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Dual effects of diadenosine polyphosphates on INS-1 cells
s24
93
95
CORRELATION BETWEEN PERMEABILITY COEFFICIENTS IN A PERFUSED RAT MODEL AND THE CACO-2 CELL MODEL e C.M. Meamy, R. Gubbfns, C. O’DrbcoU
TRANSPORT CHARACTERISTICS OF FORMOTEROL AND SALBUTAMOL ACROSS A BRONCHIAL EPITHELIAL DRUG ABSORPTION MODEL B. A.B. Lansley. Pharmacy Department, Kinds College London, Manresa Road, London SW3 6l.X, UK.
#@%?%mmceutica,
Trbrity College, Dublin,
In order to be absorbed a drug has to diffuse across a series of separate barriers. These mclude the mucus layer. the mtesthtal epithelial cell the lamina propria and t8f’ e endothelium of the capillaries. The rate limmng barrier to drug absorption across the intestinal mucosa is the single layer of intestinal epithelial cells.. In the present work we have examined the correlation between
in-vivo. 1.
Kemp, I.,.Park, K.Y.. Kamani, A., Ho, N.F.H. and tgucht, W.I. (1980) Int. J. Pharm. 4249-262.
DUAL EFFECTS OF DIADENOSINE POLYPHOSPHATES ON INS-l CELLS E. J. Versoohl and B. Johanntilie Institute of Pharmaceutical Chemistry, Dep. of Pharmacology, University of MDnster. Hittorfstr. 5652.46149 M6nster. Germany Recently a no+? of diadenosine polyphosphates (ApnA) as 2nd messengers suggested for insulin secreting cells. In this study we investigated whether receptors for these compounds exist In insulin secreting ceils (INS-I) and how these extracellular compounds conttibute to insulin release. Binding of [‘H]Ap+I to INS-1 ceil homogenates was time dependent and saturable (one-half maximal after 4 min. maximal at 30 min., 12 % bound of total radioactivity). The rank order of diadenosine pdyphosphates displacing [‘H]Ap& from binding sites wss Ap& = ApsA > Apd = ApsA. Adenosine. ATP, UTP. alpha.beta-meATP. beta,gamma-meATP, ADP-betas. 2-MeSATP, and PPADS also displaced [‘H]Ap,A from binding sites whereas suramin, a compound not structurally related to Ap,A. had no displacement activity. Insulin release (RIA) WBSinhibtted dose dependently (max. by 45 %) by either of the four Ap,,A (n= 3-6) In the presence of 6.3 mM glucose during a 90 min. (long term) incubation. However, a stimulation of glucoseinduced Insulin release was observed during penfusion experiments (short term incubation), Due to these contradictory results the stability of fH]Ap&I wes tested. [‘H]Ap,A degraded in the extracellular medium to ATP, ADP. AMP, inoslne and mainly adenoslne (60 % after 90 min) wfth a half-life of about 20 min. During incubation of INS-1 cells dth [‘H]ApA an uptake of radiolabeled compounds occurred. The uptake was inhibited by NaCN and concanevalin A indicating a specific uptake mechanism. However, only small amounts of [‘H]Ap,A can be detected In INS-I cells whereas major amounts consist of [‘H]ATP. This data indicates that diadenosine polyphosphates increase insulin release via binding sites; quickly emergin9 extracellular degradation products such es adenosine inhibit insulin release. The binding sites involved may be diadenosine polyphosphate receptors albeit others, such as P, or P2 receptors cannot be ruled out.
The aim of this study was to investigate the mechanisms of transport of salbutamol and formoterol across a recently developed drug absorption model of the human bronchial epithelium. 16HBE140cells were cultured on permeable tissue culture supports at an air-liquid interface and the medium in the basoiateral chamber (DMEMHam’s F12 1 :I supplemented with 2 %v/v Ultroser G and 2 mM glutamine) was changed daily. The transport of formoterol and salbutamol across the cells (n=12) was studied at day 7 and apparent oermeabilitv coefficients fPaooI were calculated. The aolcal to basolateral permeabifhy ’‘of the drug compounds ‘ was compared to the simultaneously measured permeability of mannitol. a paracellular marker. in addition, the transport of the drugs was studied using non-physiological apical pH (range pH 6.0-9.0) conditions. The Papp of salbutamol across each cell layer was correlated strongly with the transport of mannitol (r-0.998) and the near-zero Intercept of the linear regression suggested exclusive paracellular transport (y=O.Q45x + 0.015). The transport of formoterol was less strongly correlated with mannitoi transport (r-0.914) and linear regression suggested a transcellular comoonent to the transoort tv=0.737x + 1.579). The transport of salbutamol (which is fully ionised at apical pH 6.0-9.0) was not affected by the pH in the apical chamber. However, the permeability of formoterol (for which the proportion of unionised species increases between pH 6.0 to pH 9.0) increased with apical pH, providing further evidence for a transoellular component to transport. In conclusion, the transport of salbutamol across the bronchial drug absorption model was by the paraceilular route, but formoterol utilised both the paracellular and transcellular routes.
INSULIN ABSORPTION VIA TRANSFERIUN RECEPTORMEDIATED TRANSCYTOSIS IN INTESTINAL EPITHELIUM I. Wang, D. Shah and WC. Shen Sciences, University of Southern Depattment of PharmCalifornia School of Pharmacy, Los Angeles, CA 90033, USA.
A mode1 of oral drug delivery system via transferrin receptor (TfR)-mediated transcytosis in intestinal epithelial cells was demonstrated in human enterocyte-like Caco-2 cell cultures and in streptozotocin-induced diabetic CF/l mice, using an insulintransferrin conjugate (Ins-TfJ as a drug and Brefeldin A (BFA) as a transcytosis enhancer. In filter-grown Caco-2 cells, BFA at 1.6 @ml increased the localization of TfR on the apical surface and caused a ‘I-fold enhancement of Tf-transcytosis in the apical-tobasal direction. The transcytosis of insulin across Caco-2 Cell monolayers was detected when Tf was replaced by Ins-Tf, and a 5fold enhancement of the Ins-Tf transcytosts was also observed in the presence of 1.6 @III of BFA in the culture medium. To
demonstrate that such a transport process also occurs in the gastrointestinal tract in viva, oral absorption of Ins-Tf was investigated in diabetic CW1 mice. Two crheria, i.e., the plasma levels of insulin and glucose, were used for the evaluation of the efficacy of Ins-Tf in oral absorption. When mice were orally fed with free or Tf-conjugated insulin (20 Ill/kg) using a gavage needle, the average plasma levels of insulin (n=4) were 2.3, 1.8, dO.3, and co.3 ng/0.2 ml, for Ins-Tf with and without BFA (10 mg/kg), and The insulin with aad without BFA (10 mglkg), respectively. hypoglycemic effect of the orally administered Ins-Tf could be detected as the reduction of blood glucose levels in diabetic mice treated with Ins-Tf co-administered with BFA (-35.5% n=8) and, to a lesser extent, with Ins-Tf but without BFA (-22.01, n=X). No significant decrease of glucose levels was observed in mice treated with insulin, either with or without BFA. Co-administration of a large excess of Tf together with Ins-Tf decreased the hypoglycemic effect, indicating the involvement of TfR in the absorption of Ins-Tf in the gastrointestinal tract. Therefore, our results demonstrate that the utilization of Tf-TfR system, in combination with a transcytosis eohancer, is a feasible approach for tbe enhancement of peptide and protein dNg absorption across the gastrointestinal epitbelium.
93
95
CORRELATION BETWEEN PERMEABILITY COEFFICIENTS IN A PERFUSED RAT MODEL AND THE CACO-2 CELL MODEL e C.M. Meamy, R. Gubbfns, C. O’DrbcoU
TRANSPORT CHARACTERISTICS OF FORMOTEROL AND SALBUTAMOL ACROSS A BRONCHIAL EPITHELIAL DRUG ABSORPTION MODEL B. A.B. Lansley. Pharmacy Department, Kinds College London, Manresa Road, London SW3 6l.X, UK.
#@%?%mmceutica,
Trbrity College, Dublin,
In order to be absorbed a drug has to diffuse across a series of separate barriers. These mclude the mucus layer. the mtesthtal epithelial cell the lamina propria and t8f’ e endothelium of the capillaries. The rate limmng barrier to drug absorption across the intestinal mucosa is the single layer of intestinal epithelial cells.. In the present work we have examined the correlation between
in-vivo. 1.
Kemp, I.,.Park, K.Y.. Kamani, A., Ho, N.F.H. and tgucht, W.I. (1980) Int. J. Pharm. 4249-262.
DUAL EFFECTS OF DIADENOSINE POLYPHOSPHATES ON INS-l CELLS E. J. Versoohl and B. Johanntilie Institute of Pharmaceutical Chemistry, Dep. of Pharmacology, University of MDnster. Hittorfstr. 5652.46149 M6nster. Germany Recently a no+? of diadenosine polyphosphates (ApnA) as 2nd messengers suggested for insulin secreting cells. In this study we investigated whether receptors for these compounds exist In insulin secreting ceils (INS-I) and how these extracellular compounds conttibute to insulin release. Binding of [‘H]Ap+I to INS-1 ceil homogenates was time dependent and saturable (one-half maximal after 4 min. maximal at 30 min., 12 % bound of total radioactivity). The rank order of diadenosine pdyphosphates displacing [‘H]Ap& from binding sites wss Ap& = ApsA > Apd = ApsA. Adenosine. ATP, UTP. alpha.beta-meATP. beta,gamma-meATP, ADP-betas. 2-MeSATP, and PPADS also displaced [‘H]Ap,A from binding sites whereas suramin, a compound not structurally related to Ap,A. had no displacement activity. Insulin release (RIA) WBSinhibtted dose dependently (max. by 45 %) by either of the four Ap,,A (n= 3-6) In the presence of 6.3 mM glucose during a 90 min. (long term) incubation. However, a stimulation of glucoseinduced Insulin release was observed during penfusion experiments (short term incubation), Due to these contradictory results the stability of fH]Ap&I wes tested. [‘H]Ap,A degraded in the extracellular medium to ATP, ADP. AMP, inoslne and mainly adenoslne (60 % after 90 min) wfth a half-life of about 20 min. During incubation of INS-1 cells dth [‘H]ApA an uptake of radiolabeled compounds occurred. The uptake was inhibited by NaCN and concanevalin A indicating a specific uptake mechanism. However, only small amounts of [‘H]Ap,A can be detected In INS-I cells whereas major amounts consist of [‘H]ATP. This data indicates that diadenosine polyphosphates increase insulin release via binding sites; quickly emergin9 extracellular degradation products such es adenosine inhibit insulin release. The binding sites involved may be diadenosine polyphosphate receptors albeit others, such as P, or P2 receptors cannot be ruled out.
The aim of this study was to investigate the mechanisms of transport of salbutamol and formoterol across a recently developed drug absorption model of the human bronchial epithelium. 16HBE140cells were cultured on permeable tissue culture supports at an air-liquid interface and the medium in the basoiateral chamber (DMEMHam’s F12 1 :I supplemented with 2 %v/v Ultroser G and 2 mM glutamine) was changed daily. The transport of formoterol and salbutamol across the cells (n=12) was studied at day 7 and apparent oermeabilitv coefficients fPaooI were calculated. The aolcal to basolateral permeabifhy ’‘of the drug compounds ‘ was compared to the simultaneously measured permeability of mannitol. a paracellular marker. in addition, the transport of the drugs was studied using non-physiological apical pH (range pH 6.0-9.0) conditions. The Papp of salbutamol across each cell layer was correlated strongly with the transport of mannitol (r-0.998) and the near-zero Intercept of the linear regression suggested exclusive paracellular transport (y=O.Q45x + 0.015). The transport of formoterol was less strongly correlated with mannitoi transport (r-0.914) and linear regression suggested a transcellular comoonent to the transoort tv=0.737x + 1.579). The transport of salbutamol (which is fully ionised at apical pH 6.0-9.0) was not affected by the pH in the apical chamber. However, the permeability of formoterol (for which the proportion of unionised species increases between pH 6.0 to pH 9.0) increased with apical pH, providing further evidence for a transoellular component to transport. In conclusion, the transport of salbutamol across the bronchial drug absorption model was by the paraceilular route, but formoterol utilised both the paracellular and transcellular routes.
INSULIN ABSORPTION VIA TRANSFERIUN RECEPTORMEDIATED TRANSCYTOSIS IN INTESTINAL EPITHELIUM I. Wang, D. Shah and WC. Shen Sciences, University of Southern Depattment of PharmCalifornia School of Pharmacy, Los Angeles, CA 90033, USA.
A mode1 of oral drug delivery system via transferrin receptor (TfR)-mediated transcytosis in intestinal epithelial cells was demonstrated in human enterocyte-like Caco-2 cell cultures and in streptozotocin-induced diabetic CF/l mice, using an insulintransferrin conjugate (Ins-TfJ as a drug and Brefeldin A (BFA) as a transcytosis enhancer. In filter-grown Caco-2 cells, BFA at 1.6 @ml increased the localization of TfR on the apical surface and caused a ‘I-fold enhancement of Tf-transcytosis in the apical-tobasal direction. The transcytosis of insulin across Caco-2 Cell monolayers was detected when Tf was replaced by Ins-Tf, and a 5fold enhancement of the Ins-Tf transcytosts was also observed in the presence of 1.6 @III of BFA in the culture medium. To
demonstrate that such a transport process also occurs in the gastrointestinal tract in viva, oral absorption of Ins-Tf was investigated in diabetic CW1 mice. Two crheria, i.e., the plasma levels of insulin and glucose, were used for the evaluation of the efficacy of Ins-Tf in oral absorption. When mice were orally fed with free or Tf-conjugated insulin (20 Ill/kg) using a gavage needle, the average plasma levels of insulin (n=4) were 2.3, 1.8, dO.3, and co.3 ng/0.2 ml, for Ins-Tf with and without BFA (10 mg/kg), and The insulin with aad without BFA (10 mglkg), respectively. hypoglycemic effect of the orally administered Ins-Tf could be detected as the reduction of blood glucose levels in diabetic mice treated with Ins-Tf co-administered with BFA (-35.5% n=8) and, to a lesser extent, with Ins-Tf but without BFA (-22.01, n=X). No significant decrease of glucose levels was observed in mice treated with insulin, either with or without BFA. Co-administration of a large excess of Tf together with Ins-Tf decreased the hypoglycemic effect, indicating the involvement of TfR in the absorption of Ins-Tf in the gastrointestinal tract. Therefore, our results demonstrate that the utilization of Tf-TfR system, in combination with a transcytosis eohancer, is a feasible approach for tbe enhancement of peptide and protein dNg absorption across the gastrointestinal epitbelium.