapy; Protoporphyrin IX; Skin cancer;

by Conrad Coester (445).