Oral inhalation results in pulmonary drug targeting and thereby reduces systemic side effects, ma... more Oral inhalation results in pulmonary drug targeting and thereby reduces systemic side effects, making it the preferred means of drug delivery for the treatment of respiratory disorders such as asthma, chronic obstructive pulmonary disease or cystic fibrosis. In addition, the high alveolar surface area, relatively low enzymatic activity and rich blood supply of the distal airspaces offer a promising pathway to the systemic circulation. This is particularly advantageous when a rapid onset of pharmacological action is desired or when the drug is suffering from stability issues or poor biopharmaceutical performance following oral administration. Several cell and tissue-based in vitro and ex vivo models have been developed over the years, with the intention to realistically mimic pulmonary biological barriers. It is the aim of this review to critically discuss the available models regarding their advantages and limitations and to elaborate further which biopharmaceutical questions can and cannot be answered using the existing models.
American journal of respiratory cell and molecular biology, Sep 11, 2016
Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fl... more Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and relative contributions of alveolar epithelial type II (AT2) vs type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which β1 subunit was knocked out in either AT1 cells or in both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing for the first time that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung wei...
Purpose: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva... more Purpose: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva. Methods: The transport of [14C]guanidine, the model substrate, in the excised pigmented rabbit conjunctiva was evaluated in the modified Ussing chamber. Tetraethylammonium (TEA) transport also was investigated to determine substrate specificity. Results: The apparent permeability coefficient for guanidine and TEA in the mucosal-to-serosal (ms) direction was 5.4 and 49.6 times greater than that in the serosal-to-mucosal (sm) direction, respectively. Guanidine transport in the ms (but not sm) direction revealed temperature and concentration dependency over 0.02 to 10 mM with an apparent Michaelis-Menten constant of 3.1 mM and a maximal flux of 11.4 nmol/(cm2 x h). Net guanidine transport measured at 0.1 mM across the conjunctiva was decreased by 71% or 82%, respectively, on the addition of 1 microM valinomycin (a K+ ionophore) in both bathing fluids or in a high K+ buffer in the mucosal fluid. Interestingly, net guanidine transport was reduced, rather than enhanced, by 63% upon acidifying the mucosal bathing fluid. By contrast, net guanidine transport was not affected by the serosal presence of 0.5 mM ouabain (a Na+, K+-ATPase inhibitor), by the mucosal and serosal presence of 0.1 microM monensin (a Na+ ionophore) or 0.3 microM carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, a H+ ionophore). Guanidine transport in the ms direction was polyspecific, as indicated by the 48% to 82% inhibition by structurally diverse amines. In particular, guanidine ms transport was inhibited by the antiglaucoma drugs dipivefrine (72%), brimonidine (70%), and carbachol (78%). Conclusions: A carrier-mediated organic cation transport process appears to exist in the conjunctiva, mediating the absorption of organic amines, including certain amine-type ophthalmic drugs. This process may be driven by an inside-negative apical membrane potential difference.
Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled d... more Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km ~ 50 μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibit both an high (Km < 20 μM) and a low affinity (Km > 0.6 mM) transport components, referable...
Metabolism and transport of glutathione (GSH), the endogenous thiol antioxidant, in conjunctival ... more Metabolism and transport of glutathione (GSH), the endogenous thiol antioxidant, in conjunctival tissue to date are poorly understood. The purpose of the present study was to define transport characteristics of GSH in primary cultured rabbit conjunctival epithelial cells (RCECs). RCECs were grown on membrane filters to exhibit tight barrier properties (transepithelial electrical resistance, TEER, approximately 1 k(Omega)/cm(2)). Uptake, efflux, and transepithelial transport of GSH were determined in the presence or absence of extracellular Na(+) under conditions of inhibition of GSH biosynthesis and degradation. Uptake was determined at 15 minutes after instillation of (3)H-GSH to the apical or basolateral bathing fluid. GSH efflux was estimated from the time course of release of prebiosynthesized (35)S-GSH. Transepithelial transport was assessed by instillation of (3)H-GSH in either the apical or basolateral bathing fluid, followed by sampling from respective contralateral sides. A...
The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epitheli... more The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epithelial cell monolayers was studied. At 0.1 nM donor [125I]AVP, the radiolabel flux measured in the apical-to-basolateral (AB) direction was about 10 times greater than that in the reverse (BA) direction. HPLC analyses of the basolateral receiver fluid collected at the end of these flux measurements showed that about 97% of total [125I]label represented subspecies of AVP, whereas the apical receiver fluid contained largely intact AVP (-85% of total [125I]label). Both donor fluids contained virtually no degradation products of AVP (>99%). In the presence of an excess 0.1 mM unlabeled AVP in the apical donor fluid, the Papp for radiolabeled AVP in the AB direction was decreased by ~68%, while the fraction of intact AVP in the basolateral receiver fluid was increased six-fold as compared to that observed at 0.1 nM [125I]AVP alone. Under this condition, the flux of intact AVP was approximately...
It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated ... more It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated on tissue culture-treated polycarbonate Transwell filters are tight (&gt; 2,000 ohm-cm2) and exhibit morphological and phenotypic characteristics of in vivo type I pneumocytes. We studied, utilizing these tight monolayers, the transepithelial transport of thyrotropin-releasing hormone (TRH). Either [125I]TRH or [3H]TRH was used to measure the transalveolar epithelial radiolabel fluxes across the monolayer. Radiochromatography was performed, utilizing reverse-phase HPLC techniques, to determine the presence of TRH and its subspecies in dosing, donor and receiver fluids. The apparent permeability coefficient (Papp) estimated from 125I-radiolabel fluxes was approximately 1.7 x 10(-7) cm/sec in both the apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. In contrast, the Papp for 3H-radiolabels was approximately 4.2 x 10(-7) cm/sec in both directions. Radiochromatography results indicated that neither apical nor basolateral receiver fluid collected at the end of 4 h flux studies contained metabolites of [125I]TRH or [3H]TRH. In the presence of 1,000-fold excess of unlabeled TRH in the donor fluid, the Papp of neither [125I]- or [3H]-TRH in either direction was altered. These data taken together provide evidence for restricted diffusional transport of intact TRH across the rat alveolar epithelial cell monolayer, most likely via paracellular pathways. Thus, it appears that TRH delivery via pulmonary alveolar epithelium in the distal airspaces of the mammalian lung may be feasible without significant interference from peptidase activities.
Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbi... more Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC).
To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial c... more To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial cell (RCEC) layers to low molecular weight drugs of varying lipophilicity. 3H-mannitol; hydrophilic sotalol and atenolol; moderately lipophilic metoprolol, timolol, propranolol; and highly lipophilic betaxolol were used as model compounds. The conjunctival apparent permeability coefficient (Papp) of mannitol (1 x 10(-7) cm/s) was 2.4 times lower than that of the most hydrophilic beta-blocker, sotalol (Papp = 2.4 x 10(-7) cm/s). Differences in the degree of tightness of the epithelial cell layers brought about a 30-fold difference in the transport of atenolol in favor of the leaky cell layers, while not affecting the transport of the lipophilic drug, propranolol. Within the log partition coefficient (PC) range of -0.62 (sotalol) and 3.44 (betaxolol), there was a hundred-fold difference in the Papp. A sigmoidal curve was used to depict the influence of lipophilicity on solute permeation across conjunctival epithelial cell layers. An effective half-maximal Papp was observed at a log PC value of 1.2. These findings on the lipophilicity effect on drug transport are generally similar to those reported for the isolated rabbit conjunctiva, suggesting the utility of cultured rabbit conjunctival epithelial cell layers as an in vitro model for evaluating drug transport.
Oral inhalation results in pulmonary drug targeting and thereby reduces systemic side effects, ma... more Oral inhalation results in pulmonary drug targeting and thereby reduces systemic side effects, making it the preferred means of drug delivery for the treatment of respiratory disorders such as asthma, chronic obstructive pulmonary disease or cystic fibrosis. In addition, the high alveolar surface area, relatively low enzymatic activity and rich blood supply of the distal airspaces offer a promising pathway to the systemic circulation. This is particularly advantageous when a rapid onset of pharmacological action is desired or when the drug is suffering from stability issues or poor biopharmaceutical performance following oral administration. Several cell and tissue-based in vitro and ex vivo models have been developed over the years, with the intention to realistically mimic pulmonary biological barriers. It is the aim of this review to critically discuss the available models regarding their advantages and limitations and to elaborate further which biopharmaceutical questions can and cannot be answered using the existing models.
American journal of respiratory cell and molecular biology, Sep 11, 2016
Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fl... more Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and relative contributions of alveolar epithelial type II (AT2) vs type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which β1 subunit was knocked out in either AT1 cells or in both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing for the first time that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung wei...
Purpose: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva... more Purpose: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva. Methods: The transport of [14C]guanidine, the model substrate, in the excised pigmented rabbit conjunctiva was evaluated in the modified Ussing chamber. Tetraethylammonium (TEA) transport also was investigated to determine substrate specificity. Results: The apparent permeability coefficient for guanidine and TEA in the mucosal-to-serosal (ms) direction was 5.4 and 49.6 times greater than that in the serosal-to-mucosal (sm) direction, respectively. Guanidine transport in the ms (but not sm) direction revealed temperature and concentration dependency over 0.02 to 10 mM with an apparent Michaelis-Menten constant of 3.1 mM and a maximal flux of 11.4 nmol/(cm2 x h). Net guanidine transport measured at 0.1 mM across the conjunctiva was decreased by 71% or 82%, respectively, on the addition of 1 microM valinomycin (a K+ ionophore) in both bathing fluids or in a high K+ buffer in the mucosal fluid. Interestingly, net guanidine transport was reduced, rather than enhanced, by 63% upon acidifying the mucosal bathing fluid. By contrast, net guanidine transport was not affected by the serosal presence of 0.5 mM ouabain (a Na+, K+-ATPase inhibitor), by the mucosal and serosal presence of 0.1 microM monensin (a Na+ ionophore) or 0.3 microM carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, a H+ ionophore). Guanidine transport in the ms direction was polyspecific, as indicated by the 48% to 82% inhibition by structurally diverse amines. In particular, guanidine ms transport was inhibited by the antiglaucoma drugs dipivefrine (72%), brimonidine (70%), and carbachol (78%). Conclusions: A carrier-mediated organic cation transport process appears to exist in the conjunctiva, mediating the absorption of organic amines, including certain amine-type ophthalmic drugs. This process may be driven by an inside-negative apical membrane potential difference.
Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled d... more Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km ~ 50 μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibit both an high (Km < 20 μM) and a low affinity (Km > 0.6 mM) transport components, referable...
Metabolism and transport of glutathione (GSH), the endogenous thiol antioxidant, in conjunctival ... more Metabolism and transport of glutathione (GSH), the endogenous thiol antioxidant, in conjunctival tissue to date are poorly understood. The purpose of the present study was to define transport characteristics of GSH in primary cultured rabbit conjunctival epithelial cells (RCECs). RCECs were grown on membrane filters to exhibit tight barrier properties (transepithelial electrical resistance, TEER, approximately 1 k(Omega)/cm(2)). Uptake, efflux, and transepithelial transport of GSH were determined in the presence or absence of extracellular Na(+) under conditions of inhibition of GSH biosynthesis and degradation. Uptake was determined at 15 minutes after instillation of (3)H-GSH to the apical or basolateral bathing fluid. GSH efflux was estimated from the time course of release of prebiosynthesized (35)S-GSH. Transepithelial transport was assessed by instillation of (3)H-GSH in either the apical or basolateral bathing fluid, followed by sampling from respective contralateral sides. A...
The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epitheli... more The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epithelial cell monolayers was studied. At 0.1 nM donor [125I]AVP, the radiolabel flux measured in the apical-to-basolateral (AB) direction was about 10 times greater than that in the reverse (BA) direction. HPLC analyses of the basolateral receiver fluid collected at the end of these flux measurements showed that about 97% of total [125I]label represented subspecies of AVP, whereas the apical receiver fluid contained largely intact AVP (-85% of total [125I]label). Both donor fluids contained virtually no degradation products of AVP (>99%). In the presence of an excess 0.1 mM unlabeled AVP in the apical donor fluid, the Papp for radiolabeled AVP in the AB direction was decreased by ~68%, while the fraction of intact AVP in the basolateral receiver fluid was increased six-fold as compared to that observed at 0.1 nM [125I]AVP alone. Under this condition, the flux of intact AVP was approximately...
It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated ... more It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated on tissue culture-treated polycarbonate Transwell filters are tight (&gt; 2,000 ohm-cm2) and exhibit morphological and phenotypic characteristics of in vivo type I pneumocytes. We studied, utilizing these tight monolayers, the transepithelial transport of thyrotropin-releasing hormone (TRH). Either [125I]TRH or [3H]TRH was used to measure the transalveolar epithelial radiolabel fluxes across the monolayer. Radiochromatography was performed, utilizing reverse-phase HPLC techniques, to determine the presence of TRH and its subspecies in dosing, donor and receiver fluids. The apparent permeability coefficient (Papp) estimated from 125I-radiolabel fluxes was approximately 1.7 x 10(-7) cm/sec in both the apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. In contrast, the Papp for 3H-radiolabels was approximately 4.2 x 10(-7) cm/sec in both directions. Radiochromatography results indicated that neither apical nor basolateral receiver fluid collected at the end of 4 h flux studies contained metabolites of [125I]TRH or [3H]TRH. In the presence of 1,000-fold excess of unlabeled TRH in the donor fluid, the Papp of neither [125I]- or [3H]-TRH in either direction was altered. These data taken together provide evidence for restricted diffusional transport of intact TRH across the rat alveolar epithelial cell monolayer, most likely via paracellular pathways. Thus, it appears that TRH delivery via pulmonary alveolar epithelium in the distal airspaces of the mammalian lung may be feasible without significant interference from peptidase activities.
Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbi... more Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC).
To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial c... more To evaluate the permeability characteristics of primary cultured rabbit conjunctival epithelial cell (RCEC) layers to low molecular weight drugs of varying lipophilicity. 3H-mannitol; hydrophilic sotalol and atenolol; moderately lipophilic metoprolol, timolol, propranolol; and highly lipophilic betaxolol were used as model compounds. The conjunctival apparent permeability coefficient (Papp) of mannitol (1 x 10(-7) cm/s) was 2.4 times lower than that of the most hydrophilic beta-blocker, sotalol (Papp = 2.4 x 10(-7) cm/s). Differences in the degree of tightness of the epithelial cell layers brought about a 30-fold difference in the transport of atenolol in favor of the leaky cell layers, while not affecting the transport of the lipophilic drug, propranolol. Within the log partition coefficient (PC) range of -0.62 (sotalol) and 3.44 (betaxolol), there was a hundred-fold difference in the Papp. A sigmoidal curve was used to depict the influence of lipophilicity on solute permeation across conjunctival epithelial cell layers. An effective half-maximal Papp was observed at a log PC value of 1.2. These findings on the lipophilicity effect on drug transport are generally similar to those reported for the isolated rabbit conjunctiva, suggesting the utility of cultured rabbit conjunctival epithelial cell layers as an in vitro model for evaluating drug transport.
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