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Gastrointestinal fate of wheat germ agglutinin using CACO-2 monolayers
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ABSORPTION CHARACTERISTICS OF SALMETEROL ACROSS CACO-2.16KE!E140- AND RAT ALVEOLAR CELLS &-Bap, G. P. Martin and A. B. Lansley Department of Pharmacy, King’s College London, Manresa Road, London, SW3 6LX, UK.
chanctwkrlknofdnrgtm#patIni~coliUmad isotabdwgments~rdkork~~
The P-glycoprotein eMux pump (P-gp) is reported to be present in the epithelium of the lung and might be expected to restrict the pulmonary absorption of drugs using the transcellular route. The absorption characteristics of [14C]-salmeterol across Caco-2, 16HBEl40- and rat alveolar cells cultured on microporous supports were examined in the presence and absence of verapamil, a classic inhibitor of P-gp. Transepithelial electrical resistances (TER’s) were usually in the order of 100-300, 400-500 and 1000-1500 R.cm’ for 16HBE140-, CaCo-2 and alveolar cells respectively. The apparent permeability coefficient (papp) in the apical to basolateral (A-B) and basolateral to apical (B-A) directions was calculated. The Papp of salmeterol across alveolar cells was approximately three-fold &dater B-A than A-B (78.8 +/- 5.4 x 10m7cm s-’ and 24.7 +/- 1.8 x lo_7 cm s-1 respectivcl~) (n=6). The inclusion of verapamil (0.1 mM) in the apical chamber significantly decreased the secretion (B-A) of salmeteml (Papp 40.0 +I- 2.4 x IO-7 cm s-l; P
across the pulmonaryepithelium.
c.H.~.versanhroart,~
TNGPharma,Tow
W.R. Laaman, E. Dulzarand J.P. oro(en.
DMsla~, Zelsl,TheNethalands
Dmgsir&ved in parecellular, tmnsc&lar and acllvahars3pDn m studied in the Caco-2 snd lEGl6 cell linsa in comprvisonwith Ihe banspod in isolated Ma&al aagms& from rat and pig. ParmeeMliQ of the pafacatily transpodad madrars mar&d, PEG4000 and Fo4 (lor methods see Ch.&eret al., J.CmW?eWe, 49,1997,39&) was 5 to * fold lower in C-2 than in IEC-18 cells, wheres, ths pamtaM& for lhe transc&lady transported mafkam cc&c4 and tasloetemn ~83 similar. Transport of the Povcopmtein substrat%s axtisol, rhodamme 123 and vlncMne was 1.7, 30 and B-fold, rasp., faster kum 1ha basc4aM to tha apical compdmant than fmm the apkal to basolaloralcomparbnentin Csco 2 but not in IEG18 calls, indiw!ing tha( the lEGI cells do nd ex~aaas P glycoprotein. FMarmora, calcein. a subside for the o~8& snion transpoftar MRP. waa dlklxad fnwll the cac& cans faslaf tnto the apkal than into thabasdateral compadmant. The”st~mucosalsegnen$ofrat~ndpigbvnwereplaeedinasmaC scale twocompartment transpoil device to sapan& a VnucosaP from a “samsaralmpmimant.Permeabinyofnleperscellularandt~laI markers WBSccmparabla in tat kun 8nd IEG18 &Is. Transpmt wan sknvar inMastlnalsagnaMsfrcnnpigcomparedtoralhmvavar,thapafma&iMy ralio for mannild vs. pM4lOO resembled ihe fatlo knmd in IEG18 (mM=10) falhar than In Caco-2 (&b-36) cslL Maslinal bbpla shwad a morapmnnuncadphasa1snd2tn&&ismthan~calis.Transportd ~hesubslra&forMRPam curranliyaxamkxdlntha!fMinalsagmmt3.
378 EVALUATION OF CYTOTOXICITY OF OCULARDRUGS AND EXCIPIENTS JN AN IMMORTALIZED HUMAN CORNRAL EPITHELIAL CELL LINR _ . P.‘, T. J%rvinen2, K. Araki-Sasaki’, H.
Watanabe“ and A. Urtti’. Department of ‘Pharmaceutics and ‘Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland, ‘Toyanaka Municipal Hospital, Osaka, Japan, ‘Department of Ophthalmology, Osaka University Medical School, Osaka, Japan. An SV40-immortalized human cornea1 epithelial cell line (HCE) was tested as a screening tool to predict topical ocular irritation/toxicity by pharmaceuticals. Cytotoxicity of ocular drugs, eyedrop excipients and cyclodextrins (CDs) were evaluated using the 3-(4,5diiethylthiazol-2-y1)2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and the propidium iodide assay. Cells’were used for cytotoxicity assays when they became confluent on day 3. HCE cells were exposed to various concentrations of test substances in microwell plates for 5 and 60 min. The mitochondrion-based MTT assay was a more sensitive indicator of cytotoxicity than the plasma Imembrane-based propidium iodide assay. The cytotoxicity rankinks for ocular drugs assessed by these in vitro assays was #dipivetYin > timolol > pilocqine e dexamethasone; for excipients: benzalkotium chloride (BAC) > sodii edetate (NqEDTA) > polyvinyl alcohol (PVA) = methylparaben; for CDs C&CD> dimethyl$CD (DM-PCD) > sulfobutyl ether-a CD (SBE-PCD) = hydroxypropy@CD (HP-@CD) > ?/-CD. The data suggests that HCE cell line is potentially useful in predicting the in viw cornea1 toxicity of ocularly applied compounds.
GASTROINTESTINAL
FATE OF WHEAT GERM
AGGLUTININUSING CACO-2 MONOLAYERS M.Wirtb,M.Wolf and F.Gabor Instituteof PbannacmticalTechrs~logy.the Universityof Vienna AltbamtraBe14,A-1090Vienna Austria Inordertotakeadvantageofth%cytoadhmtveanflcytoInvadlq pqetiesofwheatgermq@ttnin(WGA)fcrcraIdtugdellwry, assessmentof prueolytic staMl&yarxi intaa&ms with the lIucous layacoveringthehuninalsurfaceofentfzocytesisaprequisitefar WGA-dated oral drugdelivery. when tluores&n-tagged WGA (F-WGA) was pretreated with high ammmtsofpepsin,tq&n,chymo@qmin,e&tasearpancreatiami lrmbated with C-2 cells, ule atmnmtof cell-w F-WGAws.sas hiQasthatofthec@rolas&ermiaedbyflowcytom&y.Upon
SDS-electmphme&s, prc&se-p&e&d WGA exhibited no &?~produ~~~~~ca38nnlagprdedytic stabll&yof WGA too. As F-WGA-bind@to pig gas+&n&n @GM)CO&dW&SWaSdqmdmtand&ddbitatbychltUrk?3e as well as N-ac&yi-D-glucosan&, the PGM-WGA inter&km is attributedto m. In contrast to cytoa&~?&on, the PGM-WGA imerticm was completelyrever&Ie as indtcdtedby full release of PGM-barndF_WGAuponlrddttloaof~~wimSnlhIswenIs inQpadeacet?oroseqwlceandintervalofaddtdcmofcarbdrydrate awl F-WGA Whm S@g PGM clodCaco-2 m (4.Sxld cells) wae allowedto ccqete far binding of 2.8pg F-WGA, SO% of the lectln applied wL8 balMI to UK. cdla. corlsidafng
revemihiuty and
saturationof the PGM-WGAinteraction as wdl as cytoinvrsioa of WGA, WGA-medhueddrag de&q might @de far targeted ~IKI enhancedefficacyofconjugated drugsdespiteof the mucousbar&.
377
379
ABSORPTION CHARACTERISTICS OF SALMETEROL ACROSS CACO-2.16KE!E140- AND RAT ALVEOLAR CELLS &-Bap, G. P. Martin and A. B. Lansley Department of Pharmacy, King’s College London, Manresa Road, London, SW3 6LX, UK.
chanctwkrlknofdnrgtm#patIni~coliUmad isotabdwgments~rdkork~~
The P-glycoprotein eMux pump (P-gp) is reported to be present in the epithelium of the lung and might be expected to restrict the pulmonary absorption of drugs using the transcellular route. The absorption characteristics of [14C]-salmeterol across Caco-2, 16HBEl40- and rat alveolar cells cultured on microporous supports were examined in the presence and absence of verapamil, a classic inhibitor of P-gp. Transepithelial electrical resistances (TER’s) were usually in the order of 100-300, 400-500 and 1000-1500 R.cm’ for 16HBE140-, CaCo-2 and alveolar cells respectively. The apparent permeability coefficient (papp) in the apical to basolateral (A-B) and basolateral to apical (B-A) directions was calculated. The Papp of salmeterol across alveolar cells was approximately three-fold &dater B-A than A-B (78.8 +/- 5.4 x 10m7cm s-’ and 24.7 +/- 1.8 x lo_7 cm s-1 respectivcl~) (n=6). The inclusion of verapamil (0.1 mM) in the apical chamber significantly decreased the secretion (B-A) of salmeteml (Papp 40.0 +I- 2.4 x IO-7 cm s-l; P
across the pulmonaryepithelium.
c.H.~.versanhroart,~
TNGPharma,Tow
W.R. Laaman, E. Dulzarand J.P. oro(en.
DMsla~, Zelsl,TheNethalands
Dmgsir&ved in parecellular, tmnsc&lar and acllvahars3pDn m studied in the Caco-2 snd lEGl6 cell linsa in comprvisonwith Ihe banspod in isolated Ma&al aagms& from rat and pig. ParmeeMliQ of the pafacatily transpodad madrars mar&d, PEG4000 and Fo4 (lor methods see Ch.&eret al., J.CmW?eWe, 49,1997,39&) was 5 to * fold lower in C-2 than in IEC-18 cells, wheres, ths pamtaM& for lhe transc&lady transported mafkam cc&c4 and tasloetemn ~83 similar. Transport of the Povcopmtein substrat%s axtisol, rhodamme 123 and vlncMne was 1.7, 30 and B-fold, rasp., faster kum 1ha basc4aM to tha apical compdmant than fmm the apkal to basolaloralcomparbnentin Csco 2 but not in IEG18 calls, indiw!ing tha( the lEGI cells do nd ex~aaas P glycoprotein. FMarmora, calcein. a subside for the o~8& snion transpoftar MRP. waa dlklxad fnwll the cac& cans faslaf tnto the apkal than into thabasdateral compadmant. The”st~mucosalsegnen$ofrat~ndpigbvnwereplaeedinasmaC scale twocompartment transpoil device to sapan& a VnucosaP from a “samsaralmpmimant.Permeabinyofnleperscellularandt~laI markers WBSccmparabla in tat kun 8nd IEG18 &Is. Transpmt wan sknvar inMastlnalsagnaMsfrcnnpigcomparedtoralhmvavar,thapafma&iMy ralio for mannild vs. pM4lOO resembled ihe fatlo knmd in IEG18 (mM=10) falhar than In Caco-2 (&b-36) cslL Maslinal bbpla shwad a morapmnnuncadphasa1snd2tn&&ismthan~calis.Transportd ~hesubslra&forMRPam curranliyaxamkxdlntha!fMinalsagmmt3.
378 EVALUATION OF CYTOTOXICITY OF OCULARDRUGS AND EXCIPIENTS JN AN IMMORTALIZED HUMAN CORNRAL EPITHELIAL CELL LINR _ . P.‘, T. J%rvinen2, K. Araki-Sasaki’, H.
Watanabe“ and A. Urtti’. Department of ‘Pharmaceutics and ‘Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland, ‘Toyanaka Municipal Hospital, Osaka, Japan, ‘Department of Ophthalmology, Osaka University Medical School, Osaka, Japan. An SV40-immortalized human cornea1 epithelial cell line (HCE) was tested as a screening tool to predict topical ocular irritation/toxicity by pharmaceuticals. Cytotoxicity of ocular drugs, eyedrop excipients and cyclodextrins (CDs) were evaluated using the 3-(4,5diiethylthiazol-2-y1)2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and the propidium iodide assay. Cells’were used for cytotoxicity assays when they became confluent on day 3. HCE cells were exposed to various concentrations of test substances in microwell plates for 5 and 60 min. The mitochondrion-based MTT assay was a more sensitive indicator of cytotoxicity than the plasma Imembrane-based propidium iodide assay. The cytotoxicity rankinks for ocular drugs assessed by these in vitro assays was #dipivetYin > timolol > pilocqine e dexamethasone; for excipients: benzalkotium chloride (BAC) > sodii edetate (NqEDTA) > polyvinyl alcohol (PVA) = methylparaben; for CDs C&CD> dimethyl$CD (DM-PCD) > sulfobutyl ether-a CD (SBE-PCD) = hydroxypropy@CD (HP-@CD) > ?/-CD. The data suggests that HCE cell line is potentially useful in predicting the in viw cornea1 toxicity of ocularly applied compounds.
GASTROINTESTINAL
FATE OF WHEAT GERM
AGGLUTININUSING CACO-2 MONOLAYERS M.Wirtb,M.Wolf and F.Gabor Instituteof PbannacmticalTechrs~logy.the Universityof Vienna AltbamtraBe14,A-1090Vienna Austria Inordertotakeadvantageofth%cytoadhmtveanflcytoInvadlq pqetiesofwheatgermq@ttnin(WGA)fcrcraIdtugdellwry, assessmentof prueolytic staMl&yarxi intaa&ms with the lIucous layacoveringthehuninalsurfaceofentfzocytesisaprequisitefar WGA-dated oral drugdelivery. when tluores&n-tagged WGA (F-WGA) was pretreated with high ammmtsofpepsin,tq&n,chymo@qmin,e&tasearpancreatiami lrmbated with C-2 cells, ule atmnmtof cell-w F-WGAws.sas hiQasthatofthec@rolas&ermiaedbyflowcytom&y.Upon
SDS-electmphme&s, prc&se-p&e&d WGA exhibited no &?~produ~~~~~ca38nnlagprdedytic stabll&yof WGA too. As F-WGA-bind@to pig gas+&n&n @GM)CO&dW&SWaSdqmdmtand&ddbitatbychltUrk?3e as well as N-ac&yi-D-glucosan&, the PGM-WGA inter&km is attributedto m. In contrast to cytoa&~?&on, the PGM-WGA imerticm was completelyrever&Ie as indtcdtedby full release of PGM-barndF_WGAuponlrddttloaof~~wimSnlhIswenIs inQpadeacet?oroseqwlceandintervalofaddtdcmofcarbdrydrate awl F-WGA Whm S@g PGM clodCaco-2 m (4.Sxld cells) wae allowedto ccqete far binding of 2.8pg F-WGA, SO% of the lectln applied wL8 balMI to UK. cdla. corlsidafng
revemihiuty and
saturationof the PGM-WGAinteraction as wdl as cytoinvrsioa of WGA, WGA-medhueddrag de&q might @de far targeted ~IKI enhancedefficacyofconjugated drugsdespiteof the mucousbar&.