European Journal of Pharmaceutical Sciences 17S (2002) S87–S138 www.elsevier.nl / locate / ejps
EUFEPS 2002 Supplement PO-111 SIMULATION OF SHEAR FORCES ON TABLETS IN THE FED STOMACH B Abrahamsson 2 , A Pal 1 , W Schwizer 3 , G Hebbard 4 & J G Brasseur 1 – 1 Pennsylvania State University, University ¨ Park, PA, USA; 2 AstraZeneca R&D, Molndal, Sweden; 3 University Hospital, Zurich, Switzerland; 4 Repatriation General Hospital, Daw Park, Australia The objective was to determine in vivo surface shear stress variation on tablets moving within the stomach in the fed state, which could control drug release, e.g. from polymerbased extended release tablets by effects on tablet surface erosion . Computer simulations; a numerical code based on the ‘‘lattice Boltzmann’’ algorithm and combined with a realistic stomach geometry model was developed to predict gastric fluid motions and forces on 1-cm-diameter tablets moving in a simulated human stomach in two dimensions with fundic and antral contraction and pyloric opening representative of the fed state. MRI movies of the human stomach were used as basis for the stomach model. Antral contraction waves (CW) create recirculating gastric fluid motions which create a ‘‘mixing zone’’ of high relative surface shear stress. Frequency distributions of surface shear stress during tablet motion revealed 4 levels of surface shear stress: (i) low level shear stress (,1 dynes / cm 2 ) when tablets move within the fundus, (ii) moderate shear stress (|10 dynes / cm 2 ) when tablets move in the antrum between CWs, (iii) high shear stress (|35– 50 dynes / cm 2 ) generated as tablets encounter antral CWs, (iv) very high short-lived shear stress (.300 dyne / cm 2 ) as tablets are forced retrograde through advancing CWs. The frequency of high shear stress events from tablet-CW encounters is much lower than during the moderate shear stress periods. Nevertheless, the relative contributions to overall, time-integrated, surface shear stress of the shorterlived high stress periods vs. the longer-lived moderate stress periods were comparable. In conclusion, shear stress on tablet surface in the fed stomach is dependent of location of tablet and its position in relation to anteral CW’s. Tablets experience a wide range of shear forces from very mild treatment in the fundus to very strong, short-acting forces during retrograde CW’s in the antrum. These unique data should allow for
definition of more in vivo relevant in vitro test conditions with respect to hydrodynamic conditions. ¨  Abrahamsson B, Roos K, Sjogren J. Investigation on prandial effects on hydrophilic matrix tablets. Drug Dev Ind Pharm 1999;25:765–771.
PO-112 EFFECT OF VARIOUS TRANSMUCOSAL PERMEATION ENHANCERS ON DELIVERY OF FELODIPINE BY BUCCAL ROUTE Javed Ali, Alka Ahuja, Rinku Kalra & Roop K Khar – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110 062, India Transbuccal route has very low permeability to most of the compounds . Many permeation enhancers have been tried over the past to improve permeation of such compounds with promising results. The main purpose of our study was to compare the enhancement possibilities of different classes of enhancers across the bovine cheek pouch membrane . Sodium taurocholate, sodium lauryl sulphate, 1-menthol and b-cyclodextrins in concentration ranging from 1%–5% w / w were taken up for the comparative studies. A modified Franz-diffusion cell was used for measuring in vitro flux of drug through the mucosa. Permeability coefficients were calculated from the curves plotted between cumulative amount of drug permeated versus time. The changes in the buccal mucosa at cellular level were studied using light and electron microscopy and infrared studies. Sodium taurocholate at 5% w / w concentration brought about the maximum in vitro flux with an enhancement ratio of 5.361.2. No enhancement was observed at 1.0% w / w concentrations. Histological studies showed that the enhancers brought about significant changes at 5% w / w concentrations. Sodium taurocholate was found to have the best enhancement properties across bovine buccal mucosa.  Adams D. The mucous barrier and absorption through the oral mucosa, J. Dent. Res. 1975;54:B19–B20.  Vries M, Bodde H et al. Mechanisms of non-electrolyte penetration across dog and rabbit oral mucosa in vitro, Int. J. Pharm, 1991;76:25–29.
0928-0987 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0928-0987( 02 )00158-6
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-113 DESIGN AND CHARACTERIZATION OF RETENTIVE FIBERS FOR PERIODONTITIS Javed Ali, Alka Ahuja, Ranajoy Sarkar & Roop K Khar – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110 062, India A low dose targeted drug delivery device which could be easily and conveniently placed in the periodontal pocket was developed. Ethylene vinyl acetate fibers were loaded with amoxycillin trihydrate . In vitro studies were carried out in alkaline phosphate buffer pH 8.1 in a biological shaker at 50 r.p.m. at 37 8C. An in situ apparatus simulating the in vivo conditions of the periodontal pocket was designed for release studies . Alkaline borate buffer pH 8.1 simulating the pH of gingival crevicular fluid was continuously pumped at a flow rate of about 3.5 ml / day at 37 8C. The optimized formulation was subjected to dimensional analysis, physical characterization, weight variation, content uniformity, SEM analysis, XRD, stability studies etc. Microbiological evaluation of in-situ release study samples was carried out against the strains implicated in oro-dental infections namely S. aureus, S. mutans and B. cereus. The polymeric fibers loaded with 2% amoxycillin trihydrate were soft and elastic having a diameter of 0.5 mm. Fibers followed zero order release rate. The in situ release studies revealed that the drug released was above its minimum inhibitory concentration for a period of 12 days. The samples were effective in inhibiting the growth of strains. Results of stability studies showed that the degradation rate constant was 2.79310 24 / day.  Abu Fanas S, Drucker D et al. Evaluation of acrylic strips containing amoxycilline with clavulanic acid for local drug delivery. J Dent., 1991;19(2):92–96.  Ali J, Khar R K and Ahuja A. Formulation and characterization of a bucco adhesive erodible tablet form treatment of oral lesions. Die Pharmazie, 1998;53(5):329–333.
PO-114 CATIONIC CELLULOSE HYDROGELS: RHEOLOGICAL ANALYSIS OF THE CROSS-LINKING PROCESS AND CHARACTERIZATION AS DRUG DELIVERY SYSTEMS ´ Rodrıguez ´ Carmen Alvarez-Lorenzo, Rosalıa & Angel ´ FarConcheiro – Dept. de Farmacia y Tecnologıa ´ maceutica, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain Cationic hydroxyethylcelluloses, such as polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are hydrophilic polymers with a high affinity for the skin and mucoses. To improve their applications as drug delivery systems, the cross-linking process with ethylenglycol diglycidylether (EGDE), at different pHs and temperatures, was analyzed using oscillatory rheometry. Cationic cellulose dispersions
(3 wt.-%) were prepared in ultrapure water or in NaOH solutions (0.05 or 0.10 M) under stirring, and left to stand for 12 h at 4 8C, and 12 h at room temperature. EGDE (1 or 2 mL) was added to 10 mL polymer solution at 20 8C, stirred for a few minutes, and immediately spread on the Peltier plate of the rheometer (Rheolyst AR-1000N, TA Instruments, measuring geometry: 4 cm flat plate covered with a solvent tramp). The evolution of the elastic (G9) and viscous (G0) moduli, recorded in time-sweep experiments carried out at 0.1 rad / s, showed that the cross-linker requires a minimum of 0.05 M NaOH and 30 8C to be active. The increase in G9 and G0 followed a first order kinetic, the slopes of G’ being higher than those corresponding to G0. Apparent activation energies, estimated from the dependence of the gel time (time at which G95G0) on temperature  ranged between 70 and 90 KJ / mol. The differences in cross-linking rate between PQ-4 and PQ-10 may be attributed to their different molecular weight and content in hydroxyethyl groups . Hydrogels prepared in test tubes at the conditions found as optimum by rheometry (60 8C in 0.10 M NaOH medium) presented a smooth, continuous surface and a high capacity to absorb water and sodium diclofenac. Drug release is sensitive to pH and ionic strength of the medium. The low toxicity of the cross-linker agent, and the mechanical properties, water affinity, and drug loading and controlled release properties of these hydrogels provide them a wide range of potential applications in biomedical and pharmaceutical fields. ´  B. Fernandez et al. Rheokinetic variations during curing of a tetrafunctional epoxy resin modified with two thermoplastics. Eur. Polym. J. 2001;37:1863– 1869. ´  R. Rodrıguez et al. Rheological evaluation of the interactions between cationic celluloses and Carbopol 974P in water. Biomacromolecules, 2001;2:886–893.
PO-115 A NEW DRUG DELIVERY SYSTEM FOR METOCLOPRAMIDE HCl Saima Amin, Kanchan Kohli & Asgar Ali – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi-110062, India Objective: To develop transdermal drug delivery system of metaclopramide and improve patient compliance. Methods: In-vitro skin permeation studies were carried out using Keishery Chien diffusion cell mounted with Albino rat skin. in-vitro dissolution studies using USP Paddle Over Disc method. Skin irritation (as per Draize score test) and stability studies (as per WHO guidelines) were carried out. Results and discussion: Also the optimized formulation containing Eudragit RL-100 and Eudragit RS-100 showed 87% of drug release over a period of 48 hours. The higher
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
release and skin permeation could be attributed to high concentration of penetration enhancer. Addition of 10% w / w DMSO increased the cumulative percent of drug released from 66% to 80%. The drug release from the patch followed Higuchian matrix mechanism. The fabricated systems produced none or very mild irritation which could be well tolerated during the course during the course of therapy. Conclusion: Transdermal patches of metoclopramide HCl were successfully formulated and was found to be stable for a period of 3 years.  Al-Humayyd, M. S. Effect of metoclopramide on lamotrigine absorption in rabbits, Int. J. Pharm., 1996;144:171–175.  Hadgraft, J., Pugh, J. and Michael, W. H. Transdermal delivery of some antiemetics, Int. J. Pharm., 1999;123:285–289.
PO-116 EXPERIMENTAL DESIGN AND OPTIMIZATION OF TABLET FORMULATION REGARDING TABLET HARDNESS AND IN-VITRO DRUG RELEASE PROFILE Mattias Andersson, Anders Ringberg & Christina Gustaf¨ ¨ ¨ ¨ sson – AstraZeneca R&D Sodertalje, SE-151 85 Sodertalje, Sweden As a screening step a fractional factorial design was set up to investigate the influence and further optimization of the amount of tablet excipients on tablet hardness and in-vitro drug release profile. The experimental runs were performed in small scale, approximately 30 g. The active drug and the excipents were subjected to wet granulation before compaction in an instrumented tablet machine (Korsch EK0, Germany). A principal component analysis showed that the four responses related to tablet hardness were almost orthogonal to the seven drug release profile responses in the loading plot. Therefore further evaluation of the design was separated into two different models . These two models were then linked together. A simulated optimization was performed using a simplex optimization tool and a desirability function . Four extra experiments were then added to the design to verify the simulated results and to explore the potential effect of quadratic terms. A tablet formulation of desired hardness and a fast drug release profile was obtained with a high drug:filler ratio, and high amount of disintegrant. If these variables were set at proper levels it was possible to avoid addition of a surfactant despite its significant effect on the in-vitro drug release profile.  Eriksson L, Hermens JLM, Johansson E, Verhaar HJM, Wold S. Multivariate analysis of aquatic toxicity data with PLS. Aquatic Sciences 1995;57:217–241  Carlson R, Hansson L, Lundstedt T. Optimization in
organic synthesis. Strategies when the desired reaction is accompanied by parasitic side reactions. An example with enamine synthesis. Acta Chem Scand 1986;B40:444–452.
PO-117 DETERMINATION OF STABILITY CONSTANTS OF b-CYCLODEXTRIN COMPLEXES WITH A MEMBRANE PERMEATION TECHNIQUE ¨ & Nicolaas Schipper – Ulrika Andersson, Emma Bostrom ¨ ¨ ¨ ¨ AstraZeneca R&D Sodertalje, S-151 85 Sodertalje, Sweden The purpose of the study was to determine the stability constants for various b-CD complexes with two model compounds using a membrane permeation technique . The stability constants were determined by studying the permeation of hydrocortisone and indomethacin across cellulose ester membranes in presence of b-CD, DM-b-CD and HP-b-CD in Franz diffusion cells. Using a numerical method the stability constants were calculated from the concentration profile as a function of time. In addition, stability constants were determined with the phase solubility method. In the presence of CD in the donor chamber the permeation rate of hydrocortisone and indomethacin was reduced as compared to permeation in absence of CD. The total concentration of drug in the donor chamber is the sum of the free and complexed form of the drug. Only the free fraction of the drug can permeate the membrane. From the differences in the amount of permeated drug versus time profile in absence and presence of CD the stability constants for complex formation could be calculated. In general, stability constants determined from phase solubility diagrams and from the permeation technique were in good agreement. In phase solubility diagrams with DM-bCD and indomethacin, a linear correlation between solubility and CD concentration was not obtained, indicating formation of other types than solely 1:1 drug:CD complexes. Such a deviation from 1:1 complexes cannot easily be observed from the concentration-time profiles in the permeation method. In the present study using a numerical method to determine the stability constants from the permeation profile in absence and presence of CD a 1:1 complex was a prerequisite for correct calculation.  Ono N, Hirayama F, Arima H, Uekama K. Eur J Pharm 1999;8:133–139.
PO-118 THE MULTIPURPOSE SOLUTION FOR HYDROPHILLIC CONTACT LENSES DISINFECTS THE LENSES AT OCULAR pH A Arora, K Kohli & R K Khar – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-62, India
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
Different solutions ranging from pH 5.0–9.0 were prepared . The inhibitony zones were produced on agar plates against Pseudomonas aeruginosa. The multifunctional solution contained Polyhexanide (0.0001%). Polaxamine (0.1%). Disodium edetate (0.1%). At pH 7.4 the maximum size of inhibitory zone was produced . The same solution also gave inhibitory zones against Aspergillus fumigatus and Candida albicans . The multifunctional solution for hydrophillic contact lenses at pH 7.4 gave the maximum size of inhibitory zones .  Tang I, Wong DM, Yee DJ, Harris MG. The pH of multipurpose soft contact lens solutions. Optom Vis Sci 1996;73:746–9.  Landa AS, Vander Mei HC et al. Efficacy of ophtmalmic solution to detach adhering Psendomonas aeruginosa from contact lenses. Cornea 1998;17:293–300.  Durban JJ, Monteoliva Sanchez M et al. Antimicrobial efficiency of hydrogel contact lens soaking solution marketed in Spain. Optom Vis. Sci 1998;75:126–31.  Charles G, Lance MS, Shelly M et al. The effectiveness of several current soft contact lens care systems against Aspergillus. CLAO J 1998;24:82–4.
PO-119 INFLUENCE OF DOSING VEHICLES AND SOLUBILITY PROPERTIES ON ORAL ABSORPTION OF DANAZOL AND KETOCONAZOLE Bruce Aungst, Glenn Dobson, Laura Letendre & Ismael Hidalgo – Absorption Systems, 440 Creamery Way, Suite 300, Exton, PA 19341-2554, USA Dosing vehicle selection can be a critical determinant of the outcome of animal pharmacokinetic studies. In this study we evaluated the influence of dosing vehicle on the oral absorption of the poorly water soluble compounds danazol and ketoconazole. In addition, we examined how the absorption of these compounds relates to their solubility properties and tendency to precipitate from the vehicle. Oral absorption was assessed in fasted rats and fasted dogs, with blood samples collected via jugular vein cannulae or by venipuncture. Plasma concentrations of test compounds were determined using LS / MS. Solubilities were determined by shake flask methods. Danazol had negligible solubility in aqueous media at pH 2 or 6.5, but in simulated intestinal fluid danazol had 5 mg / ml solubility. In rats, danazol had very low plasma concentrations after oral dosing with either a methylcellulose suspension or a PEG 400 solution. However, AUC was .5-fold greater when Labrafil was used as the dosing vehicle. Inclusion of a co-surfactant in Labrafil did not significantly further increase absorption. Maximizing danazol absorption appears to require solubilization by the vehicle, as well as prevention of precipitation upon dilution, since
danazol readily precipitated when the PEG vehicle was diluted with water. A Labrafil dosing vehicle was further compared with a marketed solid-filled capsule formulation of danazol in dogs. Oral bioavailability of danazol was improved from 2% for the capsule to approximately 75% with the Labrafil solution. Ketoconazole was evaluated as a test case of a compound with pH-dependent solubility. Ketoconazole had solubilities of 5 mg / ml in pH 6.5 buffer, 9 mg / ml in simulated intestinal fluid, and .3000 mg / ml at pH 2. In rats, ketoconazole was well absorbed from a methylcellulose suspension or a PEG 400 solution (F5 64% and 88% respectively). In contrast to danazol, a Labrafil solution vehicle resulted in lower AUC of ketoconazole compared with the suspension, and absorption appeared to be prolonged. Bioavailability was reduced to 31%. Ketoconazole is apparently well absorbed when released into the acidic gastric environment. Labrafil may reduce ketoconazole absorption because some of the dose is released from the vehicle in the intestine at a pH where ketoconazole is poorly soluble. In conclusion, the selection of a dosing vehicle that maximizes oral absorption of poorly soluble test compounds requires an understanding of the solubility behavior in various media as well as upon dilution of the vehicle in an aqueous environment.
PO-120 IMPROVED DELIVERY OF MELOXICAM BY COMPLEXATION WITH CYCLODEXTRINS S Baboota & S P Agarwal, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi-110062, India Meloxicam (MLX) is a non-steroidal anti-inflammatory (NSAID) drug prescribed for the long-term treatment of musculo-skeletal complaints  which suffers from the drawback of gastrointestinal (GI) side effects, which can reduce patient compliance and discourage physician from prescribing them. There is therefore, a need for a delivery system for NSAIDs with improved GI tolerability while retaining its efficacy. The present study is an attempt to prepare tablets containing inclusion complexes of MLX with b-CD and HPb-CD to improve the aqueous solubility of the drug thus enhancing its dissolution rate, thereby showing a faster onset of action and less GI mucosal toxicity. The complexes were prepared by freeze-drying method (FD). Physico-chemical studies on the complex was done by DSC, XRD, SEM, FT-IR and NMR. The tablets were prepared by direct compression method. The ulcerogenic studies were done in wistar rats by the method reported by Nambu et al. . Anti-Inflammatory studies were carried out in carrageenan induced rat hind paw oedema model. The physico-chemical characterization indicated maximum complex formation in MLX-HPb-CD complexes. The aqueous solubility of MLX b-CD/ HPb-CD was more
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
than the pure drug i.e., MLX both in pH 1.2 and pH 7.4. Oral administration of MLX-HPb-CD complexes was found to inhibit carrageenan induced paw odema in rats to a significant extent as compared to MLX-b-CD complex and MLX. Lower ulcerogenic scores were obtained by MLX-HPb-CD complexes (0.460.41) as compared to MLX-b-CD complexes (0. 960.25) and pure MLX (2.2560.96). An inclusion complex of MLX with b-CD and HPb-CD was prepared usefully by freeze drying method, in a molar ratio of 1:2 with improved aqueous solubility, faster onset of action and a significant decrease in gastric ulceration property.  Noble, S. and Baifour, J.A. Meloxicam – A drug Profile. Drugs, 1996;51:424.  Nambu, N., Kikuchi, K., Kikuchi, T., Takahashi, Y., Ueda, H., Nagai, T. Influence of inclusion of nonsteroidal anti-inflammatory drugs with b-cyclodextrin on the irritation to stomach of rats upon oral administration’’. Chem. Pharm. Bull. 1978;26:3609.
PO-121 PHEA-THIOGLYCOLIC ACID CONJUGATE: PREPARATION AND CHARACTERISATION ´ ˇ ´ 2 , K Sankovic´ 3 M Barbaric´ 1 , A Martinac 2 J Filipovic-Grcic 1 1 & B Zorc – Department of Medicinal Chemistry; 2 Department of Pharmaceutical Technology; 3 Department of Biophysics, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Mucoadhesive polymers have received considerable attention as excipients in pharmaceutical technology. They assure an intimate and prolonged contact of drug delivery systems to the adsorbing mucosa and therefore increase uptake and subsequently the bioavailability of the drug. Recently, it has been shown that thiolated polymers (thiomers) provide enhanced adhesive properties, probably due to interactions with cysteine-rich subdomains of mucus glycoproteins via disulfide exchange reactions (1). The aim of this study was to prepare and evaluate the poly[a,b-(N2-hydroxyethyl-DL-aspartamide)]-thioglycolic acid (PHEA-TGA) conjugate. TGA was covalently attached to PHEA in coupling reaction mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC).
The conjugate was fully physico-chemically characterised (average Mw , solubility, IR, EPR spectra, amount of thiol groups). The swelling behaviour and mucoadhesive properties of conjugate were determined by a gravimetric method and by tensile studies on freshly excised porcine intestinal mucosa, respectively. The results have shown that modification of the parent PHEA molecule with TGA could lead to polymer with enhanced mucoadhesive properties.  Kast, C.E., Bernkop-Schnurch, A. Thiolated polymersthiomers: development and in vitro evaluation of chitosan-thioglycolic acid conjugates. Biomaterials 2001;22:2345–2352.
PO-122 PREDICTION OF DRUG SOLUBILITY AND PERMEABILITY BASED ON MOLECULAR SURFACE PROPERTIES. APPLICATION IN BIOPHARMACEUTICAL CLASSIFICATION ¨ 1 , Melissa Strafford 2 , Lucia Christel A S Bergstrom 1 Lazorova , Alex Avdeef 2 , Kristina Luthman 3 & Per Artursson 1 – 1 Department of Pharmacy, Uppsala University, Uppsala Biomedical Center, P.O. Box 580, SE-751 23 Uppsala, Sweden; 2 pION Inc., 5 Constitution Way Woburn, MA 01801, USA; 3 Medicinal Chemistry, Depart¨ ment of Chemistry, Goteborg University, SE-412 96 ¨ Goteborg, Sweden The objective of this study was to build in silico models for drug solubility and permeability based on molecular surface properties, in order to perform a theoretical biopharmaceutical classification. Twenty-three structurally diverse orally administered drugs, selected from the WHO list of essential drugs, were investigated. Experimental solubility (11 ng / ml to .20 mg / ml) and permeability (3310 28 to 4310 24 cm / s) values were determined using potentiometric titration and Caco-2 cell monolayers, respectively. Partitioned total surface area (PTSA) and descriptors such as polar and non-polar surface areas were calculated from low energy conformations obtained by conformational analysis using molecular mechanics calculations. Correlations between the molecular descriptors and solubility and permeability were established with multivariate data analysis (PLS), using training sets and test sets. The obtained in silico solubility model, which was mainly built on non-polar descriptors, had an R 2 50.93 and resulted in RMSE tr of 0.37 log units and RMSE te of 0.58. The developed in silico permeability model, which was mainly built on polar descriptors, had an R 2 50.93 and resulted in RMSE tr of 0.35 log units and RMSE te of 0.99. Biopharmaceutical classification into six classes based on the two models outperformed the commonly used rule-of five and resulted in the correct prediction of 20 out of the 23 compounds (87%). Thus, molecular surface area de-
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
scriptors predict permeability, solubility and biopharmaceutical class with excellent accuracy. Our results suggest that rapid and easily interpreted theoretical models based on molecular surface properties are promising tools for predicting drug absorption.
PO-123 PREDICTION OF AQUEOUS DRUG SOLUBILITY FROM 2D AND 3D MOLECULAR DESCRIPTORS ¨ 1 , Carola Wassvik 1 , Ulf Norinder 2 , Christel A S Bergstrom 3 Kristina Luthman & Per Artursson 1 – 1 Department of Pharmacy, Uppsala University, Uppsala Biomedical Center, P.O. Box 580, SE-751 23 Uppsala, Sweden; 2 Department of Medicinal Chemistry, AstraZeneca Re¨ ¨ search and Development, SE-151 85 Sodertalje, Sweden; 3 ¨ Department of Chemistry, Medicinal Chemistry Goteborg ¨ University, SE-412 96 Goteborg, Sweden. The aim of this study was to devise computational models for aqueous drug solubility using readily calculated molecular descriptors. Intrinsic solubility values for a series of 92 structurally diverse drug-like molecules were determined. Two-dimensional molecular descriptors, mainly describing electrotopology, were calculated using the AstraZeneca in-house program SASA. Three-dimensional molecular properties were obtained using molecular mechanics calculations and the programs MAREA (surface area descriptors) and VolSurf (polarity, size / shape and hydrophobicity descriptors). Correlations between the calculated descriptors and aqueous drug solubility were established using PLS, generating global (all compounds) and subset specific models (the 92 compounds divided into acids, bases, ampholytes and non-protolytes). All models were generated step-wise from each set of descriptors (SASA, MAREA and VolSurf) as well as from combinations of these descriptors. Subset specific models were successfully generated from descriptors obtained either from the SASA, MAREA or VolSurf programs; combinations of these descriptors were not needed to achieve predictive subset models. The precision of each program-specific set of descriptors varied, depending on which subset that was studied. Surface area properties were strong predictors for the solubility of bases, while VolSurf descriptors better described acids and electrotopology descriptors best described ampholytes. To obtain a predictive global model for all 92 compounds, a combination of the descriptors from the various programs had to be used, which resulted in R 2 50.82, Q 2 50.72 and RMSE tr 50.80 RMSE te 51.01. These results suggest that theoretical models for prediction of aqueous drug solubility can be obtained from rapidly calculated descriptors. The demanding dataset with large structural diversity were predicted using a more complex model than that obtained for solubility prediction of chemical subsets.
PO-124 COMPARISON OF P-GLYCOPROTEIN EFFLUX MECHANISM IN SITU AND IN VITRO FOR A NEW FLUOROQUINOLONE ´ ´ ´ M Bermejo, I Gonzalez-Alvarez, C Fernandez-Teruel, V ´ – Departamento Merino, T M Garrigues, A Ruiz-Garcıa ´ Farmaceutica, ´ Farmacia y Tecnologıa Universidad de ˜ Valencia, Espana The aim of the study was to contrast the influence of P-glycoprotein on in situ and in vitro intestinal absorption models for a 6-fluoroquinolone (CNV 97100, Cenavisa Laboratories (Spain)) . Fluoroquinolones show active efflux processes when they are studied in vitro models as CaCo-2 cultured cells , but the relevance of this process in situ or in vivo is not always significant. The absorption rate coefficient (ka) of CNV 97100 was determined in the whole intestine of the rat at five different concentrations ranging from 0.15 mM to 1500 mM. The in vitro study was developed in Caco-2 monolayers (ATCC), drug concentration ranged from 50 mM to 7500 mM. The lower concentration in both models was assayed in presence of Verapamil 4 mM. The in situ data (concentration versus time) and the in vitro data (amounts in receptor chamber versus time) in every condition were fitted to a system of differential equations of a combined passive and Michaelis-Menten kinetics using Winnonlin v.1 software. In situ parameters (Vm56.29e–4 nmol?cm 22 ?s 21 ; Km524.20 mM, Pdif5 3.41e–5 cm?s 21 ) were compared to the in vitro ones (Vm50.014 nmol?cm 22 ?s 21 ; Km513.56 mM; Pdif54e– 6 cm?s 21 ). The presence of Verapamil in the solutions produces an increase in both the in situ and the in vitro permeabilities to a maximum value that corresponds approximately with the passive component. The efflux process is relevant not only in vitro but also in situ in rats. The influence in the in vivo situation will be further studied. ˜ ´ N et al.  Sanchez-Castano G, Ruiz-Garcia A, Banon Intrinsic absolute bioavailability prediction in rats based on in situ absorption rate constants and / or in vitro partition coefficients: 6-fluoroquinolones. J Pharm Sci 2000;89:1395–403.  Griffiths NM, Hirst BH, Simmons NL. Active intestinal secretion of the fluoroquinolone antibacterials ciprofloxacin, norfloxacin and pefloxacin; a common secretory pathway? J Pharmacol Exp Ther 1994;269:496–502.
PO-125 INTESTINAL ABSORPTION OF SARAFLOXACIN AND SPARFLOXACIN: PREDICTION FROM LIPOPHILICITY VALUES AND A BIOPHYSICAL ABSORPTION MODEL ´ ´ ´ M Bermejo, C Fernandez-Teruel, I Gonzalez-Alvarez, V ´ – Departamento Merino, T M Garrigues, A Ruiz-Garcıa
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
´ Farmaceutica, ´ Farmacia y Tecnologıa Universidad de ˜ Valencia, Espana The aim of the present study was to investigate the absorption mechanism of Sarafloxacin and Sparfloxacin and to compare the Permeability values obtained in situ with the predicted ones using the Higuchi-Ho model based on the use of lipophilicity indexes . The determination of the intestinal permeability coefficient was made by Doluisio’s method in male Wistar rats. For the predictions, lipophilicity was quantified by means of n-Octanol partition-coefficient at pH 7.00. Permeability coefficient of Sparfloxacin was determined at two perfusion concentrations: 120 and 10 mg / mL. For Sarafloxacin the study was carried out at five different concentrations: 50, 25, 5, 1 and 0.5 mg / mL. Permeability values at the different concentrations for Sarafloxacin showed statistically significant differences, whereas no differences were for Sparfloxacin. The Permeability value experimentally obtained for Sparfloxacin, Peff55.7e–5 cm / s is very similar (included in the 95% confidence interval) to the predicted value by the HiguchiHo model. For this compound it is possible to predict its intestinal Permeability in rat from its partition-coefficient and to consider its absorption as an apparent passive process in the range of concentrations assayed. Sarafloxacin showed a combined passive and active absorption kinetic (Vm50.402 mg?mL 21 ?h 21 ; Km50.692 mg?ml 21 : kdif50.426 h 21 ) and the passive component (kdif) for this compound is quite lower than the predicted one by the Higuchi-Ho model. This can be due to the structural differences with the homologous compounds used to establish the absorption-partition correlation. The deviations from the absorption partition correlation found for the homologous quinolones can help to detect active components in the absorption process.  Bermejo M, Merino V, Garrigues TM et al. Validation of a biophysical drug absorption model by the PATQSAR system. J Pharm Sci 1999;88:398–405.
PO-126 TRANSPORT OF CLONIXIN ACROSS HUMAN INTESTINAL EPITHELIAL (Caco-2) CELLS ˜ Farinha 1 , Per ´ Antonio Bica 1 , Lucia Lazorova 2 , Ascensao 2 3 ´ Artursson , Henning Blume & Carlos Maurıcio ´ Barbosa 4,5 – 1 LEF – Laboratorio de Estudos Farˆ maceuticos, Lisboa, Portugal; 2 Dept. of Pharmaceutics, Biomedical Centre, Uppsala University, Sweden; c Johann Wolfgang Goethe-Universitat, Frankfurt am Main, Ger´ many; 4 CTMUP – Faculdade Farmacia Univ. do Porto; e ´ CETMED – Centro Tecnologico do Medicamento, Porto, Portugal The transport of clonixin (a nonsteroidal anti-inflammatory drug with anti-inflammatory, analgesic and antipyretic activity) was investigated in monolayers of human epi-
thelial cell line, Caco-2. The effect of various concentrations of clonixin on Caco-2 monolayers integrity was evaluated by measuring the permeability of the hydrophilic marker 14 C-mannitol and by measuring the transepithelial electrical resistance (TEER). Transport of clonixin was studied in apical-to-basal and basal-to-apical directions either in the presence or absence of a proton gradient. The integrity of Caco-2 cell monolayers was not altered in the presence of clonixin (0.5 mM to 2.5 mM). Clonixin transport across Caco-2 monolayers was very rapid, thus sink conditions could not be maintained during the full length of the experiment. Therefore, Papp values were determined by formula that takes into account the effect of back flux of clonixin from the receiver chamber . Calculated Papp values (1.12–1.74310 24 cm / s) suggest complete absorption of clonixin in humans after oral administration . ANOVA at a significance level of 5% did not detect statistically significant differences between Papp values within the tested concentration range. Similarly, the permeability of clonixin was the same in the apical-to-basal and basal-to-apical directions (1.7460.393 10 24 cm / s and 1.2060.29310 24 cm / s, respectively), indicating that this drug is transported across the Caco-2 cell monolayers by passive transcellular pathway. ˚ ¨ J, Artursson P.  Palm K, Luthman K, Ros J, Grasjo Effect of molecular charge on intestinal epithelial drug transport: pH-dependent transport of cationic drugs. J. Pharmacol. Exp. Ther., 1999;291:435–443.  Artursson P, Karlsson J. Correlation between oral drug ansorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Comm., 1999;175:880– 885.
PO-127 CRYSTAL HABIT MODIFICATION OF PHENYTOIN IN ALCOHOLIC SOLUTIONS: THE EFFECT OF CRYSTALLIZATION CONDITIONS N Bolourtchian, A Nokhodchi & R Dinarvand – School of Pharmacy, Shaheed Beheshti University of Medical Sciences, PO Box 14155-6153, Tehran, Iran Crystal habit modification of a particular drug is an important factor, which could affect a number of physical properties such as dissolution rate  and compaction behavior [2,3]. In the present study, the effect of cooling rate and method of crystallization on the crystal habit of phenytoin were studied. Phenytoin crystals were grown from ethanol solutions under various conditions (reducing the temperature, solvent evaporation and watering-out techniques). Scanning electron microscope, x-ray powder diffractometer (XRD), FTIR and differential scanning calorimeter (DSC) were used to investigate the physical characteristics of the crystals. The dissolution behavior and compaction properties of various crystals were also studied.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
Under different crystallization conditions, the shape of crystals changes from rod like for untreated particles to needle shape or thin plate. Based on XRD and DSC studies, no polymorphic changes have been occurred. The results showed that the compactibility of particles especially those made by watering-out technique was improved, producing high crushing strength tablets with lower tendency to cap. In conclusion, the method of crystallization has a major effect in phenytoin crystal habit modification. Using watering-out technique produced thin plate crystals with higher compactibility. Changing the crystallization temperature only altered the size of crystals. The dissolution rates of all treated samples were lower than that of untreated phenytoin, probably due to the high degree of crystallinity.  Watanabe A, Yamaoka Y, Takada K. Crystal habits and dissolution behavior of aspirin. Chem Pharm Bull 1982;30:2958–2963.  Di Martino P, Di Cristofaro R et al. Improved compression properties of propyphenazon spherical crystals. Int J Pharm 2000;197:95–106.  Garekani H A, Ford J L et al. Highly compressible paracetamol: compression properties. Int J Pharm 2000;208:101–110.
PO-128 PREPARATION AND CHARACTERIZATION OF MITOXANTRONE LOADED CHITOSAN MICROSPHERES AGAINST MALIGNANT GLIOMAS S Bozdag 1 , Y Capan 1 , I Vural 1 , A A Hincal 1 & P P DeLuca 2 – 1 Hacettepe University, Faculty of Pharmacy, Dept. of Pharmaceutical Technology, 06100 Ankara, Turkey; 2 University of Kentucky, College of Pharmacy, Faculty of Pharmaceutical Sciences, Lexington, Kentucky 40536, USA Mitoxantrone (MTZ) is a potentially important anticancer drug with a wide spectrum of antitumour activity. It is a potent inhibitor of DNA and RNA synthesis. The objective of this study is to prepare MTZ loaded chitosan microspheres for treatment of malignant glioma and to evaluate in vitro for particle size, yield, drug loading, surface morphology, release characteristics. Preparation of MTZ loaded microspheres was performed according to the modified method of Jameela et al. . Theoretical drug loading of microspheres with MTZ was targeted as 5%. MTZ was mixed with 4% solution of chitosan in 5% acetic acid and dispersed in a mixture of liquid paraffin and petroleum ether containing 0.85% sorbitan sesquioleate using a probe-type sonicator. The mixture was then transferred into a 100 mL round-bottomed flask and stirred using a paddle stirrer at 2000 rpm for 5 min. Then, toluene saturated with glutaraldehyde (4 mL) was added into flask. After stirring for 2 h, the hardened spheres were centrifuged, washed several times with petroleum ether, twice
with 5% sodium bisulphide, followed by water and finally with acetone and dried. Chitosan microspheres were smooth and spherical in shape as evidenced by SEM. Particle size analysis showed a volume average particle size of 1163.15 mm and MTZ analysis showed a drug loading of 0.95%. In vitro release studies in saline pH 7.4 phosphate buffered at 37 8C showed a slow diffusion profile for the entrapped drug reaching 55% in 2 weeks.  Jameela SR, Latha PG, Subramoniam A, et al. Antitumor activity of mitoxantrone-loaded chitosan microspsheres against ehrlich ascites carcinoma. J Pharm Pharmacol 1996;48:685–88.
PO-129 INFLUENCE OF SURFACTANTS ON THE CRYSTALLIZATION OF TRIGLYCERIDE NANO-PARTICLES H Bunjes 1 & M H J Koch 2 – 1 Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich¨ Jena, Lessingstr. 8, D-07743 Jena, Schiller-Universitat Germany; 2 European Molecular Biology Laboratory, Hamburg Outstation (EMBL c / o DESY), Notkestr. 85, D22603 Hamburg, Germany Solid triglyceride nanoparticles are being investigated as potential drug carriers, particularly for the administration of poorly water soluble drugs. They are preferably prepared by high-pressure homogenization of the molten lipid in an aqueous phase with adequate stabilizers and subsequent crystallization of the emulsified particles. In the case of low-melting triglycerides, the solidification of the nanoparticles can be problematic due to the pronounced supercooling tendency of colloidally dispersed triglycerides. The presence of certain surfactants may increase the crystallization temperature of the dispersed particles  and thus facilitate the crystallization of triglycerides with a low crystallization tendency. To obtain more detailed information on the influence of surfactants on the crystallization process of triglyceride nanoparticles, the effect of polysorbate surfactants (Tween 20, 40, 60, 80) on the crystallization behavior of different triglycerides (trilaurin, trimyristin, tripalmitin, tristearin) was investigated by differential scanning calorimetry and synchrotron radiation X-ray diffraction. The onset temperature of the main crystallization event remains near a lower critical limit for the specific triglyceride when a surfactant with a laurate or oleate hydrophobic tail is used. Increasing the length of the saturated surfactant chain from C12 to C16 or C18 leads to higher crystallization temperatures. The higher crystallization temperature in these dispersions indicates an influence of the surfactant layer. When higher crystallization temperatures are observed, crystallization often occurs in a multiple event with a pretransition preceding the main crystallization. The position and size of the pretransition depend on the type of triglyceride. Closer investigations of trilaurin and trimyristin dispersions reveal
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
that the pretransition in these dispersions is correlated with the formation of a structure that displays wide angle but no small angle X-ray reflections and almost no supercooling. The pretransition may thus be caused by structure formation within the interfacial layer that facilitates the subsequent crystallization of the lipid matrix.  Bunjes H, Koch MHJ, Westesen K, Effects of surfactants on the crystallization and polymorphism of lipid nanoparticles. Progr Colloid Polymer Sci., in press. PO-130 IN VITRO AND IN VIVO RELEASE OF DEXAMETHASONE AND VEGF FROM PLGA MICROSPHERES Diane J Burgess 1 , Tae-Kyoung Kim 1 , Banu S Zolnik 1 , Dakshina Chilukuri 2 & David Young 2 – 1 Department of Pharmaceutical Sciences, U. of Connecticut, 372 Fairfield Rd, Storrs, CT 06269, USA; 2 Globomax LLC, 7250 Parkway Drive, Suite 430 Hanover, Maryland 21076, USA The objective of this study was to investigate: in vitro and in vivo release of dexamethasone and vascular endothelial growth factor (VEGF) from PLGA microspheres; the mechanism of drug release and to compare these data. Dexamethasone and 14 C-VEGF loaded poly(lactic-coglycolic) acid (PLGA) microspheres were prepared using a solid-in-oil-in-water solvent evaporation technique . Due to the low loading of VEGF required, 14 C-VEGF was diluted with 3 H-RSA prior to dispersing in the methylene chloride-in-water emulsion . In vitro release was conducted in pH 7.4 PBS at 37 8C and 100 rpm for 30 days. Dexamethasone was analyzed at 246 nm using HPLC equipped with a C18 column (Waters, Nova-Pak, 3.93150 mm) and a 50:50 mixture of 2 mM acetate buffer (pH 4.8) and acetonitrile as the solvent system. The microspheres were dissolved in dimethyl sulfoxide to determine the amount of residual drug in the microspheres. For the in vivo studies, microspheres were injected s.c. into the backs of rats. The rats were serially sacrificed at 1, 3, 5, 7, 14, 21, 28, 35, 42 and 49 days, blood samples were taken and subcutaneous tissue (including microspheres), was removed from the injection site. The concentration of drug at the s.c. site was calculated by subtracting the amount of drug remaining in the microspheres from the total amount of drug injected. The degraded microspheres (in vitro and in vivo) were dissolved in distilled tetrahydrofuran to measure molecular weight using gel permeability chromatography. One and two compartment PK modeling was conducted. The in vitro and in vivo data were modelled. The drug release profiles were different in the in vitro and in vivo environments. This was considered to be due to complex biological responses to the PLGA microspheres and the lack of sink conditions at the s.c. site. However, in vitro and in vivo release did follow the same rank order as did the in vitro and in vivo degradation of the microspheres. In order to model the pharmacokinetic profile of the drugs from the microspheres the conventional one
compartment open model was modified. An additional parameter R was included to describe the sustained release profile, where R is the concentration of protein in the tissue that has been already released from the microspheres. The protein absorption at the subcutaneous site included both the release rate from microspheres, which was the dominant step, and the absorption rate at the subcutaneous site. Four pharmacokinetic parameters (AUC, T max , Cmax in blood and k el at the s.c. site) were useful to characterize the microspheres dosage form and to compare the data. This research was supported by a grant from National Institute of Health ([1R01RR14171).  Kim TK, Burgess DJ. Formulation and release characterization of PLGA microspheres containing chemically modified protein. J. Pharm. Pharmac. 2001;53:23– 31.
PO-131 TRANSDERMAL DELIVERY OF FLURBIPROFEN: EFFECT OF ISOTONIC SODIUM PHOSPHATE BUFFER OF pH 7.4, PROPYLENE GLYCOL, ISOPROPYLALCOHOL AND THEIR BINARY COMBINATIONS ON PERMEATION THROUGH RAT SKIN Naseem A Charoo, Kanchan Kohli, K K Pillai & Areeg A Shamsher – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India The freshly excised rat skin was mounted on the Keishery Chien diffusion cell with the stratum corneum side facing the donor compartment and the dermal side facing the receptor compartment. Drug solutions (1% w / v) in different solvents IPB, PG and IPA and their binary combinations (70:30, 50:50, 30: 70) were placed in intimate contact with the skin. The receptor solution (IPB of pH 7.4) were placed in as then introduced into the stirred receptor compartment maintained at 37 8C by a circulating water jacket. The samples from the receptor compartment were withdrawn at predetermined time intervals and immediately replaced by an equal volume of fresh buffer solutions. The samples were then analyzed at 247 nm by spectrophotometrically. The flux of Flurbiprofen was found to increase with increasing concentration of IPA in PG-IPA binary combination. The maximum flux of 0.2131 mg / cm 2 / h was found when PG and IPA were taken in 30:70 ratio respectively. IPA has been shown to exhibit penetrationenhancing effect  and PG acts as a sorption promoter.PG partitions into the skin and thereby promotes the movement of the drug into and through the skin. In conclusion PG-IPA exhibit excellent solvent properties for transdermal delivery of Flurbiprofen and the solvent system act as penetration enhancer as well .  Holegaard A, Mollegaard B. Dermal drug delivery improvement by choice of vehicle on drug derivative. J controlled release 1985;2:111–120.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
 Hadgraft J. Passive enhancement strategies in topical and transdermal drug delivery. Int J Pharm 1999;185:1–6.
PO-132 POLYSACCHARIDES AS MATRIXES WITH DRUG DELIVERY FUNCTIONS: SURFACE PLASMON RESONANCE STUDY V I Chegel 1 , Y M Shirshov 1 , M A Demchenko 2 , M I Mustafaev 2 – 1 Institute of Semiconductor Physic NASU, pr. Nauki, 45, Kiev 252650, Ukraine; 2 Research Institute for Genetic Engineering and Biotechnoloogy, Tubitak Mam, Gebze-Kocaeli, Turkey The method of Surface Plasmon Resonance (SPR) was ratified recently, as one of the basic methods used for research as of various type of thin-film structures, and volumetric objects with small index of light absorption. SPR spectroscopy especially attractive for techniques allowing to estimate kinetics processes in real time mode, due to high speed of measurements and stability. From this point of view polysaccharides and also various organic polymer structures received on their basis, represent convenient objects for researches with SPR application. Taking into account thus dextrane ability to form with water solution the gel structure and opportunity to immobilize in such structures the biomolecules, similar matrixes have found wide application in biomonitoring . Modification or attachment of proteins or other molecules with synthetic and natural polymers can provide many benefits for both in vivo and in vitro application. Polymers with multivalent reactive sites can be used to couple numerous small molecules for creating pharmacologically active agents that possess long half-lives in biological systems. Similar complexes can be formed to create highly potent immunogens consisting of haptenpolymer conjugates for induction of an antibody response toward the hapten. These polycomplexes provide a high level of immunological protection, which is important for immunization and vaccine production. They have been used as hapten carriers to elicit an immune response against coupled antigen molecules and also as multifunctional linkers to cross-link monoclonal antibody conjugates with chemotherapeutic agents. Many polymers have been studied for their usefulness in producing pharmacologically active complexes with proteins or drugs. Synthetic and natural polymers such as polysaccharides, poly(amino acids), poly(vinyl alcohols), polyvinylpyrrolidinones, poly(acrylic acids) derivatives have been coupled with a diversity of substances to explore their properties. Modified polysaccharides are of great interest as the carriers of antigens. One of them most often used in these applications is dextran. This polymer consists of repeating units of a single monomer-glucose. Dextran can be activated through its hydroxyl groups by a number of chemical
Fig. 1. SPR kinetic curves of adsorption of polysacchairdes to BSA layer previously formed on the gold surface (part 1). Desorption of polysaccharides in flow regime (part 2). 1 – dextran, 2 – aldehyde dextran sulfonate, 3 – aldehyde ethyl cellulose, 4 – dextran sulfonate.
methods that are efficient for coupling of other molecules. Dextran can be activated at multiple sites throughout its chain, since each monomer contains hydroxyl residues. Soluble dextran of molecular weight 10,000–500,000 has been used extensively as a modifying or cross-linking agent for proteins and other molecules. It has been used as a drug carrier to transport greater concentrations of pharmaceuticals to tumor sites in vivo, as a hapten carrier to elicit an immune response against coupled molecules, as a multifunctional linker to cross-link monoclonal antibody conjugates with chemotherapeutic agents and as a stabililizer of enzymes and other proteins. In represented work the opportunities of SPR on complex research of some linear polymers as matrixes with drug delivery functions are shown. As objects of study natural dextrane, dextran sulfate, aldehydedextrane and ethylcellulose were taken. For comparison of drug adsorption activity the binding kinetic for molecules of various polymers with molecules of bovine serum albumin (BSA) as model protein in buffer solution was observed (Fig. 1). To estimate the properties of various polysaccharides as drug holders the washing kinetic of BSA from polymer matrices was also registered.  V. Chegel, Yu. Shirshov, S. Avilov, M. Demchenko, M. Mustafaev. A novel aldehyde dextran sulfonate matrix for affinity biosensors. J. Biochem. Biophys Methods, 2002;50:201–216.
PO-133 IN VIVO APPLICATION OF MODIFIED RELEASE TABLETS CONTAINING KETOPROFEN LOADEDMICROSPONGES 2 ¨ ˘ 1 , A Savas¸er 2 , Y Ozkan ¨ ¨ 1 & T T C ¸ omoglu , N Gonul 1 1 Baykara – Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Tan˘ dogan, Ankara, Turkey; 2 Gulhane Military Academy, Department of Pharmaceutical Sciences Center, Etlik, Ankara, Turkey
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
Ketoprofen is a NSAI drug that has a short half-life (0.5–2 hours) and has a low single administration dose which makes ketoprofen a good candidate for the formulation of modified release dosage forms . For these reasons, microsponges were chosen to minimize the side effects and to provide the modified release of the drug. Microsponges were prepared by quasi-emulsion solvent diffusion method. By porosity, SEM, X-Ray, DSC and particle size analysis, drug content, production yield and in vitro release rate studies, the most suitable microsponge formulation was evaluated to prepare tablets. Tablets were composed of ketoprofen microsponges, Precirol ATO-5 and Flow-Lac 100 and prepared by direct compression method at 2000 kgf / cm 2 . In vivo studies were applied to healthy volunteers by using a cross over design with the prepared tablets and the commercially available retard tablets of ketoprofen. Serum levels of the drug were determined by HPLC. The pharmacokinetic parameters were determined from the obtained data. Tablets containing ketoprofen microsponges showed modified release of ketoprofen and yield longer ketoprofen serum concentrations than Profenid Retard .  Borsa M, Tonon GC et al. Pharmacokinetics of a slow release preparation of ketoprofen lysine in man. Arzneim. Forsch. / Drug Res. 1983;33(II), Nr. 10:1497– 1500.
PO-134 SOLUBILIZATION OF 17-b-ESTRADIOL IN CARBOPOL / SURFACTANT GELS Angel Concheiro, Rafael Barreiro-Iglesias & Carmen ´ FarAlvarez-Lorenzo – Dept. de Farmacia y Tecnologıa ´ maceutica, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain The aim of this work was to evaluate the capability of 0.25% Carbopol 934 gels containing small proportions of surfactant to solubilize hydrophobic drugs, such as 17-bestradiol. Usually surfactant concentrations above micelar concentration (cmc) are required to achieve a solubilization effect in aqueous medium. Carbopol / surfactant complexes, which appear at concentrations well below the cmc , present a low polar microenvironment that may favor the solubilization of hydrophobic drugs. In our study, 40 mg of 17-b-estradiol was added to 100 mL of 0.25% carbopol dispersions containing different proportions of Tween 80 or sodium dodecylsulfate (SDS). The dispersions were under stirring for three days, then centrifuged and the amount of estradiol solubilized was quantified spectrophotometrically at 280 nm (Fig. 1). The results clearly indicate that the total solubilization capability, obtained by fitting of Myrdal and Yawlkosky  eq., of the carbopol / Tween80 (k 50.0674) and carbopol / SDS (k 50.0345) gels were greater than in the case
Fig. 1. Solubility of 17-b-estradiol in surfactant solutions and carbopol / surfactant gels.
of the surfactant solutions (k 50.0064 and 0.0208, respectively). The amount of 17-b-estradiol solubilized opens the possibility of using these gels for therapeutical purposes. Acknowledgements: Xunta de Galicia, Spain (PGIDT 00PX120303PR and 01PX1203014IF).  R. Barreiro-Iglesias et al. Incorporation of small quantities of surfactants as a way to improve the rheological and diffusional behavior of carbopol gels. J. Controlled Release 2001;77:59–75.  P.B. Myrdal, S.H. Yalkowsky, Solubilization of drugs. In: Encyclopedia of Pharmaceutical Technology, Vol.18, J. Swarbrick and J.C. Boylan (Eds.), Marcel Dekker Inc., New York, 1996.
PO-135 SURFACE COATING OF TITANIUM DIOXIDE IN SUNSCREEN FORMULATIONS M A Dabbagh 1 , M A Shahtalebi 2 & M Moradmand Jalali 1 – 1 Ahwaz College of Pharmacy, Ahwaz Med. Sci. Univ.; 2 Esphahan College of Pharmacy, Esphahan Med. Sci. Univ. Among solar radiations, UVA and UVB are mainly responsible for cutaneous pathologies such as sunburn, pigmentation, photosensitivity, photoaging and skin cancer . Thus, topical sunscreens have become essential and extremely important in providing protection against the adverse effects of solar radiation. Titanium dioxide is recognized as a safe and effective sunscreen agent . Particle size and surface properties of TiO 2 are two key parameters that determine the sunscreen efficacy of titanium dioxide . The objective of this research was investigation of using water resistant coating materials on
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
titanium dioxide microfine and determination of SPF value of the product. In this research, two different approaches were used simultaneously to insure that the product is able to produce a sticky film on the skin, which is water-resistant. The uncoated pigment was subjected to one or more of series of operations. There are three general approaches to surface treatments, namely physical adsorption, chemical grafting and encapsulation . Then photocatalytic stability and particle size of the products were tested with direct and indirect methods. Numerous formulations were prepared from base of oil in water and water in oil emulsions and effective parameters of this research were determined and final formulations were designed. After preparing the final formulation, physical and chemical stability tests and determination of water resistance and SPF value of the product were done. According to the results, the product by chemical grafting was the most efficient in preventing the transmission of UV light (SPF57.93). The coating reduced the photo-reactivity of the product which otherwise was of reduced suitability for use, particulary in cosmetic preparation. The O / W emulsion with 5% titanium dioxide had ability to screen UV light and the W/ O emulsion with silicone content had a good water resistance.  Ramos, M.F.S. Preliminary studies towards utilization of various plant extracts as antisolar agents. Int. J. Cosmet. Sci. 1996;18:87–89.  Kobayashi M. and Kalriess W. Photocatalytic activity of titanium dioxide and zinc oxide, Cosmet. & Toilet., 1997;112:83–86.  Fairhurst D. Surface coating and the optimization of microfine oxides in sunscreen formulations. Cosmet. & Toilet. 1997;112:81–88.
PO-136 ADVANCED STRUCTURE-FUNCTION PROPERTIES OF ETHYLCELLULOSE: IMPLICATIONS FOR TABLET COMPACTIBILITY ¨ 1 , R Grasso 2 , W W Harcum 1 , S-F Lau 2 , R A T Durig Salzstein 1 & G W Skinner 1 – 1 Aqualon Division; 2 Analytical Service Division, Hercules Inc., Wilmington, DE 19808, USA The objective of the study was to understand the effects of ethoxyl content and molecular weight on ethylcellulose (EC) solid state properties at the molecular level, to enable development of a highly compactible type of EC without micronization, thus avoiding poor powder flow. A sample matrix (n534) with a wide variation in ethoxyl content (45.6 to 51.0%) and molecular weight (characterized by solvent viscosity, 7 to 190 cps) was analyzed by DSC, helium pycnometry and image analysis. Compactibility was determined on a rotary press with pure polymer tablets, pre-lubricated with 1% stearate. Selected
samples were further tested on a compaction simulator. Solid state interaction geometries and energetics were also studied by computational techniques (The Discover program). Higher ethoxyl content and lower viscosity were correlated with improved compaction, lower Tg and lower true density. No correlation was observed for particle size (45–100 mm mean length). All tested samples showed superior flow over commercial micronized EC. Compaction simulator tests further showed that while total work of compaction was similar, high ethoxyl content (.49.6%) and low viscosity (8–11 cps) EC underwent significantly less viscoelastic recovery (11–15% axial recovery) compared to other EC types (25–30% recovery). This allows lower porosity and higher crushing strength to develop. Computational modeling of two interacting EC chains showed higher binding energies for higher ethoxyl content due to a higher level of dispersion and dipole-induced dipole forces. These less directional van der Waals forces provide polymer flexibility, as compared to more localized, directional H-bonds, which predominate at lower ethoxyl content. In conclusion, a developmental grade of EC (high ethoxyl content, low viscosity) with good flowability and enhanced compactibility has been characterized. High ethoxyl content and low molecular weight provide greater free volume, increased local freedom of movement and overall higher binding energies.
PO-137 ETHYLCELLULOSE IN DIRECT COMPRESSION MODIFIED RELEASE TABLETS: IMPACT OF POLYMER STRUCTURE AND FORMULATION VARIABLES ¨ T Durig, W W Harcum & G W Skinner – Aqualon, a Division of Hercules Inc., Wilmington, DE 19808, USA The objective of the study was the evaluation of ethylcellulose (EC) grades of varying ethoxyl content, molecular weight (characterized as solvent viscosity), and particle size and the impact of formulation variables on directly compressible monolithic, modified release tablets. Binary blends of drug (phenylpropanolamine, propranolol, acetaminophen, theophylline or sodium diclofenac) and the selected EC grade or ternary mixtures of drug, EC and various directly compressible fillers were prepared in a V-blender. Stearic acid (1%) was added to the final blend, before compression on a rotary press. In addition to a developmental grade of EC with high ethoxyl (.49.6%) and low viscosity (8–11 cps), five other grades of EC were tested along with a commercial grade of micronized EC. For binary drug: EC combinations, the dissolution rate was inversely related to EC compactibility. Ethoxyl content and polymer viscosity markedly influenced this relationship. Improved compactibility and retardation of drug release was achieved with a developmental grade of EC with high ethoxyl content and low viscosity. In ternary
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
mixtures, release rate was influenced by the swellability and solubility of the filler. Insoluble, non-hydrating dicalcium phosphate significantly prolonged release. Soluble sugars (lactose, mannitol and sorbitol) showed intermediate release behaviour. Insoluble, yet swellable microcrystalline cellulose markedly accelerated release. Commercial, micronized EC provided greater release retardation. However, in binary mixtures or with further addition of flowable directly compressible fillers, micronized EC caused inadequate powder flow, resulting in large compaction force fluctuations (65 kN at nominal force of 25 kN). In conclusion, ethoxyl content and polymer viscosity are important factors in determining EC function in direct compression modified release tablets. A developmental grade of EC (high ethoxyl content and low viscosity) provides high compactibility and slower release rates while maintaining good manufacturability. Release and compactibility can be modulated by careful choice of fillers.
PO-138 IMPACT OF ETHYLCELLULOSE TYPE IN COMPRESSION COATED TABLETS: TIME-CONTROLLED PULSED-RELEASE DOSAGE FORMS ¨ T Durig, W W Harcum & G W Skinner – Aqualon, a Division of Hercules Inc., Wilmington, DE 19808, USA The objective of the study was the evaluation of a developmental grade of ethylcellulose (EC) for compression coating, where high ethoxyl content and low viscosity provide good powder flow, high compactibility and modulation of drug release. The study variables included ethoxyl content, polymer molecular weight (characterized as solvent viscosity), compaction force, coating level and core swellability (due to disintegrant incorporation). Dry blends of 87% theophylline, 0, 2% or 4% croscarmellose disintegrant and q.s. microcrystalline cellulose were prepared. Stearic acid (1%) was then added to the final blend. Immediate release tablet cores (100 mg) were compressed on a rotary press using 1 / 40 FFBE tooling. Cores were manually coated with EC powder in a 3 / 80 die cavity, then compressed under power at 5–25 kN on a rotary press. In addition to the high ethoxyl level (.49.6%) and low viscosity (8–11 cps) developmental grade, five other commercial and experimental EC grades were evaluated. It was found that drug release was triggered by circumferential failure of the EC coat, in ‘‘clamshell manner’’. Without disintegrant, non-swelling coated cores yielded prolonged lag times and prolonged, irregular or incomplete drug release. With 2% disintegrant, lag times were correlated with compactibility of the various EC polymers. The developmental grade was more compactible by .25% than other EC grades. This provided greater resistance to coating failure and provided a wide range of lag times (3–18 hours) and greater control when compared to the other EC grades (0–2 hour lag times). A near linear
correlation between compaction force and lag time ranging from 3 hours at 5 kN to 16 hours at 25 kN coat compression force was observed. Increased coating weight increased lag time. In conclusion, EC compactibility and core swellability were found to be critical variables. The developmental grade of EC achieved superior
PO-139 POWDER FLOW CHARACTERISTICS OF DIRECTLY COMPRESSIBLE HYDROXYPROPYLCELLULOSE MODIFIED RELEASE MATRIX SYSTEMS ¨ T Durig, K M Lusvardi, W W Harcum & G W Skinner – Aqualon Division, Hercules Inc., Wilmington, DE 19808, USA The effect of fillers on the powder flow and tablet weight variability of directly compressed hydroxypropylcellulose (HPC) based matrix systems  was studied. Dry blends of 19% phenylpropanolamine, 30% Klucel HXF hydroxypropyl cellulose and 50% filler of various types were prepared. Magnesium stearate (1%) was then added to the final blend. Powder flow properties were evaluated using the TSI Aero-Flow姠 Analyzer to measure the mean time between avalanches of powder flowing in a rotating drum. 400 mg tablets were compressed on a rotary press. Tablet weight uniformity was based on the relative standard deviation of the weight of 10 tablets. Using the TSI Aero-Flow姠 Analyzer, mean avalanche time of the powder blends correlated relatively well with tablet weight variability. Visual observation of the powder bed motion of all blends was typical of ‘‘slumping’’ flow, whereas those of the pure powders within the blends ranged from ‘‘rolling’’ to ‘‘cataracting’’ behavior. The blends which produced tablets with the lowest weight variability and shortest mean avalanche times contained high density silicified microcrystalline cellulose (SMCC), spray-dried lactose and granulated mannitol as fillers. Of these, the HPC formulation containing SMCC, yielded the most robust tablets with the greatest retardation in drug release (t80%5380 minutes). The tablets containing lactose and mannitol suffered from marginal release profiles and low hardness, respectively. Other fillers tested included standard MCC, sorbitol, maltose, calcium carbonate and dicalcium phosphate. It is concluded that by choosing appropriate fillers, fine particle HPC-based matrix systems can be optimized for superior flowability and low tablet weight variability. These attributes coupled with controllable release profiles, excellent crushing strength, and low friability make HPC a versatile, robust choice for directly compressible matrix systems.  Alderman D, Sustained release compositions comprising hydroxypropyl cellulose ethers. US Patent 1987, 4,704,285.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-140 ARTIFICIAL MEMBRANE PERMEATION OF A SERIES OF BENZODIAZEPINES G Ecker, C Gallhuber, R Lauer & C R Noe – Institute of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Wien, Austria Permeation of the blood brain barrier (BBB) is a crucial step for all drugs acting on the central nervous system. However, a lot of cns drug candidates fail due to improper uptake into the brain. Thus, a lot of effort is undertaken to get informations on the bioavailability of lead compounds as soon as possible in the drug development process. Despite in vitro cell culture based methods and in silico predictions based on calculation of logP values and polar surface areas, also a parallel artificial membrane permeation assay (PAMPA) was proposed recently  for measurement of permeability coefficients of cns drug candidates. PAMPA is based on the donor / acceptor chamber principle. A 96-well microtiter plate filled with buffer solution serves as acceptor chamber and a multiscreen filterplate impregnated with an artificial membrane material functions as donor chamber. Transport studies are carried out via transfer of a compound solution to the donor chamber and measurement of the concentration of the respective drug in the acceptor chamber after a given time interval using a plate reader. In our study we used a set of 11 benzodiazepines known as cns-active drugs and compared the results with those obtained with a cell based method. Investigation of different lipids for impregnation of the filter plate showed, that a mixture of lecithin and cholesterol dissolved in dodecane gives the most valuable results. Correlation of the results of the PAMPA assay with calculated logP values of the compounds gave a correlation coefficient of 0.6. However, no correlation was obtained between permeability coefficients measured in an cell based system and the clearance values obtained by PAMPA (r50.2). We gratefully acknowledge financial support by the Austrian Science Fund (P14582-CHE).  Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening: paralell artificial membrane permeation assay in the description of passive absorption processes. J Med Chem 1998;41:1007–10.
PO-141 TRANSPORT AND ENANTIOSELECTIVE CYP3A4-MEDIATED METABOLISM OF R /S-VERAPAMIL IN MODIFIED Caco-2 CELL MONOLAYERS Helena Engman, Christer Tannergren, Per Artursson & ¨ – Uppsala University, Dept of Pharmacy, Hans Lennernas Uppsala, Sweden The objective of this study was to investigate the passive and carrier-mediated intestinal permeability and CYP3A4mediated metabolism of R /S-verapamil with respect to stereoselectivity and dose dependency in modified Caco-2
cells , and to compare the results to historical data obtained from in situ rat , and in vivo human intestinal perfusions . Caco-2 cells were induced to express CYP3A4 by treatment with 1.25a-(OH) 2 vitamin D 3 (D3). Enantioselective HPLC analysis was used to quantify R /Sverapamil and R /S-norverapamil. The concentration dependent permeabilities in Caco-2 and rat jejunum were strongly correlated, but lower than that obtained in human jejunum. In accordance with human and rat data, R /Sverapamil was actively transported in the secretory direction in Caco-2 cells, while norverapamil was not. Neither the passive nor the carrier-mediated permeability was stereoselective in any of the three models. CYP3A4-mediated demethylation to R /S-norverapamil was stereoselective in Caco-2 cell monolayers. Thus, S-norverapamil was formed in amounts twice as high as those of R-norverapamil. Apparent Km and Vmax values for the conversion of R-verapamil were 0.72 mM and 3.22 pmol / min, respectively, and for the corresponding S-enantiomer 0.57 mM and 5.37 pmol / min, respectively. The amounts of R- and S-norverapamil formed in the modified Caco-2 cells were 29 and 19%, respectively, of those found in the human intestine and 34% and 35%, respectively, of those found in rat intestine. The enantioselective CYP3A4-metabolism found in Caco-2 cells was previously found in humans but not in rats, indicating that the Caco-2 cells better reflects the human situation in this regard. In conclusion, R /S-verapamil permeability profiles in modified Caco-2 cells closely mimics those observed in rats, but are lower than those in human intestine. In agreement with human studies, CYP3A4-mediated formation of norverapamil in Caco-2 is stereoselective, although the metabolic turnover of R /S-verapamil in intact Caco-2 cell monolayers was lower than in human and rat intestine. ¨ H, Taipalensuu J et al.  Engman H, Lennernas CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies. J Pharm Sci 2001;90:1736–1751. ¨ R, Lennernas ¨ H. Repeated oral rifampicin  Sandstrom decreases the jejunal permeability of R / S-verapamil in rats. Drug Metab Dispos 1999;27:951–955. ¨ R, Knutson TW, Lennernas ¨ H et al. The  Sandstrom effect of ketoconazole on the jejunal permeability and CYP3A metabolismof R / S-verapamil in humans. Br J Clin Pharmacol 1999;48:180–189. PO-142 INCORPORATION OF PLASMID DNA WITHIN PLGA MICROSPHERES FOR DNA BASED IMMUNIZATION 1 ¨ A Eratalay 1 , F F Arı 2 , M F Sargon 3 & F Oner – 1 Hacettepe University, Faculty of Pharmacy, Pharmaceu¨ tical Biotechnology Dept.; 2 Suleyman Demirel University, Molecular Biology Department, Isparta, Turkey; 3 Hacettepe University, Faculty of Medicine, 06100, Sıhhiye, Ankara, Turkey
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
Biodegradable poly (D,L-lactide-co-glycolide) (PLGA) microspheres have been extensively investigated as potential vaccine carriers [1,2]. In this study cationic DNA vaccine formulations were developed by using PLGA microspheres including plasmid vector encoding Hepatitis B virus envelope protein which is carrying IL-2 cytokine gene. The plasmid was propagated in E.Coli and purified by large-scale plasmid purification method. PLGA microspheres were prepared by using W/ O / W multiple emulsion solvent evaporation method and O / W emulsion solvent evaporation method. Two different plasmid DNA encapsulation methods were used when preparing O / W solvent evaporation method for PLGA microspheres. In the first formulation 10 mg plasmid DNA was added to aqueous phase of formulation during the microsphere preparation and in the second one 10 mg plasmid DNA was added to formulation after the microsphere preparation. Stability of plasmid DNA in the formulation was determined by using 0.8% agarose gel electrophoresis. DNA can be loaded into and onto microspheres without damage. The bands of extracted plasmid DNA and control plasmid DNA were observed properly. The size distribution analysis was performed using laser diffaction particle size analyzer. Avarage particle sizes of DNA loaded microspheres were determined as 38 mm for W/ O / W double emulsion solvent evaporation method and 20 mm for O / W solvent evaporation method. Surface morphology and spherical shapes of the microspheres were observed by scanning electron microscopy (SEM).  Kowalczyk D.W., Ertl H.C.J. Immune responses to DNA vaccines. CMLS 1999;55:751–770.  O’Hagan D.T., Jeffery H., Roberts, M.J.J. et al. Controlled release microparticles for vaccine development. Vaccine 1991;9:768–771.
PO-143 POLYMER-DRUG CONJUGATES TARGETED WITH ANTIBODIES ´ ´ 2 , M Kovar ´ˇ 2 , B T Etrych 1 , K Ulbrich 1 , M Jelınkova 2 1 ˇRıhova ´ ´ – Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky´ Sq. 2, 162 06 Prague 6, Czech Republic; 2 Institute of Microbiology, Academy of Sciences of the Czech Re´ ˇ ´ 1083, 142 20 Prague 4, Czech Republic public, Vıdenska In recent years we have demonstrated, that synthetic watersoluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) containing anti-cancer drug doxoru-bicin (DOX) and polyclonal and monoclonal antibodies provide a potential drug-delivery system facilitating specific drug delivery to model tumors or tumor cells in mice. In polymer-drug conjugates, drugs are attached to the nondegradable poly(HPMA) backbone via enzymatically degradable oligopeptide spacers or via pH-sensitive hydrazone bonds. Attachment of the targeting moiety
(antibody) to the polymer conjugates with DOX bound via oligopeptide spacers improved the antitumor activity of the conjugates. Here we describe the synthesis and properties of new structures of antibody targeted water-soluble polymer conjugates with DOX attached via hydrazone bonds. Three different structures of the antibody-targeted polymer conjugates were synthesized: one with the antibody bonded through periodate-oxidised saccharide units situated in the F C part of its molecule and two other conjugates of the iminothiolane (ITH)-modified antibody with polymers containing maleimide or pyridyldisulfide groups. Conjugation of oxidised antibody to the polymer bearing in side chains hydrazide groups resulted in a considerable preservation of the binding ability of the antibody to the antigen (70%), but the number of polymer chains attached to the molecule of antibody was limited. Conjugation of ITH-modified antibody to the polymer made it possible to attached a higher number of polymer chains to the antibody with 50% preservation of the binding ability of the antibody to the antigen. The cytotoxic effect of conjugates with ITH-modified antibody was more pronounced than that of the nontargeted conjugates and conjugates with oxidised antibody. The anti-cancer activity tests in vivo showed that the activity of the conjugates with ITH-modified antibody is superior to that of free DOX, nontargeted conjugates or conjugate targeted with oxidised antibody. Acknowledgment: This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (grants S5020101 and IAA4050201).
PO-144 THE EFFECT OF PANCREATIC JUICE AND BILE ON INTESTINAL ABSORPTION AND BIOAVAILABILITY OF DIGOXIN ION PIGS ¨ 1 , S G Pierzynowski 1 , J L L Evilevitch 1 , B Westrom 2 Valverde Piedra , K H Elwanger 3 , M Tatara 2 & H ¨ 4 – 1 Department of Cell and Organism Biology. Lennernas Animal Physiology, Lund University, Lund, Sweden; 2 Department of Animal Physiology, Agricultural University of Lublin, Lublin, Poland; 3 Faculty of Veterinary Science, University of Zimbabwe, Harare, Zimbabwe; 4 Department of Pharmacy, Uppsala University, Sweden The activity of various transport proteins (such as Pglycoprotein) in the enterocyteis assumed to be regulated by different factors, such as food intake, disease and other drugs. Our aim is to study if pancreatic and biliary secretion might act as an impulse for a short term regulation of the activity of the efflux proteins localised at the apical entercocyte membrane. The study was performed on pigs that had chronicle catheters to the pancreatic duct, the bile duct and jugular vein and a chronicle fistula in the duodenum. Digoxin was used as the model drug for Pgp activity and was give
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
intravenously and through the duodenal fistula at single dose of 10 mg / kg to the pigs. Repeated blood samples were taken which were assayed with an enzyme multiplied immuno test. Pharmacokinetic parameters were calculated by using noncompartmental analysis. The area under the plasma concentration-time curve (AUC) was calculated by the linear / logarithmic trapetzoidal rule. The same dos of Digoxin was administered to the duodenal fistula, either with the pancreatic juice (pj) and bile present, or with the pj and bile excluded for 12 hours before the experiment. The secretion was kept interrupted during the 8 hours after the administration. After iv dose the plasma AUC of digoxin was 25.4 mmol / l / hrs. After the duodenal administration to pigs with intact secretion the plasma AUC was 13.5 mmol / l / hrs. After the administration of the duodenal dose to pigs with interrupted secretion the plasma AUC of digoxin was 26.7 mmol / l / hrs. It suggests that the presence of bile and pancreas secretory products in the intestinal lumen play an important role for the intestinal absorption of digoxin.
PO-145 DIFFERENTIAL SCANING CALORIMETRY (DSC) TO INVESTIGATE THE COMPATABILITY OF CIPROFLOXACIN HCL WITH SOME EXCIPIENTS M Fatthy, M A Hassan & F A Mohammed – Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt The comparability between ciprofloxacin hydrochloride (CFX) and some excipients was evaluated by using differential scanning calorimetry (DSC). Physical mixtures, coground mixtures, compressed blends and kneaded mixtures were prepared to study the effect of sample manipulation. In addition, the samples of physical mixtures were also accelerated at 55 8C for three weeks to obtain more reliable conclusions. Different types of excipients that currently used in tablets and / or capsule formulations namely, calcium phosphate dibasic dehydrate (Emcompress), magnesium strearate, lactose, sorbitol, mannitol, croscarmellose sodium (Ac-Di-Sol), sodium carboxymethyl starch (Primojel), microcrystalline cellulose (Avicel PH 101, Emcocil) were examined. The DSC scans of CFX showed two endothermic peaks corresponding to a fusion process followed by degradation process. Based on the DSC thermograms, CFX appeared to interact with sorbitol, mannitol, croscarmelose sodium (Ac-Di-Sol), Primojel and microcrystalline cellulose (Avicel PH 101, Emcocil).
PO-146 APPLICABILITY OF CELLACTOSE 80 FOR DIRECT COMPRESSION ` A Ferlan, S Perc & A Puneuh Kolar – Research and ˇ ˇ cesta 6, 8501 Development Division, Krka, d.d., Smarjeska Novo mesto, Slovenia
Cellactose 80, designed especially for direct compression, is a spray-dried compound consisting of 75% alpha-lactose monohydrate (Ph. Eur. / USP-NF / JP) and 25% cellulose powder (Ph. Eur.) dry matter. Therefore it combines filling and binding properties of the two excipients, which have been synergistically combined providing better compression performance at lower cost. The aim of this study was to evaluate physical properties of compression mixtures and tablets of two different formulations which contain the same part of Cellactose 80. In the first formulation 2-methyl-1,2,3,4,10,14b-hexahydrobenzo [c] pyrazino [1,2-a] pyrido [3,2-f] azepine and in the second one 2-methyl-4-(4-methyl-1-piperazinyl)10H-thieno-[2,3-b] [1,5] benzodiazepine were used as low dose drugs. Flowability time, angle of repose, bulk volume, tapped volume, Hausner ratio and a / a ref. on compression mixture of each pilot batch were determined. Tablets were controlled of mass, thickness and hardness [1,2]. In order to check reproducibility of the physical properties of tablets, different tablet sizes and compression speeds for each formulation were tested. Different tablet shapes between formulations were used. We can confirm that compression mixtures, which contain Cellactose 80 as a filler-binder for direct compression, have good flowability properties and therefore provide high weight consistency at various compression speeds. Physical properties of the tablets were reproducible apart from size or shape of the tablets. It has excellent compactibility. According to our analitical results it provides high content uniformity of both low dose drugs in the tablets.  Wells JI. Pharmaceutical Preformulation. The Physicochemical Properties of Drug Substances. 1 st ed. Chichester: Ellis Horwood Limited, 1988.  Gibson M. Pharmaceutical Preformulation and Formulation. A Practical Guide from Candidate Drug Selection to Commercial Dosage Form. 1 st ed. Englewood: IHS Health Group, 2001.
PO-147 STUDY ON THE PIROXICAM RELEASE FROM PARAFFIN MATRIX MICROCAPSULES PREPARED USING DIFFERENT QUANTITIES OF ETHYLCELLULOSE AND EXCIPIENTS Domingos C Ferreira 1 , Bruno Sarmento 1 , Pedro M B Coelho 1 , M H Amaral 1 , F Veiga 2 , A Ribeiro 3 & J M Sousa ´ Lobo 1 – 1 Faculdade Farmacia da Universidade do Porto – 2 ˆ ´ Dep. Tecnologia Farmaceutica; Faculdade Farmacia da Universidade de Coimbra – Dep. Tecnologia Far3 ˆ ˆ ´ maceutica; Instituto de Ciencias da Saude-Norte – Dep. ˆ ´ Tecnologia Farmaceutica e Biofarmacia The aim of this work was to evaluate the influence of the quantity of ethylcellulose on the piroxicam release from
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
paraffin matrix microcapsules. The effect of adding coexcipients (magnesium stearate and dioctyl ftalate) was also evaluated. Three different quantities of ethycellulose (2.25 g, 2.5 g and 3.75 g) were used respectively in formulation 1, 2 and 3. For each formulation, a mixture of 250 mg of piroxicam, acetone and of respective amount of ethylcellulose were placed in a polytron homogenizer and stirred for 2 hours in a paraffin matrix. After this process, the mixture was filtered under pressure and the microcapsules obtained were washed with ciclohexane. In order to study the effect of adding excipients, 0.15 g of magnesium stearate was added to formulation 2 (Formulation 4) and 0.25 g of dyoctil ftalate was added to Formulation 3 (Formulation 5). For each formulation a representative sample was weighed and the piroxicam release profile was determined during 8 hours according to the USP specifications . After 8 hours Formulation 1 microcapsules had released 91% of their piroxicam content, Formulation 2 microcapsules had released 94% while Formulation 3 microcapsules had released 99%. In the same period of time the Formulation 4 microcapsules prepared using magnesium stearate released 41% of their piroxicam content while Formulation 5 microcapsules (prepared using dioctyl ftalate) released only 22%. The quantity of ethylcellulose used affects the piroxicam release profile from the paraffin matrix microcapsules in this way: The more ethylcelulose used the less piroxicam is released after 8 hours. Adding magnesium stearate or dioctyl ftalate to the microcapsules formula decreases considerably the piroxicam release. The microcapsules prepared using 2.25 g of ethylcellulose show a good release profile and can be used in the preparation of 8 hours extended release piroxicam formulations.  United States Pharmacopoeia 23, US Pharmacopoeial Convention, Rockville.
PO-148 STARCH BASED MELT-FILMS AND THEIR ENZYMATIC DIGESTIBILITY C Fertig & F Podczeck – The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK Drug targeting is an important requirement for efficient drug delivery. Numerous systems have been designed differing in material, drug release principle and rate as to comply with the unique properties of both the administered drug and the target site. Starch is a common component in staple food, and the 1,4-glycosidic linkages of the amylose fraction are naturally hydrolysed by a-amylases in the digestive system. Its fusion with a matrix former, such as glyceryl monostearate, yields a melt-film, which can be used as a bio-
degradable coating for solid dosage forms. Since starch degradation occurs mainly in the lower part of the intestine these films are of particular interest for drug delivery in the colon. The aim of this work was the application of starches of various amylose content in an unprocessed state for the use in films applied to colon delivery formulations. This approach deviates from the commonly held belief that in such films the amylose fraction has to be in a glassy state to allow enzymatic digestion . Therefore, a melting technique using native starch will not only allow the formulation of solvent sensitive drugs but will also eliminate the heat intensive step of transforming amylose into its glassy state prior to processing. Films were prepared using 7 starches of different amylose content ranging from less than 1% in waxy corn starch to up to 100% in purified corn starch (PURE). These were blended with the matrix former Dynasan 114 in ratios from 10 to 50% of starch. In order to obtain different film thicknesses, between 20 and 50 mm, calculated amounts of blends were applied onto cellulose nitrate filter membranes. The latter were chosen to mimic the surface of a solid dosage form and yet to be able to test the films per se. The membranes were then exposed to 85 8C over 1 hour as to allow the film to form. The integrity of the films was determined by testing their permeability using a diffusion cell with norephedrine as the model drug. Subsequently, the films were incubated in a-amylase (B. licheniformis) at 37 8C overnight after which their integrity was reassessed – this time for an increase in permeability due to digestion of the amylose in the starch. Highest permeabilities were achieved for films of 20% starch content and 30 mm thickness for all 7 starches, which was particularly surprising for the low-amylose starches waxy and corn starch. Hence, the marked digestion of these two starches showed that the amylose content in the starch is not critical for starch degradation in the colon. In the next set of experiments the films were tested with 3 more a-amylases (B. liquefaciens, human saliva, hog pancreas), which are similar to those encountered in the human digestive system, as well as in porcine pancreatin. a-amylase from hog pancreas showed highest digestibility
Fig. 1. Degree of enzymatic digestion expressed as maximum permeability of films after 120 min.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
in most cases whereas that of pancreatin is due to the digestion of the lipid matrix glyceryl monostearate. It can be concluded that the glassy state of amylose is not required for a-amylase digestion. The results showed that the hydrolysis of the scarcely present 1,4-glycosidic linkages in the non-amylose fraction of starch is sufficient to increase film permeability by enzymatic digestion. However, the observed digestibility of the lipid matrix will require the film to be protected against lipases by means of a lipase-resistant pre-coating if used in colon drug delivery.  Milojevic, S., Newton, J.M., Cummings, J.H., Gibson, G.R., Botham, L.R., Ring, S.G., Stockham, M., Allwood, M.C. J Control Release 1994;38:75–84.
PO-149 DEVELOPMENT AND CHARACTERIZATION OF BIOADHESIVE NATAMYCIN VAGINAL TABLETS Gamal El-Din Abdel Fattah El-Gindy, Ahmed Mostafa El-Sayed, Abdel-Razzak Abdel-Magied & Ghareb Mohamed Abdel-Aal – Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Bioadhesive vaginal tablets of natamycin were prepared using different bioadhesive polymers such as carbopol 971p, carbopol 974p, hydroxy propylmethyl cellulose and sodium carboxymethyl cellulose. Soluble excipients like polyethylene glycol 4000 and 6000 and lactose were added to the tablets to increase the release rate of the drug. Preformulation studies were carried out on binary mixtures of the drug and different carrier systems using DSC and IR techniques to ensure compatibility between the drug and different carrier systems. The prepared tablets were evaluated for drug content, hardness, friability and in vitro drug release. The swelling behaviour of the tablets was investigated in citrate buffer pH 4.5. The bioadhesive strengthens of the tablets were evaluated using cow vagina as the model membrane and were found to increase with increasing the concentration of the bioadhesive polymers. Stability studies on the final formulations were carried out to determine the effect of the presence of formulation additives on the stability of the drug and also to determine the physical stability of the formulations under accelerated storage conditions of temperature.  Gursoy A., Sohtorik I., Uyanik N. and Peppas N.A. S.T.P. Pharma, 1989;5(12):886–892.  Ceschel G.C., Maffi P., Lombardi Borgia S., Ronchi C. and Rossi S. Drug Dev. Ind. Pharm. 2001;27(6):541– 547.  Genc L., Oguzlar C. and Guler E. Pharmazie, 2000;55(4).
PO-150 EVALUATION OF BSA LOADED PLGA MICROSPHERES PREPARED BY DOUBLE EMULSION W/ O / W METHOD; INFLUENCE OF THE FORMULATION VARIABLES K Goracinova 1 , K Mladenovska 1 , A Porjazoska 2 & M Cvetkovska 2 – 1 Ss. Cyril and Methodious University, Faculty of Pharmacy, Vodnjanska 17, 1000, Skopje, Macedonia; 2 Ss. Cyril and Methodious University, Faculty of Technology and Metallurgy, Ruger Boskovic 16, 1000 Skopje, Macedonia Series of DL-PLGA and L-PLGA copolymers were prepared by bulk ring opening polymerization of lactide / glycolide and were characterized by GPC, FTIR, 1 H-NMR and DSC analyses. Polymers with different molecular mass and lactide / glycolide ratio were used to prepare BSA loaded microspheres by double emulsion w / o / w method [1,2]. The influence of some preoperative variables such as the type of polymer (different molecular mass), the polymer concentration in the dispersed phase, the volume of the dispersed phase, the concentration of the stabilizing agent (polyvinyl alcohol) and the viscosity of the external and internal phases of dispersion, have been investigated in order to optimize the process. All formulations were spherical and porous (SEM, Hitachi S-450), while the mean particle size (in a range of 2 to 94 mm), yield (up to 65%), drug content (up to 9% vs. theoretical 10%), and loading efficiency (up to 35%) were influenced by the formulation variables. Biodegradation data of blank microspheres suggest that the particle size, as well as crystallinity and the molecular mass of the copolymers dominantly influence the rate of polymer degradation. In the in vitro release assay, BSA showed polyphasic release profile. Following the initial burst release, there was a phase of continuous much slower release at DL-PLGA microspheres. At L-PLGA microspheres after the initial release, a phase of very low or no release was noticed, followed by the constant BSA release due to the higher crystallinity of the polymer and thus slower biodegradation rate.  Uchida T, Yoshida K, Ninomiya A, Goto S. Optimization of preparative conditions for Polylactide (PLA) microspheres containing ovalbumin. Chem Pharm Bull. 1995;43 (9):1569–1573.  Painbeni T, Venier-Juliene MC, Benoit JP. Internal morphology of poly(DL-lactide-co-glycolide) BCNU loaded microspheres. Influence on drug stability. Eur J Pharm Biopharm 1998;45:31–39.
PO-151 A NEW PRINCIPLE FOR PULSATILE DELIVERY OF PLASMID DNA TO EPITHELIAL TISSUES IN VIVO ¨ ¨ ˚ ¨ H Guan, M Koping-Hoggard, K Regnstrom, R Ek & P Artursson – Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
The aim of the present project is to improve the delivery of pDNA to epithelial tissues, specifically to the nasal epithelium. For this purpose, microcrystalline cellulose particles were hydrolyzed, spray-dried and sieved to give particles with an average diameter of about 30 mm. pDNA encoding for the reporter genes luciferase or beta-galactosidase was then adsorbed to the surface of the porous cellulose particles. In vitro studies showed that the pDNA was released from the cellulose powder in a pulsatile manner and more than 70% of the plasmid was released within the first 2 min. The expression of the reporter genes were studied in mice after intranasal administration of the dried cellulose powder formulation. Naked DNA in free form, formulated with dried lactose powder or as PEI polyplexes were used as controls. Kinetic studies using luciferase as a reporter showed that intranasal administration of the cellulose formulation gave a transient gene expression with a Cmax of about 500 pg / mg protein already after 24 h and a duration of at least 5 days. X-gal staining showed that the cellulose formulation was highly effective as compared to the controls. Surprisingly, naked DNA and the PEI-formulation, gave no X-gal staining at all. These results were confirmed using a luciferase reporter. We also studied the immune response to the beta-galactosidase reporter both at the mRNA and protein levels. Many genes involved in the immune response were upregulated after administration of the cellulose formulation as compared to the naked DNA formulation. This was confirmed at the protein level in spleenocyte cultures from the immunized animals. Restimulation of the cultures using a beta-galactosidase specific peptide resulted in strong induction of IFN-gamma only in spleenocytes from mice immunized with the cellulose formulation, indicating a strong induction of the cellular immune response. In conclusion, our new particulate delivery system for intranasal administration outperformed one of the most efficient non-viral gene delivery systems, PEI polyplexes. Moreover, our immunization experiments suggest that the cellulose particles are promising as vehicles for DNA vaccines.
PO-152 THE IMPORTANCE OF USING PHYSIOLOGICALLY RELEVANT SOLUBILITY VALUES FOR PREDICTING THE IN VIVO ABSORPTION BEHAVIOUR FOR POORLY SOLUBLE DRUGS G Hanisch, E S Kostewicz, A S Carlsson & B Abrahamsson – Biopharmaceutics, AstraZeneca R&D, S-431 83 ¨ Molndal Sweden In the initial characterisation of a new compound, solubility measurements to predict in vivo absorption behaviour have previously been performed in simple buffers in the pH 2–7 range. However, human small intestinal fluid (SIF) is the media of choice, but due to the high costs and limited availability, it is of interest to examine more
simpler and easily accessible alternatives. FaSSIF media has been recently suggested as an alternative to fasted SIF for solubility studies. The aim of the present study was to test the hypothesis that solubility determinations in simulated and real intestinal fluid quantitatively predicts the plasma profile of a model BCS class II basic compound by use of the computer program GastroPlus . The resulting solubility value in FaSSIF closely resembled the value determined in SIF, however in the plain buffer at similar pH, the value was significantly lower. Due to the low solubility determined in the buffer, in vitro studies were performed using a model recently developed  to examine the likelihood for precipitation in the proximal fasting small intestine. Results indicated no significant precipitation. The measured solubility values and the results from the precipitation study were then used to simulate the plasma profile using GastroPlus . The simulations using the solubility and precipitation results from both SIF and FaSSIF most closely resembled the plasma profile obtained in a bioavailability study in man. In conclusion, it is more useful to use physiologically relevant GI media to measure solubility in order to perform in vivo predictions. Furthermore, for this model BCS class II compound, FaSSIF can be substituted for real human fasting intestinal juice for solubility measurements.  Kostewicz ES et al. In vitro model to predict the precipitation of a poorly soluble weak base following its transition out of the stomach and into the intestine. AAPS Pharm Sci 1999;Suppl 1.
PO-153 FORMULATION AND EVALUATION OF CIPROFLOXACIN HYDROCHLORIDE AND NORFLOXACIN TOPICAL GEL M A Hassan 1 , F A Mohammed 1 & F A Sabour 2 – 1 Pharmaceutical Department, Faculty of Pharmacy, Assiut University; 2 Microbiological Department, Faculty of Medicine, Assiut University Topical gel formulations containing Ciprofloxacin Hydrochloride and Norfloxacin for the treatment of skin infections were made. These antibiotics were not systematically absorbed, when applied locally. Different Gel bases were used for the preparation of antibiotics gel and evaluated for its release rate. To evaluate the clinical use of ciprofloxacin hydrochloride and norfloxacin as topical gel formulation, different gel bases were used include carboxymethylcellulose, pluronic acid, carbapol 934, and o / w cream. These formulation were evaluated for drug release from cellophane membrane,skin permeability through hairless mice skin and antibacterial and antifungal activity. Different concentration of the drug were also used (0.25%, 0.5%, 1%, 2%). The results revealed that maximum drug release obtained from CMC.Pluronic acid.Carbapol.o /
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
w cream respectively for both drugs. Also the results indicated that the release rate depend on the type of the drug and its concentration as well as the type of the base. The antibacterial activity of ciprofloxacin hydrochloride and norfloxacin in the prepared gel bases were tested against Pesudomonous, Staph areaus, and salmonella using the agar diffusion method. Results indicating excellent activity against all the tested microorganisms. The inhibition zone was dependent on the type of the gel base and concentration of the drug.Norfloxacin showed more pronounced inhibition when compared with ciprofloxacin hydrochloride aginst all the tested microorganisms. The antifungal activity was tested against candida albicans. The results obtained based on the inhibition zone indicated that both antibiotics exhibited excellent antifungal activity. In conclusion, the authors recommended the two antibiotics at 0.5% and 1% w / w as topical antibacterial and / or antifungal gels.
PO-154 AZD3582, A COX-INHIBITING NITRIC OXIDE DONATOR (CINOD), SHOWS GASTROINTESTINAL PROTECTIVE EFFECTS IN THE RAT ˚ ¨ Anneli Hallgren ¨ ¨ Janet Hoogstraate, Erica Astrom, & Goran ¨ ¨ ¨ ¨ ¨ Ojteg – AstraZeneca R&D Sodertalje, S-151 85 Sodertalje, Sweden The objective of this study was to compare the effects of orally administered AZD3582 [4-(nitrooxy)butyl-(2S)-2(6-methoxy-2 naphthyl)propanoate], a cyclooxygenase-inhibiting nitric oxide donator (CINOD), and naproxen on the gastrointestinal (GI) tract of rats. AZD3582 or naproxen (doses of 0–120 mmol / kg b.i.d. as an oil-in-water emulsion) or vehicle, were administered for 4.5 days. The abdomen was opened and the presence and degree of ascites and adhesions were noted, as were the number of perforations along the GI tract. Segments from the stomach, duodenum, jejunum and ileum were removed and prepared for light microscopy. Four to five representative micrographs were chosen from each GI segment and given an integrity score (0–5, where higher scores indicate less damage) by a single-blind assessment. Body weight was recorded once daily throughout the study period. Blood samples were taken from each rat prior to examination to determine the haematocrit. Naproxen at doses $60 mmol / kg caused statistically significant impairment of body weight gain and decreased haematocrit compared with the vehicle-treated group. Macroscopic examination of the GI tract revealed the presence of ascites, adhesions and visible perforations of the small intestine in rats treated with naproxen at doses $60 mmol / kg. Neither AZD3582 nor vehicle caused any visible intestinal damage, except at the highest dose of AZD3582 (120 mmol / kg). At a naproxen dose of 100 mmol / kg, integrity scores for the intestinal samples were between 1.5 and 2.5, indicating substantial damage to the
mucosa. All doses of AZD3582, including the 120 mmol / kg dose, resulted in high integrity scores, suggesting that AZD3582 spares the GI mucosa. The findings of this study confirm observations in clinical studies that naproxen is associated with GI damage. The superior GI integrity observed with AZD3582 may be attributable to its multi-pathway mechanism of action.
PO-155 THE GASTROINTESTINAL STABILITY AND PERMEABILITY OF AZD3582, A COX-INHIBITING NITRIC OXIDE DONATOR (CINOD) ˚ ¨ 1 , Kirsti Gjellan 1 , Stellan Janet Hoogstraate 1 , Erica Astrom 1 Swedmark , Margareta Diczfalusy 2 , Stefan Alexson 2 & ¨ ¨ Urban Fagerholm 1 – 1 AstraZeneca R&D Sodertalje, S-151 ¨ ¨ 85 Sodertalje, Sweden; 2 Karolinska Institute, 14186 Huddinge, Sweden The objective of this study was to determine the fate of AZD3582 [4-(nitrooxy)butyl-(2S)-2-(6-methoxy-2 naphthyl)propanoate], a COX-inhibiting nitric oxide donator (CINOD) developed for the treatment of acute and chronic pain and inflammation, in the gastrointestinal (GI) tract during oral administration to rats and humans. AZD3582 was formulated as an oil-in-water emulsion and incubated in fluids and mucosal scrapings from the rat duodenum, jejunum, ileum and colon, and in human gastric and jejunal fluid. Hydrolysis of AZD3582 at various concentrations was investigated in samples from the incubation media with and without BNPP (an inhibitor of esterase activity). Samples were taken during a 2 h incubation at 37 8C and analysed by HPLC-UV for the level of AZD3582 and its active metabolite (S)-2-(6methoxy-2 naphthyl) propanoic acid (naproxen). Western blot analysis, using antibodies against esterases, was performed on the GI protein samples. Ussing chamber experiments with rat intestinal tissue were performed to determine transport across the small intestine. During 3 h incubation at 37 8C, samples were taken from the receiver compartment and analysed for content of AZD3582 and naproxen. Parallel experiments were performed with naproxen. Transport was investigated in both directions to determine if AZD3582 is a substrate for transport proteins. The half-life of AZD3582 in the GI environment of rats was between 0.6–6 h, shortest in the duodenal fluid and mucosa and longest in ileal fluid. AZD3582 was stable in human gastric fluid (half-life: 1163 h) and less stable in human jejunal fluid (half-life: 362 h). Hydrolysis of AZD3582 into naproxen was inhibited by BNPP, indicating that it is mediated by carboxylesterases in these tissues. Highest esterase activity was seen in the duodenum and colon tissues from the rat. AZD3582 concentrations could only be measured in the donor compartment of the Ussing chamber since it was hydrolysed before and during absorption and resulted in metabolite at the receiver side. The
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
resulting permeability for the metabolite was 7.063.23 10 26 cm / s. The permeability in the opposite direction was similar. Naproxen has a high passive intestinal permeability of 26060.82310 26 cm / s. AZD3582 is partially hydrolysed into (2S)-2-(6methoxy-2 naphthyl)propanoate before absorption in rat and to a lesser extent in humans. This hydrolysis occurs via esterases. The extent of hydrolysis allows for part of the dose to be absorbed as the highly permeable (2S)-2-(6methoxy-2 naphthyl)propanoate and as the intermediately permeable AZD3582, thereby allowing distribution of AZD3582 to target therapeutic sites. AZD3582 is not a substrate for transport proteins.
PO-156 A MULTIVARIATE DATA ANALYSIS APPROACH TO INVESTIGATE THE IMPORTANCE OF RHEOLOGICAL PARAMETERS FOR MUCOADHESION OF POLYMER GELS ¨ ¨ ¨ & Katarina EdsHelene Hagerstrom, Christel Bergstrom man – Department of Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden In this study a series of 24 polymer gels were investigated with respect to their rheological behaviour and their mucoadhesive properties. The rheological diversity was identified with principal component analysis (PCA) . The mucoadhesive properties were assessed using a tensile strength method, utilising a texture analyser and porcine nasal mucosa . As an indicator of the mucoadhesive strength the tensile work was used, recorded as the area under the force–distance curve. The cohesiveness of the gels and the mucus layer was assessed in a similar way . Partial least square projection to latent structures (PLS)  was performed to investigate important descriptors for the mucoadhesion measurements. Two principal components were extracted when analysing the rheological properties. The selected series of gels did not contain any outliers. For some gels the tensile work of the mucoadhesion (tw mucoad ) did not differ from the cohesive works of the gel (tw gel ) and the mucus (tw muc ). For others the tw gel and the tw mucoad were approximately the same, but higher than the tw muc , implying that a strengthening of the mucus had occurred. Further, for a few gels, the tw mucoad was not only higher than the tw muc , but also lower than the tw gel , indicating that the failure recorded really occurred in the strengthened mucus layer. In the PCA score plot these gels were located in the right half, whereas the ones giving no strengthening were located to the left. To identify descriptors important for the tw mucoad a PLS analysis of the difference between tw mucoad and tw gel (D) was performed. The PLS loading plots showed that for most of the gels the rheological properties of the gels had the same influence on D. However, G9 and G0 also contained specific information needed to explain D. Furthermore, the deformation parameters from the
tensile measurements were also important descriptors. We concluded that multivariate data analysis seems to be a valuable tool for evaluation of properties important for tensile work related to the mucoadhesion of gels. The rheological parameters and the deformation properties of the gel proved to be important, one reason might be that the gel was often the weakest region of the system.  Jackson EJ. A users guide to principal components. 1991, New York: Wiley. ¨ ¨ H, Edsman K. Interpretation of mucoadhe Hagerstrom sive properties of polymer gel preparations using a tensile strength method. J. Pharm. Pharmacol. 2001;53:1589–1599. ¨  Hoskuldsson, A. PLS regression methods. J. Chemometrics 1988;2:211–228.
PO-157 THE EFFECT OF MOBILE ION SELECTION ON THE RELEASE OF SALICYLIC ACID AND 5CARBOXYLSALICYLIC ACID FROM ANION-EXCHANGE FIBER ¨ Kaisa Hanninen, Katri Lahtinen, Ann Marie Kaukonen & Jouni Hirvonen – Pharmaceutical Technology Division, and Viikki Drug Discovery Technology Center, Department of Pharmacy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland Ion-exchange fibers are an efficient way to control the release and permeation of drugs from transdermal drug delivery systems [1,2]. The release of drugs from ionexchange fiber is a stoichiometric process that makes this kind of drug delivery system easily controllable. When one charged molecule binds to the fiber, another charged molecule is released from the fiber. We have studied the effect of the choice of mobile ion (chloride (single charge), sulphate (double charge) and citrate (triple charge)) and its concentration on drug release from the anion-exchange fiber (viscose-g-trimethyl). Model drugs used in the study were salicylic acid (single charge) and 5-carboxylsalicylic acid (double charge). The drugs were bound to the fiber by immersing the fiber in the drug solution (0.5% (m / V), pH 7.4) two times consecutively. The amount of substance of the extracting ion was the same or ten times higher than the amount of substance of the drug bound to the fiber, the volume of extracting solution was constant (15 ml). The amount of drug released from the fiber increased with the number of charged groups in the mobile ion being the highest with citrate-ion and the lowest with chloride-ion. The released amount of drug from the fiber increased with a higher mobile ion concentration of all the extracting ions. The amount of salicylic acid bound to the fiber was about two times higher as compared to 5-carboxylsalicylic acid. The amount of released drug at 24 h and the release rate of salicylic acid from the fiber was also significantly higher as compared to 5-carboxylsalicylic acid. The choice and the
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
concentration of extracting mobile ion, and the number of charged groups in the drug have a profound effect on the drug release from the ion-exchange fiber. One can precisely control the release of drug from the fiber by selecting optimal extracting conditions for drug delivery.  Jaskari T, Vuorio M, Kontturi K, Urtti A, Manzanares JA, Hirvonen J. Controlled transdermal iontophoresis by ion-exchange fiber. J Controlled Release 2000;67:179–190.  Jaskari T, Vuorio M, Kontturi K, Manzanares JA, Hirvonen J. Ion-exchange fibers and drugs: an equilibrium study. J Controlled Release 2001;70:219–229.
PO-158 PERMEATION OF THE ATYPICAL NEUROLEPTICS AMISULPRIDE AND CLOZAPINE ACROSS AN ARTIFICIAL BLOOD–BRAIN- AND BLOOD–LIQUOR BARRIER 1 ¨ Sebastian Hartter , Amal Abou El-ela 2 , Peter Langguth 2 , 3 ¨ Sabine Huwel , Tina Lohmann, Christoph Hiemke 1 & Hans-Joachim Galla 3 – 1,2 Dept. of Psychiatry and Dept. of Pharmacy, University of Mainz, Mainz, Germany; 3 Dept. of Biochemistry, University of Muenster, Muenster, Germany The purpose of this study was to evaluate the disposition of the hydrophilic amisulpride (AMS) and the highly lipophilic clozapine (CLZ) and its main metabolite Ndesmethylclozapine (DCLZ) to their target structures in the CNS by applying an in vitro blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCB) based on monolayers of porcine brain microvessel endothelial cells (PMEC) or porcine choroid plexus epithelial cells (PCEC). Primary brain endothelial cells were isolated from porcine brain homogenate and cell monolayers were prepared . Epithelial cell monolayers from porcine choroid plexus were obtained by an established preparation . Transport studies through PMEC- and PCEC-monolayers were conducted for 60 min at concentrations of 1, 5, 10, and 30 mM applied to the donor compartment. Samples from the acceptor side were taken 15, 30, 45, and 60 min after application of the drug to the donor compartment. Drug concentrations were measured by HPLC. PMEC were almost impermeable for AMS (permeation coefficient, P, 1310 27 cm / s) in resorptive direction. Interestingly, a transport of AMS in the secretory direction was observed with a P (6SD) of 1.460.1310 25 cm / s. The resorptive P of CLZ and DCLZ were 5.061.1310 25 and 3.161.73 10 25 cm / s, respectively. For the permeation across PCEC in the resorptive direction a P of 4.363.3310 26 cm / s was found for AMS and a P of 274643 and 61624310 26 cm / s was calculated for CLZ and DCLZ, respectively. Both, CLZ and DCLZ could easily pass both barriers with a 5 fold higher permeation rate of CLZ at the BCEC. The permeation of AMS across the BBB is restricted partly due
to an efflux transport. It is, thus, suggested that AMS reaches its target structures mainly via transport across the BCB.  Franke H, Galla H-J, Beuckmann CT. Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood–brain barrier in vitro. Brain Res Protoc 2000;5:248–56.  Hasselbach M, Wegener J, Decker S et al. Porcine choroid plexus epitthelial cells in culture: Regulation of barrier properties and transport processes. Microsc Res Techniq 2001;52:137–52.
PO-159 DISSOLUTION FIT FACTORS AS RESPONSE VARIABLES IN EVALUATION OF INFLUENCE OF FORMULATION VARIABLES ON AGEING OF ASPIRIN EXTENDED RELEASE TABLETS Svetlana Ibric´ 1 , Milica Jovanovic´ 1 , Zorica D « uric´ 1 , Jelena 1 1 ˇ ´ , Marija Stanojevic´ , Slobodan D. Petrovic´ 2 & Parojcic Biljana Stupar 2 – 1 Dept. of Pharmaceutical Technology, Faculty of Pharmacy, Belgrade, Yugoslavia; 2 Hemofarm ˇ Yugoslavia a.d., Beogradski put bb., Vrsac, The objective of this study was to evaluate influence of % of Eudragit RS PO as matrix substance and compression pressure (i.e. tablets hardness), as independent variables, on changes in drug release profiles after one year of storage of aspirin tablets and to evaluate usefulness of dissolution fit factors in dissolution profile comparisons. Ten formulations of aspirin extended-release tablets were prepared, according to Central Composite design. Percent of Eudragit RS PO was varied from 2% to 6%, while tablet hardness was from 25 N to 90 N. Drug release from these formulations was investigated according to USP 23 (Test 2 – water, 1000 ml, 30 rpm) immediately after preparation and one year after storage in 20% RH and 20 8C in glass containers. Similarity ( f1 ) and difference factors ( f2 ) were used for dissolution profile comparisons . Preparation and optimisation of extended / controlled release tablets can be simplified by Artificial Neural Networks (ANN) . ANN is a learning system based on a computational technique which can simulate the neurological ability of a human brain and could be applied to quantifying a non-linear relationship between causal factors and pharmaceutical responses by means of iterative training of data obtained from a designed experiment. Generalised Regression Neural Network (GRNN) was applied in optimisation , using 6 neurones in second radial layer, Smoothing factor 0.1, and K-means algorithm. % of Eudragit RS PO and tablet hardness were selected as causal factors in regression network, while similarity ( f1 ) and difference factor ( f2 ) were outputs of the network. GRNN was trained using 10 training formulations, accord-
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
ing to Central Composite Design, while two test formulations were used for testing the network abilities. For two tested formulations there was very good agreement between the GRNN predicted and observed values of dissolution fit factors. Both similarity ( f1 ) and difference factor ( f2 ) demonstrate that there was no significant changes in dissolution profiles for formulations with tablet hardness greater than 60 N, while for formulations with tablet hardness smaller than 60 N, significant changes in dissolution profiles occurred. % of Eudragit RS PO had no effect on changes in drug release after one year of storage. This work illustrates that dissolution fit factors could be successfully used as response variables in stability studies.  Shah V.P., Tsong Y., Sathe P., Williams R.L. Dissolution profile comparison using similarity factor, f2 , Dissolution Technol. 1999;6:15.  Achanta A.S., Kowalski J.G., Rhodes C.T. Artificial Neural Networks: Implications for Pharmaceutical Sciences, Drug Dev. Ind. Pharm., 1995;21(1):119– 155.  Speckt D.F. A Generalized Regression Neural Network, IEEE Transactions on Neural Networks, 1991;2(6):568–576.
PO-160 FORMULATION AND DEVELOPMENT OF OCULAR IN-SITU GEL FORMING SOLUTIONS OF CHLORAMPHENICOL Kanchan Kohli, Rahul Jain & Asgar Ali – Department of Pharmaceutics, F / o Pharmacy, Jamia Hamdard, New Delhi-62, India Methods used: Fifteen different formulations of Methyl cellulose K4M and Carbopol 974P were prepared. Rheological studies were done by Brooks field viscometer . Dissolution studies were carried using Flow through apparatus. Mucoadhesion was studied using rabbit stomach mucosa tied on teflon blocks in between of which gel was placed . In vitro corneal permeation studies were carried out using goat cornea mounted in modified Franz diffusion cell . Results obtained: Out of different formulations prepared, formulations containing 1.5% Methylcellulose and 0.3% Carbopol was selected as optimized formulation. Viscosity of sol and gel at pH 4.7 and pH 7.4 at 37 8C were 2540 and 7580 cps respectively. Mucoadhesion was found to be 55 gms. In vitro dissolution showed 81% cumulative release of drug. In vitro transcorneal permeation showed a cumulative 78% drug release. Stability studies, according to ICH Guidelines, allow to give formulation a shelf life of 2 years. Conclusion: Formulation showed a great change in viscosity, hence,forming gel in eye, giving prolonged release with good corneal permeability as suggested by In
vitro transcorneal permeation studies. Drug release is also prolonged as formulation showed excellent mucoadhesion. Lastly, Stability studies showed that formulation prepared is stable up to a period of 2 years.  Edsman K, Harju K et al. Rheological evaluation and ocular contact time of some carbomer gels for ophthalmic use. Int. J. Pharm. 1996;137:233–41.  Saettone M, Chetoni P et al. Evaluation of mucoadhesive properties and in vivo activity of ophthalmic vehicles based on hyaluronic acid. Int. J. Pharm. 1989;51:203–4.  Malhotra M, Majumdar D K. In vitro transcorneal permeation of ketorolac from oil based ocular drops and ophthalmic ointment. Indian J. Exp. Bio. 1997;15:1324–30.
PO-161 N-IN-ONE PERMEABILITY STUDIES FOR INCREASED THROUGHPUT IN Caco-2 CELLS Ann Marie Kaukonen 1,2 , Leena Laitinen 1,3 , Heli Kangas 4 , Kati Hakala 1 , Tapio Kotiaho 1 , Risto Kostiainen 1,4 & Jouni Hirvonen 1,2 – 1 Viikki Drug Discovery Technology Center, Department of Pharmacy, University of Helsinki, Helsinki, Finland; 2 Division of Pharmaceutical Technology, Department of Pharmacy, University of Helsinki, Helsinki, Finland; 3 Division of Biopharmaceutics and Pharmacokinetics, Department of Pharmacy, University of Helsinki, Helsinki, Finland; 4 Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Helsinki, Helsinki, Finland In order to increase the throughput of Caco-2 cell permeability studies, the use of n-in-one cocktails of up to 10 compounds was evaluated. Compounds transported via both passive (antipyrin, midazolam) and different active mechanisms were chosen, with potential interactions regarding both efflux (verapamil, propranolol, hydroxyzine, timolol, buspirone, procaine) and active transport (naproxen, ketoprofen). Permeability experiments were performed at 50 mM concentration with single compounds and cocktails of 5–10 compounds (total concentration 0.25–0.5 mM) both in the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) direction. Compounds were quantified utilising an LC-ESI / MS / MS method . In most cases, individual permeabilities in cocktails correlated well with those obtained with single compounds. All basic compounds exhibited higher permeabilities in the B-A direction, while Papp values for the two acids were higher in the A-B direction. Part of the polarized permeability behaviour of both bases and acids could, to varying degrees, be contributed to the pH gradient present during experiments (pH 5.5 in the apical and pH 7.4 in the basolateral chamber). Naproxen and ketoprofen showed decreased A-B permeabilities when present in cocktails, indicating a shared active transport
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
mechanism. Overall, the use of cocktails caused only minor changes in the ranking and / or classification of compounds, showing that cocktails could be used to obtain permeability data in a more time-efficient way.  Kangas H, Hakala K, Kotiaho T et al. N-in-one analysis of Caco-2 absorption kinetics by LC-ESI / MS / MS. Drug Analysis 2002 Symposium, 21–25 April 2002, Bruges, Belgium.
PO-162 DEVELOPMENT AND EVALUATION OF BUCCOADHESIVE TABLETS OF AMOXYCILLIN FOR THE TREATMENT OF PYORRHOEA K Kohli – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India Buccoadhesive tablets of amoxycillin were prepared each containing 25 mg. of the drug along with polymers HPMC K15M in various combinations i.e. 1:2, 1:4, 2 3, 3:2. Invitro dissolution studies were carried out in modified version of Woods intrinsic dissolution apparatus, invitro permeation studies  were carried out in Franz diffusion cell, insitu release studies were carried out in flow through apparatus simulating the in-vivo conditions. Among all the formulations, formulation containing drug and polymer in ratio 2:3 exhibited the maximum drug release of 90% in in vitro experiments over 6 hours. The drug permeation across the bovine cheek pouch followed ist order permeation profile and only 2.28% of the drug permeated in 6 hours. In situ release studies revealed that drug concentration remained well above the MIC of the drug (1–10 mcg / ml). Drug was found active in the formulation against bacteroid fragilis and fusobacterium. The correlation between cumulative percent drug release and % wet weight recovered was found to be 0.9432 which suggested that swelling is a potential factor for drug release. In conclusion drug could be successfully loaded in the buccoadhesive delivery system.  Jimenez C, Zia M, Rhodes C. Muccoadhesive drug delivery systems. Drug Dev Ind Pharm 1993;19(1&2):143–194.
PO-163 DEVELOPMENT AND EVALUATION OF BUCCOADHESIVE FILMS OF SECNIDAZOLE K Kohli, S Arora, A Ali & A Arora – Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India The system was designed to ensure intimate contact of the drug with the oral mucosa for prolonged period of time. In-vitro dissolution rate studies were carried out in a modified version of Woods intrinsic dissolution apparatus. For carrying out the in-vitro permeation studies, a modified
version of a ‘‘Franz Diffusion Cell’’ was used. In order to carried out the in-situ release studies, a ‘‘Flow through apparatus’’ simulating the in-vivo conditions was designed in order to established the in-vitro in-vivo correlation both for the release studies and for the measurement of duration of bio-adhesion / erosion. The optimized formulation was then subjected to various evaluation tests including & finally subjected to stability studies. Among all the formulations developed formulation containing CP-934P, HPMC-E15 and Eudragit NE-30 exhibited the maximum drug release in vitro i.e. 92.7%. The drug release from the buccoadhesive films occurred for a period of 90 minutes. A maximum in-situ drug concentration of 9 mg / ml. was obtained. In-vitro skin permeation studies revealed only 2.28% of drug permeated in 90 minutes. Stability studies showed no significant changes in drug content, polymers, plasticizer and solvents required for the formation the stable films.  Jimenenz C., Zia, H. and Rhodes C.T. Muco-adhesive drug delivery systems, Drug Dev Ind Pharm, 1993;19(1&2):143–194.  Ishida M., Nambu N., and Nagai T. Mucosal dosage form of lidocaine for toothache using hydroxy propyl cellulose and Carbopol, Chem Pharm Bull 1982;30:980–984.
PO-164 RELEASE PROPERTIES OF DIFFERENT DRUGS FROM STARCH ACETATE MATRIX TABLET Ossi Korhonen 1 , Pauliina Ahokanto 1 , Petteri Paronen 1 , Mika Vidgren 2 & Jarkko Ketolainen 1 – 1 Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN70211 Kuopio, Finland; 2 Polymer Corex Kuopio Ltd., Sammonkatu 10, FIN-70500 Kuopio, Finland The aim of the study was to investigate how starch acetate (ds. 2.7) works with physicochemically different model drugs (propranolol, diltiatzem, verapamil, theophylline, ibuprofen, atenolol, diclofenac and metoprolol) and how certain formulation and process factors affect drug release properties. The drug release mechanisms and kinetics were also studied. The weighed amounts of drug (50 mg) and starch acetate were blended and the blends (15, 20 and 25% (w / w) of drug) were compressed at the force of 20 kN. Blend 20% (w / w) was also compressed at the forces of 15 and 25 kN. Drug release from the matrix tablets was evaluated using rotating basket dissolution method at 100 rpm. The dissolution medium (USP SGF and SIF without enzymes) of 900 ml was maintained at 137 8C. The amount of dissolved drug was analysed by an HPLC assay. Dissolution data were fitted using different release equations. Results pointed out that the main drug release mechanism from the starch acetate matrix is diffusion. The relaxation of matrix structure was observed to be negligible. The increased amount of starch acetate, higher
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
compression force and lower drug water solubility decreased the Higuchi rate constant. The results were in good agreement with diffusion-controlled Higuchi theory , where the initial concentration of drug, solubility of the drug, porosity and tortousity are the factors that affect the release kinetics of drugs from the matrix. As a conclusion, starch acetate controlled the release of physicochemically different model drugs from the matrix tablets. However, the release rates of different model drugs differed from each other depending on their water solubility. Also, the formulation factor (initial concentration of drug in the matrix) and process factors (compression forces and subsequent porosity and tortousity) affected the release kinetics in the diffusion-controlled release system.  Higuchi T. Mechanism of sustained-action medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J. Pharm. Sci. 1963;52:1145–1149.
PO-165 THE APPROACHES TO CREATION IMMUNOLIPOSOME – DRUG FORMULATION M Kortava 1 , N Oborotova 1 , A Polozkova 1 , A. Surovoy 2 , T Zabotina 1 , O Orlova 1 & A Baryshnikov – N.N. Blokhin Memorial Cancer Research Center, 24, Kashirskoye sh., 115478, Moscow, Russia; 2 Institute of Bioorganic Chemistry, 16 / 10, Miklukho-Maklaya st., 117997, Moscow, Russia The investigation of new more efficient methods of the drugs delivery to target tumors, is an important present tendency in oncology. The immunoliposomes are the most promising way in the investigation of antitumor agents delivery methods. This research aim to obtain immunoliposomes using the high affinity of streptavidin to biotin [1,2]. The liposomes were composed of egg phosphatidilcholine, cholesterol, cardiolipin (12:6:1) and biotinilated PEG. The liposomes were prepared using sonification after vacuum evaporation of spirituous lipids solution and hydration dry lipid films in PBS pH 7,4 buffer. Rhodamin was used as dye of liposomes. The liposomes were passed through 100 nm polycarbonate membranes to give primarily unilamellar vesicles of approximately 100 nm in diameter . To target tumor tissue anti-CD5-antibodies were used. AntiCD5-Ab was aim with FITS and N-Hydroxysuccinimidobiotin and streptavidin. After each incubation the unbound substances were separated by gel filtration over a Sephadex G-50 column using PBS pH 7,4 buffer. By flow citometry it was demonstrated that 90% T-lymphoblastic Jurkat cells were specifically bound with antiCD5-FITS-biotin-streptavidin-mAb complex . We detected that 84% of T-lymphoblastic Jurkat cells had been spesifically bound with immunoliposomes. The Raji cells line (B-cell limphoma) was used as a negative control.
 Helen Loughrey, Marcel B.Bally and P.R. Cullis, Biochim. Biophis. Acta 1987;901:157–160.  Christian B. Hansen, Grace Y. Kao, Elaine H. Moase, Biochim. Biophis. Acta 1995;1239:133–144.
PO-166 COMPARISON OF DOG AND HUMAN INTESTINAL FLUID AND ITS IMPACT ON SOLUBILITY ESTIMATIONS E S Kostewicz 1 , A S Carlsson 1 , G Hanisch 1 , R G 2 ¨ Nilsson 2 , L Lofgren & B Abrahamsson 1 – 1 Departments of Biopharmaceutics, AstraZeneca R&D, S-431 83 ¨ Molndal, Sweden; 2 DMPK & Bioanalytical Chemistry, ¨ AstraZeneca R&D, S-431 83 Molndal, Sweden The aim of the present study was to characterize dog intestinal fluid (IF) for comparison with human IF in order to examine its potential influence on the solubilization capacity of poorly soluble compounds. A newly developed HPLC method was used to characterize human and dog IF with respect to type of bile acid, concentration and protein content. Using different batches of IF collected from fasted subjects by a jejunal intubation (man) and mid jejunal fistula (dog), solubility measurements of poorly soluble drug compounds were performed. The total amount of bile acids in human IF was approximately 2 mM, whereas in the dog, the concentration varied between 5–8 mM. Differences in the type of major bile acid present was observed between species. For example, taurocholic acid (TCA) is the most common bile acid (about 75%) in the dog, whereas glycocholic acid (GCA), which is not detectable in the dog, is the most predominant bile acid in man (about 35–50%). The solubility was significantly higher in the dog, most probably due to the different bile composition compared to man. The difference between man and dog in solubilization capacity due to variations in bile compositions should be considered in extrapolation of animal data to man.
PO-167 EFFECT OF PEG ON DRUG RELEASE FROM ETHYL CELLULOSE COATED RESIN–DRUG COMPLEX M Kouchak F Atyabi – Faculty of Pharmacy, Ahwaz University of Medical Sciences, Ahwaz, Iran The maintaining drug–resin beads in the swollen state during coating process can prevent disruption of the coat on rehydration. Treatment of resin beads with polyethylene glycol 4000 (PEG) before coating can avoid rupture of the dried particles [1,2]. In this research, the effect of PEG treatment on drug release from coated resin–drug complex was studied. Diclofenac sodium was loaded on Amberlite IRA-900, an anionic resin. The beads were treated with different
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
amounts of PEG 4000 and coated with ethyl cellulose by the emulsification-solvent evaporation method. The total drug release of the coated beads in a phosphate buffer solution of pH 6.8 was measured for 11 hours using USP dissolution apparatus method II. The results showed that the higher the PEG:EC ratio in the formulation, the slower the drug release. One probable reason for this decrease is the plasticising effect of PEG in improving the coating efficiency and thereby retarding the drug release. Furthermore, in dichloromethane solution containing EC (during microencapsulation process) most of PEG is adsorbed on the hydrophilic surface of resin beads and is surrounded by EC. As the system is placed into contact with aqueous medium, water penetrates through the film and dissolves the trapped PEG that results in an increase in the viscosity of the solvent-pores of the film and decrease the drug diffusion coefficient. This study has shown that the addition of hydrophilic polymers to hydrophobic films cannot always increase drug release from systems.  Cuna M, Vila-Jato JL and Torres D. Controlled-release liquid suspensions based on ion-exchange particles entrapped within acrylic microcapsules. Int J Pharm 2000;199(2):151–58.  Irwin WJ, Belaid KA and Alpar HO. Drug-delivery by ion–exchange. Part IV: Coated resinate complexes of ester pro-drugs of propranolol. Drug Dev Ind Pharm 1988;14(10):1307–25.  Raghunatan Y, Amsel L, Hinsvark O and Bryant W. Sustained-release drug delivery system I: Coated ion exchange resin system for phenylpropanolamine and other drugs. J Pharm Sci 1981;70(4):379–384.
PO-168 THE RELEASE OF TRICLOSANE FROM BIODEGRADABLE POLYMER MATRICES ´ Kubıcek, ´ˇ ´ ´ & Milan Dittrich – Vladimır Petra Patkova Charles University Prague, Faculty of Pharmacy, ´ ´ ´ Czech ReHeyrovskeho 1203, 500 05 Hradec Kralove, public Polyesters of lactic and glycolic acids and manitol in three different ratios were prepared. Using laboratory balances polymer, triclosane and triethylcitrate (plasticizer) were mixed to prepare studied samples of determined composition. These mixtures were thawed and stirred to reach homogeneous solutions. TRIZMA buffer (pH57.4) and phosphate buffer (pH55.5) were utilized as release medium. Every 24 hours the medium was removed and the triclosane content in the medium was determined by UV– VIS spectrophotometer. Fresh buffers were then poured into the vessels again. The samples were stored at 37 8C. Complete release of triclosane was observed after 9–30 days according to the composition of the studied sample.
Hence the period of the complete release is influenced by the composition of the polymer, the plasticizer content and the drug load. If the triclosane load was low (up to 10%) the burst effect (in % of the total triclosane load) was found larger in comparison with the burst effect of higher loaded samples. This phenomenon could be attributed to the inhibition effect of triclosane during the polymer degradation and is in an agreement with our previous observations. The concentrations of triclosane found in the release medium are higher than MIC (minimal inhibition concentration) . The presented results include basic information about the biodegradable polymer-plasticizertriclosane mixtures necessary for the further development of the polymeric delivery systems. This investigation was supported by Research Project MSM 111600001 and by Research Centre Project LN00B125; both of Czech Ministry of Education.  Russel A D, McDonell G. Concentration: a major factor in studying biocidal action. J Hosp Infection 2000;44:1–3.
PO-169 IS ABSORPTION OF DRUGS AFFECTED BY CO-ADMINISTRATION OF LAXATIVES, AN IN VITRO STUDY USING Caco-2 CELL MONOLAYERS L Laitinen 1,2 , E Takala 2 , A M Kaukonen 1 & M Marvola 2 – 1 Viikki Drug Discovery Technology Center (DDTC), Department of Pharmacy, University of Helsinki, Helsinki, Finland; 2 Division of Biopharmaceutics and Pharmacokinetics, Department of Pharmacy, University of Helsinki, Helsinki, Finland The aim of this study was to evaluate the potential effects of three laxatives (a mixture of sennosides A and B, senna hot water extract, and danthrone) on the absorption of drugs. The permeability of acetaminophen (passive transcellular diffusion), ketoprofen (active transport), and propranolol (passive diffusion and active efflux) without or with co-administration of the laxatives was studied across Caco-2 cell monolayers. Mannitol was used as passive paracellular marker. Permeability experiments with the drugs were performed at 0.1 mM concentration in the apical-to-basolateral (AP-BL) direction at apical pH 6.8 without or with the sennosides (0.095 and 0.19 mg / ml), the senna extract (4 and 8 mg / ml) or danthrone (0.3 and 0.6 mg / ml). The viability of Caco-2 cells was determined by using MTT- and LDH-tests. The integrity of Caco-2 monolayers was determined by measuring the transepithelial electrical resistance (TEER) of the monolayers and by permeability experiments with 14 C-mannitol. In control monolayers, about 0.22% of the mannitol dose was transported over 120 minutes to the acceptor compartment. With the studied laxatives, the paracellular permeability of mannitol was slightly increased during this
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
time period. Sennosides had hardly, if any, effect on acetaminophen, propranolol or ketoprofen permeability. The senna extract decreased the permeability of acetaminophen and propranolol (15 and 30%), while ketoprofen transport across Caco-2 monolayers was increased by approximately 30%. Danthrone decreased ketoprofen transport clearly (30%), and had a slight decreasing effect on transport of acetaminophen (10%) and propranolol (5–10%). None of the laxatives showed any toxicity at studied concentrations. The studied laxatives may affect passive and / or active transport of other, co-administered, drugs. Although their main site of action is the colon, laxatives may disturb the normal functions of the upper parts of the intestine and thus affect concomitant absorption of drugs.
PO-170 THE EFFECT OF TREHALOSE DIHYDRATE ON STABILITY OF ASPIRIN IN DICALCIUM PHOSPHATE DIHYDRATE BASED TABLETS STORED UNDER ACCELERATED CONDITIONS ´ M Landin, M J Fontao & R Martınez-Pacheco – Dpto. ´ Farmaceutica, ´ Farmacia y Tecnologıa Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain Trehalose dihydrate (TD) is the most chemically unreactive and stable sugar in nature. While the use of TD as preservative of biomolecules has received a considerable attention , little work has been done about its applications as an excipient in solid dosage forms probably due to the high price of the material. The recent commercialisation of a low cost enzimatically produced trehalose obliges to raise the usefulness of that interesting product. This work studies the stabilising effect of TD in aspirin (ASA) tablets, a drug which is known to be readily hydrolysed. Dicalcium phosphate dihydrate (DCPD) has been used as the base excipient. It is well documented that changes in the physical properties of DCPD tablets on storage are frequent and they have been attributed to the dehydration process of DCPD . Tablets of a 10% ASA, DCPD and 0, 10 and 20% w / w of TD were elaborated by direct compression and stored at 35 8C and 82.9% RH for 6 months . Additionally, in order to compare the effect of TD with the commonly combined excipient to DCPD to improve stability, a formulation including 20% mannitol (M), was evaluated. At predetermined time intervals, formulations were tested for drug content, weight and tensile strength (TS). ASA degradation curves have been best fitted to Weibull equation (r.0.99). Results reveal that both sugars decrease ASA degradation rate constant in tablets. As a consequence, at 6 months, ASA in the TD formulations was around 10% less degraded than in the one containing just DCPD regardless TD tablet content. The mannitol has
significantly minor effect (aprox. 5%). The presence of TD or M reduces the weight loss characteristic of DCPD tablets  but it does not protect them from a decrease in TS (.50%) on ageing. Authors thanks Xunta de Galicia (PGIDT 01PX120304IF) for financial support.  Miller DP et al. Pharm. Res. 1997;14:578–590.  Shiromani PK and Bavitz JF. Drug Dev. Ind. Pharm. 1988;14:1375–1378. ´ M et al. Int. J. Pharm. 1994;107:247–249.  Landın
PO-171 THE EXCITATORY AMINO ACID TRANSPORT SYSTEMS EAAT2 AND EAAT3 OF AN INVITRO MODEL OF THE BLOOD–BRAIN BARRIER R Lauer, B Dampier, G Ecker & C R Noe – Instiute of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Wien, Austria The blood–brain barrier acts as a barrier between bloodstream and the extracellular space of the central nervous system, permeable only for small and lipophilic molecules. Other compounds gain acess to the brain only via active transport systems. In this way the blood–brain barrier impedes the entrance of substances that could interfere with brain activities. Glutamate is not only one of the most important neurotransmitters but also one of the most neurotoxic substances. Dysfunctions in glutamate metabolism lead to overexcitation of cells and serious brain damage. Our gain was to show the expression of the high affinity glutamate transporters EAAT2 and EAAT3 in an established PBMEC cell line (porcine brain microvasculatur endothelial cells) using transport studies, immunocytochemistry and Western blotting. In the transport studies of glutamate through our blood–brain barrier model it was shown that endothelial cells transport glutamate faster than it can be explained by diffusion driven movement alone. An explanation for this result is the existence of glutamate transporters at the blood–brain barrier. This assumption was substantiated by an inhibition experiment: Kainic acid significantly decreases clearance rates of glutamate. On the protein level immunostaining of EAAT2 and EAAT3 was unsuccsessfull, but Western blot analysis confirmed that EAAT2 transporter proteins exist on the endothelial cells. Due to the fact that the established blood–brain barrier model does express excitatory amino acid transporters it is now possible to study the influence of drugs on the glutamate transporter and so our in vitro model can be used as a specific tool in drug research. We gratefully acknowledge the financial support provided by the Austrian Science Fund (project FWF P14582CHE)
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-172 PERMEATION OF A SET OF BENZODIAZEPINES ACROSS AN IN VITRO BLOOD– BRAIN BARRIER MODEL R Lauer, G Ecker & C R Noe – Institute of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, 1090 Wien, Austria The passage of compounds from blood to brain is regulated in the cerebral capillaries. The endothelial cells of these capillaries form the blood–brain barrier. To facilitate investigations of functional and transport characteristics of brain endothelial cells, we developed an in vitro blood– brain barrier model. Immortalized porcine brain microvascular endothelial cells (PBMEC)  form a monolayer on a porous filter insert, which is placed in a six well plate. We performed transport studies with 12 benzodiazepines to investigate the influence of lipophilicity and plasma protein binding on the permeability across our in vitro model. The transport studies were done in serum free medium as well as in serum containing medium. To prove the tightness of the monolayers we measured the transendothelial electrical resistance and the permeability of the monolayers for FITC-Dextran. The concentrations of the drugs were determined by HPLC and permeability coefficients (PEs) were calculated. Though we did not achieve full resolution of all 12 benzodiazepines, we could see three clusters of drugs, depending on their permeability. The two drugs with the highest permeability had a nitro group in position 7, while the benzodiazepines that showed medium permeation had a methyl group in position 1. The other drugs, which exhibit relatively low permeability coefficients were unsubstituted in position 1. PEs measured in medium containing serum showed nearly the same pattern as those measured in serum free medium, but they were significantly lower. Our investigations showed that this in vitro model is a good tool to perform at least semiquantitave separation of drugs which belong to the same substance class. We gratefully acknowledge the financial support provided by the Austrian Science Fund (project FWF P14582CHE).  Teifel M, Friedl P. Establishment of the Permanent Microvascular Endothelial Cell Line PBMEC / C1-2 from Porcine Brains. Exp. Cell Res. 1996;228:50–57. PO-173 DETERMINATION OF THE BIOEQUIVALENCE OF TWO OMEPRAZOLE PREPARATIONS S E Leucuta, C Iuga, R Capalneanu, A Popa & M Preda – Bioequivalence Laboratory, University of Medicine and Pharmacy ‘‘Iuliu Hatieganu’’, 3400 Cluj-Napoca, Romania The problem of bioequivalence in Romania is characterized by the simultaneous presence on the market of very big multinational companies along with medium and small
Romanian companies, with contrasting economic interests. Romania needs to reduce the continuously increasing social cost of therapy and growth in the generic drug market would appear to be the most appropriate. Applicants for Romanian generic drugs must however guarantee that their formulation is interchangeable with the formulation from the innovator company and must submit a dossier on the drug’s chemistry, pharmaceutical features as well as a pivotal bioequivalence study. The bioequivalence of two oral preparations of the omeprazole, the tested generic preparation Omeran (Glaxo Smithkline-Europharm, Brasov, Romania) and the reference innovator product, Losec (Astra), both in a dose of 20 mg omeprazole, was assessed in an open, cross-over, randomised trial in 12 healthy male volunteers. Written, informed consent was obtained from all study participants before they accepted to participate the study. The University Ethical Committee approved the study protocol. Blood samples were withdrawn at 0; 0.5; 1; 1.5; 2; 2; 5; 3; 4; 6 and 8 hours after an overnight fast for at least 10 hours. For quantification of omeprazole a high-pressure liquid chromatographic method was developed (HP 1100 Series liquid chromatograph). Calibration curves in plasma were linear over the range of 15–500 ng / ml. The limit of quantification: 15 ng / ml. Intra-day and inter-day validation: 0.3–7.3 (C.V.%) for precision and accuracy. Recovery: 81–99%. The pharmacokinetic characteristics for omeprazole were determined from the plasma concentration-time data . The maximum plasma concentrations (Cmax) and time to reach maximum plasma concentrations (Tmax) were obtained directly by inspection of the individual drug plasma concentration-time data. The area under the plasma concentration-time curve (AUCt) up to the last time showing a measurable concentration (Ct) was determined by using the linear trapezoidal rule. A computer program (Kinetica 3, Inna Phase) calculated the apparent elimination rate constant Kel. The parametric 90% confidence intervals of the mean log values of the ratio Omeran / Losec of pharmacokinetic parameters were 0.81–1.04 and 0.80–1.18 for AUC 0 – 8 hours and Cmax respectively, i.e. were in each case within the bioequivalence acceptable range (Schuirman’s two one-sides t-test) . The time to maximum concentration Tmax difference of the mean values of the products studied was analyzed assuming an additive model, and was found to be not significant (Friedman test). In conclusion, the tested product Omeran, was bioequivalent with the reference product Losec, both as amount and rate of absorption, so these products are interchangeable.  Marzo A., Balant L.P. Bioequivalence. An updated reappraisal addresed to applications of interchangeable multi-source pharmaceutical products. Arzneim. Forsch. / Drug Res., 1995;45(I):109–115  Schuirmann D.J. J. Pharmacokin. Biopharm., 1987;15:657
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-174 DRUG DISSOLUTION OF POORLY SOLUBLE COMPOUNDS IN HUMAN INTESTINAL FLUID (HIF) ¨ 1 , A Carlsson 2 , G Hanisch 2 , H. Lennernas ¨ 1 &B F Liback 2 1 Abrahamsson – Uppsala University, Dept. of Pharmaceutics, SE-751 23 Uppsala, Sweden; 2 AstraZeneca R&D, Dept. of Preformulation & Biopharmaceutics, S-431 ¨ 83 Molndal, Sweden
Intestinal solubility of poorly soluble drugs can be much higher than indicated by testing in plain buffers due to the micellar solubilisation by bile components. However, studies in simulated intestinal fluids has shown that the dissolution rate, which is the key variable with respect to drug absorption, could be decreased in the presence of mixed micelles in spite of the improved solubility . The purpose of the present study was to study the drug dissolution rate and solubility of poorly soluble drugs in real HIF. The study included two poorly soluble, non-ionic model drugs; felodipine (F) with a ClogP of 4.9, which is completely absorbed in man in doses up to at least10 mg, and griseofulvin (G) with a ClogP of 2.2, which absorption is strongly reduced by dissolution at 500 mg. Saturation solubility and dissolution rate studies were performed at 37 8C in undiluted, fasting jejunal fluid from healthy volunteers obtained by oral intubations and in phosphate buffer pH 6.8. The saturation solubility was determined after 24 h and the dissolution rate was determined by the rotating disc method at 1000 rpm. The drug concentrations were determined by HPLC with UV detection. The low water solubility of the two compounds was verified by solubility values of 2 and 8 mg / ml for F and G, respectively, in the buffer. The solubility of F was increased six times in HIF whereas no significant enhancement was obtained for G. This is in accordance with earlier data obtained in simulated intestinal media where no solubilisation was obtained for drugs with log P,2 whereas more lipophilic compounds were increasingly solubilised by increasing lipophilicity . The initial dissolution rate at a constant surface area was 0.3 and 1.1 mg / cm 2 for F and 3.7 and 6.8 mg / cm 2 for G in buffer and HIF, respectively. Thus, the dissolution rate for F was improved in HIF compared to buffer but not to the same extent as the solubility. The faster dissolution rate for G in HIF compared to buffer, despite lack of any significant solubilisation, was probably due to wetting effects. Additionally, the poor oral absorption of G, in contrast to F, are clearly related to the much higher dose of G since the dissolution was actually faster for G than F and the solubility was similar in HIF. In conclusion, the present study indicates that the dissolution rate in HIF should not be extrapolated from solubility data but needs to be experimentally determined for accurate estimations of dissolution limitations of oral drug absorption.
 Higuchi WI, Prakongpan S, Surpuriya V, Young F. Cholesterol dissolution rate in micellar bile acid solutions: retarding effect of added lecithin. Science 1972;178:633–4.  Mithani SD, Bakatselou V, TenHoor CN, Dressman JB. Estimation of the increase in solubility of drugs as a function of bile salt concentration. Pharm Res. 1996;13:163–7.
PO-175 IN VITRO EVALUATION OF VARIOUS HYDROPHILIC POLYMERS AS BARRIER COATINGS FOR COLON SPECIFIC DRUG DELIVERY A Lindahl, E Persson, B Abrahamsson & M Wikberg. – Pharmaceutical and Analytical R&D, AstraZeneca R&D ¨ ¨ Molndal, S-431 83 Molndal Sweden The aim was to evaluate the potential of Guar gum NF, Pectin USP and HPMC 50 cPs as barrier coatings for colon specific delivery of drugs, using diluted human faeces slurry, purified enzyme solutions or buffer solution pH 6.8 as test media. Two or three layer discs were compacted at 10 kN in a die with a surface area of 1 cm 2 . The die was vertically attached to a laboratory stirrer and lowered into a thermostated titercup containing test medium (37 8C). The release of the model drug, metoprolol succinate, from discs that were either un-coated or press-coated with a 1 mm thick layer of the polymers, was examined in the various media at various stirring rates (60–240 rpm). The release of metoprolol from the uncoated discs was fast (,5–10 min) and independent of stirring rate in phosphate buffer pH 6.8 (representing the small intestine). In diluted faeces slurry (representing the colon) the release was significantly slower and dependent on the stirring rate. At the highest stirring rate the release rate was similar in both media. In phosphate buffer pH 6.8, Guar gum NF and HPMC 50 cPs coatings completely prevented drug release for up to 5 h, while Pectin USP released about 10–20% with an onset of release already after 3 h. In diluted faeces slurry, the release after 10 h varied from 1 to 37% and 13 to19% for Guar gum NF and Pectin USP (onset of drug release after 6 h), respectively, while HPMC 50 cPs showed almost no release. The release rate in enzyme solutions (Gamanase and Pectinase), also representing the colon, was significantly faster compared to phosphate buffer pH 6.8 and diluted faeces slurry for both Guar gum NF and Pectin USP. In conclusion, only for Guar gum NF was the drug release specifically triggered by the human diluted faeces slurry. The dependence of stirring rate in diluted human faeces indicates that other factors rather than solely enzymatic degradation of a coating need to be considered when evaluating colon specificity in vitro.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-176 SOLUBILITY MEASUREMENTS IN DIFFERENT MEDIA – IMPACT ON MAXIMUM ABSORBABLE DOSE (MAD) Tuulikki Lindmark 1 , Helena Flatow 2 , Ulla Wrange 1 & Carina Ekholm 1 – 1 Preclinical R&D, Biovitrum, Stockholm, Sweden; 2 Uppsala University, Faculty of Pharmacy, Uppsala, Sweden The aim of this study was to investigate if the choice of medium for solubility measurements, i.e. the use of more physiological media that more accurately mimic the intestinal fluid composition rather than a simple phosphate buffer , could impact MAD. Chlorothiazid, danazol, hydrochlorthiazid, ibuprofen, ketoconazole, ketoprofen, and phenytoin were used as model compounds. MAD was calculated according to MAD5S*K a* SIWW*SITT where S5solubility (mg / ml); Ka5transintestinal absorption rate constant (min 21 ); SIWV5small intestinal water volume (250 ml) and SITT5small intestinal transit time (270 min) . Solubility measurements were performed in four different media: phosphate buffers pH 6.5 and pH 7.4 or two media simulating the intestinal fluids at fed and fasted state respectively . Permeability (Papp ) was measured using Caco-2 cells and subsequently, K a was calculated from the measured Papp values. All analytical work was performed using LC-UV. For compounds that were ionised, and thus expected to have a good solubility, throughout the pH interval used (pH 5.0–7.4), the choice of media did not influence MAD. For poorly soluble compounds, the choice of dissolution medium resulted in an up to 100-fold change in MAD. The results were in good agreement with reported data on oral absorption in humans. The choice of dissolution medium can substantially influence the prediction of MAD. Therefore, when predicting MAD of poorly soluble compounds, the use of a broader range of media is recommended prior to rejecting a compound at an early development stage.  Curatolo W. Physical chemical properties of oral drug candidates in the discovery and exploratory development settings, Pharm. Sci. Technol. Today 1998;1;387–393.  Galia E. et al., Evaluation of various dissolution media for predicting in vivo performance of class I and class II drugs, Pharm. Res. 1998;15;698–705.
PO-177 PREPARATION OF THE CONTROLLED RELEASE HYDROPHILIC MATRIX TABLETS OF KETOROLAC TROMETHAMINE WITH CELLULOSE DERIVATIVES USING FACTORIAL DESIGN ¨ Lutfi Genc¸ & Esmaeil Jalvand – Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 26470 Eskis¸ehir, Turkey
Ketorolac tromethamine (KT) was absorbed rapidly (T max ,1.0 hr) and efficiently (.87%) following oral and intramuscular doses. It was 36 times more potent than phenylbutazone, approximately twice as potent as indomethacin and 3 times more potent than naproxen in systemic anti-inflammatory activity. Its analgesic activity was stronger than aspirin. Clinical studies indicated that single dose efficacy of KT was greater than that of morphine, pethidine and pentazocine in moderate to severe postoperative pains [1,2]. For this reason, KT was used in our study. Controlled release matrix tablets of KT were prepared by direct compression technique using cellulose derivatives as hydroxypropylmethyl cellulose (HPMC), hydroxyethyl cellulose (HEC) and carboxymethyl cellulose (CMC) in different concentrations (10–20%). The influence of polymer type and concentration on tablet specifications and dissolution rate of matrix tablets was investigated. The objective of this work is to outline 2 3 factorial design and to study the effect of three factors; HPMC (A), HEC (B) and CMC (C) on the dissolution rate of KT in matrix tablets. In this case there are eight treatment combinations: (1), a, b, ab, c, ac, bc, abc . For the quality control of matrix tablets, weight deviation, hardness, friability, diameter-high ratio, content uniformity of KT (these results were in accordance with the pharmacopoeia limits) and in vitro dissolution technique were performed. UV Spectrophotometric method was used to detection of KT in matrix tablets . This method was validated [5–8]. An important step in the validation of any analytical method is the establishment of the relationship between released % (y) and the concentration of the analyse (x) and the method may be calibrated. When correlation coefficient is above 0.9990 and the RSD for the sample preparation step might be approximately 1%, the assay method was acceptable. Our results are above 0.9990 and these results are suitable for method validation. Dissolution profiles of the formulations were plotted and evaluated kinetically. The ideal matrix tablet formulation was found by evaluating of these findings. According to dissolution results, tablets prepared with 10% HPMC1 10% HEC110% CMC (F8) were found to be the most suitable formulation for KT. The release of KT from formulations F2, F3, F4, F7 and F8 were found to be 97.82%, 94.57%, 94.32%, 100.7% and 93.27%, respectively in 4.5 h. F1, F5 and F6 were disintegrated after 1.0, 1.5 and 2.0 h, respectively. The in vitro release of KT was changed as the polymer ratio and mixture changed. As for the kinetic evaluations the highest determination coefficient and the best linear relation were observed for matrix tablet (F8) by Higuchi equation . Graphically Higuchi distribution gave a straight line. Mechanisms for drug release from matrices of cellulose esters (CEs) imply water penetration in the matrix (with drug dissolution on the surface, causing its immediate release), hydration and swelling of CEs, diffusion of the dissolved drug, and the erosion of gelatinous polymer layer [10,11]. The influence
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
of polymer type and concentration on dissolution rate of matrix tablets was investigated.  Mroszczak, E.J., Lee, F.W., Combs, D., Sarnquist, F.H., Huang, B-L., Wu, A.T., Tokes, L.G., Maddox, M.L., Cho, D.K. Ketorolac tromethamine absorpsition, distrubition, metabolism, excretion and pharmacokinetics in animals and humans. Drug Metabolism and Disposition 1987;15(5):616–626.  Buckley, M.M-T., Brogden, R.N. Ketorolac, A rewiew of ist Pharmacodynamic and pharmacokinetic properties and therapeutic potential. Drugs 1990;39(1):86–109.  Cohran, W.G., Cox, G.M. Experimental Designs, John Wiley and Sons, New York, 1957.  Kamath, B.V., Shivram, K., Vangani, S. Spectrophotometric determination of Ketorolac tromethamine by charge transfer and ion-pair complexation. Analytical Letters 1994;27(1):103–112.  Reiley, C.M., Fell, F. Development and validation of analytical methods. Progress in Pharmaceutical and Biomedical Analysis 1996;3:20–22.  Beuving, G. Validation of Analytical Methods, Validation Seminar, 31 May–01 June 2001, Istanbul, 2001.  Caporal-Goutier, L., Nivet, J.M., Algranti, P., Guilloteau, M., Histe, M., Lallier, M.. N’Guyen-Huu, J.J., Russotto, R. Guide de validation analytique Rapport ´ d’une commission SFSTP I. Methodologie. S.T.P. Pharma Pratiques 1992;2(4):205–226.  USP 24-NF 19 1998, 3th Edition, pp. 948–949.  Higuchi, T. Mechanism of sustained-action medication. J. Pharm. Sci. 1963;52(12):1145–1149.  Handbook of Pharmaceutical Excipients, 1986, p. 141.  Ford, J.L., Rubinstein, M.H., Mclaul, F., Hogan, J.E., Edgarp, J. Int. J. Pharm. 1987;40:223.
PO-178 NEUTRON ACTIVATION OF SAMARIUM OXIDE AND PHARMACO-SCINTIGRAPHIC EVALUATION OF DRUG RELEASE FROM VARIOUS ORAL FORMULATIONS J Marvola, H Korpela, L Slot, H Hietanen, K Lindevall & ¨ M Marvola – Remedium Clinical Research, Sinimaentie 14, 02630 Espoo, Finland; Department of Pharmacy, P.O.B. 56, 00014 University of Helsinki, Finland It has become evident that in vitro studies alone are not enough during development of drug formulations. In vivo behaviour needs to be monitored at an early stage. One technique commonly used for this purpose is gamma scintigraphy. In the case of complicated formulations the marker used should be a stable isotope. Samarium oxide is a good candidate for this purpose. In addition, access to a proper nuclear reactor and a gamma camera is required.
The drug products studied were 1) ethyl-cellulosecoated capsules, 2) enteric-coated (Eudragit L ) capsules and 3) the Egalet constant-release model (Egalet a / s, Denmark). The capsules contained lactose q.s. and 6 mg of Sm 2 O 3 . Egalet contained 50 mg of caffeine and 8 mg of Sm 2 O 3 . Samarium was activated using the FiR-1 250 kW TRIGA nuclear reactor (Technical Research Centre of Finland) and a neutron flux of 1.1?10 12 n cm 22 s 21 . The temperature in the irradiation space did not exceed 40 8C. Stability of the formulations was confirmed in vitro. Ten healthy male volunteers participated in the study. Forty-eight hours were allowed to elapse before drug administration, to allow unwanted radioisotopes to decay. Activity levels did not exceed 1.3 MBq, resulting in effective absorbed doses of below 1 mSv. Gamma images were taken in the Diacor Hospital using a Multispect 2 dual-head camera (Siemens AG, Germany). Collimators were of the LEHR type. Images were taken at intervals of 0.5 to 3 hours, for 24 hours. Blood samples were taken for determination of caffeine levels. The study protocol had been approved by the Ethics Committee for Helsinki University Hospital. Imaging of all formulations over the 24-hour period proved possible without difficulty. Marked differences in the fate of the formulations were detected in the stomach, intestine and colon. A correlation was found between counts and the location of the formulation in the GI-tract. Changes in count rates correlated with changes in plasma caffeine levels. Rapid transits resulted in smaller AUC values and vice versa. After an initial lag time, release of 153 Sm 2 O 3 from Egalet was linear but biphasic, higher in the small intestine than in the colon.
PO-179 HAEMOLYTIC EVALUATION OF NANOSPHERES PREPARED FROM AMPHIPHILIC bCYCLODEXTRINS MODIFIED ON THE PRIMARY OR SECONDARY FACE ˆ 2 ˘ 1 , Amelie ´ Bochot 2 , Dominique Duchene Erem Memis¸oglu 1 1 & A. Atilla Hıncal – Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara, Turkey; 2 Universite´ de Paris-Sud, Faculte´ de Pharmacie, UMR CNRS 8612, 92296 ChatenayMalabry Cedex, France Nanospheres designed for parenteral administration prepared from b-CDC6, modified on the secondary face, and 6-N-CAPRO-b-CD , modified on the primary face were assessed for their haemolytic properties on whole blood and erythrocyte suspension. Nanospheres were prepared within a concentration range of 0.5 to 4 mM. Whole blood and erythrocytes separated by centrifugation were treated with increasing concentrations of nanospheres prepared in PBS. Samples were incubated and centrifuged. Supernatant was analysed for haemoglobin content at 543 nm UV spectrophotometrically. Haemolysis percentage was then
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
calculated as ratio of UV absorbances of samples treated with nanospheres to those treated with distilled water . All results with compared to corresponding data of natural b-CD. It was demonstrated that the more lipophilic the derivative the less haemolytic. Order of haemolytic activity varied as follows; b-CD.6-N-CAPRO-b-CD.b-CDC6. Amphiphilic cyclodextrins were non-haemolytic in the concentration range actually applied for therapeutic use.
¨  Schroder H. Thromb Res 1990;57:839–45.  Bassi B. Degree Thesis, University of Florence, 2001.  Minghetti P, Casiraghi A, Cilurzo F et al. J. Pharm. Pharmacol., 1999;51:673–8.  Bilia AR, Bergonzi MC, Bassi B. et al., 6 th International ESCOP Symposium-Herbal Medicinal Products Scientific Strategies in Europe, 10–11 May 2001, Bonn.
ˆ ˘ E, Bochot A, S¸en M, Charon D, Duchene  Memis¸oglu ´ D, Hyncal AA. Amphiphilic b-cyclodextrins modified on the primary face. Synthesis, characterization and evaluation of their potential as novel excipients in the preparation of nanocapsules. J. Pharm. Sci., 2002;91(5):1214–1224.  Leroy-Lechat F, Wouessidjewe D, Andreux JP, ˆ D. Evaluation of the cytotoxicity Puisieux F, Duchene of cyclodextrins and hydroxypropylated derivatives. Int. J. Pharm., 1994;101:97–103.
PO-181 PROTEIN LOADED PLGA MICROSPHERES; MICROPARTICLE / POLYMER DEGRADATION AND THE IN VITRO RELEASE PROFILES K Mladenovska 1 , K Goracinova 1 , A Porjazoska 2 & M Cvetkovska 2 – 1 Faculty of Pharmacy, Ss. Cyril and Methodious University, Vodnjanska 17, 1000, Skopje, Macedonia; 2 Faculty of Technology and Metallurgy, Ss. Cyril and Methodious University, Ruger Boskovic 16, 1000 Skopje, Macedonia
PO-180 SKIN PERMEABILITY OF SESQUITERPENES OF A NEW ARNICA MONTANA L. EXTRACT P Minghetti 1 , M C Bergonzi 2 , F Cilurzo 1 , A Casiraghi 1 , A R Bilia 2 , F Vincieri 2 & L Montanari 1 – 1 Istituto di Chimica Farmaceutica e Tossicologica, Milano, Italy; 2 Dipartimento Scienze Farmaceutiche, Firenze, Italy Sesquiterpene lactones of Arnica (Arnica montana L.) are secondary plant metabolites of potential medical interest because of their potent anti-inflammatory activity [1,2]. Recently, a supercritical carbon dioxide extract of Arnica flower, having higher sesquiterpenes content and better stability  in comparison with tinctures and conventional dried extracts usually used as ingredient of topical preparations, has been developed. The ability of sesquiterpenes to permeate the skin was evaluated by using solutions of the supercritical CO 2 extract (2% m / v) in the following permeation enhancers: oleic acid (OA), dimethylsulfoxide (DMSO), Lauroglycol (LG), isopropylmiristate (IPM), and Tween 80 (TW). The skin permeation study was performed by using modified Franz diffusion cell and the human stratum corneum and epidermis as a membrane . An HPLC-DAD and HPLC-MS method was adapted to perform the analysis and identification of each component of the extract . The sesquiterpene permeated amounts through the skin after 24 h by using LG, IPM and TW solutions were lower than the quantification limits of the analytical method. In the case of DMSO, there was a lag time of about 3 h, then the sesquiterpene permeated amounts increased linearly. The OA increased the sesquiterpene flux in a very significant way so that the equilibrium between the donor and the receptor phase was reached within the first hour. Therefore, among the selected skin permeation enhancers DMSO and OA can be considered as good candidate for topical preparations.  European Pharmacopoeia, III Suppl., 3 rd Ed., 2000, pp. 396–398.
Series of DL-PLGA copolymers were prepared by bulk ring opening polymerization of lactide / glycolide and were characterized by GPC, FTIR, 1 H-NMR and DSC analyses. Polymers with different molecular mass and fixed lactide / glycolide ratio (85 / 15 wt%) were used to prepare BSA loaded microspheres by double emulsion w / o / w method [1,2]. Scanning electron microscopy (Hitachi S-450) confirmed porous structure of all formulations, while the observed parameters were influenced by the copolymer’s molecular weight. Mean particle size (laser diffractometry; Fritsch particle size analysette DLAB / 22) was in a range of 11 to 13 mm, and the microspheres’ yield up to 60%. The BSA content, determined by measuring the radioactivity of radiolabelled 131 I-BSA, was up to 3% (theoretical 10%) with loading efficiency up to 17%. The molecular mass determinations and the weight loss of the blank microspheres vs. time suggest that the molecular weight of the copolymers has a dominant influence on the rate of polymer degradation; e.g. during 20 days no degradation was observed at the particles (mean diameter 12.89 mm) prepared from copolymers with Mw 83 789 gmol 21 , while in the same period, 25% of microspheres (mean diameter 11.47 mm) prepared from copolymers with Mw 13 290 gmol 21 were degraded. The BSA release is thought to depend largely on the biodegradation rate, but also on microspheres size and porosity. Following the initial burst release (60% released during 8–9 h), there was a phase of continuous much slower release.  Ikada Y, Tsuji H. Biodegradable polyesters for medical and ecological applications. Macromol Rapid Commun 2000;21:117–132.  Pushpa GS and Leonard CB. Porcine insulin biodegradable polyester microspheres: stability and in vitro release characteristics. Pharm Dev Tech. 2000;5(1):1–9.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-182 AN INVESTIGATION INTO THE EFFECT OF VARIOUS CARBOMERS ON THE RELEASE PROFILE OF IBUPROFEN FROM MATRIX-TYPE TABLETS S A Mortazavi & H Tabandeh – School of Pharmacy, Shaheed Beheshti University of Medical Sciences and Health Services, P. O. Box 14155-6153, Tehran, Iran For the purpose of this study, tablets containing 400 mg Ibuprofen, colloidal silicon dioxide, magnesium stearate, microcrystaline cellulose, and various carbomers including 934P (C934), 971P (C971), 974P (C974), and Pemulen TR-1 (PEM), either alone or in combination, were prepared by direct compression. The dissolution medium was 0.1 N HCl for the first 2 h, and phosphate buffer with a pH of 6.8 for the next 10 h. The amounts of drug released after 12 h, were in the following order for the polymers studied: C934.C974. PEM.C971. The Swelling rates of the tablets were also in the same order. The lower degree of swelling in the two latter polymers, could effect in more areas of low microviscosity in gel structure and the less reduced release rate. The Higuchi model was the best fitted kinetic model for drug release in all formulations. However, in some cases zero-order model was also found suitable, suggesting the possibility of both barrier and diffusion-controlled mechanisms for release . The presence of erosion-promoting ingredients may help the formulations to show this behavior . The addition of PEM to C934, caused the formulation to show a more sustained drug release profile with a lower total amount of polymer. This effect has previously been reported for the addition of C971 to C934 . A combination of C934 and PEM is suggested to be the best formulation for Ibuprofen sustained release tablets.  Meshali, M. M., El-Sayed, G. M., El-Said, Y. and El-Aleem, H. M. Preparation and evaluation of theophylline sustained release tablets. Drug Dev. Ind. Pharm. 1996;22:373–376.  Pather, I., Russell, I., Syce, J. A. and Neau, S. H. Sustained release theophyline tablets by direct compression. Part 1: formulation and in vitro testing. Int. J. Pharm. 1998;164:1–10.  Khan, G. M. and Zhu, J. B. Studies on drug release kinetics from Ibuprofen-carbomer hydrophylic matrix tablets: influence of co-exipients on release rate of the drug. J. Contr. Rel. 1999;57:197–203.
PO-183 DRUG DISTRIBUTION IN THE URINARY BLADDER WALL AFTER INTRAVESICAL INSTILLATION: ASSESSMENT OF CHITOSAN AND POLYCARBOPHIL INFLUENCES BY ADVANCED COMPARTMENTAL MODEL A Mrhar 1 , I Grabnar 1 , M Bogataj 1 , A Belicˇ 2 & R Karba 2 – 1 ˇ ` Faculty of Pharmacy, Askereeva 7, 1000 Ljubljana, ˇ ˇ 25, Slovenia; 2 Faculty of Electrical Engineering, Trzaska 1000 Ljubljana, Slovenia
Intravesical instillation of a drug solution is a common way of treating superficial bladder cancer. It aims to optimize drug delivery near the site of action while minimizing systemic exposure. Even though drug concentrations in the bladder wall are frequently sub-therapeutic mostly due to low permeability of the bladder mucosa and systemic absorption. Our objective was to assess bladder permeability enhancing effects of chitosan (CH) and polycarbophil (PC). Mathematical model was developed to simulate time courses of drug concentration in the urine, bladder tissue and plasma. Excised porcine bladder wall was exposed to a solution of a model drugpipemidic acid (PPA) or polymer dispersion (0.5 and 1% w / v) in PPA solution for up to 2 hours. It was rapidly frozen and sectioned parallel to luminal surface. Tissue PPA concentrations were determined by HPLC . To the obtained drug concentration-tissue depth profiles biphasic diffusion model was fitted, taking into account lower permeability of urothelium compared to lamina propria and muscular layers. Compartmental diffusion model was connected to urine dynamics model and linked to PPA pharmacokinetic model through compartments representing blood perfused lamina propria and muscular layers. Considering the intravesical dose administered, urine formation rate, residual urine volumes, bladder voiding, systemic absorption rate from the bladder wall and identified diffusion parameters, time courses of drug concentration in urine, bladder tissue at various depths and plasma were simulated. Permeability of urothelium for PPA increased 3.6 and 3.2 times in the presence of CH and PC, respectively, while permeability of the underlying connective and muscular tissue remained unchanged. This resulted in about two-fold increase in bladder drug exposure and systemic availability. The proposed experimental model is relevant for design and evaluation of the subsequent in vivo studies.  Wientjes MG, Dalton JT, Badalament RA et al. A method to study drug concentration-depth profiles in tissues: Mitomycin C in dog bladder wall. Pharm Res 1991;8:168–173.
PO-184 IMPLICATIONS OF pH EFFECTS ON FALSE AND TRUE UPTAKE AND EFFLUX OF DRUGS ACROSS Caco-2 MONOLAYERS Sibylle Neuhoff 1,2 , Anna-Lena Ungell 1 , Ismael Zamora 1,3 & Per Artursson 2 – 1 AstraZeneca R&D, DMPK & BAC, ¨ S-431 83 Molndal, Sweden; 2 Dep. of Pharmacy, Uppsala University, S-751 23 Uppsala, Sweden; 3 Lead Molecular Design, S.L., E-081 90 Barcelona, Spain The aim of this study was to investigate the effect of ionisation on passive diffusion and carrier-mediated transport of drugs across the intestinal epithelium. Therefore, pH-dependent transport rates of weak basic, acidic and
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
neutral drugs were investigated bi-directionally across Caco-2 monolayers. In addition, the pH-dependency of the clinically relevant digoxin / quinidine-interaction was studied. A net secretory flux was observed for atenolol, a low permeable weak base, and metoprolol, a high permeable weak base, when a pH-gradient was applied across the monolayers (apical pH lower than the basolateral pH). However, in the absence of a pH-gradient, the transport rates were equal in both directions for each of the compounds. In contrast, the acids, salicylic acid and foscarnet, showed a net uptake when the apical pH was lower than the basolateral pH. Finally, efflux ratios of the neutral compound, digoxin, were pH-independent. This indicates that the observed net efflux of the investigated weak bases was not due to a real carrier-mediated efflux and that the net uptake of the investigated acids was not only caused by a real carrier-mediated uptake. Instead, the observed asymmetric transport is probably caused by a pH-dependent concentration of the uncharged species of the drugs in the apical compartment. Further support for this hypothesis was obtained when the corresponding experiments were performed with weak basic drugs, that are known P-glycoprotein substrates, and for which a clear efflux was also observed in the absence of a pH-gradient. Furthermore, a clear pH-dependence of the clinically important digoxin / quinidine-interaction was observed, indicating that the interaction is dependent on the amount of charged and uncharged species of quinidine available at the binding site of P-glycoprotein. Our results indicate that investigations on carrier-mediated efflux and uptake should be studied in the presence and absence of a pH-gradient. In addition, pH-dependence should be considered in studies of drug / drug-interactions involving P-glycoprotein and possibly also other transporters.
PO-185 PHYSICAL CHARACTERIZATION OF SOLID DISPERSIONS FOR A RATIONAL PHARMACEUTICAL DEVELOPMENT Carole Neves 1 , Hagit Elmaleh 1 & Anna Svensson 2 – 1 Aventis Pharma, Paris Research Center, Paris, France; 2 Lund University, Lund, Sweden The number of poorly soluble drug candidates has risen sharply and the formulation of poorly soluble compounds for oral delivery now presents one of the most frequent and greatest challenges to formulation scientists in the pharmaceutical industry. Various formulation and chemical approaches can be taken to improve the solubility or to increase the dissolution rate. The choice of a more soluble polymorph, the use of amorphous form and complexation of the drug are among the physical approaches. The compound can also be presented in a solid dispersion that has the benefit of a local increase in the solubility within the carrier. By judicious selection of a carrier, the dissolution rate of the drug can also be increased by up to several
orders of magnitude. For the manufacture of solid dispersions, polyethylene glycols (PEGs) or mixture of PEGs and modified PEGs are often used. Their solubility in water and melting points can be advantageous for the manufacture of solid dispersions by the melting method. The drug / carrier ratio in a solid dispersion is one of the main factors influencing the performance of the product. There are three main types of stability that need to be monitored for solid dispersions: chemical stability of the drug, physical stability of the drug and physical stability of the product. The physical properties of carriers are known to change over time and during melting process and aging. A physical change in the core of the matrix can influence and modify the release mechanism of the drug from the matrix. Physical characterization of the carrier and the product, investigation of the key parameters such as cooling rate and storage temperature are thus needed to control and prevent physical change of the matrix and to define the ability to scale up the manufacturing process. A PEGs-based carrier was physically studied: its crystalline / amorphous organization and physical stability were defined. Among methods that have been used are thermoanalytical, small angle X-ray diffraction and optical microscopy. The impact of water content showed the need to control humidity levels during manufacturing and storage processes. Solid dispersions obtained by the melting process using the PEGs-based carrier and drug substance A were manufactured by varying drug / carrier ratio. Physical characterization of the products was used to differentiate between solid solutions (solubilized drug in the carrier) and solid dispersions (dispersed drug). The drug state in the matrix was studied during manufacturing and under different storage conditions. A drug polymorph change had a huge impact on physical stability and organization of the solid dispersion. For a rational pharmaceutical development of solid dispersions, carrier and product physical characterizations are needed in order to avoid physical instability, to help scale-up processes and to anticipate product variability.
PO-186 THE PREPARATION OF PELLETS WITH SILICIFIED MICROCRYSTALLINE CELLULOSE BY EXTRUSION AND SPHERONISATION J Michael Newton – The School of Pharmacy, University of London, 29 / 39 Brunswick Square, London WC1N 1AX, UK Background: The major excipient used to prepare pellets by extrusion / spheronization microcrystalline cellulose (MCC). Surface modification of MCC with silicon dioxide or silicic acid provides a product, (silicified microcrystalline cellulose, Prosolv, SMCC), which has been shown to provide different rheological properties to MCC when mixed with water .
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
Aim: To investigate whether the differences in rheological properties of wet masses of MCC and SMCC caused any differences in the ability to produce pellets by the process of extrusion / spheronization. Materials and methods: MCC was Avicel grade PH101 (FMC corp. Cork, Ireland), while the SMCC was Prosolv 50 (Penwest, Nastola, Finland). The model drugs, in order of ascending water solubility, were of EP quality were ibuprofen (I) (BASF, Ludwigshafen, Germany), theophylillin (T) (Knoll, Nottingham, UK), paracetamol (P) (Knoll, Nottingham, UK), propanolol hydrochloride (Luscochemica, Milan, Italy), boric acid (B) (BDH, Poole UK), aminophyllin, anhydrous (A) (Boehringer Ingelheim, Bracknell, UK) ascorbic acid (Ac) (BDH, Poole, UK) and sodium salicylate (SS) (Merck, Poole, UK). Fifty g of each drug was mixed with an equal weight of either MCC or SMCC in a planetary mixer (Kenwood Chef, Kenwood, London, UK). Sufficient water was added to ensure that the wet mass-could be extruded at 200 mm / min through a die 1 mm in diameter and 5 mm in length attached to the barrel of a ram Acer extruder (Rheometrics, Leatherhead, UK). The extrudate was spheronized at 1800 rpm on the plate of a 12 cm diameter spheronizer (Caleva Process Solutions, Dorset, UK). The wet pellets were dried for 30 mins at 60 8C in a fluid bed drier (Uniglatt, Glatt, Binzen, Germany) The size of the pellets was determined by sieve analysis with British Standard sieves and the crushing strength of 20 pellets determined with a CT5 strength tester (Engineering Systems, Nottingham, UK). Results: The values for the amount of water needed to prepare the pellets, the modal size, and the % by weight in the model size, the median size and the load required to break the pellets are presented in Table 1. Conclusion: It is clear that, while the use of SMMC to replace MCC can change the pellet size and mechanical strength, it is possible to make pellets with drugs whose solubility ranges from 0.1 to 1000 g / l by using SMCC.
PO-187 CITRIC ACID AS TABLET-MATRIX EXCIPIENT IN MULTIPLE-UNIT ENTERIC-COATED TABLETS FOR COLON-SPECIFIC DRUG DELIVERY 1 2 ¨ ¨ P Nykanen , T Sten 1 , H Jurjenson , P Veski 2 & M 1 1 Marvola – Division of Biopharmaceutics and Pharmacokinetics, Department of Pharmacy, University of Helsinki, FIN-00014 Helsinki, Finland; 2 Institute of Pharmacy, University of Tartu, EE-2400 Tartu, Estonia
 Luukkonnen P et al. Use of a capillary rheometer to evaluate the rheological properties of microcrystalline cellulose and silicified microcrystalline cellulose. Int. J. Pharm 2001;216:147–157.
¨ ¨ ¨ S, Aaltonen M-L, Jurgenson ¨  Nykanen P, Lempaa H, Veski P and Marvola M. Citric acid as excipient in multiple-unit enteric-coated tablets for targeting drugs on the colon. Int J Pharm 2001;229:155–162.
We have prepared enteric-coated colon-specific multipleunit formulations by means of granules that are also enteric-coated. In the formulations, citric acid has been used as a pH-regulating additive . The aims of the study reported here were to prepare tablets containing citric acid as an excipient in the tablet matrix only, and to investigate whether onset of drug liberation and absorption could be controlled by varying the amount of citric acid in the tablet matrix from none to 15%. Drug release rates were studied at pH 6.8 and 7.4. Drug absorption was studied by means of bioavailability tests. Rate of release of the model drug declined in vitro as percentages of citric acid in the tablet matrix were increased from zero to 10 to 11.7. Increasing the percentage of citric acid in the tablet matrix to over 11.7 did not reduce release rate further. It was concluded that when a tablet matrix contained 11.7% or less of citric acid the rate of dissolution of the enteric-coating declined as the amount of citric acid present increased. Citric acid is water-soluble. When its percentage in a tablet matrix exceeds 11.7 it can promote degradation of the tablet matrix. A lag time of one to two hours was observed when the citric acid content of the tablet matrix equalled or exceeded 10%. Percentages of citric acid of between 10 and 15 in a tablet matrix might be appropriate for preparation of colonspecific formulations. With such formulations, fairly rapid absorption after a lengthy lag time might be achieved. This would however need to be demonstrated by visualization, e.g. using gammascintigraphy, before a conclusion could be reached.
Table 1 Properties of pellets produced with model drugs and MCC or SMCC Drug
I T P Pr B A Ac SS
Water g 65 65 65 60 60 30 50 30
Modal size range (mm)
% in Modal size
Median size (mm)
Breaking load (kg)
1.4–1.7 1.0–1.4 1.0–1.4 1.4–1.7 1.0–1.4 1.0–1.4 1.4–1.7 1.0–1.4
0.71–1.0 1.0–1.4 1.4–1.7 1.0–1.4 0.71–1.0 1.0–1.4 1.0–1.4 1.0–1.4
95.6 55.8 52.5 69.3 79.4 60.1 56.6 48.9
82.3 55.4 85.0 67.6 65.8 92.6 65.2 51.1
1.542 1.051 1.028 1.750 1.285 1.330 1.662 1.394
0.864 1.051 1.565 1.295 0.818 1.216 1.295 1.372
1.13 1.44 1.46 0.27 1.38 0.82 0.95 1.60
0.42 1.49 2.62 0.38 0.63 0.80 0.82 1.61
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-188 PREDICTION OF INTESTINAL ABSORPTION USING PHYSIOLOGICALLY BASED APPROACHES: COMPARATIVE ASSESSMENT OF COMMERCIALLY AVAILABLE SOFTWARE Neil Parrott & Thierry Lave´ – F. Hoffmann – La Roche AG, Pharmaceuticals Division, CH-4070 Basel, Switzerland We have assessed two commercial software tools employing physiologically based models for prediction of intestinal absorption in human. iDEA姠 2.0 and GastroPlus姠 3.1.0 were compared both in their ability to predict fraction absorbed for a set of 28 drugs and in terms of the functionality offered. The emphasis was placed on the practical usefulness to pharmaceutical drug discovery. Predictions were assessed for 3 levels of input data (i) pure in silico input, (ii) thermodynamic solubility and in silico permeability, (iii) thermodynamic solubility and human colon carcinoma cell line (CACO-2) permeability. We found the pure in silico prediction ability of the tools to be comparable with 70% correct classification rate. With measured input data the iDEA姠 prediction rate improved to 79% while GastroPlus姠 stayed at 70%. In terms of functionality GastroPlus姠 is a powerful system for the trained user. Open access to model parameters, diagnostic tools and the ability to integrate data make it particularly suitable for the later stages of discovery and development. iDEA姠 is web based and presents a simple interface suitable for widespread use with minimal training. However the limited functionality and inconvenient handling of multiple compound batches currently restrict the usefulness of version 2.0 for drug discovery.
PO-189 ASSESSING PRECIPITATION OF PRE-CLINICAL FORMULATIONS IN RAT GI FLUIDS R S Paterson & J G Murray – AstraZeneca R&D Charnwood, Preformulation and Biopharmaceutics, Bakewell Road, Loughborough, LE11 5RH, UK The advent of high throughput screening in drug discovery has resulted in many candidates with high potency, but with poor biopharmaceutical properties. Obtaining high plasma concentrations after oral administration (e.g. during toxicity testing) can be challenging when absorption is limited by low GI solubility. High dose suspensions (30 mg / mL), with a high solubilised fraction, were assessed for their ability to maintain solubility in the rat GI tract. Selected pre-clinical formulations (e.g. HP-b-cyclodextrin / HPMC / 0.2 M meglumine (10 / 0.5 / 89.5 w / w)) were used to solubilise the Na salt and free acid of a candidate drug. The two objectives of this study were to 1) assess extent and site of drug precipitation in the rat GI tract and 2) correlate solubility values with high dose in vivo data in the rat (250 mg / kg, 10 ml / kg, n53). The formulations
were mixed (150 rpm) with relevant quantities of rat gastric or SI fluid (biological and simulated fluids) at 37 8C for times that approximated the transit in the stomach (45 min) and SI (2 hr). The samples were centrifuged, diluted and assayed by HPLC. Solubility measurements in simulated fluids correlated with biological fluids in that increased precipitation occurred in SI fluid. Increased precipitation in the SI was unexpected as the compound is a weak acid (pKa 6.8). The alkaline formulations (pH11) maintained higher gastric solubility due to a low stomach volume containing only dilute HCl (pH 3). The formulations solubilised the Na salt to a greater extent than the free acid (e.g. 24 mg / ml and 5 mg / ml respectively in HP-b-cyclodextrin / HPMC / 0.2 M meglumine (10 / 0.5 / 89.5 w / w)), which resulted in higher solubility values upon dilution in GI fluids. In order to gain further information on sites of GI precipitation the quantities and locations of contents in the rat SI were determined (n5 12). The duodenum and upper jejunum (first 40 cm) were mostly empty in comparison to greater amounts in the lower jejunum and ileum (next 60 cm). These observations suggest that following gastric emptying the formulations could be less sensitive to precipitation in the upper SI regions (containing fewer contents) before precipitating to a greater extent in the lower SI regions (greater contents). In vivo data showed much higher Cmax and AUC values for the Na salt in comparison to the free acid; the Cmax and AUC of the different forms correlated with the solubility values in the GI compartments. The experiments were useful for predicting the site and extent of precipitation in the rat GI tract. The subsequent in vivo exposure data in the rat was consistent with the ex vivo solubility data. The rat GI solubility values could be useful inputs for future simulations of the effect of formulation on oral drug absorption.
PO-190 POLYMERIC NANOPARTICLES PREPARED FROM POLYLACTIC ACID OF TWO DIFFERENT MOLECULAR WEIGHTS Leena Peltonen, Piritta Koistinen & Jouni Hirvonen, Pharmaceutical Technology Division and Viikki Drug Discovery Technology Center, PL 56 (Viikinkaari 5 E), 00014 University of Helsinki, Finland. The aim of this work was to formulate polymeric nanoparticles for pulmonary drug administration. The polymer used was polylactic acid (PLA), which is widely used in drug formulations as a biodegradable and biocompatible polymer. As a model drug was selected sodium cromoglycate, which is used as an antiallergic drug in the treatment of bronchial asthma. Sodium cromoglycate has a short half-life and frequent dosing schedule. A long acting sodium cromoglycate formulation is, therefore, desirable to improve patient compliance. In this study, the effect of
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
molecular weight of the PLA polymer on the nanoparticle preparation, particle size and morphology were studied. Nanoparticles were prepared by a modified interfacial polymer deposition method. The method was performed as follows: the drug substance was dissolved in water and after that acetone was added to this solution. PLA as a biodegradable polymer and propylenglycol as a surfaceactive agent were dissolved in chloroform and this solution was added to the drug solution to form dispersion. This dispersion was added to an aqueous ethanol solution and the organic solvents were removed by evaporation under reduced pressure. The molecular weight of PLA was either appr. 2000 g / mol or 100 000 g / mol. With both the PLAs it was possible to formulate nanoparticles. The approximate particle size was 300–500 nm. Particles were smooth and round-shaped. The composition with lower molecular weight PLA demanded more surface-active agent in order to form particles. Also the efficiency of particle formation was not that good with the lower molecular weight PLA, and the particles were more prone to aggregate.
PO-191 SODIUM CROMOGLYCATE LOADED POLYLACTIDE NANOPARTICLES: THE EFFECT OF COSOLVENTS Leena Peltonen, Piritta Koistinen & Jouni Hirvonen, Pharmaceutical Technology Division and Viikki Drug Discovery Technology Center, PL 56 (Viikinkaari 5 E), 00014 University of Helsinki, Finland. Polylactic acid (PLA) is widely used polymer in pharmaceutical formulations, because it is biodegradable and biocompatible. However, lower molecular weight PLA (Mw 2000 g / mol), which degrades faster in human body as compared to the higher molecular weight PLA, has rarely been utilized in nanoparticle formulations. Sodium cromoglycate is an antiallergic drug, which is used in the treatment of bronchial asthma. With water-soluble drug substances, like sodium cromoglycate, a co-solvent is needed to make the aqueous drug solution and the organic polymer solution dispersible with each other. In this present work the effect of co-solvent selection on the properties of the nanoparticles was studied. Preparation technique for nanoparticles was a modified interfacial polymer deposition method. Sodium cromoglycate was dissolved in water and after that acetone or ethanol was added to this solution. PLA as a biodegradable polymer and propylenglycol as a surface-active agent were dissolved in chloroform and this solution was then added to the drug solution to form dispersion under vigorous stirring. The dispersion was added to an aqueous ethanol solution, the suspension was filtered and the organic solvents were removed by evaporation. With both acetone and ethanol it was possible success-
fully prepare sodium cromoglycate loaded PLA nanoparticles with this method. However, after the nanoparticle formation the aggregation of nanoparticles was more prone with acetone as a co-solvent. On the other hand, the formulations with ethanol tended to aggregate more during the evaporation process. As a conclusion it could be said that the filtration and evaporation steps are very important in order to get stable and individual nanoparticles.
PO-192 RELEVANCE OF COSMETIC SKIN TYPES IN CUTANEOUS HEALTH CARE ´ M. Rodrigues Ana Gago Pereira, Ana Rita Ribeiro & Luıs – UCTF, Laboratory of Cutaneos Biology, Faculdade de ´ Farmacia Universidade de Lisboa, Av. Forc¸as Armadas, 1640-019 Lisboa, Portugal Cosmetics are a principal component of an healthy skin. The many developments noted from formulation technology to claims created growing expectations regarding the effects, while transformed the choice and counselling into a very complex process. The present study aims to clarify the relative importance of the known ‘‘cosmetological skin types’’ as conditioning factors of cosmetic’s efficacy. Data was collected from healthy women (n520) 25–55 y.o., after informed written consent. The experimental methodology involved the long term ‘‘in-use test’’ of a standard hydration formulation in 3 different anatomical areas after which (1) an auto-evaluation questionnaire including the self-appreciation of the skin type and (2) biometrical assessments of the most representative variables considering skin basic physiology (hydration and biomechanics) took place. Measurements occurred at inclusion (t0), 15 days (t15) and 30 days (t30) and were compared with contra-lateral non-treated areas. A confidence interval of 95% was adopted. Results suggest that cosmetic skin ‘‘types’’ may only be discussed when considering the areas where sebaceous glands are distributed. Otherwise, the sole particular conditions found was ‘‘dry’’ skin, meaning that all the other classifications may be considered within a ‘‘normal’’ condition. In any case, regular cosmetic care improved all the physiological variables measured, independently from cosmetic skin types or the anatomical area tested.  Le Fur, I., Lopez, S., Morizot, F., Guinot, C., Tschaler, E. Comparison of cheek and forehead regions by bioengineering methods with different self-reported ‘‘cosmetic skin types’’, Skin Res. & Technol. 1999;5:182–188.  Rodrigues, L., Marcelo-Pereira, L. Basal Transepidermal Water Loss Dependence on Right / Left Forearm Differences and Motoric Dominance, Skin Res. & Technol. 1998;4:135–137.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-193 INTESTINAL ABSORPTION AND METABOLISM OF SULFORAPHANE AND QUERCETIN AS WELL AS REGULATION OF PHASE 2 ENZYMES IN HUMAN JEJUNUM IN VIVO AND IN Caco-2 CELLS N Petri 1 , C Tannergren1, R N Bennett 2 , B Holst 2 , Y Bao 2 , G W Plumb 2 , J Bacon 2 , K O’Leary 2 , L Knutson 3 , P ¨ 1 & G Williamson 2 – Forsell 3 , T Eriksson 3 , H Lennernas 1 Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden; 2 Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK; 3 Department of Surgery, University of Uppsala, Enkoping Hospital, SE-745 25 Enkoping, Sweden To study permeability (Peff ) and metabolism of sulforaphane and quercetin-3,49-diglucoside in the human jejunum. Shed enterocytes were also collected in order to investigate the short term regulation of phase 2 enzyme gene expression. Using a human in vivo jejunal perfusion (Loc-I-Gut ) sulforaphane and quercetin-3,49-diglucoside were applied in their natural, but protein free, matrix to six (3 male and 3 female) healthy volunteers. The compounds were perfused for 90 min and shed cells were collected before and after the perfusion. A control study was also performed. Total RNA from the enterocytes was extracted using the RNeasy mini kit and mRNA was analysed using Taq-man RT-PCR. The average (6SEM) Peff and %Absorbed were 1764.9e–04 cm / s and 74611% for sulforaphane and 8.962.9e–04 cm / s and 60612% for quercetin-3,49-diglucoside. The conjugates sulforaphane-glutathione and quercetin-39-glucuronide were excreted back into the lumen. The gene expression analysis in exfoliated enterocytes showed 1.860.5-fold induction of glutathionetransferase-A1 (GSTA1) mRNA and 2.161.3 induction of UDP-glucuronosyl-transferase-1A1 (UGT1A1) mRNA. The control study did not show any changes in mRNA levels. The changes in gene expression were also seen in Caco2 cells, where it was shown that quercetin is responsible for induction of UGT1A1 and sulforaphane for induction of GSTA1. For the first time the human intestinal permeability of phytochemicals in a complex matrix using an in vivo perfusion system has been studied. The jejunal epithelial cells provide not only the capacity for absorption and first pass metabolism of these compounds but also a direct pathway for excretion of conjugates.
PO-194 TRANSPORT CHARACTERISTICS OF FEXOFENADINE IN THE Caco-2 CELL MODEL COMPARED TO IN VIVO PERMEABILITY DATA ¨ 1 – N Petri 1 , C Tannergren 1 , L Knutson 2 & H Lennernas 1 Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden; 2 Department of Surgery, University of Uppsala, Enkoping Hospital, SE-745 25 Enkoping, Sweden
To investigate the transport mechanisms of fexofenadine in the Caco-2 model. A jejunal perfusion study with fexofenadine and verapamil was also conducted to compare the in vitro results with human in vivo data. Transport studies were performed in Caco-2 cell monolayers 21–25 days after seeding. The permeability of fexofenadine was determined in the concentration range from 10 to 1000 mM in both apical-to-basolateral (a-b) and basolateral-to-apical (b-a) direction. The effect of 10–200 mM P-glycoprotein (P-gp) (ketoconazole, verapamil, erythromycin) and MRP inhibitors (indomethacin, probenecid) on the bi-directional transport of 150 mM fexofenadine was also studied. A jejunal single-pass perfusion study in healthy volunteers was performed using the Loc-IGut technique, where 100 mg / L (186 mM) fexofenadine was given either alone or with 500 mg / L (1018 mM) verapamil. Fexofenadine displayed polarized transport, with the apparent b-a permeability (Papp) being several-fold higher compared to the a-b direction. The Papp in the b-a direction was concentration dependent, suggesting saturation of an apical efflux transporter. The P-gp inhibitors had a strong, concentration dependent effect on the Papp of fexofenadine. For example 100 mM verapamil, increased the a-b Papp 5-fold (from 0.17 to 0.93310 26 cm / s) and decreased the b-a Papp 2-fold (from 6.9 to 2.8310 26 cm / s) ending at an efflux ratio of 3. The MRP inhibitors did not affect the Papp of fexofenadine. Co-administration with verapamil did not result in a significant change in the human effective jejunal permeability (Peff ) of fexofenadine. The mean (6SEM) Peff was 0.06 (60.02) and 0.04 (60.02)?10 24 cm / s without or with verapamil, respectively. Based on the in vitro results, fexofenadine seems to be effluxed by P-gp but not by MRP. However, the strong effect of the P-gp inhibitor verapamil was absent in vivo in human jejunum even at a high concentration.
PO-195 THE JEJUNAL PERMEABILITY (Peff ) AND PRESYSTEMIC METABOLISM OF ISOTRETINOIN IN HUMANS N Petri 1 , C Tannergren 1 , L Knutson 2 , U Wiegand 3 & H ¨ 1 – 1 Department of Pharmacy, Uppsala UniLennernas versity, Box 580, SE-751 23 Uppsala, Sweden; 2 Department of Surgery, University of Uppsala, Enkoping Hospital, SE-745 25 Enkoping, Sweden; 3 F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland The objectives of the study were to assess the intestinal permeability (Peff ) and presystemic metabolism of isotretinoin in healthy young male volunteers using a jejunal perfusion technique (Loc-I-Gut ). A regional single-pass perfusion experiment of the jejunum was performed in six healthy male volunteers using Loc-I-Gut . The subjects were to receive an isotreti-
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
noin dose of 0.5 mg / kg body weight as jejunal perfusion during a time period of 90 minutes. Five of the six subjects obtained a placebo solution as a control in a randomised crossover fashion. 14 C-radiolabelled PEG 4000 was included in the formulation as a non-absorbable volume marker to assess the net water flux in the segment. The concentrations of isotretinoin in the perfusate leaving the jejunal segment and plasma were measured by HPLC with column switching technique. The analysis of the outlet perfusate samples showed that isomerisation of isotretinoin to retinoic acid occurred in the intestine. In average (6SEM), 2.3561.30% of the given dose of isotretinoin was converted to retinoic acid. No 4-oxo-isotretinoin or 4-oxo-retinoic acid was detected in the jejunal perfusate. In plasma were retinoic acid and both of the 4-oxo-metabolites detected. The average (6SEM) Peff for isotetinoin was 0.9960.11 e–04 cm / s and % absorbed of the drug was 23.7962.13%. It is evident from the data that only isomerisation, and no oxidation, occurred in the jejunal fluid. However, this does not exclude the possibility that oxidation may occur in cells of the gut wall besides the liver as the plasma concentrations of the oxidative metabolites clearly document. It was also found that isotretinoin is a high permeable drug.
PO-196 ASSESSING THE CUTANEOUS ‘‘BARRIER’’ FUNCTION BY COMPARTMENTAL ANALYSIS OF TRANS-EPIDERMAL WATER LOSS Pedro C Pinto, Jose´ M Magro, M a] M Mouzinho, Ricardo Ferreira, Jose´ Silva, M a] A Almeida, Luis M Rodrigues – Unit of Experimental Dermatology, ULHT Campo Grande 376 1700 Lisboa, & Lab. of Cutaneous Biology, FFUL, Av das Forc¸as Armadas, 1600 Lisboa, & Plastic and Recon´ 1150 Lisboa, Portugal structive Surgery Service, H.S. Jose, Mathematical modelling of physiological variables such as Trans-epidermal water loss (TEWL) has been proposed as a practical assessment strategy to quantitatively describe this water-mass driven process . The present study aims to demonstrate the usefulness of this methodology to describe the in vivo cutaneous ‘‘barrier-function’’ in grafted patients following cutaneous loss by thermal injury. Thermal burns patients (n58) with autologous splitthickness skin grafting were submitted to a POST (Plastic occlusion stress test) methodology that included a 24 H occlusion period with proper material on week 8, 12 and 16 after surgery. Desorption TEWL curves obtained on lesions areas (graft and donor) were compared with normal contra-lateral controls. This TEWL profiles were used to obtain the useful parameters from the mathematical bicompartmental model previously developed [1,2]. Compartmental analysis of TEWL is useful to characterise and describe different conditions of the skin barrier.
This non-invasive methodology also provides quantitative results based in physiological assumptions, which may be used to follow-up the process in order to further identify its complexities
 Rodrigues, L.M., Pinto, P., Galego, N., Amores da Silva, P., Marcelo Pereira, L. Transepidermal water loss Kinetic Modeling Approach for the Parameterization of Skin Water Dynamics, Skin Res. & Technol. 1999;5:72–82.  Rodrigues, L.M., Pinto, P.C., Mouzinho, M.M., Ferreira, R., Pereira, J., Magro, J.M., Almeida M a] . A. Modelling Cutaneous water dynamics in Allografted Skin, Burn Care & Rehab., Proceed Am Burn Assoc., 2002;23(2);S139.
PO-197 INFLUENCE OF THE OCCLUSION TIME OVER THE IN VIVO CUTANEOUS WATER KINETICS DESCRIBED BY COMPARTMENTAL ANALYSIS OF TEWL ´ 2 , Luis M Pereira 1 , Luis M Pedro C Pinto 1 , Rui Minhos 2 1 Rodrigues – UDE, Unit of Experimental Dermatology, ULHT Campo Grande 376 1700 Lisboa, Portugal; 2 Lab. of Cutaneous Biology, FFUL, Av das Forc¸as Armadas, 1600 Lisboa, Portugal
Recent methodologies used to describe in vivo cutaneous water dynamics are based in the compartmental analysis of the Transepidermal Water Loss (TEWL) profiles following the Plastic Occlusion Stress Test (POST) . However, proposal of the useful comparative parameters must result from the characterisation of all the conditionings such as the occlusion time. Thus, the present work aims to study the influence of the occlusion periods over this analytical strategy. The study was performed in 40 healthy volunteers, after informed written consent. All patients were submitted to a POST methodology (Plastic occlusion stress test) with adequate material, following 4 different occlusion periods (1H, 6 H, 12H 24 H). TEWL decays were obtained for 30 min starting immediately after patch removal. A previously developed compartmental model was adjusted to data and quantitative dynamical parameters (half-life times) were calculated. Results demonstrate that the occlusion time does influence the parameters obtained. Two clusters of parameters may be identified. One corresponding to fast evaporation processes (1 H of occlusion) and other corresponding to slow processes (6 H, 12 H and 24 H of occlusion). Results also demonstrate the capacity of the compartmental analysis to produce quantitative parameters that correlated well to the skin water and, therefore, useful to study cutaneous water dynamics.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
 Beradesca, E., Elsner P. Dynamic Measurements: The Plastic Occlusion Stress Test (POST) and the Moisture Accumulation Test (MAT), In: Elsner P., Berardesca E., Maibach H. (Eds.), Bioengineering of the skin: Water and the Stratum corneum, CRC Press, 1994, pp. 97–102.  Rodrigues, L., Pinto, P., Galego, N., Amores da Silva, P., Marcelo Pereira, L.. Transepidermal water loss Kinetic Modeling Approach for the Parameterization of Skin Water Dynamics, Skin Res. & Technol. 1999;5:72–82.
PO-198 A PHARMACOGENOMIC ANALYSIS OF LUNG TISSUES AFTER INTRATRACHEAL ADMINISTRATION OF NON-VIRAL GENE DELIVERY SYSTEMS. ¨ ¨ ˚ &K E G E Ragnarsson, P Artursson, M Koping-Hoggard ¨ – Department of Pharmacy, Uppsala UniversiRegnstrom ty, Sweden The objective was to compare two cationic gene delivery systems, polyethylenimine (PEI) and ultra pure chitosan (UPC), with respect to different gene expression profiles and stabilization of the luciferace reporter after in vivo administration. The two polymers (PEI and UPC) were formulated with the reporter plasmid pLuc and were injected intratracheally to mice. After 24 or 72 hours the animals were sacrificed and total RNA was extracted from the lungs. cDNA probes labeled with 32 P were made with the RNA as a template. The probes were hybridized to an array with genes involved in stress and toxic reactions. Selected results from the array were confirmed on the protein level by Western blotting. The genes with a significant change in expression were clustered using self-organizing maps, SOMs. Six different SOM clusters were obtained. Most of the clusters contained genes which were strongly affected after administration of PEI, giving PEI a more toxic ‘‘fingerprint’’ than the biodegradable polymer UPC. The genes affected by PEI provide a likely explanation for the mechanism behind the more toxic effect of PEI as compared to UPC . The PEI-formulation also up-regulated several chaperone subunits. Chaperones are known to stabilize and facilitate the folding of proteins, including luciferace, in cells. The up-regulated expressions of some distinct subunits were confirmed with Western blot analysis of lung tissues. This study identifies important differences in the gene expression profiles obtained after lung administration of two non-viral gene delivery systems, based on PEI and UPC. PEI showed a more toxic expression profile than UPC and also up-regulated a luciferas-stabilizing chaperone. We speculate that this up-regulation, at least partly, explain the prolonged gene expression of the luciferase reporter gene after administration by PEI. We
also conclude that gene expression profiling has a great potential for evaluation of gene delivery systems.  Koping-Hoggard M, Tubulekas I, Guan H et al. Chitosan as a nonviral gene delivery system. Gene Ther 2001;8(14):1108–21.
PO-199 PREDICTION OF THE MOISTURE LOSS OF DIFFERENT HUMECTANTS USING AN IN VITRO METHOD Helena M Ribeiro, Joana Ogando & Eduardo Barata – ´ Faculdade de Farmacia da Universidade de Lisboa, Av. Forc¸as Armadas, 1600 Lisboa, Portugal Humectants are hygroscopic materials that are added to cosmetic creams particularly of oil in water type, to reduce the rate of dry-out. The aim of this work is to characterise, by an in vitro method (gelatine method) the rate of loss moisture of six different humectants (S193 , NMF , Lactil , Hydrolact , Lubragel , and Liponic ) using glycerine, dimethicone and liquid paraffin as reference substances. Some physical and chemical properties of the raw materials were evaluated: pH determination, rheology characterisation, spreadbility, and moisture content. Ten o / w emulsions were processed using the same ingredients, except the humectants and the references. These preparations were also analysed (pH determination, viscosity evaluation, moisture content and stress stability) . To predict the in vitro rate of loss moisture all the materials and the creams were placed in a surface with gelatine (that acts as the Sratum corneum). Under the gelatine there is always 50 mL of water to create an environment of 100% humidity. This system was placed in a temperature and humidity controlled conditions. Weighting the total amount of water, gelatine and ingredients or creams before and after 24 h and 48 h the results can predict the differences between them . The results obtained allowed to identify differences regarding the water dynamics expressed in terms of rate of of loss moisture. We can conclude that this in vitro method can discriminate the different humectants used in cosmetic creams.  Ribeiro, H. M., Afonso, R., Barata, E., Morais, J. A. Correlation of physical and formulation properties on different emollients, Proc., 2 nd World Meeting, APGI / APV, 1998, pp. 751–752.  Barata, E. A cosmetologia, Lidel Ed., 2002, p. 228.
PO-200 SKIN PERMEATION ASSESSED IN VIVO BY CYANOACRYLATE SURFACE BIOPSY Catarina Rosado & Luis Rodrigues, Laboratory of Cuta´ neous Biology & UCTF, Faculdade de Farmacia da Universidade de Lisboa, Av das Forc¸as Armadas, 1600 Lisboa, Portugal
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
The use of solvents that affect the skin barrier function is one of the classic strategies of penetration enhancement. Some of these solvents have well characterised actions on the stratum corneum, but the majority is still selected using empirical criteria. The objective of this study was to compare differences in the permeation of a hydrophilic and a lipophilic dye after treatment of the skin with different vehicles. The solvents were applied to the volar forearm of healthy volunteers using an occlusion chamber. After 30 minutes the chamber was removed and treated and untreated (control) sites were exposed to either solutions of methylene blue or Sudan III for 1 hour . Four consecutive skin surface biopsies of each site were taken and the cumulative amounts of the dyes in the stripped stratum corneum were measured by reflectance colorimetry [2,3]. Results showed that the two model permeants were influenced differently by the various treatments and that it was possible to cause more significant changes in the permeation of the hydrophilic molecule. There was no simple correlation between the properties of the solvent and permeation enhancement. This study has only superficially explored the potential of the combination of skin surface biopsy and colorimetry, but the encouraging results obtained confirm that the technique can be extended to the study of more complex formulations.  Zhai H, Maibach HI. Effect of barrier creams: human skin in vivo, Contact Dermatitis 1996;35:92–96.  Marks R, Dawber RP. Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Derm 1971;84:117–123. ´  Pierard GE. EEMCO guidance for the assessment of skin colour, J Eur Acad Dermatol Venereol 1997;10:1– 11.
PO-201 DIFFERENCES IN SKIN PERMEATION ASSESSED IN VIVO BY THE POST OCCLUSION STRESS TEST Catarina Rosado 1 , Pedro C Pinto 2 & Luis M Rodrigues 1 – Laboratory of Cutaneous Biology & UCTF, Faculdade de ´ Farmacia da Universidade de Lisboa, Av das Forc¸as Armadas, 1600 Lisboa, Portugal; 2 UDE, Unit of Experimental Dermatology, ULHT Campo Grande 376 1700 Lisboa, Portugal The mathematical modelling of transepidermal water loss (TEWL) curves that result from a post occlusion stress test (POST) is a useful approach to describe and quantify the cutaneous ‘‘barrier’’ function . An obvious potential interest may be found in applying this methodology to in vivo permeation studies. The objective of this study was to compare differences in the desorption curves obtained from different anatomical sites in order to perceive differences in the hydration
dynamics and to predict differences in the permeation. Ten volunteers participated in this study after informed consent. Subjects were submitted to a POST, involving the application of an occlusive patch to the mid volar forearm, the wrist and the forehead. Occlusion took place for one hour, after which the desorption curves were recorded. Data points were continuously registered for 30 minutes. The mathematical model was adjusted to the data points using a specially modified simplex routine and the software ADAPT written in Fortran. Calculated parameters considered relevant to the study were Kevap (water evaporation at skin surface), t 1 / 2evap (evaporation half-life) and DWM (dynamic water mass). Results seem to indicate differences in the parameters obtained for the three anatomical sites, which suggests differences in the water dynamics and that the method is valid for the objectives proposed. ´ R, Rodrigues LM.  Pinto PC, Pereira LM, Minhos Testing the discriminative capacity of compartmental modelling for the analysis of the in vivo epidermal water content changes following topical application under occlusion, IFSCC Magazine 2002;5:1–6.
PO-202 STUDY ON THE INFLUENCE OF SEBUM CASUAL LEVELS ON THE EPIDERMAL PHYSIOLOGY Martha Oviedo Salazar, Nuno Furtado & Luis M Rodrigues – Laboratory of Experimental Physiology & UCTF, ´ Laboratory of Cutaneous Biology, Faculdade de Farmacia da Universidade de Lisboa, Av das Forc¸as Armadas, 1600 Lisboa, Portugal The forehead sebum measurement has proved to be useful for cosmetological efficacy studies. These are particularly important to search for better practical approaches of special dermatological conditions such as acne vulgaris. The aim of this study was to evaluate the relative contribution of cutaneous sebum, expressed by sebum casual levels (SCL), on the stratum corneum physiology. Fifteen healthy women were included after informed written consent. The SCL was evaluated by a photometric method (Sebumeter SM810PC from C1K electronics, FRG) and expressed in mg / cm 2 . Other physiological variables such as skin hydration and biomechanical descpritors were also evaluated by non-invasive methodologies. The forehead was divided into two areas, right and left, then sub-divided into ten adjacent square of 1 cm 2 . The forehead was treated with an organic solution to remove the lipid components of the skin. The SCL was evaluated in times 0, 1, 3, and 5 hours after the cleaning treatment. A significant level of P,0.05 was adopted. SCL through 8 hours conserved a symmetrical production over the opposite site, suggesting that a contralateral evaluation of cosmetic products can be used as a
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
reference control, at least for the ‘‘oily skin’’ group. However, cosmetic efficacy tests should also be supported by complementary assessments including, clinical evaluation, and consumer’s preference questionnaires. ´ ´  Pierard, G.E., Pierard-Franchimont C., Marks, R., Paye, M., Rogiers, V., Berardesca, E., Elsner, P., ´ ` Leveque, J.L., Loden, M., Masson, Ph., Parra, J.L., Rodrigues, L. EEMCO Guidance for the in vivo assessment of skin greasiness, Skin Pharmacol. Appl. Skin Physiol., 2000;13:372–389.  Rodrigues, L., Jaco I., Melo M., Silva R., M.Pereira L., Catorze N., Barata E., Ribeiro H., Morais J. About Claim Substantiation for Topical Formulation; an Objective approach to Skin Care Products Biological Efficacy, J. Appl. Cosmetol., 1996;14:93–98.
PO-203 INTERPRETATION OF DISSOLUTION DATA OF TABLETS MADE FROM BINARY MIXTURES OF ETHYLCELLULOSE AND A MODEL DRUG IN LIGHT OF THE PERCOLATION THEORY ˜ F Pinto & Paulo J Costa – Dept. ´ Paulo J Salustio, Joao ˆ ´ Tecnologia Farmaceutica, Faculdade de Farmacia da Uniˆ versidade de Lisboa and Dept. Tecnologia Farmaceutica, ´ Faculdade de Farmacia da Universidade do Porto, Portugal Evaluation of changes in drug release, based on the percolation theory. Different mixtures of ethylcellulose (EC) and propranolol. HCl (PRO) were produced in a tumble mixer for 10 minutes. PRO content in the mixture ranged from 0 (0P) up to 100% (10P). Tablets containing each one 400 mg of the mixture were tabletted at 50 MPa using a mechanical press. The dissolution profiles of PRO in the tablets were obtained from dissolution tests run according to the EP (paddle, 150 rpm, 900 mL, pH51.0, 37 8C). Samples were quantified online by UV spectrophotometry (248 nm). Higuchi and Peppas models were considered upon analysis of the results. The release of the drug was faster when less amount of EC was present in the formulation (Fig. 1, 5P to 10P). As anticipated, the amounts of drug released increased accordingly with the amount of drug in each formulation (Fig. 2). Moreover, lower loads of PRO in the formulations led to an incomplete release of the drug. Figs. 1 and 2 show that for low amounts of PRO (1P and 2P) the amount of PRO released were identical and much lower than expected. A possible
interpretation to this observation may arrive from ceasing a continuous cluster of PRO, for percentages below 30% (3P), which was percolating the tablet. It can be assumed that this threshold reflects the presence of discontinuous clusters of PRO embedded into a continuous network of EC. This observation was confirmed by the drug’s release mechanism, which changed from Higuchi (1P and 2P) to zero order (3P–5P). The study underlined the usefulness of the theory of percolation on the explanation of tablet’s structure and its effect on the bio-availability of the drug considered.
PO-204 PREPARATION OF THE TOPICAL FORMULATIONS WITH ANTIMICROBIAL ACTIVITY FROM SOME LABIATAE SPECIES ¨ ¸ er 2 , A Akın 3 & H Demir 3 – ¨ ¨ 2 , A Olc E S¸arer 1 , N Gonul 1 Department of Pharmacognozy; 2 Department of Pharmaceutical Technology; 3 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Ankara University, ˘ 06100 Tandogan, Ankara, Turkey The members of the Labiatae family are found in the tropics, subtropics and temperate parts of the world. A large number of secondary metabolites have been described from the aerial parts of several members of the family. Some of them have shown interesting biological activities. Aerial parts extracts (24 extracts) of four Labiatae viz. Rosmarinus officinalis, Teucrium polium, Teucrium chamaedrys and Melissa officinalis have been tested for antimicrobial properties against some gram(1) and gram(2) bacteria and a yeast-like fungus. Efficient extracts were determined and used at 1% ratio in the topical semisolid formulations  such as cold cream, emulsion, emulgel, hydrophilic ointment, carbopol 934 and chitosan gels. Release of the secondary metabolites from the different vehicles and also the release of the active substances from the commercially available topical antimicrobial preparations were studied and evaluated. Maximum release was observed from emulgel and carbopol 934 gel.  Nairn JG. Solutions, emulsions, suspensions and extracts. in: Gennaro AR (Ed.), Remington: the Science and Practice of Pharmacy 2000:721–51.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-205 PROSOLV SMCC CUSTOM PROCESSING TO CONTROL HYGROSCOPICITY OF PHARMACOLOGICALLY ACTIVE COMPOUNDS David Schaible, Bob E Sherwood, Joseph A. Zeleznik & Theodore Montalto – Penwest Pharmaceuticals Co., 2981 Route 22, Patterson, NY 12563, USA Purpose: To evaluate moisture sorption and examine powder properties associated with hygroscopicity of model active compounds compared to those compounds Custom Processed with PROSOLV SMCC. Methods: Several model active compounds having known moisture sorption issues were prepared with PROSOLV姠 SMCC in a proprietary custom process. Moisture sorption was evaluated by mass increase as a function of time at various relative humidities. Materials were evaluated for powder flowability on a Hanson Flodex姠 for flow rates and bridging aperture. Qualitative comparisons were also made on powder beds after moisture exposure over time. Results: Custom Processed model active compounds showed little moisture sorption compared to untreated raw materials and raw materials treated by traditional processing. Flow properties showed improvement regardless of moisture exposure for custom processed materials. Compound ‘‘B’’, which did not flow at any aperture on the Flodex姠 flowed through 9 mm a aperture after PROSOLV SMCC Custom Processing. Exposure of this Custom Processed to high humidity showed little change in flowability. Other model actives showed similar flow improvements after PROSOLV custom processing. Conclusion: Custom processing with PROSOLV SMCC significantly reduced hygroscopicity and improved flowability. Custom processing allowed longer term moisture exposure providing for greater material stability during tablet manufacture.
PO-206 PHARMACEUTICAL ASSAYS OF LIPOSOMES ENCAPSULATED DOXORUBICIN Z Shprakh, A Polozkova, O Orlova, I Shoua, N Oborotova & A Baryshnikov – N.N. Blokhin Memorial Cancer Research Centre Russian Academy of Medical Science, Kashirskoye sh., 24, 115478 Moscow, Russia Liposomes are effective carries of anticancer antibiotics. In this research liposomes encapsulated Doxorubicin (LED) were prepared to assay the possibility of cancer multidrug resistance overcoming by new pharmaceutical dosage form. LED was prepared by evaporation to dryness of spirituous solution of cholesterol, cholesterol and lecithin (12:6 molar correlation) or cholesterol, lecithin and cardiolipin (12:6:1). To encapsulate Doxorubicin to LED uniform lipid film was hydrated by drug aqueous solution. A red multilamellar LED emulsion was obtained. Correlation between Doxorubicin and lipids were 1:5, 1:10 and 1:15 . LED size was measured on Nanosizer NICOMP
380 and was determined as about 5006120 nm. Small liposomes were created by extruding through polycarbonate membrane with 100 nm pore. This LED size was 120630 nm. The entrapment efficiency measurement involved measurement of nonentrapped drug solubilized in the aqueous milieu. The liposome emulsion was centrifuged at 30 0003g for 45 min. A red pellet containing lipid vesicles was obtained. The supernatant was collected to determine the amount of drug by UV-spectrophotometry at wavelength of 252 nm. Has been determined that LED composition influenced on Doxorubicin entrapment, which was the most effective (more than 70%) in LED including cardiolipin Investigation of stability of liposomes consisted of observation of aggregate stability and measuring the entrapment efficiency in different points of time after liposomes formation. Has been shown that LED of all compositions were aggregate stable during 4 days at 37 8C. But Doxorubicin release from cardiolipin including LED was the slowest (about 6% for 4 days).  R. Perez-Soler, W. 1990;50:4260–4266.
PO-207 IN VITRO EVALUATION OF DRUG-LOADED P(MAA-g-EG) HYDROGELS 2 ¨ Oya Sipahigil 1,2 , Ayla Gursoy & Nicholas Peppas 1 – 1 Purdue University, School of Chemical Engineering, West Lafayette, IN 47907-1283, USA; 2 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 81010 Istanbul, Turkey The objective of this work was to prepare and characterize hydrogel carriers composed of poly(methacrylic acid) grafted with poly(ethylene glycol) [P(MAA-g-EG)] loaded with diltiazem HCl, diclofenac Na, ciprofloxacin HCl and izoniazid. P(MAA-g-EG) hydrogels were prepared by free radical solution polymerization. The monomers were used in a molar ratio of 1:1 repeating units. Tetra(ethylene glycol) dimethacrylate was used as a crosslinking agent and Irgacure 184 was the photoinitiator. After diltiazem HCl, diclofenac Na, ciprofloxacin HCl and izoniazid were loaded into P(MAA-g-EG) hydrogels as small molecular weight model drugs, their loading efficiencies and in vitro release profiles were studied in pH 1.2–5.8–6.8 and 7.4. The loading efficiencies of diltiazem HCl, diclofenac Na, ciprofloxacin HCl and izoniazid were 44.7264.27%, 23.2362.80%, 13.4061.39% and 52.2761.51% respectively. Approximately 75% of the drugs were released in 10 hours in a pH 7.4 phosphate buffer solution. Drug loaded P(MAA-g-EG) hydrogels were stored at 40 8C / 75% relative humidity for three months to evaluate the effect of accelerated conditions on the release profiles of these systems. Finally, Fourier transform infrared spectroscopy and differential scanning calorimetry studies were performed before and after the stability study.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
PO-208 FORMULATION STUDIES OF HYDROXYAPATITE TABLETS FOR DIRECT COMPRESSION. ˜´ ´ ´ J M Sune-Negre, E Garcıa-Montoya, P Perez-Lozano, A ˜ Orriols, M Minarro & J R Tico´ – Pharmaceutical Technology Unit, School of Pharmacy, University of Barcelona, Av Joan XXIII, s / n 08028 Barcelona, Spain Purpose: To study the obtention of tablets with chondroitin calcium sulphate, hydroxyapatite and calcium carbonate, for use as a source of calcium in deficiency states, by direct compression due to the great sensitivity of chondroitin to the humidity. The direct compression makes possible a faster manufacturing process (with less exposure to environmental humidity) and a greater cost-effectiveness. The difficulty lies in the poor compression capacity of chondroitin and in the high proportion of actives that must be included in the tablet (830 mg in a total tablet weight of 1000 mg), leaving a very small margin to incorporate direct compression (DC) excipients. Methods: Several DC formulations were prepared using the following different DC diluents: Avicel PH101, Avicel PH102, Avicel PH200, Destab , and Prosolv . These formulations were completed with a disintegrant (Ac-Di-Sol ) and lubricants (magnesium stearate and Aerosil 200). The same process was followed in each formulation: The mixture (except of magnesium stearate) was blended (biconic-blender 5 minutes), sifted (0.84 mm mesh sieve), and mixed again (biconic-blender 20 minutes). Magnesium stearate was sifted (0.84 mm mesh sieve) and added to the previous mixture. The final powder was mixed (biconic-blender 5 minutes) and compressed (Manesty rotatory tablet press, 20.5 mm38.5 mm capsuleshaped punches). With the final mixture of the powder, flowability tests (angle of repose, flow rate, bulk and tapped density) were carried out. Tablets were checked for: weight and hardness uniformity, friability, and disintegration. Results: Due to the fact that the actives represent a 83% of the total tablet weight, there were serious difficulties to achieve a suitable formulation for DC. When one type of Avicel was used in the formulation the compression was not possible. When a combination of two types of Avicel were used the compression was possible, but the obtained tablets presented exfoliation during the process and they do not passed a friability test. Controversely, the addition of Prosolv HD90 improved the flowability properties and compactibility of the mixture, either combined with Avicel or used alone, considering the last case as the optimal for being the one that provides the simplest formulation (less excipients), favours disintegration (difficult due to physical properties of chondroitin and hydroxyapatite) and gives the best flowability, compressibility and final tablet properties. The final formulation had the following characteristics: angle of repose and flow rate values of the final mixture 28,068 and 20 g / s, respectively, bulk density 0.504 g / ml, tapped density 0,659 g / ml, mean
weight uniformity of 999 mg (CV50.017%, 10 tablets), mean hardness 172.37 N, friability 0.02% and disintegration time 12 min. Conclusions: A formulation has been developed to obtain tablets of chondroitin calcium sulphate (330 mg), hydroxyapatite (250 mg) and calcium carbonate (250 mg), with a final tablet weight of 1000 mg, by DC. The utilization of Prosolv HD90, excipient for direct compression, provides a mixture of appropiate compressibility, suitable flowability properties, and final tablets with satisfactory pharmaco-technical characteristics. Acknowledgment: This study is part of the project number 301213 from Bosch i Gimpera Foundation, supported by Laboratories Bioiberica, S.A.
PO-209 MICROCRYSTALLINE CHITOSAN AS A MUCOADHESIVE EXCIPIENT IN STOMACH-SPECIFIC SLOW-RELEASE FORMULATIONS OF FUROSEMIDE 1 2 ¨ ¨ Mia Sakkinen , Heidi Jurjenson , Peep Veski 2 & Martii 1 1 Marvola – Department of Pharmacy, Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland; 2 Institute of Pharmacy, University of Tartu, Estonia The bioavailability of furosemide from slow-release oral formulations is often poor, obviously because furosemide is absorbed to the greatest extent in the upper regions of the gastrointestinal tract, mainly in the stomach. The focus of the study described here was on development of slowrelease formulations of furosemide from which bioavailability is better than from existing formulations. Use of chitosan as an excipient could increase amounts of drug absorbed, through prolongation of gastric residence times of slow-release formulations. The bioavailability of furosemide from matrix granules containing microcrystalline chitosan (MCCh) was studied in human volunteers. The granules acted as slow-release dosage forms of furosemide. The amounts of furosemide absorbed were, in general, substantial. The highest AUC 0 – 8 value (3050 mg / l?h) was achieved with a formulation containing 80% of MCCh of molecular weight (Mw) 150 kDa. In a previous study, markedly lower AUC values had been obtained with slow-release formulations of furosemide . For example, with a commercial slowrelease formulation the AUC 0 – 8 was about 20% lower than for the granules in the present study, taking account of differences in drug doses. The high bioavailability of furosemide can be regarded as indirect evidence that granules containing MCCh had adhered to the gastric mucosa. If a substantial amount of the furosemide dose had been released in the small intestine, the AUC value would have been substantially lower. The rate of absorption of furosemide decreased as the amount or Mw of MCCh increased. An important finding was that amounts of
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
furosemide absorbed decreased as the rate of absorption declined. Mucous turnover may limit gastric residence times of mucoadhesive granules. ¨  Marvola M, Nykanen P, Rautio S, Isonen N, Autere AM. Enteric polymers as binders and coating materials in multiple-unit site-specific drug delivery systems. Eur J Pharm Sci 1999;7:259–267.
PO-210 ASSESSMENT OF THE USEFULNESS OF SUGAR SURFACTANTS AS SOLUBILIZING AGENTS IN PARENTERAL FORMULATIONS ¨ Erik Soderlind, Maria Wollbratt & Christian von Corswant ¨ ¨ – AstraZeneca R&D Molndal, SE-431 83 Molndal, Sweden The objective of the present study was to assess the usefulness of a number of sugar based surfactants as solubilizers for sparingly soluble substances in parenteral formulations, and compare this group of surfactants with existing polyoxyethylene based surfactants. For this purpose, the solubilization capacity, k , for felodipine and the haemolytic activity of the surfactants were determined. The haemolytic activity of each surfactant was assayed in a static haemolysis method in which 100 ml surfactant solution was mixed with 400 ml freshly collected blood from Beagle dogs. The degree of haemolysis was determined from the release of haemoglobin. In a homologous series of sugar surfactants the solubilization capacity increased with increasing chain length, due to growing size of the micelle core. The structure of the headgroup also influences k, which was illustrated by the C 12 -chain surfactants sucrose monododecanoate (SMD), dodecyl maltopyranoside (C 12 G 2 ), and polyoxyethylene (23) lauryl ether (Brij 35). The k of those surfactants increased in the order k(SMD),k(C 12 G 2 ),k(Brij 35). Polyoxyethylene based surfactants caused haemolysis at concentrations well above their respective critical micelle concentration, cmc. The sugar surfactants were more haemolytic and caused haemolysis at or below their corresponding cmc:s. The haemolytic activity of the sugar surfactants increased with increasing hydrocarbon chain length. The structure of the headgroup also influenced the haemolytic activity. Disaccharide surfactants were shown to be less haemolytic than the corresponding monosaccharide surfactants. The sugar surfactants investigated here are less suitable as solubilizing agents in parenteral formulations than the existing polyoxyethylene based surfactants. The solubilizing capacities are lower although the haemolytic activities are higher.  Yalkowsky SH. In: Solubility and solubilization in aqueous media. Oxford University Press, New York, 1999.
PO-211 THE EFFECT OF ETHYLCELLULOSE, EUDRAGIT S100, AND EUDRAGIT RS100 ON RELEASE OF ASPIRIN AND ITS SENSITIVITY TO TABLET HARDNESS H Tabandeh & S A Mortazavi – School of Pharmacy, Shaheed Beheshti University of Medical Sciences and Health Services, P.O. Box 14155-6153, Tehran, Iran In this study, formulations with polymers in different amounts were prepared. The dissolution profile was studied on acceptable formulations, as well as on counterparts with a hardness value of 3 units lower, by sampling at 1, 2, 4, and 8 hours. For formulations containing each of the three polymers, the Higuchi model for release seems to be the best fitted model. However, in ethylcellulose-containing formulation, zero-order model is also found suitable. This could be due to the presence of the erosion-promoting ingredients causing the profile to resemble zero-order model . Changing the hardness is suggested to affect the release rate through changing the porosity of tablets . The Eudragit S100 formulation was completely sensitive to change in tablet hardness, with regard to release. The Eudragit RS100 series was not so sensitive, and ethylcellulose series was completely insensitive. It has been reported that in ethylcellulose matrix tablets, the hardness and porosity are of little importance on release rate in hardness values over a certain limit . It is suggested that matrix tablet containing ethylcellulose could be a valuable candidate for sustained-release aspirin formulation.  Pather, I., Russell, I., Syce, J. A. and Neau, S. H. Sustained release theophylline tablets by direct compression. Part 1: formulation and in vitro testing. Int. J. Pharm. 1998;164:1–10.  Katikaneni, P. R., Upadrashta, S. M., Neau, S. H. and Mitra, A. K. Ethylcellulose matrix controlled release tablets of a water-soluble drug. Int. J. Pharm. 1995;123:119–125.  Stamm, A. and Tritsch, J. C. Some considerations on the liberation of drugs from inert matrices. Drug Dev. Ind. Pharm. 1986;12:2337–2353.
PO-212 A LIPOSOMAL DOSAGE FORM OF EBSELEN WITH POOR WATER SOLUBILITY S Tajiri, N Suzuki, K Ebihara & H Kikuchi – Drug Metab. & Physicochem. Property Res., Daiichi Pharm., Co., Tokyo, Japan Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a selenium-containing, heterocyclic, neuroprotective antioxidant that inhibits lipid peroxidation caused by glutathione peroxidase-like mechanisms. Although it has been developed as an oral dosage form, an injectable formulation is required to treat unconscious patients with acute is-
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
chemic stroke. Conventional aqueous formulations of ebselen, however, cannot be made because of its poor water solubility (ca. 0.003 mg / mL). Efforts to improve the water solubility of ebselen with injectable surfactants such as polyoxyethylene sorbitan mono-oleate, for a instance, were not successful. In contrast, ebselen was solubilized in a liposomal formulation containing 100 mg / mL of egg lecithin at a concentration of 1 mg / mL. In-vitro protein binding studies showed the immediate quantitative transfer of ebselen from the liposomes to rat plasma proteins because of its extremely high protein binding. The pharmacokinetic profile of ebselen after intravenous administration of its liposomal formulation was evaluated in rats (1.0 mg of ebselen / kg). An ebselen aqueous solution including tetrahydrofurfuryl alcohol polyethyleneglycol ether was used as a control formulation. There was no difference in the plasma concentration of ebselen between both formulations. The phramacokinetic date indicated that ebselen was promptly transferred to plasma proteins from the liposomes in blood. It was also confirmed that 1% of ebselen was distributed to the brain, which was the target tissue of ebselen, after administration of liposomes containing radiolabeled one. Liposomal ebselen was demonstrated to be a useful injectable formulation for improving apparent water solubility of lipophilic ebselen without any change in phamacokinetic profile.
PO-213 MULTIPLE EMULSION BASED THREE PLY WALLED MICROCAPSULES FOR CONTROLLED DELIVERY OF THEOPHYLLINE S Talegaonkar & P R Mishra – Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India The preparation of three-ply walled microcapsules (TPMC) is essentially based on the technique of multiple emulsion (w / o / w), wherein formation of polymeric interfaces is followed by rigidization of the wall on evaporation of solvent [1,2]. The Polyvinylalcohol / Ethylcellulose / Polyvinylalcohol (PEP) based TP-MC was selected. Poloxamer coating was done by dispersing the TP-MC into poloxamer solution. The characterization of TP-MC for physico-chemical properties is summarized below:
PEP Coated PEP
4–6 m 8–12 m
In-vitro drug release in 12 h (Mean6 s.e.m.)
Organ Distribution (after 0.5 h) Liver
The observations suggest that the poloxamer coating could sustain and prolong the release of encapsulated theophylline. The plasma-level profiles revealed that in
absence of surfactant coating particles have been cleared rapidly from blood and therefore plasma levels are low. In contrast, presence of poloxamer sustained the plasma levels up to 24 h. It is noted that uncoated microparticles were deposited mainly in the liver due to their rapid clearance from blood stream (phagocytosis). While the poloxamer coating lowers the angel of contact and therefore prevents phagocytosis resulting in sustained drug plasma level. The high levels of drug in lungs from coated particles may also be attributed to larger particle size.  Morris NJ and Warburton B. Three-ply-walled w / o / w microcapsule formed by a multiple emulsion technique. J Pharm Pharmacol 1982;34:475–79.  Bhatnagar S, Nakhare S and Vyas SP. Poloxamer coated three-ply walled microcapsules for controlled delivery of diclofenac sodium. J Microencap 1995;12:13–22.
PO-214 TABLETS OF AMIRON – ORIGINAL COMPOUND WITH HIGH ANTITUMOR ACTIVITY N Tchikineva, N Oborotova, O Orlova, A Polozkova, N Yavorskaya, I Golubeva, L Smirnova & A Baryshnikov – N. Blokhin Memorial Cancer Research Center of RAMS, Kashirskoye sh., 24, 115478 Moscow, Russia Amiron is a new representative of a group of chloroethylamines , which was synthesized according to the hypothesis of addressing therapeutic action for the treatment of the tumors of the thyroid gland as it contains tyrosine. The aim of this study was to create and study suitable amiron drug formulation. As amiron is insoluble in water, the oral way of its administration looks optimal. So tablets of amiron were decided to be prepared. Technological study of amiron substance pointed out the necessity of additional ingredients in order to increase the stability and quality of tablets. As the process of hydrolysation characterizes chloroethylamines during granulation, natrii chloride was chosen as stabilizating agent to be added to the mixture for tablet preparation. The starting dose of amiron in tablets was taken on the base of toxicological experiments on mice (LD 10 ). So for humans it was 4.26 mg / kg or 255 mg per individual. It means that every tablet must contain 0.25 mg of amiron and 0.25 mg of additional compounds. The antitumor activity of these tablets was studied on the model of murine mamarian adenocarcinoma Ca-755. Amiron tablets and substance were administered 5 / 24 h in 1% starch solution in dose of 100 mg / kg (the dose was chosen in previous experiments as the most effective one). On the 7-th day after tumor inoculation TGI caused by both amiron substance and tablets was about 91%, by the 18 day of the experiment TGI was 59 and 65% respectively (statistically significant if compared with control). So amiron demonstrated high and prolonged antitumor activity in mice, this effect did not decrease after tablet
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
preparation. Amiron tablets were recommended for further toxicological investigations.  Tchikineva N., Yavorskaya N., Golubeva I. et al. Determination of Activity of New Chloroethylamine – Amiron and its Derivatives. Pharm Chem J 2001;35(9):8–10.
PO-215 PHYSICOCHEMICAL PROFILING OF INHALED DRUGS – INVESTIGATIONS OF THE PULMONARY ABSORPTION RATE AND BIOAVAILABILITY OF DRUGS IN VIVO IN RATS ´ 2 , Hans Marchner 3 , Anna-Karin Ann Tronde 1,2 , Bo Norden 2 ¨ 1 & Ursula Hultkvist Bengtsson 2 Wendel , Hans Lennernas 1 – Department of Pharmacy, Uppsala University, Sweden; 2 AstraZeneca R&D Lund, Sweden; 3 AstraZeneca R&D ¨ ¨ Sodertalje, Sweden Improved understanding of the influence of drug physicochemical properties on the absorption rate and bioavailability from the lungs are important to aid design of inhaled drugs for local and systemic action. The objectives of this study were to identify the inhaled drugs on the market 2001, calculate their physicochemical parameters, and to make a profile of the most significant properties of these compounds. Furthermore, the influence of the drug properties on the pulmonary absorption rate and bioavailability in vivo in rats was investigated, as well as the permeability characteristics in Caco-2 cell monolayers. Selected drugs (cromolyn, cyanocobalamin, formoterol, imipramine, losartan, metoprolol, talinolol, and terbutaline) were administered to anesthetized rats by intratracheal nebulization . Plasma concentrations of the drugs were determined by LC-MS / MS. We found 29 inhaled compounds on the market 2001, exclusive of volatile anesthetic agents, pulmonary surfactants, and polypeptides (e.g. DNase). The low number of inhaled compounds, dominated by corticosteroids and bagonists, made comparisons with oral drugs difficult. However, the inhaled drugs tended to be less lipophilic and more polar than oral drugs. The pulmonary bioavailability (F%) of the selected drugs was generally found to be high. F% for the p-gp substrate talinolol was 81% in 6 h, indicating an insignificant role for p-pg in limiting the absorption from the rat lung. The drug absorption across the lung barrier was rapid, as shown by the short t max (2–13 min) and absorption half-life (T 50% ) (1–17 min). The apparent absorption rate constant across the lung barrier in vivo was found to correlate to the apparent permeability in Caco-2 cells.  Tronde et al. Miniaturized nebulization catheters – A new approach for delivery of defined aerosol doses to the rat lung. J Aerosol Med 2002, in press.
PO-216 INFLUENCE OF PHYSICOCHEMICAL PROPERTIES ON DRUG PERMEATION ACROSS THE ISOLATED AND PERFUSED RAT LUNG AND Caco-2 CELL MONOLAYERS ´ 2 , Ann-Britt Jeppsson 2 , Per Ann Tronde 1,2 , Bo Norden 2 ¨ 1 & Ursula Brunmark , Elisabeth Nilsson 2 , Hans Lennernas 2 1 Hultkvist Bengtsson – Department of Pharmacy, Uppsala University, Sweden; 2 AstraZeneca R&D Lund, Sweden Besides the earlier work performed by Schanker and coworkers, there are few systematic investigations of the absorption characteristics of low molecular weight drugs from the lungs . Yet, investigations of the permeability characteristics of the respiratory tract epithelium in relation to the drugs’ physicochemical properties would be beneficial to aid the design of inhaled drugs for local or systemic action. In this study, the pulmonary absorption of nine structurally diverse low molecular weight drug compounds (enalaprilate, atenolol, terbutaline, formoterol, losartan, enalapril maleate, propranolol, metoprolol, and budesonide) and one high molecular weight compound (FITC-dextran 10 000 Da) were investigated in the isolated, perfused, and ventilated rat lung model (IPL) after aerosol delivery . The pulmonary transport characteristics were compared with the permeability of the intestinal Caco-2 cell monolayers. A multivariate data analysis was performed to evaluate the influence of the drugs’ physicochemical properties on the transport in the two applied absorption models. The apparent first-order absorption rate constant in IPL (ka lung ), for the investigated compounds, showed a positive correlation to the cLogD and to the apparent permeability (Papp) across the Caco-2 cell monolayers, and a negative correlation to the polar surface area (PSA) of the compounds. No relationship was found between the Pappvalues or the ka lung and the extent of absorption across the lung barrier of the IPL.  Schanker LS. Drug absorption from the lung. Biochem Pharmacol 1978;27:381–385.  Tronde A, Krondahl E, von Euler-Chelpin H et al. High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery. Peptides 2002;23:469–478.
PO-217 INFLUENCE OF SIZE OF CUSHIONING PELLETS ON DRUG RELEASE FROM COATED DRUG PELLETS ˚ Tunon ´ & G. Alderborn – Department of Pharmacy, A. Uppsala University, Box 580, SE-751 23 Uppsala, Sweden Drug pellets consisting of salicylic acid and microcrystalline cellulose (MCC) were prepared by extrusion and
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
spheronisation. Cushioning pellets consisting only of MCC were prepared in two different size fractions. The drug pellets were spray coated with ethyl cellulose in a Wurster based fluid bed and mixed with cushioning pellets of the different sizes. After lubrication with magnesium stearate the pellets were compacted in an instrumented single punch press. The pellets were characterised in terms of intragranular porosity, bulk density, intergranular voidage and external surface area. Directly after compaction the tablets were manually deaggregated. The degree of deformation of the retrieved drug pellets was determined in terms of the circularity, assessed by image analysis. The drug release was studied in an automated USP basket apparatus. The intragranular porosity of the two types of cushioning pellets was similar, while the porosity of the drug pellets was much higher. Compaction of the coated drug pellets with the small size cushioning pellets gave a similar drug release while compaction with large size pellets gave a faster release. The compaction pressure had unexpectedly low effect on the drug release. The degree of deformation of the drug pellets, reflected by the circularity values, was only slightly influenced by the size of the cushioning pellets and the compaction pressure. Large size cushioning pellets and high pressure gave a higher degree of deformation. The mode of deformation, however, was highly dependent on the size of the cushioning pellets. The size of the cushioning pellets had a marked effect on the drug release from and the mode of deformation of the drug pellets, but a lesser effect on the degree of deformation. Compaction with small cushioning pellets seems to be better than large cushioning pellets, giving a release profile similar to that of uncompacted drug pellets.
PO-218 MEASURING SKIN HYDRATING EFFECT OF ASCORBYL PALMITATE IN VARIOUS FORMULATIONS ¨ ¨ Gonullu, ¨ ¨ ¨ G Yener, T ~Incegul ¨ & T Altınkurt – M Uner, U Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Istanbul, Beyazıt 34452 Istanbul, Turkey Ascorbic acid is a natural antioxidant that combats the reactive oxygene species that can cause damage to cells endangering tissue integrity. The study of ascorbic acid in dermocosmetic formulations is important because of its physological functions as free radical scavenger activity, inhibitory effect on melanogenesis and anti-aging properties. The use of ascorbic acid in cosmetic formulations is very difficult due to its low stability in aqueous solutions. To solve this problem of stability, derivatives of vitamin C have been synthesized having an action similar to ascorbic acid, but with improved chemical stability. It is also known that ascorbyl palmitate penetrates from skin more easily. The aim of this study is to use one of this derivatives,
lipophilic ascorbyl palmitate to determine its skin hydrating effect and penetration properties compared with ascorbic acid, for the first time. For this purpose, gel, o / w emulsion and oleagenous creams were prepared and applied to 10 volunteers inner arms. Moisture content of the skin were measured by using Corneometer (Courage Khazaka, Germany). Percutaneous penetration studies were performed by using excised human skin on Franz diffusion cells.
PO-219 HUMAN PHYSIOLOGICALLY BASED IN VITRO DIGESTION MODELS SIMULATING FASTING AND FED CONDITIONS C Versantvoort, A Oomen & A Sips – Centre for Substances and Integrated Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, The Netherlands It is known that the fasted or fed status can have a large impact on the oral bioavailability of compounds. The presence of food markedly alters the conditions in the gastrointestinal tract. The effect of food on the oral bioavailability of compounds is often due to the dissolution (bioaccessibility) of compounds in the chyme and their transport across the intestinal epithelium. We present in vitro digestion models reflecting the conditions of the gastrointestinal tract for the fasted and fed state of man in order to study the bioaccessibility of compounds from their matrix. The method involves a three-step procedure simulating human digestion for the fasted cq fed state in subsequently mouth, stomach and small intestinal compartment. Infant formula feedings were used as food and physiologically based conditions i.e. composition of media, pH and residence time periods typical for each compartment were applied. The effects of fasted vs. fed conditions were studied on the bioaccessibility of different types of compounds from soil: metals (lead, cadmium, arsenic (metalloid)) and a lipophilic compound (benzo[a]pyrene). The bioaccessibility of lead, cadmium, arsenic and benzo[a]pyrene from soil was on average 22%, 30%, 44% and 6%, respectively, for fasting conditions. The fed state had no (arsenic and cadmium) or little (lead,factor 2) effect on the bioaccessibility of the metals. In contrast, the bioaccessibility of benzo[a]pyrene was 3 to 8 fold increased under fed conditions compared to the fasted conditions. Prolongation of the residence time in the stomach from 2 h to 16 h had no effect on the bioaccessibility of either compound. Doubling the bile concentration increased the bioaccessibility of only benzo[a]pyrene by a factor of 1.5 to 2. Thus, the bioaccessibility from soil was compound specific, whereas fed conditions had a major effect on the bioaccessibility of the lipophilic compound benzo[a]pyrene.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
The in vitro digestion models, which closely simulate fasted and fed conditions, can be used to model events occurring during digestion and solubilisation of compounds (including pharmaceuticals) in the gastrointestinal tract. Furthermore, the digestion models in combination with the Caco-2 transport model (data will be presented at the meeting) can assist in more accurate predictions of absorption of compounds.
PO-220 RELEASE OF DRUGS FROM THERMOSENSITIVE POLY(N-VINYL CAPROLACTAM) NANOPARTICLES Henna Vihola 1 , Antti Laukkanen 2 , Jouni Hirvonen 1 & Heikki Tenhu 2 – 1 Division of Pharmaceutical Technology, Viikki Drug Discovery Technology Center, PB 56, FIN00014, University of Helsinki, Finland; 2 Laboratory of Polymer Chemistry, Department of Chemistry, PB 55, FIN-00014 University of Helsinki, Finland Poly(N-vinyl caprolactam)-particles (PVCL) in water exist in a swollen state at room temperature, but collapse and precipitate upon heating above the lower critical solution temperature, LCST. PVCL has a LCST near body temperature, and it may have several biomedical applications. The PVCL nanoparticles were synthesised by batch emulsion polymerisation . Interactions of the PVCL particles with three different drugs, nadolol, propranolol and tacrine, were studied. Dynamic light scattering was used to measure the particle sizes as a function of temperature. Binding and release of the drugs into and from the nanoparticles were studied at 20 8C and 40 8C, when PVCL was either in a swollen or collapsed state. Drug release tests were performed using Franz-type diffusion chambers. The amount of drug diffused into the reciever chamber was measured by HPLC. The results of light scattering showed that the more hydrophobic drugs, propranolol and tacrine, swelled considerably the PVCL-particles. The more hydrophilic drug, nadolol, decreased the transition temperature of PVCL particles, whereas with the other drugs it was quite similar to pure PVCL. Drug release tests showed that the bblockers nadolol and propranolol were strongly bound to the polymer, and this was more evident at higher temperatures. Tacrine permeation across the membrane increased clearly, when the temperature was raised and the PVCLparticles were present. As a conclusion, both physical and chemical properties of the drugs clearly affected their binding to the PVCL particles and the release of drugs was affected by temperature.  Laukkanen, A., Hietala, S., Maunu, S-L., Tenhu, H. Poly(N-vinylcaprolactam) microgel particles grafted with amphiphilic chains. Macromolecules 2000;33:8703–8708.
PO-221 SOLID FORM GENERATION AND CHARACTERIZATION USING NON-AMBIENT XRAY DIFFRACTION Z Ping Wu, Ryan Hart & Dave Stirling – Pharmaceutical Sciences, Pfizer Global Research and Development, 3565 General Atomics Court, San Diego, CA 92121, USA The purpose of this presentation is to demonstrate the utility of a rapid method for generating and characterizing polymorph forms for drug candidates and for identifying the lower energy form of a given candidate, often more suitable for use in development.  The technique used here is the Non-ambient X-ray Diffraction method that allows the X-ray powder pattern of a sample to be recorded at selected temperatures and relative humidities, where new forms may be formed . p-Nitrophenol was used as a model compound to demonstrate the utility of the method. Two polymorphs of the model compound (Form a and b) were heated to melting temperature and allowed to cool in the sample cell of the X-ray diffractometer. Solid structure changes were monitored by X-ray diffraction. Different heating and cooling conditions were tested and forms generated characterized. Form b was shown to convert to Form a, the thermodynamically more stable form, under these conditions. The results suggest that Non-ambient X-ray Diffraction can be a powerful tool in generating and identifying low energy polymorphs for drug candidates.  Chemburkar S R, Bauer J, Deming K el al. Dealing with the impact of Rotonavirpolymorphs on the late stage of bulk drug process development. Org Proc Res Dev 2000;4:413–17.  Fawcett T G, Martin E J, Crowder C E et al. Analysis of multi-phase pharmaceuticals using simultaneous differential scanning calorimetry and x-ray diffraction. Adv X-ray Anal 1986;29:323–332.
PO-222 EXTRUSION ABILITY OF MIXTURES CONTAINING POLY(ETHYLENE OXIDE) FOR THE PRODUCTION OF PELLETS ˜ F Pinto – Dept. Tecnologia Kathrin Wunder & Joao ˆ ´ Farmaceutica, Faculdade de Farmacia da Universidade de Lisboa, Av. Prof. Gama Pinto, P-1649-003 Lisboa, Portugal The aim of the work is the assessment of the extrudability of mixtures containing high amounts of poly(ethylene oxide) for the production of pellets by extrusion and spheronisation. Different mixtures of poly(ethylene oxide), lactose monohydrate, LAC and propranolol hydrochloride, PRO and demineralised water, W were made in a mixer, for 10 minutes. A ram extruder with different dies ranging
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
from 1 up to 9 mm diameter and constant length to diameter ratio of 4. The extruder was fitted to a mechanical press able to control the displacement of the ram and the force applied to the mass. The extrusion process and the quality of extrudates were evaluated according to the shape of the extrusion profiles, the force at steady state, the expansion of the extrudates after extrusion, smoothness of the extrudate’s surface and dissolution profiles of the model drug. The results have shown as anticipated, an increase on the speed of extrusion, decrease on the diameter of the die, reduction of water content of the formulation increased the extrusion force at steady state. Less obvious was the behaviour of the masses under pressure. Three different extrusion profiles were observed depending on the amount of water: a smooth steady state was observed for high ratios of PEO / W, for medium PEO / W ratios an elastic peak was clear and for low PEO / W a negative slope of the curve at steady state was observed. Release of the drug was higher for thin extrudates (high specific surface area) than for thicker ones. Comparison with tablets with the same surface area has shown a faster release rate than extrudates. In conclusion, the study has shown that up to 40% of PEO content in the formulation and a well-defined amount of water extrudates can be produced. The relationship between the W and PEO contents is not linear. The viscoelastic behaviour and, particularly the elastic component of PEO and its mixtures, turn the interpretation of the results complex. Even though, no deviations to the theoretical aspects of extrusion were observed. The elastic recovery of extrudates increased with the ratio PEO / W, W content and the speed of extrusion but decreased with the diameter of the die.
PO-223 INFLUENCE OF PREPARATION PARAMETERS ON MONOOLEIN / POLOXAMER / WATER-DISPERSIONS ¨ 1 , B Siekmann 2 & H Bunjes 1 – 1 Department of G Worle Pharmaceutical Technology, Institute of Pharmacy, Fried¨ Jena, Lessingstrasse 8, D-07743 rich-Schiller-Universitat ¨ Jena; 2 Camurus AB, Ideon Science Park, Solvegatan 41, S-22370 Lund, Sweden Colloidal dispersions of bicontinuous cubic phases of the system monoolein / poloxamer / water  are discussed as potential drug carriers for, e.g., intravenous (i.v.) administration . Against this background, the influence of different preparation parameters was investigated to test the possibility of producing long term stable dispersions suitable for i.v.-administration with respect to particle size. Equilibrated coarse dispersions (95% water, 4.6% monoolein, 0.4% poloxamer 407) were homogenized with a Microfluidizer (MF, 15 minutes) at different temperatures (20 / 40 / 60 / 80 8C) and 350 bar or different pressures (280 / 350 / 500 bar) at 40 8C. Alternatively, an APV Micron Lab (ML, 5 cycles) was used at 350 bar and 20 or 60 8C.
Homogenization at 20 and 80 8C did not lead to the desired colloidal dispersions (macroscopically visible particles in the dispersions). At 40 and 60 8C (MF), homogenous dispersions were obtained with mean particle sizes in the colloidal range (analyzed by laser light diffraction including polarization intensity differential scattering, LD1 PIDS). At 60 8C, particles were distinctly smaller and ’ring formation’ in the storage vial was reduced compared to 40 8C. Small angle X-ray scattering (SAXS) indicated the presence of cubic phase in both cases, some particles up to 5–10 mm were detected in the light microscope. The use of the alternative homogenizer (ML) at 60 8C gave similar results to those obtained with the MF at this temperature. Variation of the homogenization pressure (MF) led to a decrease in particle size with increasing pressure but had no effect on ’ring formation’ and the occurrence of cubic reflections (SAXS) which were observed in all these dispersions. In conclusion, the temperature during homogenization has much stronger influence on the properties of the resulting dispersions than the type of homogenizer or the homogenization pressure.  Gustafsson J et al. Submicron particles of reversed phases in water stabilized by a nonionic amphiphilic polymer. Langmuir 1997;13:6964–6971. ¨  Engstrom S, et al. A Cubosome formulation for intravenous administration of Somatostatin. Proc Int Symp Controlled Release Bioact Mater 1996;23:89– 90.
PO-224 THE EFFECT OF AMOUNTS OF LACTOSE IN THE DRUGS RELEASE CHARACTERISTICS OF MATRIX TABLETS OF ACETAZOLAMIDE PREPARED FROM DIFFERENT GRADE VISCOSITS OF HYDROXY PROPYL METHYL CELLULOSE M H Zarrintan – Forouzesh, Elham Several different matrices has been used to dedicating suitable sustained release characteristics to acetazolamide tablets. At present study the release of the drug from acetazolamide tablets prepared from hydroxy propyl methyl cellulose (K15 M, K4 M) locate and magnesium stearate was compared. The results showed that increasing of the lactose amount increased the drug release. This effect probably arose from elevating of matrix tortosity due the dissolving of lactose in the matrix body. Increasing of viscosity (in the case of HPMC K15M) decreased the drug release. The effect seemed to be related to increasing of Jell layer thickness performed around tablets resulted decreasing of water penetration in the matrix.
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
By choosing suitable amount of lactose sustained released tablets of acetazolamide can be produced with creating different amount of lactose for each type.
PO-225 PROSOLV SMCC CUSTOM PROCESSING TO ENHANCE PHYSICAL AND FUNCTIONAL PROPERTIRES OF PHARMACOLOGICALLY ACTIVE COMPOUNDS Joseph A Zeleznik, Bob E Sherwood, David Schaible & Theodore Montalto – Penwest Pharmaceuticals Co., 2981 Route 22, Patterson, NY 12563, USA Purpose: To evaluate the effectiveness and functional performance of model active compounds Custom Processed with PROSOLV姠 SMCC . Methods: Several model active compounds were combined with PROSOLV姠 SMCC in a proprietary Custom Process. Materials were evaluated for powder flowability on a Hanson Flodex姠 for rates and bridging aperture. Blends were prepared by mixing Custom Processed materials with disintegrant and lubricant in a twin shell V-blender and compacted on a Korsch PH-106 rotary tablet press. Results: Custom Processed model compounds showed a significant improvement in flow over traditionally prepared formulations. Compound ‘‘A’’ improved to 4 mm from 18 mm in flow bridging aperture. Compound ‘‘B’’, which did not flow at any aperture on the Flodex姠 flowed through a 9 mm aperture after Custom Processing with PROSOLV SMCC. Other model actives showed similar flow improvements with Custom Processing. Compaction profiles illustrated from 50% to 90% improvements in compactibility over conventional processing methods such as granulation and direct compression. Conventionally blended formulations increased excipient requirements from as little as twice to as much as five times that required by PROSOLV SMCC Custom Processed actives. Conclusion: Custom Processing with PROSOLV SMCC improved flowability and compactibility. Custom Processing also resulted in smaller tablets for the same active dose. Tablet size reduction ranged from two to five times.
PO-226 APPLICATION OF REFLECTANCE SPECTROSCOPY FOR IN-PROCESS CONTROL OF PELLET FORMATION AND COATING EFFECTIVENESS Zs Zsigmond 1 , I Antal 2 , P Fekete 1 & S Marton 2 – 1 Egis ´ 30; Pharmaceutical Ltd., H-1106 Budapest Kereszturi 1 Pharmaceutical Institute, Semmelweis University, H-1092 ˜ Budapest, Hogyes 7 Coated multiparticulate dosage forms are of great interest considering controlled drug release with improved safety
and efficacy . The final quality of the product is related to the dissolution profile test, which can take several hours in the case of sustained release preparations. Benefits of near-infrared spectroscopy (NIRS) lead to the growing interest of the pharmaceutical industry . Diffuse reflectance spectra can be gathered within minutes for both chemical and physical information, close to the process at line or on line. The objective of this study was to follow the formation of pellets containing potassium chloride in a pilot rotofluid equipment (Glatt GPCG 15) as well as study the coating procedure with tangential spraying method in the same equipment. Changes in diffuse reflectance spectra of the product were analyzed according to the critical parameters of pelletization (composition, particle size, moisture content) as well as to the extent of coating. Results demonstrated the suitability of NIRS for the in-process control of pellet size and the coating process. Applying mathematical analysis, the in vitro drug release profile can be predicted from the in-process test and a correlation was found between dissolution fit factors  and spectral data. From 120 minutes coating time, a sufficient coating level was achieved which met the requirements for the dissolution profile. Although dissolution tests are essential to qualify modified release products during production, NIRS may have benefits as an alternative method for in-process control of coating.  Ghebre-Sellassie, Pharmaceutical Pelletization Technology, Drugs and Pharmaceutical Sciences, Vol. 37, Marcel Dekker Inc, New York, 1989, pp. 1–13.  M. Day, Optimizing the manufacture of solid dosage forms with NIR spectroscopy. Pharmaceutical Technology Europe, 2001;4:22–27.  Jeffry W. Moore and Henry H. Flanner, Mathematical comparison of dissolution profiles Pharmaceutical Technology 1996;6:64–72.
PO-227 PREPERATION OF GEL FORMULATIONS OF MELOXICAM ¨ ¸ er, C Hasc¸ic¸ek, N Gonul ¨ ¨ – Department of PharmaA Olc ceutical Technology, Faculty of Pharmacy, Ankara Uni¨ versity, 06100 Ankara, Turkiye Meloxicam is a new nonsteroidal antiinflammatory drug (NSAID) of enolic acid class compounds. The mechanism of action of meloxicam, like that of other NSAIDs, may be related to prostoglandin synthase inhibition. It is also selective COX-2 inhibitor. It has antiinflammatory, analgesic, and antipyretic activities [1,2]. In this study different semisolid dosage forms of meloxicam were attempted to develop. For this aim C 934 gel, HEC gel and, Emulgel formulations of meloxicam were prepared. The drug release experiments were employed by
EUFEPS 2002 Supplement 17 S (2002) S87 –S138
using modified Franz diffusion cells system (1.32 cm 2 area) and synthetic membrane (cellulose acetate, pore size 0,22 mm). A phosphate buffer solution (pH 7.4) was used as receptor phase. The pH and viscosity values of these formulations were measured. According to in vitro release profiles obtained the maksimum release was observed from HEC gel formulation.
 Garcia SM, Pedrone CS, Albero I, Marti J. Spectrophotometric methods for determining meloxicam in pharmaceuticals using batch and flow-injection procedures. Eur J Pharm Sci 2000;9:311–16.  Physican’s Desk Reference Copyright 2002 by Medical Economics Company, Inc. Montvalle, NJ, pp. 1–11.