European Journal of Pharmaceutics and Biopharmaceutics (v.70, #1)

APV Diary (S1-S2).

Evaluation of promoter strength in mouse and rat primary hepatocytes using adenovirus vectors by Eri Arita; Masuo Kondoh; Katsuhiro Isoda; Hikaru Nishimori; Takeshi Yoshida; Hiroyuki Mizuguchi; Kiyohito Yagi (1-6).
Primary cultured hepatocytes are widely used in the studies of basic and clinical hepatology. Finding an efficient method for gene transfer into primary hepatocytes will be an important advance for these studies. In the present study, we evaluated the activity of an adenovirus vector including promoters for the Rous sarcoma virus (RSV), elongation factor 1α, and cytomegalovirus (CMV) as well as the β-actin promoter/CMV enhancer (CA) using β-galactosidase as a reporter gene. Although RSV and elongation factor 1α promoters had low transcriptional activity in hepatocytes, the CA and CMV promoters had high activity. The CA promoter was the most active, mediating 50.3- and 204.4-fold more activity than the RSV promoter in mouse and rat hepatocytes, respectively. Dose-response studies revealed that transgene activity can be controlled by as much as 1000-fold, by selection of the promoter and the number of infectious particles per cell. These findings should help in the construction of adenovirus vectors for expressing genes of interest in rodent primary cultured hepatocytes.
Keywords: Hepatocyte; Adenovirus vector; Promoter; Transgene activity;

Novel cationic liposome formulation for the delivery of an oligonucleotide decoy to NF-κB into activated macrophages by Giuseppe De Rosa; Daniela De Stefano; Valeria Laguardia; Silvia Arpicco; Vittorio Simeon; Rosa Carnuccio; Elias Fattal (7-18).
Nuclear factor-κB (NF-κB) is involved in several pathological processes, such as inflammation. Pro-inflammatory genes expression can be down-regulated by using an oligonucleotide (ODN) decoy to NF-κB. Cationic liposomes are largely used to improve ODN uptake into cells, although a higher transfection efficiency and a lower toxicity are required to use them in therapy. In this work, we investigated the potential of a novel liposome formulation, based on the recently synthesised cationic lipid (2,3-didodecyloxypropyl) (2-hydroxyethyl) dimethylammonium bromide (DE), as the delivery system for a double stranded ODN decoy to NF-κB. Liposomes composed of DE or DE mixed with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine or cholesterol as helper lipids were complexed with ODN at different +/− charge ratios. In vitro uptake and the effect of ODN, naked or complexed with DE-containing liposomes, were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. The use of helper lipids increased liposome physical stability up to 1 year at 4 °C. ODN complexed with DE/cholesterol liposomes, at the +/− charge ratio of 8, showed a limited cytotoxicity and the highest inhibition of nitrite production, inducible nitric oxide synthase protein expression and NF-κB/DNA binding activity. Confocal microscopy confirmed a high ODN cell uptake obtained with DE/cholesterol liposomes at the highest +/− charge ratio.
Keywords: Nuclear factor-κB; Cationic liposomes; Decoy oligonucleotide; RAW 264.7 macrophages;

Reversible pegylation of insulin facilitates its prolonged action in vivo by Y. Shechter; M. Mironchik; S. Rubinraut; H. Tsubery; K. Sasson; Y. Marcus; M. Fridkin (19-28).
We attempted to engineer a novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude supraphysiological concentrations shortly after injection; (ii) maintenance of low-circulating level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating insulin through its amino side chains to a 40 kDa polyethylene glycol containing sulfhydryl moiety (PEG40-SH), employing recently developed hetero-bifunctional spacer 9-hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc-0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by spontaneous chemical hydrolysis has been created. PEG40-Fmoc-insulin is a water-soluble, reactivatable prodrug with low biological activity. Upon incubation at physiological conditions, the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear fashion, releasing the nonmodified immunologically and biologically active insulin with a t 1/2 value of 30 h. A single subcutaneous administration of PEG40-Fmoc-insulin to healthy and diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar doses of the native hormone. The beneficial pharmacological features endowed by PEGylation are thus preserved. In contrast, nonreversible, “conventional” pegylation of insulin led to inactivation of the hormone.
Keywords: Diabetes; Glucose-lowering profiles; Long-acting insulin; Normoglycemia; Pro(drug)-insulin;

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery by Sabrina Höbel; Robert Prinz; Anastasia Malek; Beata Urban-Klein; Johannes Sitterberg; Udo Bakowsky; Frank Czubayko; Achim Aigner (29-41).
Background: Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies. Aim: For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals. Results: We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22 kDa linear PEI (L-PEI), the low-molecular weight (4–10 kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection. Conclusions: Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.
Keywords: RNAi; siRNAs; PEI; Polyethylenimine; Frozen complexes; Nucleic acid delivery;

Glycation during storage and administration of monoclonal antibody formulations by Stefan Fischer; Joerg Hoernschemeyer; Hanns-Christian Mahler (42-50).
Covalent modifications of therapeutic proteins are of interest for the biotech industry as they potentially impact the quality of the material. This study focuses on covalent protein modifications by the reducing monosaccharide glucose via the glycation reaction. In particular, we examined (i) to which extent different therapeutic monoclonal antibodies are glycated, (ii) the glycation during storage in sucrose-containing formulation buffers where non-reducing sucrose potentially could hydrolyze into its reducing constituent monosaccharides and (iii) the risk of glycation in the course of short-term incubation in Dextrose infusion bags in simulated administration testing. A boronate affinity chromatography method was employed to detect and quantify glycation products in different antibody formulations. For confirmation and to determine the degree of glycation per molecule, selected samples were analyzed via LC–ESI-MS. We could demonstrate that different antibodies differed drastically regarding the degree of glycation, probably a result of their respective fermentation conditions and protein glycation susceptibility. We also demonstrated that sucrose is a non-critical excipient with respect to glycation when stored long-term at intended storage conditions (2–8 °C). Finally, we could show that short-term incubation of antibodies in Dextrose infusion bags might lead to huMAb glycation, suggesting to test on glycated products when considering diluting protein drug products in infusion media containing reducing sugars.
Keywords: Antibody; Formulation; Administration; Glycation; Glucose; Sucrose; Compatibility;

Two poorly soluble, potent anti-cancer drugs, paclitaxel and camptothecin, were successfully solubilized by mixed micelles of polyethylene glycol–phosphatidyl ethanolamine (PEG–PE) and vitamin E. Drug-containing micelles were additionally modified with anti-nucleosome monoclonal antibody 2C5 (mAb 2C5), which can specifically bring micelles to tumor cells in vitro. The optimized micelles had an average size of about 13–22 nm and the immuno-modification of micelles did not significantly change it. The solubilization of both drugs by the mixed micelles was more efficient than by micelles made of PEG–PE alone. Solubilization of camptothecin in micelles prevented also the hydrolysis of active lactone form of the drug to inactive carboxylate form. Drug-loaded mixed micelles and mAb 2C5-immunomicelles demonstrated significantly higher in vitro cytotoxicity than free drug against various cancer cell lines.
Keywords: Camptothecin; Paclitaxel; Immunomicelles; Monoclonal antibody; Polyethylene glycol–phosphatidyl ethanolamine; Vitamin E; Polymeric micelles;

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy by Claudia Colonna; Bice Conti; Paola Perugini; Franca Pavanetto; Tiziana Modena; Rossella Dorati; Paolo Iadarola; Ida Genta (58-65).
Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6 ± 2.1%) as well as the suitable physical characteristics (mean diameter of 365.5 ± 35.1 nm and a positive zeta-potential of 17.94 ± 0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 μg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).
Keywords: Chitosan nanoparticles; Cytotoxicity; Cellular uptake studies; Enzyme replacement therapy; Prolidase Deficiency;

Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin–biotin technology: Formulation development and in vitro anticancer activity by Mika Pulkkinen; Jere Pikkarainen; Thomas Wirth; Tommy Tarvainen; Vesa Haapa-aho; Harri Korhonen; Jukka Seppälä; Kristiina Järvinen (66-74).
Despite recent advances in cancer therapy, many malignant tumors still lack effective treatment and the prognosis is very poor. Paclitaxel is a potential anticancer drug, but its use is limited by the facts that paclitaxel is a P-gp substrate and its aqueous solubility is poor. In this study, three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin–biotin technology was evaluated in vitro as a way of enhancing delivery of paclitaxel. Paclitaxel was incorporated both in biotinylated (BP) and non-biotinylated (LP) PEG-PLA nanoparticles by the interfacial deposition method. Small (mean size ∼ 110 nm), spherical and slightly negatively charged (−10 mV) BP and LP nanoparticles achieving over 90% paclitaxel incorporation were obtained. The successful biotinylation of nanoparticles was confirmed in a novel streptavidin assay. BP nanoparticles were targeted in vitro to brain tumor (glioma) cells (BT4C) by three-step avidin–biotin technology using transferrin as the targeting ligand. The three-step targeting procedure increased the anti-tumoral activity of paclitaxel when compared to the commercial paclitaxel formulation Taxol® and non-targeted BP and LP nanoparticles. These results indicate that the efficacy of paclitaxel against tumor cells can be increased by this three-step targeting method.
Keywords: Paclitaxel; Nanoparticle; Biotin–avidin technology; Anti-tumoral activity; Glioma;

Targeted delivery of tacrine into the brain with polysorbate 80-coated poly(n-butylcyanoacrylate) nanoparticles by Barnabas Wilson; Malay Kumar Samanta; Kumaraswamy Santhi; Kokilampal Perumal Sampath Kumar; Nallupillai Paramakrishnan; Bhojraj Suresh (75-84).
The purpose of the present study was to investigate the possibility of targeting an anti-Alzheimer’s drug tacrine in the brain using polymeric nanoparticles. Rats obtained 1 mg/kg of tacrine by intravenous injection in the form of three preparations: (1) a simple solution in phosphate buffered saline, (2) bound to poly(n-butylcyanoacrylate) nanoparticles, and (3) bound to poly(n-butylcyanoacrylate) nanoparticles coated with 1% polysorbate 80 (Tween® 80). After 1 h of post injection the rats were killed by decapitation and tacrine concentration in brain, liver, lungs, spleen and kidneys was analyzed by HPLC. A higher concentration of drug tacrine was observed in liver, spleen and lungs with the nanoparticles in comparison to the free drug. The accumulation of drug tacrine in the liver and spleen was reduced, when nanoparticles were coated with 1% polysorbate 80. In the brain a significant increase in tacrine concentration was observed in the case of poly(n-butylcyanoacrylate) nanoparticles coated with 1% polysorbate 80 compared to the uncoated nanoparticles and the free drug. In conclusion, the present study demonstrates that the brain concentration of intravenously injected tacrine can be enhanced by binding to poly(n-butylcyanoacrylate) nanoparticles, coated with 1% the nonionic surfactant polysorbate 80.
Keywords: Brain targeting; Blood–brain barrier; Tacrine; Nanoparticles; Poly(n-butylcyanoacrylate);

Preparation, characterization and in vitro cytotoxicity of indomethacin-loaded PLLA/PLGA microparticles using supercritical CO2 technique by Yunqing Kang; Jiang Wu; Guangfu Yin; Zhongbing Huang; Yadong Yao; Xiaoming Liao; Aizheng Chen; Ximing Pu; Li Liao (85-97).
In this work, indomethacin-loaded poly(l-lactic acid)/poly(lactide-co-glycolide) (IDMC-PLLA/PLGA) microparticles were prepared using solution-enhanced dispersion by supercritical fluids (SEDS) technique in an effort to obtain alternative IDMC formulation for drug delivery system. Surface morphology, particle size and particle size distribution, drug encapsulation efficiency, drug release kinetics, in vitro cytotoxicity and the cellular uptake of drug-loaded microparticles were investigated. The drug-loaded microparticles exhibited sphere-like shape and small particle size with narrow particle size distribution. IDMC was amorphously dispersed within the PLLA/PLGA matrix after the SEDS process. In vitro release studies revealed that the drug-loaded microparticles substantially enhanced the dissolution rate of IDMC compared to the free IDMC, and demonstrated a biphasic drug release profile. In vitro cytotoxicity assays indicated that drug-loaded microparticles possessed longer sustained inhibition activity on proliferation of the non-small-cell lung cancer A549 cell lines than did free IDMC. Fluorescence microscopy and transmission electron microscopy identified the phagocytosis of drug-loaded microparticles into the A549 cells and characteristic morphology of cell apoptosis such as the nuclear aberrations, condensation of chromatin, and swelling damage in mitochondria. These results collectively suggested that IDMC-PLLA/PLGA microparticles prepared using SEDS would have potentials in anti-tumor applications as a controlled drug release dosage form without harmful organic solvent residue.
Keywords: Supercritical CO2; Indomethacin; Poly(l-lactic acid); Poly(lactide-co-glycolide); Microparticles; Drug delivery system;

Co-encapsulation of an antigen and CpG oligonucleotides into PLGA microparticles by TROMS technology by Beatriz San Román; Juan M. Irache; Sara Gómez; Nicolas Tsapis; Carlos Gamazo; María Socorro Espuelas (98-108).
It seems well established that CpG oligonucleotide Th1-biased adjuvant activity can be improved when closely associated with a variety of antigens in, for example, microparticles. In this context, we prepared 1-μm near non-charged poly(lactic-co-glycolic) acid (PLGA) 502 and PLGA 756 microparticles that loaded with high-efficiency antigen (50% ovalbumin (OVA), approximately) into their matrix and CpG–chitosan complexes (near to 20%) onto their surface maintaining OVA and CpG integrity intact. In the intradermal immunization studies, whereas OVA microencapsulated into PLGA 756 alone induced a strong humoral immune response assisted by a very clear Th1 bias (IgG2a/IgG1 = 0.88) that was decreased by CpG co-delivery (IgG2a/IgG1 = 0.55), the co-encapsulation of CpG with OVA in PLGA 502 particles significantly improved the antibody response and isotype shifting (IgG2a/IgG1 = 0.73) in comparison with mice immunized with OVA-loaded PLGA 502 (IgG2a/IgG1 = 0). This improvement was not correlated with the cellular immune response where the effect of co-encapsulated CpG was rather negative (2030 and 335 pg/mL IFN-γ for OVA PLGA 502 and OVA CpG PLGA 502, respectively). These results underscore the critical role of polymer nature and microparticle characteristics to show the benefits of co-encapsulating CpG motifs in close proximity with an antigen.
Keywords: Microparticles; Antigen; CpG sequences; Th1/Th2 immune response; Poly(lactic-co-glycolic) acid;

Preparation of amorphous cefuroxime axetil nanoparticles by sonoprecipitation for enhancement of bioavailability by Ravindra S. Dhumal; Shailesh V. Biradar; Shigeo Yamamura; Anant R. Paradkar; Peter York (109-115).
The aim of the present work was to prepare amorphous discreet nanoparticles by sonoprecipitation method for enhancing oral bioavailability of cefuroxime axetil (CA), a poorly water-soluble drug. CA nanoparticles (SONO-CA) were prepared by sonoprecipitation and compared with particles obtained by precipitation without sonication (PPT-CA) and amorphous CA obtained by spray drying. Spray drying present broad particle size distribution (PSD) with mean particle size of 10 μm and low percent yield, whereas, precipitation without sonication resulted in large amorphous aggregates with broad PSD. During sonoprecipitation, particle size and yield improve with an increase in the amplitude of sonication and lowering the operation temperature due to instantaneous supersaturation and nucleation. The overall symmetry and purity of CA molecule was maintained as confirmed by FTIR and HPLC, respectively. All the three methods resulted in the formation of amorphous CA with only sonoprecipitation resulting in uniform sized nanoparticles. Sonoprecipitated CA nanoparticles showed enhanced dissolution rate and oral bioavailability in Wistar rat due to an increased solubility attributed to combination of effects like amorphization and nanonization with increased surface area and reduced diffusion pathway.
Keywords: Cefuroxime axetil; Sonoprecipitation; Amorphous; Nanoparticles; Bioavailability;

Supramolecular organization and release properties of phospholipid-hyaluronan microparticles encapsulating dexamethasone by Carolina Gómez-Gaete; Nicolas Tsapis; Lídia Silva; Claudie Bourgaux; Madeleine Besnard; Amélie Bochot; Elias Fattal (116-126).
We describe the supramolecular organization of hybrid microparticles encapsulating dexamethasone (DXM) prepared by spray drying 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and hyaluronic acid (HA). The effect of DXM concentration on size distribution and encapsulation efficacy was evaluated as a function of HA concentration. In the absence of HA, DXM leads to a strong particle aggregation, whereas in the presence of HA, the aggregation is practically suppressed. DXM percentage of encapsulation is high (95 ± 6%), independently of composition. Drug-excipient interactions were analyzed by differential scanning calorimetry (DSC) and X-ray diffraction. DSC demonstrates that only a small fraction of DXM interacts with DPPC, whereas X-ray diffraction does not detect this interaction. Finally, in vitro release studies show that HA does not influence DXM release kinetics. In all cases, a burst release of DXM is observed during the first hour. Under sink conditions, powder concentration in the release medium governs the extent of the burst. Under non sink conditions, DXM release is mostly governed by DXM solubility in the release medium. In the dry microparticles, DXM is probably mostly in amorphous domains within the DPPC-HA matrix. Upon hydration, the majority of the drug is released and only a small amount of DXM interacts with DPPC.
Keywords: Microparticles; Spray drying; Dexamethasone; Hyaluronic acid; DPPC;

Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres by Alexandra Giteau; Marie-Claire Venier-Julienne; Stéphane Marchal; Jean-Luc Courthaudon; Michèle Sergent; Claudia Montero-Menei; Jean-Michel Verdier; Jean-Pierre Benoit (127-136).
Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, α-chymotrypsin, peroxidase and β-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.
Keywords: Protein precipitation; Protein stability; Microspheres; Poly(lactic-co-glycolic acid) (PLGA); Solid-in-oil-in-water (s/o/w) encapsulation procedure;

Poly(d,l-lactic-co-glycolic acid) (PLGA) microparticles encapsulating therapeutic proteins were prepared under a water-free formulation condition. Bovine serum albumin (BSA) and recombinant human growth hormone (rhGH) were homogeneously solubilized as nano-scale complexes in methylene chloride phase by using polyethylene glycol (PEG) as a complex-forming agent. The organic phase containing dissolved PLGA and PEG/protein complexes was directly spray dried to obtain PLGA microparticles encapsulating proteins. They exhibited sustained release profiles of BSA and rhGH up to 30 days with reduced initial bursts. The released protein molecules from the microparticles maintained structural integrity without aggregation, suggesting that the current single-step protein microencapsulation method without using water could be potentially applied for sustained delivery of a wide range of therapeutic protein drugs that are not soluble in organic solvents.
Keywords: Poly(d,l-lactic-co-glycolic acid) (PLGA); Microparticles; Protein; Spray drying; Sustained release;

Three in vitro methodologies were evaluated as models for the analysis of drug release from controlled release (CR) microparticulates for inhalation. USP Apparatus 2 (dissolution model), USP Apparatus 4 (flow through model) and a modified Franz cell (diffusion model), were investigated using identical sink volumes and temperatures (1000 ml and 37 °C). Microparticulates containing DSCG and different percentages of PVA (0%, 30%, 50%, 70% and 90%) were used as model CR formulations. Evaluation of the release profiles of DSCG from the modified PVA formulations, suggested that all data fitted a Weibull distribution model with R 2  ⩾ 0.942. Statistical analysis of the t d (time for 63.2% drug release) indicated that all methodologies could distinguish between microparticles that did or did not contain PVA (Students t-test, p  < 0.05). However, only the diffusion model could differentiate between samples containing different PVA percentages. Similar results were observed when analysing the data using similarity and difference factors. Furthermore, analysis of the release kinetic profiles for all samples suggested the data fitted the Higuchi diffusion model (R 2  ⩾ 0.862 for the diffusion methodology data set). Due to the relatively low water content in the respiratory tract and the lack of differentiation between formulations for USP Apparatus 2 and 4, it is concluded that the diffusion model is more applicable for the evaluation of CR inhalation medicines.
Keywords: Inhalation; In vitro analysis; Controlled release; DPI; Dissolution apparatus; Flow trough cell; Modified Franz cell;

Liposomal vasoactive intestinal peptide for lung application: Protection from proteolytic degradation by Brigitte Stark; Fritz Andreae; Wilhelm Mosgoeller; Michael Edetsberger; Erwin Gaubitzer; Gottfried Koehler; Ruth Prassl (153-164).
Inhalative administration of vasoactive intestinal peptide (VIP) is a promising approach for the treatment of severe lung diseases. However, the clinical use of VIP is limited by the fact that the peptide is prone to rapid degradation mechanisms and proteolytic digestion. Accordingly, VIP exhibits a very short period of activity in the lung. To overcome this problem, we have designed a liposomal drug delivery system for VIP and characterized it in terms of its potential to protect VIP from enzymatic cleavage. The proteolytic conditions of the lung, the target site of aerosolic administered VIP, were mimicked by bronchoalveolar lavage fluid (BALF), a lung surfactant solution, obtained by fiberoptic bronchoscopy. Thus, the stability of VIP was assessed by its resistance to enzymatic degradation in BALF, using a combination of high pressure liquid chromatography with mass spectrometry. We found that free VIP was rapidly digested, whereas liposomal-associated VIP remained intact. By fluorescence spectroscopic techniques using fluorescent-labelled VIP we got strong indications that the tight association of VIP with the lipid membrane is only minimally affected upon incubation with BALF. Loading capacity and stability of EtCy3-VIP loaded liposomes were measured by fluorescence fluctuation spectroscopy. Finally, the protective properties of the liposomes were also expressed in the maintained biological activity of the peptide incubated with BALF.
Keywords: Liposomes; VIP; Pulmonary delivery; Proteolytic degradation;

The release behavior of doxorubicin hydrochloride from medicated fibers prepared by emulsion-electrospinning by Xiuling Xu; Xuesi Chen; Ping’an Ma; Xinri Wang; Xiabin Jing (165-170).
The release behavior of a water-soluble small molecule drug from the drug-loaded nanofibers prepared by emulsion-electrospinning was investigated. Doxorubicin hydrochloride (Dox), a water-soluble anticancer agent, was used as the model drug. The laser scanning confocal microscopic images indicated that the drug was well incorporated into amphiphilic poly(ethylene glycol)–poly(l-lactic acid) (PEG–PLA) diblock copolymer nanofibers, forming “core-sheath” structured drug-loaded nanofibers. The drug release behavior of this drug-loaded system showed a three-stage diffusion-controlled mechanism, in which the release rate of the first stage was slower than that of the second stage, but both obeyed Fick’s second law. Based on these results, it is concluded that the Dox-loaded fibers prepared by emulsion-electrospinning represent a reservoir-type delivery system in which the Dox release rate decreases with the increasing Dox content in the fibers.
Keywords: Emulsion; Electrospinning; Nanofiber; Drug delivery; Diffusion;

The formation of a novel interpolyelectrolyte complex (IPEC) between basic butylated methacrylate copolymer and kappa-carrageenan was investigated and the product formed was characterized. Turbidity measurements and elemental analyses pointed to a 1:1 interaction of the repeating units. These results and FT-IR confirmed IPEC formation. Electronic microscopy images, particle size determination by image analysis and N2 (77 K) adsorption measurements were consistent with a porous material. This IPEC formed presented very good flowability and compactibility. Two maxima were observed in the swelling behaviour as a function of pH. The performance of the IPEC as a matrix for controlled release of drugs was evaluated, using ibuprofen as a model drug. Release profiles were properly represented by a mathematical model, which indicates that the system releases ibuprofen in a zero-order manner. These profiles could be controlled by conveniently modifying the proportion of the IPEC in the tablets.
Keywords: Kappa-carrageenan; Basic butylated methacrylate copolymer; Interpolyelectrolyte complexes; Ibuprofen; Matrix systems; Controlled release;

A novel design of one-side coated biodegradable intrascleral implant for the sustained release of triamcinolone acetonide by Yu-Mi Kim; Jeong-Ok Lim; Hong-Kyun Kim; Si-Yeol Kim; Jae-Pil Shin (179-186).
The purpose of this study was to evaluate the efficacy and safety of biodegradable intrascleral implants for the slow release of triamcinolone acetonide (TA). Intrascleral implant (1 mm thick; 3 mm diameter) was made of PLA (poly(d,l-lactide)) containing 6.4 mg of TA with one-side coating of high molecular weight PLA to render unidirectional drug absorption through the sclera. In vitro TA release was evaluated by liquid chromatography–mass spectroscopy for 90 days. In vivo release of TA was measured in aqueous humor, vitreous, and retina–choroid at 1, 2, 4, 8, and 12 weeks after intrascleral implantation in 20 rabbit eyes. Implant toxicity and biocompatibility were evaluated by slit lamp examinations, indirect ophthalmoscopy, intraocular pressure measurements, electroretinography, and histological examinations. In vitro studies demonstrated that the implants released TA in a controlled manner over 90 days. In vivo, TA was detected in aqueous humor until 4 weeks and in retina–choroid until 8 weeks after implantation, but was detected constantly over 12 weeks in vitreous. No significant retinal toxicity was observed. These results suggest that the devised intrascleral implant offers a promising controlled release system for the delivery of TA to the posterior segment of the eye.
Keywords: Triamcinolone acetonide; Poly(d,l-lactide) (PLA); Intrascleral implant; One-side coating;

The objective of this study was to investigate the potential of various formulation strategies to achieve 1-month continuous (improved) release of the novel anti-cancer drug, 2-methoxyestradiol (2-ME), from injectable cylindrical poly(dl-lactide-co-glycolide) (PLGA) implants. PLGA implants were prepared by a solvent extrusion method. PLGA 50:50 (M w  = 51 kDa, end group = lauryl ester) (PLGA–lauryl ester) implants loaded with 3–30 wt% 2-ME exhibited a pronounced lag phase (i.e., corresponding to induction time to polymer mass loss) and triphasic release profile. Incorporation of 5 wt% hydroxypropyl-β-cyclodextrin (HP-β-CD) (∼57% release after 28 days) or Pluronic® F127 (∼42% release after 28 days) in PLGA–lauryl ester implants reduced the lag-phase and improved the drug release moderately over a period of 28 days. The formation and the incorporation of a 2-ME/polyethylene glycol (PEG) 8000 solid dispersion in PLGA–lauryl ester implants further increased drug release (∼21% and 73% release after 1 and 28 days, respectively), attributable to improved drug solubility/dissolution, higher matrix porosity, and accelerated polymer degradation. Blending of PLGA 50:50 (M w  = 24 kDa, end group = COOH) (PLGA–COOH) with the PLGA–lauryl ester also provided moderate enhancement of 2-ME release over a period of 28 days. PLGA–COOH (M w  = 24 kDa) implants with 3–5% w/w pore-forming MgCO3 exhibited the most desirable drug release among all the formulations tested, and, demonstrated 1-month slow and continuous in vitro release of ∼80% 2-ME after a minimal initial burst. Hence, these formulation approaches provide several possible avenues to improve release rates of the hydrophobic drug, 2-ME, from PLGA for future application in regional anti-cancer therapy.
Keywords: 2-Methoxyestradiol; Controlled drug release; Cylindrical PLGA injectable implants; Effect of water soluble additives; Solid dispersion; Polymer blending; Effect of MgCO3;

A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: Rheological, mucoadhesive and in vitro release properties by Laura Mayol; Fabiana Quaglia; Assunta Borzacchiello; Luigi Ambrosio; Maria I. La Rotonda (199-206).
The influence of hyaluronic acid (HA) on the gelation properties of poloxamers blends has been studied with the aim of engineering thermosensitive and mucoadhesive polymeric platforms for drug delivery. The gelation temperature (T gel), viscoelastic properties and mucoadhesive force of the systems were investigated and optimised by means of rheological analyses. Poloxamers micellar diameter was evaluated by Photon Correlation Spectroscopy (PCS). Moreover in order to explore the feasibility of these platforms for drug delivery, the optimised systems were loaded with acyclovir and its release properties studied in vitro.By formulating poloxamers/HA platforms, at specific concentrations, it was possible to obtain a thermoreversible gel with a T gel close to body temperature. The addition of HA did not hamper the self assembling process of poloxamers just delaying the gelation temperature of few Celsius degrees. Furthermore, HA presence led to a strong increase of the poloxamer rheological properties thus indicating possible HA interactions with micelles through secondary bonds, such as hydrogen ones, which reinforce the gel structure. These interactions could also explain PCS results which show, in systems containing HA, aggregates with hydrodynamic diameters much higher than those of poloxamer micelles. Mucoadhesion experiments showed a rheological synergism between poloxamers/HA gels and mucin dispersion which led to a change of the flow behaviour from a quite Newtonian one of the separate solutions to a pseudoplastic one of their mixture. In vitro release experiments indicated that the optimised platform was able to prolong and control acyclovir release for more than 6 h.
Keywords: Poloxamer; Hyaluronic acid; Viscoelastic properties; In situ forming gel; Drug delivery; Thermosensitive gel; Mucoadhesion;

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan for novel effective gene carriers by Theerasak Rojanarata; Maleenart Petchsangsai; Praneet Opanasopit; Tanasait Ngawhirunpat; Uracha Ruktanonchai; Warayuth Sajomsang; Supawan Tantayanon (207-214).
The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM47-Bz42-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM43-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM47-Bz42-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM47-Bz42-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM43-CS/DNA and CS/DNA were formed at a ratio of above 1. TM47-Bz42-CS showed superior transfection efficiency to TM43-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM47-Bz42-CS and TM43-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM47-Bz42-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.
Keywords: Trimethylaminobenzyl chitosan; Gene delivery; Huh7 cells; Transfection efficiency;

Comparative evaluation of interpolyelectrolyte complexes of chitosan with Eudragit ® L100 and Eudragit ® L100-55 as potential carriers for oral controlled drug delivery by Rouslan I. Moustafine; Evgeniya B. Margulis; Liliya F. Sibgatullina; Vera A. Kemenova; Guy Van den Mooter (215-225).
With a view to the application in oral controlled drug delivery systems, the formation of interpolyelectrolyte complexes (IPEC) between chitosan (CS) and Eudragit® L100 (L100) or Eudragit® L100-55 (L100-55) was investigated at pH 6.0, using elementary analysis. The interaction or binding ratio of a unit molecule of CS with Eudragit® L copolymers depends on the molecular weight of CS, and changes from 1:0.85 to 1:1.22 (1.17 <  φ  < 0.82) for L100 and from 1:1.69 to 1:1.26 (0.60 <  φ  < 0.79) for L100-55, respectively. Based on the results of FT-IR, the structure of the IPECs can change substantially as a function of pH (from 5.8 till 7.4). Swelling behavior of physical mixtures (PM) is definitely different, and potential interactions between the two polyelectrolytes were not observed. The release of the model drug diclofenac sodium (DS) was significantly delayed from tablets made up of the IPEC and can be modified by two ways: choosing Eudragit® L copolymer types and/or changing the molecular weight of CS in the IPECs composition.
Keywords: Interpolyelectrolyte complex; Eudragit® L100; Eudragit® L100-55; Chitosan; Infrared spectroscopy; Elementary analysis; Diclofenac sodium; Oral controlled drug delivery;

Formation of drug/excipient complex through ionic interactions has proven to be very effective for both controlled release and taste masking. Unfortunately, the ionic interactions between drugs and small molecule excipients are usually weak, and the stability of the formed complexes can be greatly influenced by solution ionic strength. In this study, we explored to formulate diphenhydramine (DPH), a very bitter tasting drug, using small molecular weight and carboxyl group containing polymers. Studies showed that DPH interacted with α-helical poly(glutamic acid) specifically to produce DPH/poly(glutamic acid) complexes, mostly spherical in shape with a diameter of around 1.0 μm. Other drugs with similar chemical structures as DPH, such as phenylephrine and pseudoephedrine, could not form complexes with poly(glutamic acid) or other polymers under the same conditions. Although DPH in DPH/poly(glutamic acid) complexes existed amorphously, it showed increased stability.In vitro studies using electronic tongue demonstrated that poly(glutamic acid) might be as effective as sucralose for DPH bitter taste blocking. In addition, DPH/poly(glutamic acid) complexes were not stable in neutral or weak acidic (pH > 5) environments and dissolved rapidly and completely. Therefore, DPH/poly(glutamic acid) complex may serve as a new formulation for taste masking and controlled DPH release in gastrointestinal tract. This is the first report that small molecule drugs can interact with peptides of specific secondary structures to form stable complexes. In addition to greatly expanded ion-pairing excipient pool, application of peptides in drug formulation may also solve the selectivity and stability problems faced by current small molecule excipients.
Keywords: Peptide; Formulation; Nanoparticles; Controlled release; pH-sensitivity; Taste masking;

Differential stripping demonstrates a significant reduction of the hair follicle reservoir in vitro compared to in vivo by Alexa Patzelt; Heike Richter; Rolf Buettemeyer; Hans Joachim Roewert Huber; Ulrike Blume-Peytavi; Wolfram Sterry; Juergen Lademann (234-238).
Penetration studies are commonly performed on in vitro models, presumably due to a lack of non-invasive in vivo methods. To date, it is not clear whether in vitro models are suitable to reflect the in vivo conditions for percutaneous penetration. Apart from inter and intraspecies skin differences, the excision of a skin sample may influence the penetration rate inter alia as a result of the contraction of the elastic fibres in the skin during excision. Therefore, the aim of the present study was to investigate the follicular reservoir of the hair follicles of human skin in vivo and in vitro utilizing the method of differential stripping. The results obtained revealed a significantly reduced follicular reservoir in vitro, which was only 9.5 ± 10.6% of the in vivo reservoir. These results are important for the interpretation of earlier and future penetration investigations. It can thus be assumed that excised skin models are suitable for penetration studies only to a limited extent, as follicular penetration is greatly diminished due to the contraction of the elastic fibres of the skin.
Keywords: Follicle penetration; In vivo; In vitro skin model; Hair follicle reservoir; Elastic fibres;

Modulation of Dihydroavenanthramide D release and skin penetration by 1,2-alkanediols by Sandra Heuschkel; Johannes Wohlrab; Gerhard Schmaus; Reinhard H.H. Neubert (239-247).
If a semisolid vehicle does not allow for the sufficient penetration of the incorporated drug, the addition of enhancers, e.g. glycols, is an option. Propylene glycol is most frequently applied in dermal products. Other 1,2-alkanediols like pentylene glycol were found to exhibit moisturizing effects and good anti-microbial activity. In the present study, the influence of propylene glycol and mainly butylene glycol (BuG) and pentylene glycol (PeG) on release and skin penetration of Dihydroavenanthramide D (DHAvD) was investigated. DHAvD release increased twice up to fourfold within 30 min if 2% of a mixture of BuG and PeG was added to a lipophilic as well as to a hydrophilic cream. Incorporation of single 1,2-alkanediols into the hydrophilic cream resulted in a linear slope of the released DHAvD amount with increasing chain length of the glycol. Trends found in the release model were also reflected in penetration studies on full thickness human breast skin using Franz diffusion cells. Here, the hydrophilic cream containing the BuG/PeG mixture was compared to the glycol-free reference. Already within 30 min the amount that penetrated into the viable skin layers doubled using the glycol-containing vehicle. After 300 min 12% of the applied dose was detected in the viable epidermis and dermis following application of the pure cream compared to 41% from the improved formulation. Dermal availability was further enhanced by administration of a polymer-stabilized hydrodispersion gel which also contained the glycol mixture. Due to their favorable biopharmaceutical and technological properties, longer chain 1,2-alkanediols represent a valuable class of ingredients for dermal products.
Keywords: Pentylene glycol; Butylene glycol; Skin penetration; Release; Enhancer; Dihydroavenanthramide D;

The influence of cationic lipid type on in-vitro release kinetic profiles of antisense oligonucleotide from cationic nanoemulsions by Tal Hagigit; Taher Nassar; Francine Behar-Cohen; Gregory Lambert; Simon Benita (248-259).
Novel formulations of cationic nanoemulsions based on three different lipids were developed to strengthen the attraction of the polyanionic oligonucleotide (ODN) macromolecules to the cationic moieties on the oil nanodroplets. These formulations were developed to prolong the release of the ODN from the nanoemulsion under appropriate physiological dilutions as encountered in the eye following topical application. Increasing the concentration of the new cationic lipid exhibiting two cationic amine groups (AOA) in the emulsion from 0.05% to 0.4% did not alter markedly the particle size or zeta potential value of the blank cationic nanoemulsion. The extent of ODN association did not vary significantly when the initial concentration of ODN remained constant at 10 μM irrespective of the cationic lipid nature. However, the zeta potential value dropped consistently with the low concentrations of 0.05% and 0.1% of AOA in the emulsions suggesting that an electrostatic attraction occurred between the cationic lipids and the polyanionic ODN molecules at the o/w interface. Only the nanoemulsion prepared with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (DOTAP) remained physically stable over time. DOTAP cationic lipid nanoemulsion was the most efficient formulation capable of retaining the ODN despite the high dilution of 1:100 with simulated tear solution (STS). Less than 10% of the ODN was exchanged in contrast to 40–50% with the other cationic nanoemulsions. The in-vitro release kinetic behavior of ODN exchange with physiological anions present in the STS appears to be complex and difficult to characterize using mathematical fitting model equations. Further pharmacokinetic studies are needed to verify our kinetic assumptions and confirm the in-vitro ODN release profile from DOTAP cationic nanoemulsions.
Keywords: Cationic; Nanoemulsions; AMD; Oligonucleotide; Stability; In-vitro release; Kinetic profile; Ion-exchange process;

In the present study, viscous solutions of cysteamine hydrochloride (CH) were prepared by using 0.5%, 1.0%, 1.5% or 3.0% of hydroxypropylmethylcellulose (HPMC) and were evaluated for their in-vitro characteristics and stability. Osmolalities, pH and viscosity of the formulations were determined. The influence of benzalkonium chloride and autoclave sterilization on solution characteristics was also investigated. For stability assessment, the viscous solutions were stored at +4 and +25 °C over 12 months. In-vitro characteristics and CH contents of the stored solutions were monitored. Irritation tests for the formulations were evaluated on rabbit eyes. Dialysis sac technique was used to perform in vitro release study of the solutions containing 1.0% and 1.5% HPMC. All of the viscous solutions tested showed non-newtonian (dilatant) flow behavior. Osmolality values were ranked between 351.2 ± 6.2 and 355.1 ± 7.9 mOsm kg−1, and pH values were between 3.97 ± 0.1 and 3.98 ± 0.2 for all the solutions. Furthermore, no significant changes in dilatant behavior, osmolality or pH values of the pure HPMC solutions were observed. After addition of the excipients or CH-excipients, increased viscosity values were noted in these formulations. Neither benzalkonium chloride nor autoclave sterilization had any influence on viscosity, pH or osmolality values of the solution containing 1.5% HPMC. Stability studies showed that a faster decrease in the concentration of CH was observed in the formulations stored at 25 °C compared to those kept at 4 °C; no changes were determined in osmolality values of the solutions at all storage conditions. Increased pH and decreased viscosity values were noted in HPMC solutions containing CH and excipients, while no changes in these values were observed for pure HPMC solutions kept at 4 and 25 °C. In vitro release tests revealed that 81.2% and 85.3% of CH were released from the viscous solutions containing 1.5% and 1% HPMC, respectively, in 8 h. No irritation was observed when the viscous solutions were tested on rabbit and human eyes.
Keywords: Corneal cystinosis; Cysteamine hydrochloride; HPMC viscous solutions; In-vitro characterization; Stability;

Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells by A.M.M. Sadeghi; F.A. Dorkoosh; M.R. Avadi; M. Weinhold; A. Bayat; F. Delie; R. Gurny; B. Larijani; M. Rafiee-Tehrani; H.E. Junginger (270-278).
In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50 ± 5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC > DMEC > DEMC = TEC > chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC > DMEC > DEMC > TEC > chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.
Keywords: Caco-2 cells; Insulin nanoparticles; Trimethyl chitosan; Dimethylethyl chitosan; Diethylmethyl chitosan; Triethyl chitosan; Uptake; Transcellular transport; Paracellular transport; Mass balance studies;

Pharmaceutical excipients inhibit cytochrome P450 activity in cell free systems and after systemic administration by Xiuhua Ren; Xinliang Mao; Luqin Si; Lei Cao; Hui Xiong; Jun Qiu; Aaron D. Schimmer; Gao Li (279-288).
Excipients are largely used as inert vehicles in formulation. Recent studies indicated that some excipients could affect drug transport and disposition. But the effects of most excipients on drug metabolism are yet to be unveiled. To evaluate the actual action of pharmaceutical excipients in biotransformation, we examined the effects of 22 common excipients on cytochrome P450 3A4, the main CYP in intestinal and liver, using midazolam as the probe. The results showed that 15 of 22 (68.2%) tested excipients could inhibit the activity of CYP3A4 more than 50% in vitro, particularly the surfactants and polymers. To further understand these effects in vivo, five excipients were selected to study the effects on CYP3A4 in rats through the pharmacokinetics of midazolam and its primary metabolite 1′-hydroxymidazolam. In in vivo studies, most selected excipients significantly inhibited the activity of CYP3A4 by increasing the midazolam AUC0–∞ and decreasing the midazolam CL/F as well as decreasing the ratio of AUC0–∞ (1′-hydroxymidazolam)/AUC0–∞ (midazolam). For examples, single and multiple dose administration of PEG400 increased intraduodenally dosed midazolam AUC0–∞ to 1.78- and 1.51-fold, decreased midazolam CL/F from 8.86 to 5.25 and 6.28 L/h/kg and decreased the ratio of AUC0–∞ (1′-hydroxymidazolam)/AUC0–∞ (midazolam) from 1.14 to 0.34 and 0.39, respectively (p  < 0.05). This study indicated that some excipients could change drug metabolism through the effects on cytochrome P450 activity, such as CYP3A4, and thus this kind of inhibition should be taken into consideration in drug formulation and administration.
Keywords: Cytochrome P450; Excipient; Midazolam; 1′-Hydroxymidazolam; Pharmacokinetics;

This study attempted to characterize the influence of core and coating formulations on the release profiles to establish in vitro/in vivo correlations of pulsatile pattern for a pulsatile drug delivery system activated by membrane rupture based on three core tablet formulations (A-core: HPMC 50+4000 cps, B-core: E10M, and C-core: K100M) coated with various thicknesses of a semipermeable ethylcellulose membrane plasticized with HPMC 606 (Pharmacoat 606) at different ratios with/without adding various amounts of water to dissolve it in the coating solution. Drug release behaviors were investigated using apparatus II in four media of pH 1.2 solution, pH 6.8 buffer, deionized water, and a NaCl solution rotated at 75, 100, and 150 rpm. Pilot studies of the in vivo pharmacokinetics were conducted as well for comparison with the in vitro results. Results demonstrated that drug release from the three kinds of core tablets in deionized water increased with an increasing stirring rate, and decreased with an increasing viscosity grade of HPMC used in the core formulations. A significant promotion of drug release from core tablets was observed for the three levels of NaCl media in comparison with that in deionized water. Results further demonstrated that a slightly slower release rate in pH 1.2 solution and a faster release rate in pH 6.8 buffer than that in deionized water were observed for the A-core and B-core tablets, with the former being slower than the latter. However, similar release rates in the three kinds of media were observed for C-core tablets, but they were slower than those for the A- and B-core tablets. Dissolution of coated tablets showed that the controlling membrane was ruptured by osmotic pressure and swelling which activated drug release with a lag time. The lag time was not influenced by the pH value of the release medium or by the rotation speeds. The lag time increased with a higher coating level, but decreased with the addition of the hydrophilic plasticizer, Pharmacoat 606, and of the water amount in the coating solution. The lag time also increased with a higher concentration of NaCl in the medium. The release rate after the lag time was determined by the extent of retardation of gelation of HPMC in the core tablet based on the ionic strength of the medium. Results of the three pilot crossover studies for the exemplified pulsatile systems indicated that the lag time for the in vivo plasma profile was well correlated with that determined from the in vitro release profile in pH 1.2 solution and the in vivo release rate was better reflected by that performed in pH 6.8 buffer.
Keywords: Ethylcellulose; Hydroxypropylmethylcellulose (HPMC); Pulsatile Release; Membrane Rupture; Lag time;

In-vitro and in-vivo evaluation of enteric-coated starch-based pellets prepared via extrusion/spheronisation by Aleksandra Dukić-Ott; Thomas De Beer; Jean Paul Remon; Willy Baeyens; Paul Foreman; Chris Vervaet (302-312).
Pellet cores containing modified starch (high-amylose, crystalline and resistant starch) as the main excipient were enteric-coated with an Eudragit® L30 D-55 based dispersion. The polymer weight gain was from 15% to 30% (w/w). Pellet cores were prepared using piroxicam (2.5% w/w, poor water solubility) and anhydrous theophylline (2.5% and 25% w/w, coarse and micronised powder, medium water solubility) as model drugs. Next to the water solubility, particle size and concentration of the model drugs, the influence of sorbitol (0% and 10%, w/w) and drying method (oven and fluid-bed) on pellet yield, size (Feret mean diameter), sphericity (aspect ratio, AR and two-dimensional shape factor, e R), friability, surface morphology and drug release were evaluated. Binder (HPMC) and granulation liquid (water) concentration were optimised to obtain maximum yield (size fraction between 900 and 1400 μm) and acceptable sphericity (AR < 1.2). Pellet friability was <0.01% for all formulations, while the mean pellet diameter was lower for pellets with sorbitol and the ones dried in an oven. Mercury intrusion porosimetry combined with scanning electron microscopy revealed an influence of drying method and sorbitol level on the surface structure: the surface of fluid-bed dried pellets without sorbitol and with 2.5% of model drug was cracked, which correlated with a Hg-intrusion peak at the 6–80 μm pore size range. Due to improved mechanical properties of the wet mass, sorbitol addition smoothened the pellets as the main peak of Hg-intrusion shifted to a smaller pore size range. Using a higher drug concentration and micronised theophylline shifted the main peak of Hg-intrusion further towards the smaller pore size range. Oven-dried pellets showed no Hg-intrusion and no cracks were observed. When applying the highest coating thickness (30% weight gain), all theophylline pellet formulations were successfully coated (<10% drug release after 2 h in acid dissolution medium), while pellets with the lowest coating thickness (15% weight gain) released from 5% to about 30% theophylline. The extent of drug release depended on the pellet composition and drying method as these factors determined the surface properties. Piroxicam release in acid medium was less than 1% irrespective of the surface characteristics, due to its poor water solubility. In basic medium (phosphate buffer, pH 6.8) all pellets released the drug in less than 45 min. The bioavailability of coated and uncoated piroxicam pellets was determined after oral administration to six dogs. Values of AUC0→72h, C max and t max after oral administration of piroxicam pellets to dogs were not significantly different from the values obtained for immediate release capsules (P  > 0.05).
Keywords: Extrusion/spheronisation; Pellets; Starch; Enteric-coating; Bioavailability;

Mechanistic understanding of time-dependent oral absorption based on gastric motor activity in humans by Kazutaka Higaki; Sally Y. Choe; Raimar Löbenberg; Lynda S. Welage; Gordon L. Amidon (313-325).
The relationship of gastric motor activity and gastric emptying of 0.7 mm caffeine pellets with their absorption was investigated in the fed state in healthy human subjects by simultaneous monitoring of antral motility and plasma concentrations. A kinetic model for gastric emptying-dependent absorption yielded multiple phases of gastric emptying and rate constants (k g) with large inter-individual differences and large variability in onset of gastric emptying (50–175 min). The model suggests that 50% of the dose is emptied in 1–2 h and over 90% emptied by 3.5 h following dosing, in all subjects. The maximum values of k g (k g(max)) were much greater than those reported for emptying of liquids in the fasted state and were comparable to k g values in the late Phase II/III of the migrating motor complex (MMC). The model described the observed irregular absorption rate–time and plasma concentration–time profiles adequately but not in detail. The model was more successful at simulating double-peak phenomena in absorption rate profiles and onset of caffeine absorption. The results suggest that gastric emptying regulates drug absorption of small particles in the fed state. Further, estimates of k a derived using the time-dependent absorption model were closer to the intrinsic absorption rate constant for caffeine.
Keywords: Time-dependent absorption; Gastric motor activity; Gastric emptying; Fed state; Absorption rate constant; Absorption rate; Plasma concentration–time profile; Caffeine;

A comparative investigation of the interplay between starch botanical source, micromeritics and their functionality in formulated paediatric paracetamol granules for reconstitution was conducted using starches extracted from five local yam (Dioscorea spp.) species. Significant differences were observed in the physicochemical properties of the different starches studied (p  < 0.05). The observed differences in paracetamol dissolution correlated well with the physicochemical properties of the starches. Granules formulated with Chinese yam and Bitter yam starches showed the fastest rate of paracetamol dissolution with T 80 of 2.2 and 2.6 min, respectively, at 2.5%w/w, and 3.75 and 4.00 min, respectively, at 10% w/w binder concentrations. Those formulated with Round leaf yellow yam starch had the slowest dissolution rate with T 80 of 4.5 and 8.75 min, respectively, at 2.5% and 10% binder concentrations. The results generally indicate a significant dependence of the rate of paracetamol dissolution from granules on starch surface tension, viscosity, swelling power and water binding capacity, particle size distribution, specific surface and porosity. These, in turn, were found to be starch botanic source-dependent suggesting careful consideration of botanic source when substituting one starch product for another in granule formulation.
Keywords: Yam; Starch; Excipient; Granules for reconstitution; Dissolution; Physiochemical properties; Moisture sorption; Particle size;

Formation of shellac succinate having improved enteric film properties through dry media reaction by Sontaya Limmatvapirat; Danuch Panchapornpon; Chutima Limmatvapirat; Jurairat Nunthanid; Manee Luangtana-Anan; Satit Puttipipatkhachorn (335-344).
The aim of this study was to improve enteric properties of shellac by the formation of succinate derivative through dry media reaction. Shellac and succinic anhydride were mixed and then co-ground by planetary ball mill. The ground mixture was then activated by heating for various times and washed for removal of excess succinic anhydride. The ground mixtures and the heat-activated mixtures were characterized by physical and chemical tests, including acid value, FTIR spectroscopy, 1H NMR and 13C NMR spectroscopy, thermal analysis and film properties. The results demonstrated that acid values of heat-activated shellac mixtures increased with the increase of annealing time, suggesting the presence of carboxylic acid moieties of succinate at shellac molecules. The results were in good agreement with the DSC thermograms. The melting peak of shellac disappeared after heating, while melting peak of succinic anhydride gradually decreased, suggesting the utilization of succinic anhydride for the esterification. The shellac succinate formation was also confirmed by 1H NMR and 13C NMR spectroscopies. Film prepared from shellac succinate showed improved solubility, especially at the pH of small intestine (5.8–6.7), as compared to native shellac. The shellac succinate film also demonstrated better mechanical property, in terms of increased flexibility. In conclusion, solid-state formation of shellac succinate ester, which had improved enteric properties, was easily accomplished under the concept of “green approach”.
Keywords: Shellac; Shellac succinate; Enteric polymer; Co-grinding; Mechanochemistry;

Influence of crystal hydration on the mechanical properties of sodium naproxen by Etienne Joiris; Piera Di Martino; Ledjan Malaj; Roberta Censi; Christine Barthélémy; Pascal Odou (345-356).
The aim of this work is to establish a correlation between water uptake by anhydrous sodium naproxen (ASN) at two different relative humidities and modifications in tableting and densification behaviour under hydration. Water uptake was evaluated at different relative humidities. Models for the hydration kinetics of ASN at 55% and 86%, corresponding to the formation of the dihydrated and tetrahydrated forms, respectively, were evaluated assuming Eyring’s dependence on temperature. Tabletability, compressibility, compactibility, and densification behaviour were determined using an instrumented single punch tablet machine. Kinetic data are consistent with a model where water molecules enter the crystal preferentially along hydrophilic tunnels existing in the crystal structure and corresponding to the propionate side chain. Water inclusion perturbs the crystallographic structure, causing slight structural changes according to the amount and associated to an increase in entropy. The interposition of water molecules between sodium naproxen molecules weakens intermolecular bonds, and these sites can behave like sliding planes under compression. Such structural changes may explain the improved compression behaviour and modified densification propensity mechanism. Kinetic data describing the water hydration mechanism of ASN explain in an original way the improved tableting and densification properties under hydration.
Keywords: Sodium naproxen; Hydration; Kinetics; Tableting; Densification;

Material and tablet properties of pregelatinized (thermally modified) Dioscorea starches by Oluwatoyin A. Odeku; Wolfgang Schmid; Katharina M. Picker-Freyer (357-371).
The material and tablet formation properties of pregelatinized (thermally modified) forms of four Dioscorea starches have been investigated. Dioscorea starches were pregelatinized followed by either oven drying (PS) or freeze drying (FD) and used as excipient in direct compression. The physicochemical, morphological and material properties of the pregelatinized starches have been investigated. The tablet formation properties were assessed using the 3-D modeling parameters, the Heckel equation and the force–displacement profiles. The tablet properties were evaluated using the elastic recovery, compactibility plots and the disintegration test. The results indicate that pregelatinization improved the compressibility and flowability of the Dioscorea starches. The high bulk and tap densities of PS coupled with their good flowability offer a unique possibility of the starches being used as filler in capsule formulations. The modified starches generally showed differences in their time and pressure dependent deformation behaviour. PS exhibited higher elasticity during tableting. FD Chinese and FD Bitter showed higher plasticity and low fast elastic deformation than the PS forms of the starches indicating that the FD starches undergo the highest plastic deformation. However, FD starches generally showed higher compactibility compared to the PS forms of the Dioscorea starches. While FD White and FD Water showed fast disintegration time and high compactibility, FD Chinese and FD Bitter were non-disintegrating and showed high compactibility. The high compactibility observed with the FD starches appears to be as a result of material change occurring during tableting probably due to the effect of temperature or pressure or a combination of both factors. Thus, FD White and FD Water starches could be useful when high crushing force and fast disintegration are of concern while FD Chinese and FD Bitter, which were non-disintegrating, could find application as excipients for controlled drug delivery.
Keywords: Pregelatinized starch; Material properties; Tableting; Dioscorea starches; Yam;

Roll compaction/dry granulation is a widely used technique for granulation. A major drawback is the reduction of tablet tensile strength compared to other granulation methods. The purpose of this study was to determine the reasons for the partial loss in compactibility.Microcrystalline cellulose of different particle sizes was roll-compacted/dry-granulated. The granules were sieved to obtain two sieve cuts and then compressed into tablets. The particle-size distribution within the sieve cut was determined using image analysis. The specific surface area of sieve cut was obtained by nitrogen adsorption. Heckel equation was used to determine the change in compressibility.The work-hardening phenomenon was found to be caused by a combination of particle-size enlargement and hardening of material. Although particle size of granules was equal, the use of smaller particles as raw material resulted in tablets with higher tensile strength due to higher specific surface area.Both work-hardening and particle-size enlargement cause the partial loss in compactibility. The reduction in tensile strength could be compensated by producing smaller granules or using raw materials with small particle sizes.
Keywords: Roll compaction/dry granulation; Tablets; Dry binder; Tensile strength; Particle size; Microcrystalline cellulose; Work Hardening; Compression; Compactibility;

Comparison of physical and inhalation properties of spray-dried and micronized terbutaline sulphate by Thanh Huong Hoang Thi; Florence Danède; Marc Descamps; Marie-Pierre Flament (380-388).
Terbutaline sulphate particles, for use in dry powder inhaler formulations, were prepared by spray-drying, using a Büchi 190 mini spray dryer. Spray-drying conditions were chosen to allow the production of spray-dried terbutaline sulphate with a size similar to micronized terbutaline sulphate, that is to say about 2.9 μm of volume mean diameter. The physical properties and in vitro inhalation behaviour of micronized and spray-dried terbutaline sulphate were compared. X-ray diffraction, DSC, SEM and laser size analysis were investigated. Spray-dying produced spherically shaped particles with amorphous structure.After blending with different lactoses, adhesion and aerodynamic properties were investigated. Evaluation of adhesion was carried out with a mechanical sieve and an Alpine air-jet sieve. The adhesion of terbutaline sulphate on the lactoses tested was lower in the case of the spray-dried drug. Aerodynamic evaluation of fine particle dose and emitted dose was conducted using a twin stage impactor. The emitted doses and the fine particle doses were higher with the spray-dried terbutaline sulphate.The Alpine air-jet sieve assays showed that there was a correlation between drug separation from a carrier by sieving and that obtained from longer in vitro deposition studies. There was a linear relationship between the adhesion characteristics and the fine particle dose.
Keywords: Terbutaline sulphate; Spray-drying; Dry powder inhaler; Physicochemical properties; Adhesion; Aerodynamic behaviour;

Effect of the spraying conditions and nozzle design on the shape and size distribution of particles obtained with supercritical fluid drying by Andréanne Bouchard; Nataša Jovanović; Anne H. de Boer; Ángel Martín; Wim Jiskoot; Daan J.A. Crommelin; Gerard W. Hofland; Geert-Jan Witkamp (389-401).
In the perspective of production of dry therapeutic protein formulations, spray drying of lysozyme (as a model protein) into supercritical carbon dioxide was studied. The effects of the nozzle (i.e., co-current coaxial converging and converging–diverging, and T-mixer impinging) and process conditions (i.e., flow rates, pressure) on the drying of the lysozyme prepared in aqueous solution dried with supercritical carbon dioxide enriched with ethanol were investigated. The particle size distribution, width of particle size distribution and morphology were used to determine the effect of the various parameters assessed. Particles with a median size of ∼1.5, ∼5 or ∼25 μm were produced depending of the nozzle selected. A basic comparative study of the nozzle was done by computational fluid dynamics, but the differences in particle size could not be depicted by these computations. The proportional increase of the flow rates (up to fivefold) caused a decrease in particle size (7- to 12-fold), and doubling the pressure caused a moderate decrease of the size (5–20%). The individual effect of the supercritical carbon dioxide, ethanol and solution streams was explained with a mass transfer model. Changing the ratio between flow rates slightly affected the particle size in various ways because of the swelling and shrinking stages of the drying droplet in supercritical carbon dioxide enriched with ethanol.
Keywords: Atomisation; Lysozyme; Particle size distribution; Mass transfer; Computational fluid dynamics;

The consequences of granulate heterogeneity towards breakage and attrition upon fluid-bed drying by Florentine Nieuwmeyer; Kees van der Voort Maarschalk; Herman Vromans (402-408).
High-shear granulated lactose granulates were dried in a fluid-bed dryer at various conditions. Granules were characterized by water content and size analysis. It is shown that the drying process is very dynamic in terms of growth and breakage phenomena. Granular size heterogeneity, composition and water content determine the granule behavior upon drying. Large granules consist of small primary particles and contain more water than small granules that consist of large primary particles. This differentiates the drying rate and extent of size reduction of the different granule size classes. The results enable a critical evaluation of process control and process monitoring. Understanding of granule behavior and continuous monitoring of the fluid-bed drying process enables process and product optimization.
Keywords: Breakage; Composition; Fluid-bed drying; Granule; Heterogeneity; High- shear; Lactose; Water;

Raman and thermal analysis of indomethacin/PVP solid dispersion enteric microparticles by Adamo Fini; Cristina Cavallari; Francesca Ospitali (409-420).
Indomethacin (IMC) and three types of poly-(vinylpyrrolidone) (PVP 12PF, PVP K30 and PVP K90) were studied in the form of solid dispersion, prepared with the solvent evaporation method, by spectroscopic (Raman, FT-IR, X-ray diffraction), thermal (differential scanning calorimetry, thermogravimetry, hot-stage microscopy), fractal and image analysis. Raman and FT-IR micro-spectroscopy indicated the occurrence of drug/polymer interaction and the presence of an amorphous form of IMC, as also resulting from X-ray diffractometry. Hot-stage microscopy suggested that the interaction between IMC and the polymer occurring on heating of a physical mixture, is common to other acidic compounds and causes a depression of the temperature of the appearance of a molten phase. Co-evaporated particles were coated by spray-congealing process with molten stearic acid for gastroprotection, but also for stabilization of the amorphous structure of the drug: the final particles were spherically shaped. Dissolution tests carried out on the final microparticles showed that the coating with stearic acid prevents IMC release at acidic pH and also protects against recovery of the IMC crystallinity, at least after 9 months of aging: the extent and mode of the release, before and after aging, overlap perfectly. The test revealed a notable improvement of the drug release rate from the solid dispersion at suitable pH, with respect to pure IMC. The comparison of the present solid dispersion with IMC/PVP (surface) solid dispersion obtained by freeze-drying of an aqueous suspension, where IMC maintained its crystalline state, revealed that there was no difference concerning the release rate, but suggested a superior quality of this last process as a mean of improving IMC availability for the easiness of preparation and stability, due to the absence of the amorphous state of the drug, as a possible instability source of the system. Finally, the coating with stearic acid is discussed as a determining process for the practical application of solid dispersions.
Keywords: Indomethacin/poly-(vinylpyrrolidone) co-evaporated particles; Raman and FT-IR micro-spectroscopy; Thermal analysis; Co-evaporated and freeze-dried system comparison;