International Journal of Pharmaceutics (v.250, #1)

The purpose of this study was to develop a technique to enhance the dissolution rate of poorly water-soluble drugs with hydroxypropyl methylcellulose (HPMC) without the use of solvent or heat addition. Three poorly water-soluble drugs, naproxen, nifedipine, and carbamazepine, were studied with low-viscosity HPMC USP Type 2208 (K3LV), HPMC USP Type 2910 (E3LV and E5LV), and methylcellulose. Polymer and drug were dry-blended, compressed into slugs on a tablet press or into ribbons on a roller compactor, and then milled into a granular powder. Dissolution testing of the milled powder was performed on USP Apparatus II, 100 rpm, 900 ml deionized water, 37 °C. Drug distribution vs. particle size was also studied. The compaction processes enhanced drug dissolution relative to drug alone and also relative to corresponding loosely mixed physical mixtures. The roller compaction and slugging methods produced comparable dissolution enhancement. The mechanism for dissolution enhancement is believed to be a microenvironment HPMC surfactant effect facilitated by keeping the HPMC and drug particles in close proximity during drug dissolution. The compaction methods in this study may provide a lower cost, quicker, readily scalable alternative for formulating poorly water-soluble drugs.
Keywords: Hydroxypropyl methylcellulose; Poorly water-soluble; Drug dissolution; Roller compaction;

Artificial neural networks (ANNs) and modeling of powder flow by K. Kachrimanis; V. Karamyan; S. Malamataris (13-23).
Effects of micromeritic properties (bulk, tapped and particle density, particle size and shape) on the flow rate through circular orifices are investigated, for three pharmaceutical excipients (Lactose, Emcompress and Starch) separated in four sieve fractions, and are modeled with the help of artificial neural networks (ANNs). Eight variables were selected as inputs and correlated by applying the Spearman product-moment correlation matrix and the visual component planes of trained Self-Organizing Maps (SOMs). Back-propagation feed-forward ANN with six hidden units in a single hidden layer was selected for modeling experimental data and its predictions were compared with those of the flow equation proposed by . It was found that SOMs are efficient for the identification of co-linearity in the input variables and the ANN is superior to the flow equation since it does not require separate regression for each excipient and its predictive ability is higher. Besides the orifice diameter, most influential and important variable was the difference between tapped and bulk density. From the pruned ANN an approximate non-linear model was extracted, which describes powder flow rate in terms of the four network's input variables of the greatest predictive importance or saliency (difference between tapped and bulk density (x 2), orifice diameter (x 3), circle equivalent particle diameter (x 4) and particle density (x 8)): W=b0+{a1[1+exp(b2x2+b3x3+b4x4+b8x8)]−1+a2[1+exp(b′3χ3+b′8x8)]−1}.
Keywords: Artificial neural networks (ANNs); Powder flow rate; Self organizing maps (SOMs); Pharmaceutical excipients;

Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery by Indrajit Roy; Susmita Mitra; Amarnath Maitra; Subho Mozumdar (25-33).
Calcium phosphate nanoparticles present a unique class of non-viral vectors, which can serve as efficient and alternative DNA carriers for targeted delivery of genes. In this study we report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 80 nm in diameter. The DNA encapsulated inside the nanoparticle is protected from the external DNase environment and could be used safely to transfer the encapsulated DNA under in vitro and in vivo conditions. Moreover, the surface of these nanoparticles could be suitably modified by adsorbing a highly adhesive polymer like polyacrylic acid followed by conjugating the carboxylic groups of the polymer with a ligand such as p-amino-1-thio-β-galactopyranoside using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a coupling agent. We have demonstrated in our studies that these surface modified calcium phosphate nanoparticles can be used in vivo to target genes specifically to the liver.
Keywords: DNA; Calcium phosphate; Non-viral vector;

Rheological behavior and the SPF of sunscreens by L.R Gaspar; P.M.B.G Maia Campos (35-44).
Due to a large variety of sunscreens, it is important to study among other things, the effect of the vehicle on the thickness and uniformity of sunscreen films. In this study, we determined the physical stability of five sunscreens SPF 15 (FA to FG), containing or not PVP/eicosene crosspolymer (PVP/EC), and two different self-emulsifying bases (SEB), and also evaluated the influence of the vehicle in their SPF. In the study of physical stability, formulations were stored at 25, 37 and 45 °C, for 28 days. Viscosity and rheological behavior of the formulations were determined using a Brookfield rheometer. Investigations of the SPF were carried out in a group of 30 volunteers (COLIPA methodology). The FC samples (phosphate-based SEB), with a lower thixotropy, showed statistically higher SPF (13.6) when compared with FB (non-ionic SEB), which presented higher thixotropy and a SPF of 9.84. The FE sample (phosphate-based SEB+PVP/EC) presented the same SPF as the FC, but had a higher thixotropy. The FB formulation (stable with higher thixotropy) showed the lowest SPF while FC (an unstable formulation with lower thixotropy) presented a higher SPF. We concluded that FE was the best formulation showing a higher SPF and stability and the study of rheology can help the development of sunscreens.
Keywords: Sunscreens; Rheology; Thixotropy; SPF; Stability;

Based on the clinical fact that paclitaxel injection (Taxol®) frequently causes hypersensitivity reactions, we prepared an alternative paclitaxel microemulsion with small particle size (17.2 nm). The hypersensitivity evaluation and pharmacokinetic behavior in rats were conducted to assess the new microemulsion. The results showed that the new microemulsion was negative and the placebo Taxol® solution was positive with regard to allergic reactions. In the pharmacokinetic study, five rats were administrated Taxol® or paclitaxel microemulsion. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 h and paclitaxel determined by HPLC. The area under the curve (AUC) was significantly higher in the microemulsion group (34.98 μg ml−1 h) than that in the Taxol® group (21.98 μg ml−1 h). Also, the K 10 was much smaller in the microemulsion group (0.57 h−1) compared with the Taxol® group (1.29 h−1), showing the elimination rate was much slower in the former than in the latter. Compared with Taxol®, the paclitaxel microemulsion caused less toxicity and had a longer circulation time in rats.
Keywords: Paclitaxel; Microemulsion; Taxol; Cremophor EL; Hypersensitivity; Pharmacokinetics;

Sampling and characterization of pharmaceutical powders and granular blends by F.J Muzzio; C.L Goodridge; A Alexander; P Arratia; H Yang; O Sudah; G Mergen (51-64).
We use a variety of experimental results to illustrate issues and challenges involved in the sampling and characterization of pharmaceutical mixtures. Accurate and reliable characterization of granular mixtures is hindered by both the complexity of granular systems and the lack of validated and reliable sampling technology and techniques. Both sampling tools and sampling protocols are critically important for accurate characterization. Using cohesive and free-flowing powders and four thief probe designs, we reveal a large potential for extremely misleading results as well as severe disturbance of the granular bed. We also discuss results from several experiments designed to test the validity of various sampling protocols by varying parameters such as sampling location and frequency of sampling. These experiments illustrate the importance of effective sampling procedures to achieve the best and most efficient results.
Keywords: Powder blending; Powder sampling; Mixing; Thief sampling;

Formulation of electrically conducting microemulsion-based organogels by Shilpa Kantaria; Gareth D Rees; M.Jayne Lawrence (65-83).
Gelatin-containing, electrically conducting, rigid water-in-oil (w/o) microemulsion-based organogels (MBG), both with and without the presence of a model drug, have been prepared using pharmaceutically acceptable oils and surfactants. As a precursor to MBG formation, preliminary formulation work was carried out investigating the factors affecting the preparation of w/o microemulsions containing large amounts of dispersed aqueous phase. From these studies isopropyl myristate (IPM) was favoured as oil due to its ability to support w/o microemulsion formation over a wide range of compositions. The single most effective surfactant for stabilising the w/o microemulsions was found to be Aerosol-OT (AOT), although synergistic effects on the extent of w/o microemulsion formation were observed upon its combination with a variety of non-ionic surfactants. Upon addition of gelatin to the w/o microemulsion, MBG could be formed when using AOT as stabiliser with most of the oils investigated (with the exception of the medium and large triglyceride oils, Miglyol 812 and soybean oil, respectively) and with a number of AOT/non-ionic surfactant/IPM combinations (both in the presence and absence of model drugs such as sodium salicylate). MBG could not however be formed with non-ionic surfactants alone, or when used in combination with another non-ionic surfactant (regardless of the oil used). This latter observation was found to be not only a result of the inadequate level of water available for hydration of the surfactant head group and any gelatin present but also a consequence of the inability of these systems to form, upon heating, the percolated microstructures necessary to facilitate the supramolecular assembly of gelatin at the macroscopic level, a pre-requisite for MBG formation.
Keywords: Formulation; Microemulsion; Organogel; Phase behaviour; Transdermal drug delivery;

Phenytoin crystals having different types of habits, were prepared by recrystallization from ethanol and acetone solutions under different conditions (cooling rate or crystallization temperature, solvent evaporation and watering-out techniques). Scanning electron microscopy, X-ray powder diffractometry, FT-IR spectrometry and differential scanning calorimetry were used to investigate the physical characteristics of the crystals. The dissolution behavior and compaction properties of various batches of crystals were also studied. It was found that using watering-out technique as a crystallization method, produced thin plate crystals, while the crystals obtained by other methods were needle shape for alcoholic solutions and rhombic for acetone solutions. X-ray diffraction spectra and differential scanning calorimetry studies, did not show any polymorphic change. The dissolution rate of different crystals was lower than that of untreated samples. The compacts of phenytoin crystals produced from alcohol or acetone (especially those made by watering-out method) had higher crushing strengths than untreated phenytoin compacts due to the lower porosity and the lower elastic recovery.
Keywords: Phenytoin; Crystallization; Crystal habit; Crystal shape; Dissolution rate; Compaction;

Measurement of agitation force in dissolution test and mechanical destructive force in disintegration test by Masaharu Kamba; Yasuo Seta; Nao Takeda; Takeshi Hamaura; Akira Kusai; Hisanori Nakane; Kenji Nishimura (99-109).
The purpose of this study was to investigate the effect of the agitation force and mechanical destructive force on the drug dissolution of a tablet in the paddle rotation dissolution test and in the disintegration test. The agitation in the paddle method and the mechanical destructive force in the disintegration test were considered to be conclusive factors for drug dissolution. The dissolution rate of planar-constant-release tablets increased with increasing paddle rotation speed and increased with increasing distance from the center of the vessel bottom. Separately, the fluid resistance (agitation force) in the vessel was measured using a modified paddle method apparatus equipped with a fluid resistance sensor. The fluid resistance was 0.03×10−3 N/(64 mm2) when the paddle rotation speed was 50 rpm at a position 4 mm away from the center. A considerable position-dependent change in agitation force intensity was seen with the fluid resistance sensor. The impulsive force (mechanical destructive force) in the disintegration test apparatus was measured using a modified basket-rack assembly with a strain gauge transducer. The fluid resistance was measured using the basket-rack assembly with a different sensor probe and amplifier. The impulsive force applied by the auxiliary disk was 0.31 N and the fluid resistance at the bottom of the basket-rack assembly was 1.66×10−3 N/(64 mm2).
Keywords: Dissolution test; Disintegration test; Agitation force; Fluid resistance; Destructive force;

Possibility of a patch system as a new oral delivery system by Sudarat Eaimtrakarn; Y.V Rama Prasad; Shivanand P Puthli; Yukako Yoshikawa; Nobuhito Shibata; Kanji Takada (111-117).
A new oral patch system has been designed to increase the residence time of model drugs within the gastrointestinal tract. The system consisted of three layers (1) water-insoluble backing layer (2) drug-carrying adhesive layer composed of a model drug, fluorescein (FL) or fluorescein isothiocyanate-dextran (FD), and gel-forming polymer and (3) pH-sensitive enteric polymer. These three layers system was prepared as 3.0 mm diameter patches. As references, tablet containing FL or FD was prepared. In vitro dissolution studies showed that the mean dissolution time (MDT) of model drugs from patch preparation was 0.739±0.021 h for FL and 0.407±0.021 h for FD, which were longer than from tablet, 0.327±0.008 h for FL and 0.270±0.019 h for FD. The two test preparations were orally administered to beagle dogs in a crossover manner at a FL dose of 30 mg/dog and the measured plasma FL concentrations were used for pharmacokinetic analysis. With FL patch preparation, area under the plasma drug concentration vs. time curve (AUC) was 2.12±0.24 μg h/ml and mean residence time (MRT) was 4.60±0.18 h, which were greater than those of tablet, AUC was 1.52±0.16 μg h/ml and MRT was 3.18±0.09 h, respectively. Oral patch preparation also increased both AUC and MRT of FD, a model macromolecular drug, which was formulated into both patches and tablets and administered to dogs (30 mg/dog). The AUC and MRT of FD from the patch preparation were 1.11±0.13 μg h/ml and 5.58±0.55 h and from tablets were 0.53±0.08 μg h/ml and 4.09±0.29 h, respectively. These results suggest that oral patch preparation has as a potential a new oral delivery system to obtain a long residence time of drug in the gastrointestinal tract.
Keywords: Patch preparation; Oral delivery; Absorption; Mean residence time; Fluorescein; Fluorescein isothiocyanate-dextran; Rats;

Enhanced transdermal delivery of phenylalanyl-glycine by chemical modification with various fatty acids by Akira Yamamoto; Kazunari Setoh; Masahiro Murakami; Masaru Shironoshita; Takumi Kobayashi; Kayoko Fujimoto; Naoki Okada; Takuya Fujita; Shozo Muranishi (119-128).
We synthesized three novel lipophilic derivatives of phenylalanyl-glycine (Phe-Gly), C4-Phe-Gly, C6-Phe-Gly and C8-Phe-Gly by chemical modification with butyric acid (C4), caproic acid (C6) and octanoic acid (C8). The effect of the acylation on the stability, permeability and accumulation of Phe-Gly in the skin was investigated by in vitro studies. The stability of Phe-Gly in skin homogenates was low, but was significantly improved by the acylation. In the transport studies, a Franz-type diffusion cell was used for the permeability experiments with Phe-Gly and its acyl derivatives. The permeability of acyl-Phe-Gly derivatives across the intact skin was higher than that of native Phe-Gly. Of all the acyl-Phe-Gly derivatives, C6-Phe-Gly was the most permeable compounds across the intact skin. On the other hand, the permeability of acyl-Phe-Gly derivatives across stripped skin was less than that of native Phe-Gly in the initial time period of transport studies, but their permeability was higher than that of native Phe-Gly at the end of the transport studies. When the skin was pretreated with ethanol, which could inactivate the peptidases responsible for the degradation of Phe-Gly, the permeability of native Phe-Gly was higher than that of acyl derivatives. These findings indicated the involvement of peptidases on the permeability of Phe-Gly across the skin. The relationship between the lipophilic indexes of Phe-Gly derivatives and the permeability coefficients indicated that there is an optimal carbon number of fatty acid for improving the transdermal permeability of Phe-Gly by the acylation. A good correlation was found between the accumulation of these acyl-Phe-Gly derivatives in the intact skin and their lipophilicity. These results suggest that the stability and permeability of Phe-Gly were improved by chemical modification with fatty acids and this enhanced permeability of Phe-Gly by the acylation may be attributed to the protection of Phe-Gly from the enzymatic degradation in the skin and the increase in the partition of Phe-Gly to the stratum corneum.
Keywords: Percutaneous absorption; Skin permeation; Peptide drug delivery; Chemical modification; Skin metabolism; Transdermal drug delivery;

Equilibrium properties and kinetics of metoclopramide release of carbomer-metoclopramide (C-M) hydrogels are reported. A set of (C-M)X (x=moles percent of M=50, 75, 100) that covers a pH range between 6.49 and 8.40 was used. Hydrogels exhibited a high negative electrokinetic potential (ζ). Concentrations of ion pair [R-COOMH+] and free species [M] and [MH+] were determined by the selective extraction of M with 1,2-dichloroethane (DCE) together with pH measurements. The system (C-M) is characterized by a high proportion of drug present in the form of ion pairs and a negative ζ potential that attracts MH+ and H+ and repeals OH, providing a microenvironment of higher acidity than the bulk medium. Delivery rates of M were measured in a Franz type bi-compartimental device using water and NaCl 0.9% solution as receptor media. (C-M) hydrogels behave as a reservoir that releases the drug at a slow rate to water; the rate increases 14 times as water is replaced by NaCl solution. The pH effect on delivery rate suggests that, under the main conditions assayed, the rate of dissociation of R-COOMH+ together with the low change of pH in the polyelectrolyte environment are the factors that control releasing rates.
Keywords: Carbomer hydrogels; Metoclopramide; Carbomer–Drug complexes; Ion pair interaction; Drug delivery; Mechanism of kinetics release;

This article presents a novel drug release model that combines drug dissolution, diffusion, and immobilization caused by adsorption of the drug to the tablet constituents. Drug dissolution is described by the well-known Noyes–Whitney equation and drug adsorption by a Langmuir–Freundlich adsorption isotherm, and these two processes are included as source and sink terms in the diffusion equation. The model is applicable to tablets that disintegrate into a number of approximately spherical fragments. In order to simplify the analysis it is assumed that liquid absorption, matrix swelling, and tablet disintegration are much faster than drug dissolution and subsequent drug release. The resulting model is shown to yield release characteristics in good agreement with those observed experimentally.
Keywords: Drug release; Diffusion; Modelling; Tablets;

Assessment of oral bioavailability enhancing approaches for SB-247083 using flow-through cell dissolution testing as one of the screens by Cherng-Yih Perng; Albert S Kearney; Nagesh R Palepu; Brian R Smith; Leonard M Azzarano (147-156).
SB-247083 is a potent, nonpeptidic, orally active, ETA-selective, endothelin receptor antagonist. The diacid form and three salts (monoarginine, diarginine and disodium) of SB-247083 were evaluated during the pre-clinical phase of development. The developability attributes (i.e. hygroscopicity, thermal behavior, aqueous solubility, and drug-excipient compatibility) of these compounds were evaluated. In addition to these attributes, the flow-through cell (FTC) dissolution testing (using USP Apparatus 4) was used as a screening technique to evaluate several SB-247083 formulations of the diacid and its salts. FTC dissolution testing offers two distinct advantages over the more traditional static-condition dissolution testing: (1) maintenance of sink conditions; and (2) the ability to change the dissolution medium during a dissolution run. The former advantage is especially important for poorly aqueous soluble drugs having associated dissolution-rate-limitations, and the latter advantage allows one to more closely simulate the pH gradient associated with transit through the GI tract. Based on the comparative dissolution data, three formulations were chosen for oral dosing in dogs. The reasonable correlation found between the FTC dissolution results and the oral bioavailability data demonstrate that FTC dissolution testing can be a valuable tool for aiding in salt (solid-state form) and formulation selection in the early stages of development of drug candidates.
Keywords: SB-247083; Salt selection; Flow-through cell dissolution; Bioavailability;

Enhanced transport of a novel anti-HIV agent—cosalane and its congeners across human intestinal epithelial (Caco-2) cell monolayers by Chandrasekhar Udata; Jignesh Patel; Dhananjay Pal; Elzbieta Hejchman; Mark Cushman; Ashim K Mitra (157-168).
Purpose: Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, oral bioavailability of this highly lipophilic compound is extremely poor (<1%). The purpose of this study is to screen a variety of permeation enhancers (cyclodextrin derivatives, cremophor EL, bile salts and mixed micelles) for their ability to enhance the transport of cosalane and its analogs/prodrugs across Caco-2 cell monolayers. Methods: Cosalane and its different analogs/prodrugs were synthesized and their physicochemical properties were determined. Caco-2 cells were cultured at a density of 66 000 cells/cm2 either on collagen coated clear polyester membranes or Transwell® inserts. Side-bi-side diffusion cells and Transwell® inserts were employed to study for the transport of cosalane and its analogs/prodrugs with various permeation enhancers across Caco-2 cell monolayers. Results: Permeabilities of EH-3-39, EH-3-55 and EH-3-57 significantly improved compared to that of cosalane in the presence of bile salt, sodium desoxycholate. Among the various cyclodextrins studied, hydroxypropyl beta cyclodextrin (HP-β-CD) and dimethyl beta cyclodextrin (DM-β-CD) exhibited 22.3-fold and 19-fold permeability enhancement of cosalane respectively across Caco-2 cell monolayers. Sodium desoxycholate (10 mM) also showed a remarkable (105-fold) enhancement on the permeability of cosalane (P app 11.72±3.31×10−6 cm/s) without causing any measurable cellular damage. Cremophor EL resulted in higher transport of 14C mannitol. The mechanism of enhancement effect can be mainly attributed to the alteration of membrane fluidity by cyclodextrin and opening of tight junctions by cremophor EL. Conclusions: Among the enhancers tested, 10 mM sodium desoxycholate and HP-β-CD appear to be viable candidates for further development of an oral formulation of cosalane and its congeners.
Keywords: Cosalane; HIV; Bile salts; Mixed micelles; Cyclodextrins; Caco-2 cell monolayers; Permeation enhancers;

This study compared the rate and amount of absorption from aqueous solutions and pure powders of 3- and 4-cyanophenol (CP) into silicone rubber (SR) membranes. SR membranes were cast directly onto a zinc-selenide attenuated total reflectance (ATR) crystal, which was then mounted on a Fourier transform infrared (FTIR) spectrometer. CP was presented to the SR membrane from aqueous solutions or powders sufficient to completely cover the membrane surface. The concentration of CP in the membrane at the interface with the crystal was determined by IR absorption of the CN stretch. The amount of CP in the SR membrane at steady state was determined by extraction and UV absorption measurements of the extract. The concentration of CP in SR membranes was found to depend weakly on the concentration of the aqueous solution. The amount of CP in SR membranes equilibrated with pure powders was essentially the same as for the CP-saturated solutions. Diffusion coefficients for 3 and 4-CP (2.8±0.2 and 2.5±0.6×10−7 cm2 s−1, respectively) were not statistically different. The absorption rate of CP into SR membranes was almost the same from the powder and aqueous solution indicating that the rate of mass transfer from the powder to membrane was larger than 0.04 cm h−1.
Keywords: Silicone rubber; ATR-FTIR; Membrane; Cyanophenol; Absorption; Diffusion coefficients;

Enhanced intestinal absorption of vancomycin with Labrasol and d-α-tocopheryl PEG 1000 succinate in rats by Y.V.Rama Prasad; S.P Puthli; Sudarat Eaimtrakarn; Makoto Ishida; Yukako Yoshikawa; Nobuhito Shibata; Kanji Takada (181-190).
Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic used for the treatment of infections caused by methicillin-resistant staphylococci. It is water soluble, having a high molecular weight, and poorly absorbed from the gastrointestinal tract. Mixtures of VCM with Labrasol and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) were prepared to improve oral absorption of VCM. Administration of VCM solution to rat ileum at a dose of 20 mg/kg did not result in detectable plasma VCM concentration. Formulation containing 50% of Labrasol resulted in a Cmax value of 5.86±0.97 μg/ml and an AUC0–6h value of 16.06±1.78 μg h/ml. Addition of TPGS to VCM solution at 12.5% concentration also increased the plasma VCM concentration with a Cmax value of 4.98±0.45 μg/ml. But the AUC0–6h (9.87±1.90 μg h/ml) was significantly lower than that obtained with Labrasol. The addition of 5.0 and 25.0% TPGS to solutions of VCM containing 50% of Labrasol did not result in any significant increase either in Cmax or AUC0–6h of VCM. Whereas the addition of 12.5% of TPGS has resulted in an increase in Cmax and AUC0–6h by 2.2 and 2.4 times, respectively, suggesting that this concentration of 50% Labrasol and 12.5% TPGS (1:0.25) was optimum for improving intestinal absorption of VCM. A dose dependent decrease in the Cmax and AUC0–6h values was observed when the dose of absorption enhancers was decreased by 50% with formulation containing Labrasol and TPGS in 1:0.25 ratio. The results of the study indicate that formulations containing Labrasol and TPGS improve intestinal absorption of hydrophilic macromolecular drug, VCM.
Keywords: Vancomycin hydrochloride; Glycopeptides; Antibiotic; Methicillin-resistant staphylococci; Labrasol; Vitamin E TPGS;

The purpose of this study was to develop an organotypic cornea equivalent consisting of three different cell types (epithelial, stromal and endothelial cells) and to investigate its usefulness as in vitro model for permeation studies. The different cell types of a porcine cornea were selectively isolated and a multilayer tissue construct was created step-by-step in Transwell® cell culture insert. Histology, basement membrane components (laminin, fibronectin) and surfaces of cornea construct were investigated to evaluate the degree of comparability to porcine cornea from slaughtered animals. The cornea construct exhibited similarities to the original cornea. Ocular permeation of befunolol hydrochloride from different formulations across the cornea construct was tested using modified Franz cells and compared with data obtained from excised cornea. The cornea construct showed a similar permeation behavior for befunolol hydrochloride from different formulations compared with excised porcine cornea. However, permeation coefficients K p obtained with the construct were about three to fourfold higher for aqueous formulations and same for the w/o-emulsion. The reconstructed cornea could be an alternative to excised animal tissue for drug permeation studies in vitro.
Keywords: Cornea construct; In vitro model; Drug permeation studies; Befunolol hydrochloride;

Buccal delivery of thiocolchicoside: in vitro and in vivo permeation studies by M Artusi; P Santi; P Colombo; H.E Junginger (203-213).
Thiocolchicoside, a muscle-relaxant agent, is administered by the oral, intra-muscular and topical route. After oral administration the extent of bioavailability compared with intra-muscular administration is low, due to a first pass effect. In this paper, the delivery of thiocolchicoside through oral mucosa is studied to improve the bioavailability. Thiocolchicoside in vitro permeation through porcine oral mucosa and in vivo buccal transport in humans were investigated. Two dosage forms, a bioadhesive disc and a fast dissolving disc for buccal and sublingual administration of thiocolchicoside, respectively, were designed. The in vitro permeation of thiocolchicoside through porcine buccal mucosa from these dosage forms was evaluated and compared with in vivo absorption. Results from in vitro studies demonstrated that thiocolchicoside is quite permeable across porcine buccal mucosa and that permeation enhancers, such as sodium taurocholate and sodium taurodeoxycholate, were not able to increase its flux. The in vivo thiocolchicoside absorption experiments, in which the drug loss from oral cavity was measured, indicated that both formulations could be useful for therapeutic application. The fast dissolving (sublingual) form resulted in a quick uptake of 0.5 mg of thiocolchicoside within 15 min whereas with the adhesive buccal form the same dose can be absorbed over an extended period of time.
Keywords: Thiocolchicoside; Buccal; Formulation; Permeability; Enhancers; In vitro/in vivo;

Chitosan nanoparticles (CS NP) with various formations were produced based on ionic gelation process of tripolyphosphate (TPP) and chitosan. They were examined with diameter 20–200 nm and spherical shape using TEM. FTIR confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. Factors affecting delivery properties of bovine serum albumin (BSA) as model protein have been tested, they included molecular weight (Mw) and deacetylation degree (DD) of chitosan, the concentration of chitosan and initial BSA, and the presence of polyethylene glycol (PEG) in encapsulation medium. Increasing Mws of chitosan from 10 to 210 kDa, BSA encapsulation efficiency was enhanced about two times, BSA total release in PBS (phosphate buffer saline) pH 7.4 in 8 days was reduced from 73.9 to 17.6%. Increasing DD from 75.5 to 92% promoted slightly the encapsulation efficiency and decelerated the release rate. The encapsulation efficiency was highly decreased by increase of initial BSA and chitosan concentration; higher loading capacity of BSA speeded the BSA release from the nanoparticles. Adding PEG hindered the BSA encapsulation and accelerated the release rate.
Keywords: Chitosan; Bovine serum albumin (BSA); Nanoparticles;

In vivo evaluation of matrix granules containing microcrystalline chitosan as a gel-forming excipient by M Säkkinen; A Linna; S Ojala; H Jürjenson; P Veski; M Marvola (227-237).
Interest in drug delivery to the gastrointestinal tract by means of chitosan has been increasing. In the study reported, the biopharmaceutical properties of granules containing microcrystalline chitosan (MCCh; molecular weight 150 kDa, degree of deacetylation 75%) were evaluated via bioavailability tests in human volunteers. Ibuprofen and furosemide were used as model drugs. With ibuprofen, granules containing 40% of MCCh behaved as a slow-release formulation (t max 2.9 h). With furosemide, the most marked difference between a conventional dosage form and granules containing 40% MCCh was a marked lag time (0.5 h) before absorption from the latter. This difference was reflected in t max values for furosemide. Despite the lag time, AUC values for furosemide were high, indicating that the granules containing MCCh had remained in the stomach and that drug release had taken place in the stomach rather than in the intestine. The results of the bioavailability studies indicate that MCCh matrix granules allow a simple preparation of slow-release and perhaps stomach-specific dosage forms. Use of model drugs differing in relation to sites of absorption in the gastrointestinal tract aided identification of sites of absorption of drugs from the granules. Further studies, including γ-scintigraphic evaluations, will be performed on how the granules behave in the stomach.
Keywords: Microcrystalline chitosan; Slow-release; Stomach-specific; Ibuprofen; Furosemide;

Molecular interactions between DPPC and morphine derivatives: a DSC and EPR study by M Budai; Zs Szabó; M Szőgyi; Pál Gróf (239-250).
The interaction between different morphine derivatives (morphine, codeine, N-methyl-morphine, N-methyl-codeine) and α-l-dipalmitoyl phosphatidylcholine (DPPC) liposomes was studied with differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Small unilamellar DPPC-liposomes with the given morphine-derivative were prepared by sonication. The size distribution of liposomes was checked by dynamic light scattering (DLS). The amount of entrapped morphine was determined spectrophotometrically. Our results indicate that the morphine and its derivatives principally interact with the lipid head groups, and this interaction leads to a decrease in the mobility of the polar head groups, especially in case of codeine and N-methyl-codeine.
Keywords: DPPC-liposomes; Morphine; Codeine; DSC; EPR;

Mixed films containing pectin, chitosan and HPMC, prepared by solvent casting from 0.1 M HCl (pH 1.5) and 0.1 M acetic acid (pH 2.9) were evaluated for their morphological and leaching properties. Films cast at pH 1.5 were uniform with smooth surfaces while films cast at pH 2.9 showed particle aggregation and had rough surfaces due to polyelectrolyte complex (PEC) formation between pectin and chitosan in the medium. The leaching of pectin was higher from films at cast pH 1.5 due to the absence of PEC formation. Pectin leaching was controlled in simulated upper gastrointestinal conditions but was accelerated in the presence of pectinolytic enzymes. The leaching of pectin from the mixed films was a function of the pH of the film casting solvent, pH of the incubation medium, PEC formation and HPMC content.
Keywords: Colonic delivery; Pectin; Chitosan; Mixed films; Polyelectrolyte formation; Leaching of pectin;

Chitinosan-drug complexes: effect of electrolyte on naproxen release in vitro by Pankaj R Rege; Dhruti J Shukla; Lawrence H Block (259-272).
The purpose of this study was to examine the potential use of electrolytes to control naproxen sodium (I) release from chitinosan (II) tablets. An ANOVA was employed to evaluate the effects of molecular weight (MW) of II, electrolyte valence (EV), and pH of the dissolution medium on I's release. The intrinsic dissolution rates and saturation solubilities of I were determined at each of the pHs used. Directly compressed tablets were prepared from admixtures containing: I, NaCl, CaCl2, or AlCl3, Mg stearate, and II. The tablets were characterized for their dimensions, crushing strengths, friability, disintegration times, and in vitro dissolution profiles. The slopes of the log–log cumulative percent released-time curves (t=0–5 h) were compared using ANOVA. Based on the ANOVA, each of the variables—chitinosans, EVs, and pHs—significantly affected drug release (P<0.05). Besides the poor aqueous solubility of I, the factors possibly affecting drug release included: (a) the formation of a rate-limiting II gel barrier; (b) the interaction of I with ionized amino groups of II; (c) the effect of electrolyte on the II's gel barrier formation; and/or (d) decreased aqueous solubility of I in the presence of electrolyte.
Keywords: Chitinosan; Chitosan; Excipient; Naproxen sodium; Electrolyte; Tablets;

Atovaquone-loaded nanocapsules: influence of the nature of the polymer on their in vitro characteristics by Emmanuelle Cauchetier; M Deniau; H Fessi; A Astier; M Paul (273-281).
Nanocapsules with atovaquone concentration of 1000 μg/ml were prepared according to the interfacial deposition technique using different polymers: poly-ε-caprolactone (PECL), poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLAGA). The following characteristics of nanoparticles were determined: percentage of encapsulation of atovaquone, percentage of encapsulation of benzyl benzoate (BB), nanoparticle size, nanoparticle wall thickness, suspension pH, and in vitro stability. The different formulations showed similar characteristics: maximal percentage of encapsulation (100%), particle size of approximately 230 nm, neutral pH and wall thickness of approximately 20 nm. The type of polymer used was the main factor influencing stability, in decreasing order: PECL>PLA>PLAGA. No release of atovaquone or benzylbenzoate was noted with PECL nanoparticles over 4 months. Release of atovaquone (25.9%) was found with PLA nanoparticles at 4 months. Release of both atovaquone (18.9%) and benzylbenzoate (54.2%) was noted with PLAGA nanoparticles from the third month, indicating a disruption of the nanoparticle membrane.
Keywords: Atovaquone; Nanocapsules; PLA; PLAGA; PECL;

Comparison between polyvinylpyrrolidone and silica nanoparticles as carriers for indomethacin in a solid state dispersion by Tomoyuki Watanabe; Susumu Hasegawa; Naoki Wakiyama; Akira Kusai; Mamoru Senna (283-286).
States of interaction between indomethacin (IM) and polyvinylpyrrolidone (PVP) in an amorphous solid dispersion prepared by co-grinding were compared with those between IM and silica nanoparticles. Changes in the carbon chemical states of the solid dispersions were evaluated based on the chemical shift in the 13C-CP/MAS-NMR. Hydrogen bonds between the amide carbonyl of PVP particles and the carboxyl groups of IM molecules were formed by co-grinding. Despite the wide difference in carrier properties, the apparent equilibrium solubility (AES) of IM in the ground IM–PVP mixture was predicted by solid state NMR on the basis of the relationship previously established for IM with SiO2. This indicates that AES is affected solely by the state of IM, irrespective of the carrier species, and despite carrier-dependent chemical interactions.
Keywords: Indomethacin; Interaction; Polyvinylpyrrolidone; Co-grinding; Solid state NMR;

The aim of this investigation was to study the effect of temperature on the permeation of imipramine hydrochloride (IMH) across rat skin from two different vehicles. Differential scanning calorimetry (DSC) was used to characterize the phase transitions of rat epidermis and extracted rat SC lipids, and the transition temperatures were correlated with the permeability of IMH at different temperatures. Permeability of IMH from ethanol and propylene glycol (PG) was determined at five different temperatures and observed that a significant increase in IMH permeability occurred 45 °C from both the vehicles. Further, high energies of activation for rat skin permeation suggested that IMH diffuses across intercellular lipid matrix and therefore any change in the packing of SC lipids will have an effect on IMH permeation. Three endotherms T 1, T 2 and T 3 of rat epidermis were observed in DSC thermograms at 44, 53 and 64 °C and were assigned as transitions corresponding to orthorhombic to hexagonal, hexagonal to more disordered phase and melting of lipids with high cholesterol content, respectively. The high permeability values of IMH above 45 °C were therefore reasoned to be because of orthorhombic to hexagonal phase transition in rat skin from close to that temperature.
Keywords: Stratum corneum; Temperature; Imipramine hydrochloride; Permeability; Activation energy;

Noticeboard (301-304).