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The effect of differentiation on the expression of UGT isoenzymes in Caco-2 cell line
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e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 S ( 2 0 0 8 ) S30–S41
analysis. The detection limit of ∼10 nM was reached for all analytes. doi:10.1016/j.ejps.2008.02.113 P37 Retarding the kinetics of theophylline hydrate formation using pharmaceutical excipients Miroshnyk 1,∗ ,
I. Yliruusi 1
S.
Mirza 1 ,
¨ aki ¨ 1 , J. Aaltonen 2 , J. J. Heinam
1
Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Finland 2 School of Pharmacy, University of Otago, New Zealand Hydrate formation is a common phase transformation that pharmaceutical solids may undergo during manufacturing processes or storage. The resulting solid-state changes can cause severe problems in the processing and/or performance of pharmaceutical products and thus these changes must be prevented. This study investigates the effect of pharmaceutically accepted excipients on hydrate formation kinetics of theophylline anhydrate. Theophylline anhydrate was modified by recrystallization in the presence of an excipient (HPMC, PVP or HPC) as an additive in the crystallization media. The morphology of the excipient-modified crystals was diverse and differed from that of the unmodified theophylline, as observed by optical microscopy. Furthermore, the hydrate formation kinetics in wet masses containing the modified crystals was retarded as compared with that of unmodified crystals, as revealed by quantitative Raman spectroscopy. Hence, this study demonstrates that excipient-induced surface and/or habit modification of crystals is a promising approach to controlling hydrate formation of pharmaceutical solids. doi:10.1016/j.ejps.2008.02.114 P38 In silico screening of potential crystal growth inhibitors for the stabilization of amorphous structures K. Pajula 1 , M. Lahtela-Kakkonen 2 , O. Korhonen 1,∗ 1
Department of Pharmaceutics, University of Kuopio, Finland 2 Department of Pharmaceutical Chemistry, University of Kuopio, Finland The aim of this study was to evaluate the applicability of receptors based docking softwares for the virtual screening of potential crystal growth inhibitors. In study set up, unit cells of acetaminophen and naproxen were obtained from Cambridge structural database. The fastest growing crystal faces were determined using Morphology toolbox (Accelrys). Crystal growth sites, as receptors, in several fastest growing faces were generated by deleting some molecules from faces. For model ligands, as inhibitors, were selected all amino acids and dipeptides. As an “internal standard” was used acetaminophen and naproxen. Ligands were docked to growth sites (receptor) using MOE (Chemical Computing Group) and Sybyl SurflexDock (Tripos) docking suites. Applicability of docking suites in
small molecule docking on crystal faces was evaluated according to internal standards. Surflex-Dock suite docked perfectly acetaminophen and naproxen molecules into their own crystal lattices, thus pointing out the powerful applicability of it in novel screening approach. doi:10.1016/j.ejps.2008.02.115 P39 Betulin derivatives inhibit Alphavirus replication L. Pohjala 1,2,∗ , S. Alakurtti 3,4 , J. Yli-Kauhaluoma 3 , T. Ahola 5 , P. Tammela 1 1 Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, Finland 2 Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, Finland 3 Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland 4 Technical Research Centre of Finland, VTT, Finland 5 Institute of Biotechnology, University of Helsinki, Finland
Betulin is a widespread naturally occurring triterpenene, found in large quantities in birch bark. Despite the knowledge on betulin-derived compounds as anti-HIV agents, their wider antiviral spectrum remains poorly studied. In this study, a set of 68 betulin derivatives was screened against Semliki Forest virus (SFV), a member of Alphavirus genus and a commonly used model for replication of positive-stranded RNA viruses. The basic structure of betulin was moderately active against SFV (IC50 45 M), but antiviral potency was enhanced particularly by certain cyclic ether or ester substituents, as demonstrated by 28-O-tetrahydropyranylbetulin and 3,28-betulinyl di(2-sulfobenzoate) (IC50 values 17 and 11 M, respectively). Even though some derivatives raised also toxic responses in Huh-7 cells, therapeutic indices higher than 50 were obtained for many of the tested compounds. Due to their non-nucleoside structure and relatively low toxicity, betulin-derived compounds make a competitive contribution to the few inhibitors of these pathogens. doi:10.1016/j.ejps.2008.02.116 P40 The effect of differentiation on the expression of UGT isoenzymes in Caco-2 cell line S. Siissalo ∗ , H. Zhang, J. Hirvonen, M. Finel University of Helsinki, Finland As a part of the characterization process of Caco-2 cell line, the expression of different UGT (UDPglucuronosyltransferase) enzymes was studied. The mRNA levels of 15 UGT1A and UGT2B isoforms were determined in several samples of Caco-2 cells (four passage levels: P31, P37, P43 and P49, fully differentiated cells grown on filters and undifferentiated cells obtained from culturing flasks) using quantitative real-time RT-PCR. The expression levels were normalized using -actin as a reference gene. The difference in the degree of differentiation was demonstrated with villin
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 S ( 2 0 0 8 ) S30–S41
as a control gene. All the studied UGT’s were detected in the samples, but some at very low levels. The expression levels of most genes were at their highest at P43 and declined at P49, with the exceptions of UGT2B4 and UGT2B11. The main UGT enzymes expressed in our Caco-2 cells appeared to be UGT1A6 (both differentiated and undifferentiated samples) and UGT2B17 (differentiated samples, especially P43). doi:10.1016/j.ejps.2008.02.117 P41 Automation of three different cellular assays on robotic workstation—Feasibility evaluation A. Soikkeli 1,∗ , C. Sempio 1 , A.M. Kaukonen 1,2 , A. Urtti 2 , J. Hirvonen 1 , M. Yliperttula 3
lar assays were automated on TECAN Genesis workstation, and critical parameters were evaluated and optimized. From the automation point of view, the bidirectional Caco-2 permeability assay is a complicated assay set-up, whereas the SRB cytotoxicity test and the “mix-and-measure” CaspaseGlo® 3/7 assay kit are more suitable for automation. Automation notably increases the throughput of the last two. The common HTS assay performance indicators, Z factors and signal-tobackground ratios (S/B) were used for determining quality of the automated assays. As a conclusion, characteristics of the HTS assay protocol and cell properties will determine whether the automation is feasible. Automation of simple homogeneous assays like CaspaseGlo® 3/7 is advantageous, but also heterogeneous assays without strict time schedule are worth automation. In other cases, compensatory methods should be evaluated if low throughput is the key problem.
1
Division of Pharmaceutical Technology, University of Helsinki, Finland 2 Drug Discovery and Development Technology Center, University of Helsinki, Finland 3 Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland Assay automation presents several advantages, but all the assays are not easily adapted for automation. Three cellu-
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doi:10.1016/j.ejps.2008.02.118
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 S ( 2 0 0 8 ) S30–S41
analysis. The detection limit of ∼10 nM was reached for all analytes. doi:10.1016/j.ejps.2008.02.113 P37 Retarding the kinetics of theophylline hydrate formation using pharmaceutical excipients Miroshnyk 1,∗ ,
I. Yliruusi 1
S.
Mirza 1 ,
¨ aki ¨ 1 , J. Aaltonen 2 , J. J. Heinam
1
Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Finland 2 School of Pharmacy, University of Otago, New Zealand Hydrate formation is a common phase transformation that pharmaceutical solids may undergo during manufacturing processes or storage. The resulting solid-state changes can cause severe problems in the processing and/or performance of pharmaceutical products and thus these changes must be prevented. This study investigates the effect of pharmaceutically accepted excipients on hydrate formation kinetics of theophylline anhydrate. Theophylline anhydrate was modified by recrystallization in the presence of an excipient (HPMC, PVP or HPC) as an additive in the crystallization media. The morphology of the excipient-modified crystals was diverse and differed from that of the unmodified theophylline, as observed by optical microscopy. Furthermore, the hydrate formation kinetics in wet masses containing the modified crystals was retarded as compared with that of unmodified crystals, as revealed by quantitative Raman spectroscopy. Hence, this study demonstrates that excipient-induced surface and/or habit modification of crystals is a promising approach to controlling hydrate formation of pharmaceutical solids. doi:10.1016/j.ejps.2008.02.114 P38 In silico screening of potential crystal growth inhibitors for the stabilization of amorphous structures K. Pajula 1 , M. Lahtela-Kakkonen 2 , O. Korhonen 1,∗ 1
Department of Pharmaceutics, University of Kuopio, Finland 2 Department of Pharmaceutical Chemistry, University of Kuopio, Finland The aim of this study was to evaluate the applicability of receptors based docking softwares for the virtual screening of potential crystal growth inhibitors. In study set up, unit cells of acetaminophen and naproxen were obtained from Cambridge structural database. The fastest growing crystal faces were determined using Morphology toolbox (Accelrys). Crystal growth sites, as receptors, in several fastest growing faces were generated by deleting some molecules from faces. For model ligands, as inhibitors, were selected all amino acids and dipeptides. As an “internal standard” was used acetaminophen and naproxen. Ligands were docked to growth sites (receptor) using MOE (Chemical Computing Group) and Sybyl SurflexDock (Tripos) docking suites. Applicability of docking suites in
small molecule docking on crystal faces was evaluated according to internal standards. Surflex-Dock suite docked perfectly acetaminophen and naproxen molecules into their own crystal lattices, thus pointing out the powerful applicability of it in novel screening approach. doi:10.1016/j.ejps.2008.02.115 P39 Betulin derivatives inhibit Alphavirus replication L. Pohjala 1,2,∗ , S. Alakurtti 3,4 , J. Yli-Kauhaluoma 3 , T. Ahola 5 , P. Tammela 1 1 Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, Finland 2 Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, Finland 3 Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland 4 Technical Research Centre of Finland, VTT, Finland 5 Institute of Biotechnology, University of Helsinki, Finland
Betulin is a widespread naturally occurring triterpenene, found in large quantities in birch bark. Despite the knowledge on betulin-derived compounds as anti-HIV agents, their wider antiviral spectrum remains poorly studied. In this study, a set of 68 betulin derivatives was screened against Semliki Forest virus (SFV), a member of Alphavirus genus and a commonly used model for replication of positive-stranded RNA viruses. The basic structure of betulin was moderately active against SFV (IC50 45 M), but antiviral potency was enhanced particularly by certain cyclic ether or ester substituents, as demonstrated by 28-O-tetrahydropyranylbetulin and 3,28-betulinyl di(2-sulfobenzoate) (IC50 values 17 and 11 M, respectively). Even though some derivatives raised also toxic responses in Huh-7 cells, therapeutic indices higher than 50 were obtained for many of the tested compounds. Due to their non-nucleoside structure and relatively low toxicity, betulin-derived compounds make a competitive contribution to the few inhibitors of these pathogens. doi:10.1016/j.ejps.2008.02.116 P40 The effect of differentiation on the expression of UGT isoenzymes in Caco-2 cell line S. Siissalo ∗ , H. Zhang, J. Hirvonen, M. Finel University of Helsinki, Finland As a part of the characterization process of Caco-2 cell line, the expression of different UGT (UDPglucuronosyltransferase) enzymes was studied. The mRNA levels of 15 UGT1A and UGT2B isoforms were determined in several samples of Caco-2 cells (four passage levels: P31, P37, P43 and P49, fully differentiated cells grown on filters and undifferentiated cells obtained from culturing flasks) using quantitative real-time RT-PCR. The expression levels were normalized using -actin as a reference gene. The difference in the degree of differentiation was demonstrated with villin
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 S ( 2 0 0 8 ) S30–S41
as a control gene. All the studied UGT’s were detected in the samples, but some at very low levels. The expression levels of most genes were at their highest at P43 and declined at P49, with the exceptions of UGT2B4 and UGT2B11. The main UGT enzymes expressed in our Caco-2 cells appeared to be UGT1A6 (both differentiated and undifferentiated samples) and UGT2B17 (differentiated samples, especially P43). doi:10.1016/j.ejps.2008.02.117 P41 Automation of three different cellular assays on robotic workstation—Feasibility evaluation A. Soikkeli 1,∗ , C. Sempio 1 , A.M. Kaukonen 1,2 , A. Urtti 2 , J. Hirvonen 1 , M. Yliperttula 3
lar assays were automated on TECAN Genesis workstation, and critical parameters were evaluated and optimized. From the automation point of view, the bidirectional Caco-2 permeability assay is a complicated assay set-up, whereas the SRB cytotoxicity test and the “mix-and-measure” CaspaseGlo® 3/7 assay kit are more suitable for automation. Automation notably increases the throughput of the last two. The common HTS assay performance indicators, Z factors and signal-tobackground ratios (S/B) were used for determining quality of the automated assays. As a conclusion, characteristics of the HTS assay protocol and cell properties will determine whether the automation is feasible. Automation of simple homogeneous assays like CaspaseGlo® 3/7 is advantageous, but also heterogeneous assays without strict time schedule are worth automation. In other cases, compensatory methods should be evaluated if low throughput is the key problem.
1
Division of Pharmaceutical Technology, University of Helsinki, Finland 2 Drug Discovery and Development Technology Center, University of Helsinki, Finland 3 Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland Assay automation presents several advantages, but all the assays are not easily adapted for automation. Three cellu-
S41
doi:10.1016/j.ejps.2008.02.118