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Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air-liquid interface method for nasal drug transport study
Abstract
P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates.
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method.
MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner.
These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method.
... Interestingly, Cho et al. gathered evidence of functional P-gp activity in primary HNE cells in ALI conditions [60]. The authors revealed that ALI significantly increases transcript levels of P-gp after 10-21 days, compared with only 7 days in contact with air. ...
... The authors revealed that ALI significantly increases transcript levels of P-gp after 10-21 days, compared with only 7 days in contact with air. Additionally, the functional activity of P-gp was evaluated in bidirectional transport studies with a P-gp substrate, rhodamine 123, in the presence and absence of Pgp inhibitors (verapamil and cyclosporin A) [60]. Therefore, for the transport study of P-gp substrates, the authors determined that the suitable period for HNE cells cultured in ALI is 10-14 days after seeding [60]. ...
... Additionally, the functional activity of P-gp was evaluated in bidirectional transport studies with a P-gp substrate, rhodamine 123, in the presence and absence of Pgp inhibitors (verapamil and cyclosporin A) [60]. Therefore, for the transport study of P-gp substrates, the authors determined that the suitable period for HNE cells cultured in ALI is 10-14 days after seeding [60]. ...
The intranasal route has been receiving greater attention from the scientific community not only for systemic drug delivery but also for the treatment of pulmonary and neurological diseases. Along with it, drug transport and permeability studies across the nasal mucosa have exponentially increased. Nevertheless, the translation of data from in vitro cell lines to in vivo studies is not always reliable, due to the difficulty in generating an in vitro model that resembles respiratory human physiology. Among all currently available methodologies, the air-liquid interface (ALI) method is advantageous to promote cell differentiation and optimize the morphological and histological characteristics of airway epithelium cells. Cells grown under ALI conditions, in alternative to submerged conditions, appear to provide relevant input for inhalation and pulmonary toxicology and complement in vivo experiments. Different methodologies and a variety of materials have been used to induce ALI conditions in primary cells and numerous cell lines. Until this day, with only exploratory results, no consensus has been reached regarding the validation of the ALI method, hampering data comparison. The present review describes the most adequate cell models of airway epithelium and how these models are differently affected by ALI conditions. It includes the evaluation of cellular features before and after ALI, and the application of the method in primary cell cultures, commercial 3D primary cells, cell lines and stem-cell derived models. A variety of these models have been recently applied for pharmacological studies against severe acute respiratory syndrome-coronavirus(-2) SARS-CoV(-2), namely primary cultures with alveolar type II epithelium cells and organotypic 3D models. The herein compiled data suggest that ALI conditions must be optimized bearing in mind the type of cells (nasal, bronchial, alveolar), their origin and the objective of the study.
... In contrast, functional activity of P-gp in primary human nasal epithelial cells has been confirmed in a study by Cho et al. [32], using 5 µM rhodamine123 and having the same experimental set up for the bidirectional transport studies of rhodamine123 as in our current work. Cho et al. [32] have shown strong efflux of rhodamine123 in their study, with the P app of rhodamine123 being 6.9-fold higher in the B-A than in A-B direction. ...
... In contrast, functional activity of P-gp in primary human nasal epithelial cells has been confirmed in a study by Cho et al. [32], using 5 µM rhodamine123 and having the same experimental set up for the bidirectional transport studies of rhodamine123 as in our current work. Cho et al. [32] have shown strong efflux of rhodamine123 in their study, with the P app of rhodamine123 being 6.9-fold higher in the B-A than in A-B direction. ...
The RPMI 2650 cell line has been a subject of evaluation as a physiological and pharmacological model of the nasal epithelial barrier. However, its suitability for drug permeability assays has not yet been established on a sufficiently large set of model drugs. We investigated two RPMI 2650 cell models (air-liquid and liquid-liquid) for nasal drug permeability determination by adopting the most recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. The permeability of 23 model drugs and several zero permeability markers across the cell models was assessed. The functional expression of two efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) was shown to be negligible by bidirectional transport studies using appropriate transporter substrates and inhibitors. The model drug permeability determined in the two RPMI 2650 cell models was correlated with the fully differentiated nasal epithelial model (MucilAir™). Additionally, correlations between the drug permeability in the investigated cell models and the ones determined in the Caco-2 cells and isolated rat jejunum were established. In conclusion, the air-liquid RPMI 2650 cell model is a promising pharmacological model of the nasal epithelial barrier and is much more suitable than the liquid-liquid model for nasal drug permeability prediction.
... The main advantage of tracer dyes is the simplicity of the assays and the fact that they are inexpensive. Indeed, they have no significant disadvantages and the majority of authors testing P-gp function used at least one of them [24,[37][38][39][40]. They are compatible with conventional plate readers and with flow cytometers (for instance with a 530/30 nm filter). ...
... For instance, Salomon et al. [64] cultured human alveolar type II cells (ATII) which were obtained from non-tumor lung tissue from patients undergoing lung lobectomy. Cho et al. [38] cultured human nasal epithelial (HNE) cells to simulate the nasal epithelium. Baehr et al [39] proposed an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid and vice versa. ...
Several polymorphisms have been identified in ABCB1, the gene encoding for the P-glycoprotein. This transporter alters the pharmacokinetics or effectiveness of drugs by excreting them from cells where it is expressed (e.g., blood–brain barrier, intestine or tumors). No consensus has been reached regarding the functional consequences of these polymorphisms in the transporter’s function. The aim of this review was to describe a methodology that allows the assessment of P-gp function when harboring polymorphisms. We describe how to obtain cell lines with high expression levels of the transporter with polymorphisms and several tactics to measure its expression and activity. This methodology may help elucidate the contribution of polymorphisms in ABCB1 to drug pharmacokinetics, effectiveness and safety or to cancer chemotherapy failure.
... In order to give more physiological relevance we only used primary HNE cells in our study. In vitro cell culture models of human nasal epithelium based on primary culture technologies are known to be extremely useful for permeability and transport studies in healthy and disease tissues [15]. Furthermore, cultured nasal cells are reliable models since they are known to express important biological features such as tight junctions, mucin secretion, cilia, transporters comparable to those found in vivo systems [16]. ...
... An important issue in gene delivery is the biocompability and biosafety of the nanocarrier used in the transfection procedure [46]. Lipid based formulations like cationic lipids o cationic polymers have become a successful method to transfect cells and to reach adequate transfection efficiency in vitro [15,47]. Here, this study demonstrates promising results using this lipid-based delivery by Lipofectamine TM 2000 transfection reagent in primary cultured cells. ...
Background
Cystic fibrosis (CF) is the most common life-threatening inherited disease in the Caucasian population. It is caused by genetic defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR), a cAMP regulated chloride-bicarbonate channel mainly located in the apical membrane of polarized epithelial cells. CFTR is proposed to regulate other proteins, including the epithelial sodium channel (ENaC). Recently, we successfully restored chloride current in CFTR deficient human airway epithelial cells using wtCFTR-mRNA transfection compared to non-CF cells showing similar values. The present study aimed to optimize the wtCFTR-mRNA transfection procedures in primary cultured human nasal epithelial (HNE) cells. Methods
Dose and time dependence experiments were performed. In addition, we investigated the possible impact of the wtCFTR-mRNA transfection on ENaC function in transepithelial measurements. We reduced the wtCFTR-mRNA dose stepwise and determined the minimal concentration of 0.6 μg/cm2, which is needed for the most efficient restoration of CFTR function. Furthermore, CFTR expression was evaluated 24, 48 and 72 h after transfection. ResultsUsing the minimal concentration of 0.6 μg/cm2 wtCFTR-mRNA we confirmed a positive functional CFTR restoration over a period of 72 h. Biochemical analyses confirmed these findings. Furthermore, we could not find any significant effect on ENaC after the recovery of CFTR by wtCFTR-mRNA transfection. Conclusions
Our data show that wtCFTR-mRNA transfection is an encouraging alternative “gene” therapy in human primary culture.
... As expected and recently described (Kreft et al., 2015), the PCR amplification products of differently cultured RPMI 2650 cells resulted in weaker bands compared to the positive control. Furthermore, an increasing P-gp mRNA expression level trend from day 7 to day 21 and from submerged to ALI cultivation was already demonstrated for primary human nasal epithelial cells (using RT-PCR) and RPMI 2650 cells (using quantitative RT-PCR), respectively (Cho et al., 2011;Kreft et al., 2015). The human nasal turbinate specimens also showed a distinct band of the expected size with intensity comparable to Caco-2 cells. ...
... Using rhodamine 123 (5 mM), the apical expression of P-glycoprotein was previously described for primary HNE cells (isolated from turbinate mucosa with or without chronic sinusitis) with bidirectional transport experiments (Cho et al., 2011). In this study the basolateral to apical (B ! ...
To assess the transmucosal drug transport in the development of medications for intranasal administration, cellular in vitro models are preferred over the use of animal tissues due to inter-species variations and ethical concerns. With regard to the distribution of active agents and multidrug resistance, the ABC transporter P-glycoprotein plays a major role in several mammalian tissues. The present study compares the expression of this efflux pump in optimized in vitro models based on the human RPMI 2650 cell line with specimens of human turbinate mucosa. The presence of the ABCB1 gene was investigated at the mRNA and protein levels using RT-PCR and Western blot analysis in differently cultured RPMI 2650 cells and excised human nasal epithelium. Furthermore, the localization and activity of P-gp was examined by immunohistochemical staining and functionality assays using different substrates in both in vitro and ex vivo models. Both mRNA and protein expression of P-gp was found in all studied models. Furthermore, transporter functionality was detected in both RPMI 2650 cell culture models and excised human mucosa. The results demonstrated a highly promising comparability between RPMI 2650 models and explants of human nasal tissue concerning the influence of MDR1 on drug disposition.The RPMI 2650 cell line might become a useful tool in preclinical trials to improve reproducibility and achieve greater applicability to humans of experimental data regarding passive diffusion and active efflux of drug candidates.
... Pulmonary route has also been considered as an ideal administration route for gene and vaccine delivery (Densmore et al. 2000;Lu and Hickey 2007). To predict pharmacokinetic patterns of drugs in animal models, in vitro permeation study has been performed previously with several kinds of respiratory epithelium-derived cell culture systems, including human bronchial epithelial cell line (16HBE14o-) (Forbes et al. 2003), human broncho-tracheal epithelial cell line (Calu-3) (Grainger et al. 2006), human nasal RPMI 2650 cells (Bai et al. 2008), ATI-like human alveolar epithelial cell (hAEpC) monolayers (Fuchs et al. 2003), A549 lung cancer cells (Wang and Zhang 2004), normal human bronchial epithelial (NHBE) cells (Lin et al. 2007) and human nasal epithelial (HNE) cells Cho et al. 2011). Among these cell culture systems, the characteristics of HNE and NHBE cell monolayers cultured by air-liquid interface (ALI) method have been investigated in our previous studies and these cell monolayers have several advantages over the other cell culture systems as follows: optimal transepithelial electrical resistance (TEER) values to perform in vitro drug permeation study, the formation of tight junction and primary cells originated from human respiratory tract (Lin et al. 2007;You et al. 2003). ...
... Though the coordinates of phenylalanine and naproxen in the correlation plot (Fig. 1) were slightly apart from linear line, all of model drugs, including those two drugs, seemed to be permeated by common transport mechanism across HNE and NHBE cell monolayers irrespective of BCS class. These results indicated that the model drugs were transported by identical routes (transcellular or paracellular) and they were also able to be influenced by commonly expressed drug transporters in both HNE and NHBE cell monolayers, such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATP), and organic cation transporter (OCT) (Cho et al. 2011;Lin et al. 2007;Horvath et al. 2007;Wioland et al. 2000;Kaler et al. 2006). ...
Transport studies of model drugs were conducted across the human nasal epithelial (HNE) and normal human bronchial epithelial (NHBE) cell monolayers cultured by air–liquid interface method. Physicochemical properties (e.g., molecular weight, calculated partition coefficient, dose number) of model drugs were quoted from literatures and apparent permeability coefficients (P
app) across the HNE and NHBE cell monolayers were directly measured. A linear relationship was observed between the P
app values of model drugs in the HNE and NHBE cell monolayers. As the molecular weight of model drugs increased, the P
app showed a decreasing pattern while the increase of partition coefficients resulted in the increment of P
app. These results indicated that the transport of model drugs across both cell monolayers followed mainly the passive diffusion mechanism, although substrates mediated by drug transporters showed a deviating pattern. It was also interesting to note that almost all model drugs could be grouped into the same biopharmaceutics classification system as that classified by the human intestinal permeability when the P
app was plotted as a function of dose number (D
0) of each drug.
... 10 P-gp is responsible for the efflux of many xenobiotics and plays major roles in drug absorption, distribution, and excretion. 11 −14 In a recent report, P-gp mRNA expression was confirmed in myocardial samples of the left ventricle taken during heart transplantation in patients with end-stage heart failure. Furthermore, immunochemical staining has confirmed that P-gp is localized to the endothelium of both arterioles and capillaries in myocardial tissue, 15 suggesting that P-gp is also expressed in human myocardial tissue and may limit drug distribution to the myocardium. ...
Pimozide, an antipsychotic drug, is a potent inhibitor of the hERG channel. A case of death due to cardiac arrest has been reported in a boy who received pimozide together with sertraline and aripiprazole. In this study, we focused on drug-drug interactions and investigated the relationships between transporter-mediated intracellular accumulation and the hERG inhibitory effect of pimozide. The accumulation of pimozide in cardiomyocyte-derived AC16 cells was significantly increased by sertraline and aripiprazole, which are thought to have a P-glycoprotein (P-gp) inhibitory effect, and under P-gp siRNA conditions. These results suggest P-gp inhibition increases pimozide accumulation in AC16 cells. We introduced the hERG plasmid into AC16 cells and investigated the concentration-dependent hERG inhibitory effect of pimozide from within AC16 cells. Addition of 10 nM or more pimozide significantly inhibited the hERG current with concentration dependence. These results indicate P-gp–mediated pharmacokinetic interaction increases pimozide accumulation in AC16 cells, and the subsequent elevated pimozide levels within the cells may result in an increased risk of hERG channel inhibition. Our present study calls attention to the risks associated with the combined use of cardiotoxic P-gp substrate(s) and P-gp inhibitory medicines.
... During the transport studies, 0.2 mL aliquots were taken from the receiver side at 0, 30, 45, 60, 90, 120 and 180 min, and immediately replaced with an equal volume of buffer solution. The amount of Rho123 in the receiver side was assayed by HPLC according to the method of Cho et al., with minor modifications [19]. Concentrations of inulin were colorimetrically determined as described in the literature [20]. ...
Background
Combination therapy of linezolid (LZD) and rifampicin (RFP) may be more effective than monotherapy for treating gram-positive bacterial infections, but several studies have suggested that RFP decreases LZD exposures, thereby increasing the risk of therapeutic failure and emergence of LZD-resistant strains. However, the mechanism of the drug-drug interaction between LZD and RFP is unknown.
Methods
We conducted a prospective, open-label, uncontrolled clinical study in Japanese patients receiving LZD and RFP to evaluate the effect of coadministered RFP on the concentration of LZD. In animal study in rats, the influence of coadministered RFP on the pharmacokinetics of LZD administered intravenously or orally was examined. Intestinal permeability was investigated with an Ussing chamber to assess whether coadministered RFP alters the absorption process of LZD in the intestine.
Results
Our clinical study indicated that multiple doses of RFP reduced the dose-normalized trough concentration of LZD at the first assessment day by an average of 65%. In an animal study, we found that multiple doses of RFP significantly decreased the area under the concentration-time curve, the maximum concentration and the bioavailability of orally administered LZD by 48%, 54% and 48%, respectively. In contrast, the pharmacokinetics of intravenously administered LZD was unaffected by the RFP pretreatment. However, investigation of the intestinal permeability of LZD revealed no difference in absorptive or secretory transport of LZD in the upper, middle and lower intestinal tissues between RFP-pretreated and control rats, even though RFP induced gene expression of multidrug resistance protein 1a and multidrug resistance-associated protein 2.
Conclusions
Therapeutic drug monitoring may be important for avoiding subtherapeutic levels of LZD in the combination therapy. The drug-drug interaction between LZD and RFP may occur only after oral administration of LZD, but is not due to any change of intestinal permeability of LZD.
Trial registration
UMIN, UMIN000004322. Registered 4 October 2010.
... Moreover, the fold difference between active and passive contributions of Rhod transport was largely influenced by the concentration, meaning that at low concentrations (0.3-3 μM), both transport components were significantly indifferent and, then the active component started to markedly increase (p b 0.05). We can thereby infer that Rhod transport characteristics across locust BBB, based on concentration, time, temperature and mechanism, are in accordance with the results reported for other traditionally used systems [54][55][56][57][58]. ...
Background:
ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models.
Methods:
Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and D. melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses.
Results:
A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers.
Conclusion:
Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.
... Especially, as one of the representative efflux transporters, the expression of P-gp was verified in 16HBE14o -, Calu-3, and normal human bronchial epithelial (NHBE) cell monolayer cultured by the air-liquid interface (ALI) method (Brouillard et al., 2001; Ehrhardt et al., 2003; Florea et al., 2001; Lin et al., 2007b). Recently, the functional evidence and activity of P-gp expression in passaged primary human nasal epithelial cell monolayers cultured by the ALI method were reported (Cho et al., 2011b). In that report, MDR1 gene expression according to the cell culture period was demonstrated by RT-PCR assay. ...
Growing interest in the nasal route as a drug delivery system calls for a reliable in vitro model which is cru-cial for efficiently evaluating drug transport through the nasal cells. Various in vitro cell culture systems has thus been devel-oped to displace the ex vivo excised nasal tissue and in vivo animal models. Due to species difference, results from animal studies are not sufficient for estimating the drug absorption kinetics in humans. However, the difficulty in obtaining reliable human tissue source limits the use of primary culture of human nasal epithelial cells. This shortage of human nasal tissue has therefore prompted studies on the "passage" culture of nasal epithelial cells. A serially passaged primary human nasal epithelial cell monolayer system developed by the air-liquid interface (ALI) culture is known to promote the differentiation of cilia and mucin gene and maintain high TEER values. Recent studies on the in vitro nasal cell culture systems for drug transport studies are reviewed in this article.
... P-gp is a 170-kDa glycoprotein [28,29] expressed on the apical membranes of various tissues, such as the blood-brain barrier, liver, kidney, intestine, placenta and respiratory epithelium [30]. It is encoded by the ABCB1 gene, also known as MDR1 [31]. ...
In this article, we discussed the expression of some ABC (e.g., P-glycoprortein, MRP and CFTR) and SLC (e.g., POT, DAT, OAT, OATP, OCT, EAAT2/GLT1 and GLUT) amino acid, metal and nucleoside transporters in the nasal mucosa. The localization and therapeutic targeting of these transporters are explored in detail. The wide array of transporters discovered so far in the nasal mucosa implies that a plethora of compounds can be delivered by targeting these transporters. The article concludes with a discussion of the potential challenges and delivery options for transporter-mediated drug targeting via the nasal route.
... Prior data demonstrating that P-gp expression persists in primary nasal epithelial cell cultures provides an opportunity to study its immunoregulatory capacity in vitro. 15 Our immunohistochemical and P-gp ELISA data confirm these findings indicating that P-gp was both present and functional within our PNECCs. Of note, the primary antibody used in our Q-FIHC study is specific to an extracellular loop of the P-gp protein providing further confirmation that our data reflects the activity of the membrane bound P-gp as opposed to the cytoplasmic fraction which does not participate in substrate transport out of the cell. ...
P-glycoprotein (P-gp) is an adenosine triphosphate (ATP)-dependent efflux pump that confers chemotherapeutic resistance in cancer cells. Recent studies suggest that P-gp may also function as an immunomodulator through regulation of cytokine transport. Sinonasal epithelial cells have been recognized as drivers of local innate and adaptive immunity and are known to overexpress P-gp in the setting of inflammation. The objective of this study is to therefore determine whether P-gp participates in the regulation of cytokine secretion in sinonasal epithelial cells.
Primary nasal epithelial cell cultures (PNECCs) were cultivated from 5 healthy patients. Membranous P-gp was quantified through quantitative fluorescent immunohistochemistry (Q-FIHC) and confirmed by enzyme-linked immunosorbent assay (ELISA). Sensitivity to inhibition was determined using a rhodamine 123 accumulation assay. Baseline and lipopolysaccharide (LPS)-stimulated cytokine secretion of interleukin 6 (IL-6), IL-8, granulocyte macrophage colony stimulating factor (GM-CSF), and thymic stromal lymphopoietin (TSLP) were quantified by ELISA and compared to LPS stimulated secretion in the setting of P-gp–specific inhibition. Differences in P-gp expression and cytokine secretion were compared using 2-tailed Student t tests with post hoc testing using the Bonferroni procedure.
Membranous P-gp is detectable in PNECCs and upregulated following LPS exposure. P-gp is sensitive to inhibition by both PSC 833 and verapamil in a dose-dependent fashion. LPS stimulated secretion of normalized IL-6 (mean, 95% confidence interval [CI]) (79.67, 42.26–117.07), GM-CSF (39.92, 7.90–71.94), and TSLP (6.65, 5.35–7.96) was significantly reduced following P-gp inhibition (37.60, 11.54–63.65, p = 0.023; 7.64, 2.25–13.03, p = 0.044; and 5.13, 4.44–5.82, p = 0.038; respectively).
P-gp is functionally active in PNECCs. P-gp participates in modulation of epithelial secretion of LPS stimulated IL-6, GM-CSF, and TSLP.
The nasal epithelium is a complex, highly vascularized and innervated cellular structure that, owing to a high surface area, has been extensively investigated not only for the localized delivery of drugs but also for their systemic delivery. The revelation of a direct pathway to the central nervous system that surpasses the blood–brain barrier has attracted the attention of researchers as a strategy to target relevant drugs to the brain. This pathway is especially efficient for nanostructures of up to 300 nm; its main advantages are simplicity, minimal invasiveness, and patient compliance. At the same time, because of the complexity of the structure, the biological performance of different drug delivery systems is often assessed in vivo employing animal models and techniques that include, for example, microdialysis, a procedure that enables multiple sampling at different time points. However, this approach has drawbacks. The first is that the most common animal models (e.g., rodents) do not necessarily mimic the human nasal mucosa. The second is that at the very early stages of development and when optimization of relevant features of the delivery system is addressed, these approaches are ethically questionable. In this scenario, robust and reliable in vitro and ex vivo models represent a relevant research tool to select the most promising prototypes that will continue to the preclinical stage. This chapter presents a brief overview of the most relevant structural features of the nasal mucosa and then discusses the different proposed models.
Mice and rats are commonly used to investigate in vivo nasal drug absorption, yet their small nasal cavities limit their use for in-vitro investigations. Bovine tissue explants have been used to investigate drug transport through the nasal respiratory and olfactory mucosae, yet limited information is available regarding the similarities and differences among these animal models compared to humans. The aim of this study was to compare the presence of a number of important drug transporters in the nasal mucosa of these species.
DNA microarray results for nasal samples from humans, rats and mice were obtained from GenBank, while DNA microarray and RT-PCR were performed on bovine nasal explants. The drug transporters of interest include multidrug resistance, cation, anion, peptide, and nucleoside transporters.
Each of the species (mouse, rat, cow and human) shows similar patterns of expression for most of the important drug transporters. Several transporters were highly expressed in all the species, including MRP1, OCTN2, PEPT2 and y+LAT2.
The similarities in transporter expression between species support the continued use of these animal models during pre-clinical investigation of intranasally -administered therapeutics.
P-glycoprotein (P-gp) regulates absorption of many drugs in the gastrointestinal tract and their accumulation in tumor tissues, but the basis of substrate recognition by P-gp remains unclear. Bitter-tasting phenylthiocarbamide, which stimulates taste receptor 2 member 38 (T2R38), increases P-gp activity and is a substrate of P-gp. This led us to hypothesize that bitterness intensity might be a predictor of P-gp-inhibitor/substrate status. Here, we measured the bitterness intensity of a panel of P-gp substrates and nonsubstrates with various taste sensors, and used multiple linear regression analysis to examine the relationship between P-gp-inhibitor/substrate status and various physical properties, including intensity of bitter taste measured with the taste sensor. We calculated the first principal component analysis score (PC1) as the representative value of bitterness, as all taste sensor's outputs shared significant correlation. The P-gp substrates showed remarkably greater mean bitterness intensity than non-P-gp substrates. We found that Km value of P-gp substrates were correlated with molecular weight, log P, and PC1 value, and the coefficient of determination (R2) of the linear regression equation was 0.63. This relationship might be useful as an aid to predict P-gp substrate status at an early stage of drug discovery. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
Background
Sinonasal epithelial cells are recognized as drivers of inflammation in chronic sinusitis with nasal polyps (CRSwNP) through secretion of T helper 2 (Th2)-promoting cytokines. P-glycoprotein (P-gp) is overexpressed in nasal polyps and modulates epithelial cytokine secretion in healthy mucosa. The objective of this study is to determine whether P-gp overactivity promotes Th2-associated cytokine secretion in CRSwNP. Methods
Polyp explants (n = 4) and primary epithelial cell cultures (n = 5) were cultivated from patients with CRSwNP. Explant P-gp activity was determined using a calcein assay. In culture, P-gp was quantified by enzyme-linked immunosorbent assay (ELISA) and sensitivity to PSC-833 inhibition was determined using a calcein assay. Lipopolysaccharide (LPS)-stimulated cytokine secretion of interleukin 6 (IL-6), IL-8, IL-25, and granulocyte macrophage colony stimulating factor (GM-CSF) were quantified by ELISA and compared to secretion following P-gp inhibition. Differences in P-gp expression and cytokine secretion were compared using a Mann-Whitney U test. Secretion was correlated with P-gp expression using a Pearson correlation coefficient. ResultsCalcein retention is increased in P-gp inhibited vs uninhibited polyp explants (mean ± standard deviation [SD]; 5.17 ± 1.76 vs 2.55 ± 0.62; p < 0.05) but not in controls, indicating increased nasal polyp P-gp activity. P-gp is sensitive to dose-dependent P-gp inhibition with PSC-833 in vitro. LPS-stimulated secretion of normalized GM-CSF (45.21 ± 41.39) and IL-6 (63.16 ± 36.37) were significantly reduced following P-gp inhibition (8.47 ± 3.28; p < 0.01, and 39.94 ± 31.07; p < 0.05; respectively) and secretion was highly correlated with P-gp expression(r = 0.824, p < 0.05, and r = 0.833, p < 0.05; respectively). ConclusionP-gp overactivity promotes Th2-associated epithelial cytokine secretion in nasal polyps, suggesting a novel mechanism for maintaining chronic inflammation in CRSwNP.
The nasal cavity is both a target for locally and systemically acting medications. An adequate treatment for rhinosinusitis continues to be an unmet need. With the recent approval of intranasal medications for the treatment of pain, the nasal cavity continues to be a viable route for rapid uptake into the systemic circulation. Despite the opportunities, there is still a void in the knowledge of how therapeutic entities interact with the nasal epithelium. In addition, new opportunities in mucosal immunity via nasal vaccination as well as the elusive nose to brain uptake continue to drive innovation. To facilitate understanding of the issues involved that facilitate drug delivery in the nose, a review of nasal morphology and physiology is presented.
The blood-brain barrier (BBB) limits the distribution of systemically administered therapeutics to the central nervous system (CNS), posing a significant challenge to drug development efforts to treat neurological and psychiatric diseases and disorders. Intranasal delivery is a noninvasive and convenient method that rapidly targets therapeutics to the CNS, bypassing the BBB and minimizing systemic exposure. This review focuses on the current understanding of the mechanisms underlying intranasal delivery to the CNS, with a discussion of pathways from the nasal cavity to the CNS involving the olfactory and trigeminal nerves, the vasculature, the cerebrospinal fluid, and the lymphatic system. In addition to the properties of the therapeutic, deposition of the drug formulation within the nasal passages and composition of the formulation can influence the pathway a therapeutic follows into the CNS after intranasal administration. Experimental factors, such as head position, volume, and method of administration, and formulation parameters, such as pH, osmolarity, or inclusion of permeation enhancers or mucoadhesives, can influence formulation deposition within the nasal passages and pathways followed into the CNS. Significant research will be required to develop and improve current intranasal treatments and careful consideration should be given to the factors discussed in this review.
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
We have demonstrated increased heat resistance in Chinese hamster ovary cells grown to confluence at 37 degrees C and thermally adapted at the nonlethal temperature of 40 degrees C for 24 h. Membrane viscosity, estimated by fluorescence anisotropy, was inversely related to temperature, from 5 degrees C to 45 degrees C. For a given temperature viscosity was consistently higher in thermally adapted cells than in native cells. Having demonstrated a change in membrane structure associated with thermal adaptation, we carried out a study of the Na+-K+ pump in native and thermally adapted cells as an example of a vital active transport process known to be sensitive to membrane viscosity. 86Rb uptake measured from 31 degrees C to 50 degrees C increased steadily to 46 degrees C and then decreased rapidly in both native and thermally adapted cells. Detailed measurement of ouabain-sensitive 86Rb influx demonstrated an increase in both Km and Vmax between 37 degrees C and 45 degrees C, but there was no difference between native and thermally adapted cells. We have thus demonstrated an adaptive structural change in the cell membrane of mammalian cells which may be related to the induction of thermal resistance at 40 degrees C but which is not associated with any change in this active transport system.
MDR1 P-glycoprotein (Pgp 170), a member of the adenosine triphosphate (ATP) binding cassette transporters, acts as an efflux pump for various hydrophobic agents, particularly for xenobiotics such as benzo(a)pyrene. It has also been shown to regulate cell-volume activated chloride channels. Pgp 170 could, therefore, be of particular importance in cellular mechanisms of defence in the airways and in the control of mucus layer composition. For these reasons, we evaluated the precise localization of Pgp 170 in human adult airways. Fresh non-neoplastic bronchial specimens were collected from 33 patients (26 smokers, four exsmokers and three nonsmokers) who underwent surgery for lung carcinoma. The presence of MDR1 messenger ribonucleic acid (mRNA) was demonstrated by reverse transcriptase chain reaction (RT-PCR) in bronchial epithelial cells collected by gentle scraping from either smokers, exsmokers or nonsmokers. Immunodetection of Pgp 170 using a panel of monoclonal antibodies (MRK16, JSB1, C219, C494) was performed either on cryostat or paraffin-embedded sections of histologically normal bronchial tissue. Pgp 170 was constantly detected with intense labelling at the apical surface of ciliated epithelial cells from the surface epithelium or ciliated collecting ducts, and on apical and lateral surfaces of serous cells from bronchial glands. No staining of mucus-secreting cells was observed. Pgp 170 was also demonstrated at the luminal surface of endothelial cells of bronchial capillaries. In conclusion, the expression of MDR1 P-glycoprotein in bronchial structures, particularly at the epithelial apical surface, suggests important roles for this transmembrane protein in human airways.
CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein), members of the ABC transporter superfamily, possess multiple functions, particularly Cl(-), anion, and glutathione conjugate transport and cell detoxification. They are also hypothesized to have a number of complementary functions. It is generally accepted that data obtained from nasal mucosa can be extrapolated to lower airway cell physiology. The aim of the present study was to investigate by immunohistochemistry the differential localization of CFTR, MDR1, and MRP1 in the normal mucosa of 10 human nasal turbinates. In ciliated epithelial cells, CFTR was inconstantly expressed at the apical cell surface, intense membranous labeling was observed for MDR1, and intense cytoplasmic labeling was observed for MRP1. In the glands, a higher level of expression was observed on serous cells, at the apical surface (for CFTR), on lateral membranes (for MDR1), and with an intracytoplasmic distribution (for MRP1). In conclusion, CFTR, MDR1 and MRP1 are expressed in the epithelium and glands of the nasal respiratory mucosa, but with different patterns of expression. These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells and a protective function for MDR1 and MRP1 in respiratory ciliated cells. (J Histochem Cytochem 48:1215-1222, 2000)
The aim of our study was to investigate the effects of rabeprazole, a proton pump inhibitor, on MDR1 expressed on human colon carcinoma cell line, Caco-2, and MDR1-overexpressing human cervical carcinoma cell line, HeLa cells selected by exposure to 100 nM vinblastine (Hvr100-6 cells). Inhibitory effects of rabeprazole on MDR1-mediated transport of Rhodamine123 were examined in these cells. A thousand micro molar rabeprazole increased Rhodamine 123 uptakes in Caco-2 and Hvr100-6 cells by 68% and 185%, respectively. No significant effects of rabeprazole were observed at the concentration of 1-100 microM. Since rabeprazole did not show any effects on Rhodamine 123 transport via MDR1 at the plasma levels (approximately 1 microM), it was considered that the drug interaction with MDR1 substrates would be minimal even though the interaction occurred in the patients with rabeprazole treatment.
Many tumour cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters. Two proteins, P-gp (MDR-1, ABCB1) and MRP-1 (ABCC1) have been demonstrated to pump a wide selection of the most commonly used cancer drugs and their overexpression correlates broadly with negative treatment response characteristics in many different forms of cancer. Several generations of pharmaceutical inhibitors of P-gp have been examined in preclinical and clinical studies; however, these circumvention trials have largely failed to demonstrate the anticipated increase in therapeutic efficacy. In vitro screening has identified a number of pharmaceuticals which can selectively inhibit pumps such as P-gp, or MRP-1, by virtue of their being substrates for these pumps. The use of low toxicity pharmaceuticals or agents which have anticancer properties as ABC transporter inhibitors may allow a new paradigm of clinically useful drug resistance circumvention. Our increasing understanding of the complex pharmacological interplay of drug transporter proteins indicates that the cellular pharmacokinetics of cancer drug entry into and exit from tumour cells is of prime importance in subsequent drug efficacy and a larger portfolio of pump modulators and targeted efflux inhibition strategies is necessary to effectively overcome multiple drug resistance.
Objectives: To investigate whether growing human nasal epithelium as primary cultures alters aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyldipeptidase (DPPIV) metabolic characteristics, and mRNA gene transcript expression. Methods The formation of 7-amino-methyl coumarin from specific substrates for APN (L-alanine-4-methyl-coumaryl-7-amide, APB (L-arginine-4-methyl-coumaryl-7-amide) and DPPIV (glycyl-L-proline-4-methyl- coumaryl-7-amide) was used to estimate the KM, Vmax and the effect of aminopeptidases inhibitors on the enzymes. Polymerase chain reaction was used to investigate gene expression. Key findings: Results of this study showed that: (1) both the excised tissues and primary cultures of human nasal epithelium expressed APN, APB and DPPIV activity; (2) the KM of APB, APN and DPPIV was not significantly different in cell and tissue homogenates; (3) except for APN, the Vmax was not significantly different in the two metabolism models; (4) there was no statistically significant difference in the behaviours of APB, APN and DPPIV in response to inhibition by puromycin and bestatin in the two models; (5) the mRNA transcripts that encode APB, APN and DPPIV were expressed in both cell culture and tissue homogenate. Conclusions: Based on the results of this study, it may be concluded that nasal primary culture system is suitable for investigating peptide and protein metabolism and enzymatic stability in human nasal epithelium. Except for APN, the tissue culture conditions did not significantly alter the functional and molecular expression of the aminopeptidases.
Objectives: The aim was to establish a preclinical in-vitro system of the nasal mucosa for the simultaneous evaluation of nasal absorption and effects on ciliary activity. Methods: Human nasal epithelial cells were grown in collagen-coated transport inserts with transparent polyethylene terephthalate membranes (3 μm). Transepithelial transport and ciliary beat frequency values were measured every 15 min for 1 h. Key findings: The apparent permeability coefficients (Papp) for atenolol (mainly paracellular transport) and propranolol (transcellular transport) amounted to 0.1 ± 0.1 and 23.7 ± 0.6 × 10⁻⁶ cm/s, respectively, illustrating that the system can be used to discriminate between high permeability and low permeability compounds. Transport of talinolol (substrate for the P-glycoprotein efflux carrier) did not reveal polarity (0.3 ± 0.2 and 0.2 ± 0.1 × 10⁻⁶ cm/s for absorptive and secretory transport, respectively) and was not affected by verapamil (10 μM), suggesting the absence of P-glycoprotein in the nasal cell culture. No significant effects of atenolol, propranolol and talinolol on ciliary beat frequency were observed (98 ± 20% of the control condition after 60 min). Chlorocresol significantly decreased the ciliary activity but this decrease was not accompanied by effects on the transepithelial transport of atenolol, propranolol and talinolol. Conclusions: A new system was developed which offers possibilities as a fast screening tool for studying the potential of compounds for nasal drug administration, since permeability and a possible cilio-toxic effect can be assessed simultaneously.
Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
The LLC-PK1:MDR1, LLC-PK1 and Caco-2 cell lines were used to investigate whether rhodamine-123 or doxorubicin would be the preferred substrate to study P-glycoprotein (P-gp) functionality in vitro. Both rhodamine-123 and doxorubicin showed highly polarised transport in the Caco-2 cell line and the LLC-PK1:MDR1 cell line, indicating that P-gp is actively transporting these drugs. However, for rhodamine-123 polarised transport was also seen in the monolayers of the wild-type LLC-PK1 cell line, indicating the presence of another active transporter for this compound. Polarised transport of doxorubicin in the Caco-2 and the LLC-PK1:MDR1 cell lines could be inhibited by the P-gp inhibitors SDZ-PSC 833 (PSC 833), cyclosporin A (CsA), verapamil and quinine, but not by the inhibitors for the organic cation carrier systems cimetidine and tetraethylammonium (TEA). Polarised transport of rhodamine-123 in the Caco-2 cell line could only be inhibited by P-gp inhibitors. In the LLC-PK1:MDR1 and LLC-PK1 cell lines transport was also inhibited by inhibitors for the organic cation transport systems. In conclusion, rhodamine-123 is a substrate for both P-gp and the organic cation carrier systems in the kidney cell line. This indicates that rhodamine-123 is not selective enough to study P-gp functionality in cell systems were organic cation carrier systems are also present. Doxorubicin appears to be a more selective P-gp substrate and therefore more useful in studying P-gp functionality in vitro.
Whereas in vivo studies represent the most crucial test for any nasal drug application or formulation, mechanistic aspects of nasal absorption may be more clearly approached by well defined and controlled in vitro studies. In this review the progress of nasal in vitro models to investigate drug permeation and metabolism in the epithelium is summarized and their potential and limitations are discussed. The following subjects will be covered: (i) primary cell cultures of human nasal epithelium, including sampling techniques and culture conditions, (ii) human nasal cell lines (in particular the human nasal cell line RPMI 2650), and (iii) excised nasal epithelium (rabbit, bovine, ovine, canine, human), also summarizing suitable preparation techniques and tissue characterization, test media, tissue equilibration, viability testing, and integrity tests. Furthermore, an overview on the various experimental set-ups suitable for in vitro transport studies (permeation rates; identification of permeation pathways; mechanisms and toxicity of absorption enhancers) and for metabolism studies (rates, saturation and pathways of enzymatic cleavage) is presented. Some attention is given to identify potential endocytotic uptake mechanisms. To date, the permeation and metabolic barrier function of excised nasal tissue derived from various animals has shown to mimic the in vivo situation `ex vivo' at the highest degree possible. Supply of human tissue will continue to be short. Therefore, further studies are necessary to evaluate and improve culture conditions, handling, performance and physiologic relevance of primary human cell and cell line cultures.
The objective of this work was to explore the potential and safety of trimethyl chitosan (TMC) and PEGylated TMC for improved absorption of insulin after nasal administration.
The nasal absorption of insulin nanocomplexes of TMC or PEGylated TMC was evaluated in anaesthetized rats. Concomitantly, the histopathological effects of these nanocomplexes on rat nasal mucosa were studied using a perfusion fixation technique.
All insulin nanocomplexes containing TMC or PEGylated TMC showed a 34-47% reduction in the blood glucose concentration, when the insulin absorption through the rat nasal mucosa was measured indirectly. In addition, the relative pharmacodynamic bioavailability (F(dyn)) of the formulations was found to be dependent upon the charge ratio of insulin and polymer, regardless of polymer structure. The F(dyn) apparently decreased with increasing charge ratio of insulin : polymer. Although acute alterations in nasal morphology by the formulations were affected by the charge ratio of insulin and polymer, the formulation of insulin/PEGylated TMC nanocomplexes was shown to be less toxic to the nasal epithelial membrane than insulin/TMC nanocomplexes.
PEGylated TMC nanocomplexes were a suitable absorption enhancer for nasal delivery of insulin.
Hyaluronic acid (HA)-based microspheres containing PEG 6000 and/or sodium taurocholate (NaTC) were prepared by the spray-drying method for nasal delivery of fexofenadine hydrochloride (HCl). Their physicochemical properties including particle size and drug contents were determined, while their morphology examined by a scanning electron microscope (SEM). The effects of the solubilizer (PEG 6000) and the permeation enhancer (NaTC) on the in vitro release characteristics of fexofenadine x HCl were observed. Moreover, the in vitro permeation of fexofenadine x HCl was determined using the human nasal cell (HNE) monolayers cultured on Transwell inserts. After nasal administration, fexofenadine x HCl in rabbit plasma was determined by the LC-MS/MS system. Fexofenadine x HCl-loaded microspheres were of spherical shape with 20-30 microm mean diameter. The loading efficiency was about 95%. In vitro release of fexofenadine x HCl from the microspheres was significantly increased with the addition of PEG 6000. Although NaTC did not alter the in vitro release of drug from the microspheres, its addition further increased the in vitro permeation of fexofenadine x HCl across the HNE cell monolayers. Moreover, the bioavailability of fexofenadine x HCl after nasal administration of the microsphere formulation to rabbits was increased up to about 48% while that of the control solution was only about 3%. These results indicated that the HA microsphere formulation could further be developed into a clinically useful nasal delivery system of fexofenadine x HCl.
The aim was to establish a preclinical in-vitro system of the nasal mucosa for the simultaneous evaluation of nasal absorption and effects on ciliary activity.
Human nasal epithelial cells were grown in collagen-coated transport inserts with transparent polyethylene terephthalate membranes (3 mum). Transepithelial transport and ciliary beat frequency values were measured every 15 min for 1 h.
The apparent permeability coefficients (P(app)) for atenolol (mainly paracellular transport) and propranolol (transcellular transport) amounted to 0.1 +/- 0.1 and 23.7 +/- 0.6 x 10(-6) cm/s, respectively, illustrating that the system can be used to discriminate between high permeability and low permeability compounds. Transport of talinolol (substrate for the P-glycoprotein efflux carrier) did not reveal polarity (0.3 +/- 0.2 and 0.2 +/- 0.1 x 10(-6) cm/s for absorptive and secretory transport, respectively) and was not affected by verapamil (10 muM), suggesting the absence of P-glycoprotein in the nasal cell culture. No significant effects of atenolol, propranolol and talinolol on ciliary beat frequency were observed (98 +/- 20% of the control condition after 60 min). Chlorocresol significantly decreased the ciliary activity but this decrease was not accompanied by effects on the transepithelial transport of atenolol, propranolol and talinolol.
A new system was developed which offers possibilities as a fast screening tool for studying the potential of compounds for nasal drug administration, since permeability and a possible cilio-toxic effect can be assessed simultaneously.
The purpose of this study was to find out whether nasal application of buspirone could increase its bioavailability and directly transport the drug from nose to brain.
A nasal formulation (Bus-chitosan) was prepared by dissolving 15.5 mg buspirone hydrochloride, 1% w/v chitosan hydrochloride and 5% w/v hydroxypropyl beta-cyclodextrin (HP-beta-CD) in 5 ml of 0.5% sodium chloride solution. The formulation was nasally administered to rats and the plasma and brain concentration compared with that for buspirone hydrochloride solution after intravenous and intranasal (Bus-plain) administration. The brain drug uptake was also confirmed by gamma scintigraphic study.
The nasal Bus-chitosan formulation improved the absolute bioavailability to 61% and the plasma concentration peaked at 30 min whereas the peak for nasal Bus-plain formulation was 60 min. The AUC0-480 in brain after nasal administration of Bus-chitosan formulation was 2.5 times that obtained by intravenous administration (711+/-252 ng/g vs 282+/-110 ng/g); this was also considerably higher than that obtained with the intranasal Bus-plain formulation (354+/-80 ng/g). The high percentage of direct drug transport to the brain (75.77%) and high drug targeting index (>1) confirmed the direct nose to brain transport of buspirone following nasal administration of Bus-chitosan formulation.
These results conclusively demonstrate increased access of buspirone to the blood and brain from intranasal solution formulated with chitosan and HP-beta-CD.
To investigate whether growing human nasal epithelium as primary cultures alters aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyldipeptidase (DPPIV) metabolic characteristics, and mRNA gene transcript expression.
The formation of 7-amino-methyl coumarin from specific substrates for APN (L-alanine-4-methyl-coumaryl-7-amide, APB (L-arginine-4-methyl-coumaryl-7-amide) and DPPIV (glycyl-L-proline-4-methyl-coumaryl-7-amide) was used to estimate the KM, Vmax and the effect of aminopeptidases inhibitors on the enzymes. Polymerase chain reaction was used to investigate gene expression.
Results of this study showed that: (1) both the excised tissues and primary cultures of human nasal epithelium expressed APN, APB and DPPIV activity; (2) the KM of APB, APN and DPPIV was not significantly different in cell and tissue homogenates; (3) except for APN, the Vmax was not significantly different in the two metabolism models; (4) there was no statistically significant difference in the behaviours of APB, APN and DPPIV in response to inhibition by puromycin and bestatin in the two models; (5) the mRNA transcripts that encode APB, APN and DPPIV were expressed in both cell culture and tissue homogenate.
Based on the results of this study, it may be concluded that nasal primary culture system is suitable for investigating peptide and protein metabolism and enzymatic stability in human nasal epithelium. Except for APN, the tissue culture conditions did not significantly alter the functional and molecular expression of the aminopeptidases.
Multidrug ABC transporters such as the human multidrug resistance P-glycoprotein (ABCB1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. These transport systems contain two nucleotide-binding domains (NBDs) where ATP is bound and hydrolyzed and two membrane domains (MDs) which mediate vectorial transport of substrates across the cell membrane. Recent crystal structures of the bacterial ABCB1 homologues Sav1866 from Staphylococcus aureus and MsbA from Salmonella typhimurium and other organisms shed light on the possible conformational states adopted by multidrug ABC transporters during transport. These structures help to interpret cellular and biochemical data gathered on these transport proteins over the past three decades. However, there are contradictory views on how the catalytic cycle of ATP binding and hydrolysis by the NBDs is linked to the change in drug binding affinity at the MDs, which underlies the capture (high affinity) of the transported drug on one side of the membrane and its release (low affinity) on the other. This review provides an overview of the current evidence for the different transport models and establishes the most recent structure-function relationships in multidrug ABC transporters.
Effects of cytochrome P-450 3A- and P-glycoprotein (P-gp)-related compounds, erythromycin, midazolam, ketoconazole, verapamil, and quinidine, on transport of rhodamine 123 (Rho-123), a P-gp substrate, were studied in rat intestine and in Caco-2 cells. Ileum was mainly used in rat studies because this segment showed greater P-gp-mediated Rho-123 transport. In an in vitro everted rat ileum, all the compounds examined significantly inhibited the transport of Rho-123 from serosal to mucosal surfaces across the intestine, with different inhibitory potencies among these compounds. In an in vivo rat study, the exsorption of Rho-123 from blood to the intestinal lumen, which was evaluated as exsorption clearance of Rho-123 under a steady-state plasma concentration of Rho-123, was also inhibited when these compounds were added to the intestinal lumen. Similarly, transepithelial transport of Rho-123 from the basolateral to apical side across Caco-2 cell monolayers was inhibited by these compounds. A linear relationship was observed in their inhibitory potencies on Rho-123 transport between in vitro and in vivo studies using rat ileum and between studies with rat ileum and Caco-2 cells. P-gp-mediated transport across the intestine was found to be inhibited not only by P-gp-related but also by all the cytochrome P-450 3A-related compounds examined. Within experimental error, the relative inhibitory potencies were the same between the studies with rat ileum (in vivo, in vitro) and those with Caco-2 cells. Thus, it is suggested that the function of P-gp and its sensitivity to these drugs may be similar in rat intestine and Caco-2 cells.
The inhibition of the Na+/K+-ATPase by cardiotonic drugs like ouabain deeply perturbs both the properties of the cell membrane and the ionic composition of the cytoplasm and hence alters fundamental cell reactions. These three types of reactions may be involved in the stimulation of multidrug resistance 1 (MDR-1) gene expression and the synthesis of permeability glycoprotein [P-glycoprotein (P-gp)]. We have determined whether ouabain, which binds to an extracellular motif of the Na+/K+-ATPase, stimulates MDR-1 gene expression by measuring both mRNA and protein and whether the resulting P-gp extrudes hydrophobic compounds and causes resistance to antimitotic agents. The experiments were performed on Calu-3 cells, a human cell line from a pulmonary carcinoma. Northern blotting showed that treating the cells with submicromolar concentrations of ouabain stimulated MDR-1 gene expression within 24 h. The ouabain-induced stimulation of MDR-1 expression was not restricted to Calu-3 cells but also occurred in human carcinomatous colon (T-84 and HT-29) and hepatic (H7V3) cells. However, it is not ubiquitous because it was not found in HeLa cells. The stimulation was reproduced by other Na+/K+-ATPase inhibitors and occurred via enhanced gene transcription, apparently due to the increased cytosolic calcium concentration. Ouabain also increased the membrane content of P-gp, as detected by immunoblotting and immunohistology. We have developed a microvideo assay based on the properties of acetoxymethyl ester calcein and calcein to show that this P-gp extruded the hydrophobic acetoxymethyl ester calcein. Ouabain also caused the Calu-3 cells to become resistant to doxorubicin and vinblastine. Thus, although ouabain acts extracellularly, it may stimulate MDR-1 gene expression and P-gp synthesis and make cells resistant to hydrophobic cytotoxic compounds.
The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells.
Transepithelial transport of flunisolide was studied in reconstituted cell monolayers of Calu-3, LLC-PK1 and the MDR1-P-glycoprotein transfected LLC-MDR1 cells.
Flunisolide transport was polarized in the apical (ap) to basolateral (bl) direction in Calu-3 cells and was demonstrated to be ATP-dependent. In LLC-MDR1 cells, flunisolide was transported in the bl to ap direction and showed no polarization in LLC-PK1 cells.
Non-specific inhibition of cellular metabolism at low temperature (4°C) or by 2-deoxy-D-glucose (2-d-glu) and sodium azide (NaN3) abolished the polarized transport. Polarized flunisolide transport was also inhibited by the specific Pgp inhibitors verapamil, SDZ PSC 833 and LY335979.
Under all experimental conditions and in the presence of all used inhibitors, no decrease in the TransEpithelial Electrical Resistance (TEER) values was detected. From all inhibitors used, only the general metabolism inhibitors 2-deoxy-D-glucose and NaN3, decreased the survival of Calu-3 cells.
Western blotting analysis and confocal laser scanning microscopy demonstrated the presence of MDR1-Pgp at mainly the basolateral side of the plasma membrane in Calu-3 cells and at the apical side in LLC-MDR1 cells. Mass spectroscopy studies demonstrated that flunisolide is transported unmetabolized across Calu-3 cells.
In conclusion, these results show that the active ap to bl transport of flunisolide across Calu-3 cells is facilitated by MDR1-Pgp located in the basolateral plasma membrane.
British Journal of Pharmacology (2001) 134, 1555–1563; doi:10.1038/sj.bjp.0704390
To study the expression of P-glycoprotein (P-gp), lung resistance-related protein (LRP), and caveolin-1 (cav-1) in the human bronchial epithelial cell line 16HBE14o-.
The presence of P-gp, LRP, and cav-1 in 16HBE14o- cell layers was evaluated using immunocytochemical staining and visualization with confocal laser scanning microscopy (CLSM). Functionality of P-gp was determined by bidirectional transport of rhodamine-123 with and without a P-gp inhibitor, verapamil. Caveolae were visualized using transmission electron microscopy (TEM). Flux of fluorescein-Na was also studied as a paracellular transport marker.
Immunocytochemical staining showed expression of P-gp localized at the apical membrane of 16HBE14o- cell layers. The flux of rhodamine 123 across cell layers exhibited a greater Papp value for the secretory (i.e., basolateral-to-apical) direction. This asymmetry disappeared in the presence of verapamil. CLSM provided evidence for the expression of LRP and cav-1. TEM further showed typically shaped caveolae at the apical and basolateral membranes.
Cell layers of 16HBE14o- express drug transport systems that are also present in the human bronchus in vivo, indicating that the 16HBE14o- cell line may be a suitable candidate for an in vitro model for mechanistic studies of drug transport processes involved in the smaller airways.
Previous literature has suggested the absence of an effective barrier between the nasal mucosa and the brain for compounds administered via the nasal route. These experiments were conducted to elucidate the role of the blood-brain barrier efflux transporter P-glycoprotein (P-gp) in attenuating delivery of P-gp substrates to the brain after nasal administration in mice.
Brain uptake of several radiolabeled P-gp substrates, was measured in P-gp-deficient and P-gp-competent mice following nasal instillation. Additional experiments were performed to assess the potential for enhancing brain uptake by inhibiting P-gp with intranasal rifampin.
All substrates examined were measurable in brain tissue within 2 min. Substrate accumulation in P-gp-deficient mice was higher than in P-gp-competent animals; the degree to which P-gp attenuated brain uptake after nasal administration was similar to that during in situ brain perfusion. Co-administration of rifampin enhanced brain uptake of relevant substrates, and resulted in complete elimination of P-gp-mediated transport for 3H- verapamil.
P-gp attenuates brain accumulation of intranasally-administered P-gp substrates. Thus, biochemical components of the blood-brain barrier, such as efflux transporters may influence brain penetration after nasal administration. Co-administration of a P-gp inhibitor enhances the brain uptake of relevant substrates, suggesting that the transporter barrier functions may be reversible.
The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells.
Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity).
R123 secretory P(app) (P(app,BA)) showed concentration dependence, whereas R123 absorptive P(app) (P(app,AB)) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P(app,AB), but enhanced R123 and doxorubicin P(app,BA). In calcium-free medium, R123 P(app,AB) increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable.
R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.
To evaluate the feasibility of using a serially passaged culture of human nasal epithelial cell monolayers on a permeable support for in vitro drug transport studies. The optimum conditions for passaged culture as well as the correlation between the transepithelial electrical resistance (TEER) value and drug permeability (Papp) were evaluated.
Fresh human nasal epithelial cells were collected from normal inferior turbinates and were subcultured repeatedly in serum-free bronchial epithelial cell growth media (BEGM) in petri dishes. The subcultured cells of each passage were seeded onto permeable supports at 5 x 10(5) cells/cm2 and grown in Dulbecco's modified Eagle medium (DMEM). Morphologic characteristics were observed by scanning electron microscopy (SEM). To verify the formation of tight junctions, actin staining and transmission electron microscopy (TEM) studies were conducted. In the drug transport study, [14C]mannitol and budesonide were selected as the paracellular and the transcellular route markers, respectively.
Serially passaged cells were successfully cultured on a permeable support and showed significantly high TEER values up to passage 4. After 14 days of seeding, SEM showed microvilli, and protrusions of cilia and mucin granules were detected by TEM. The paracellular marker [14C]mannitol showed a nearly constant permeability coefficient (Papp) when the TEER value exceeded 500 omega x cm2 regardless of the passage number. However, as expected, budesonide showed a higher permeability coefficient compared to [14C]mannitol and was less affected by the TEER value.
Human nasal epithelial cell monolayers were successfully subcultured on a permeable support up to passage 4. These cell culture methods may be useful in high-throughput screening of in vitro nasal transport studies of various drugs.
Experiments were performed to assess the brain distribution of [3H]-verapamil, including the influence of delivery route of inhibitor and substrate (nasal vs. systemic) on brain distribution. The anatomic location of P-glycoprotein (P-gp) at the nose-brain barrier also was investigated.
Separate groups of mice were pretreated with rifampin or vehicle nasally or intravenously. [3H]-verapamil was administered either nasally or via in situ brain perfusion, and dose-response profiles were constructed for P-gp inhibition. Localization of P-gp in freshly obtained brain slices and olfactory tissue was evaluated by confocal microscopy.
Rifampin inhibited the P-gp-mediated efflux of [3H]-verapamil, regardless of delivery route (Imax = 62 +/- 6%). The ED50 for enhancement of [3H]-verapamil uptake by nasal rifampin was approximately 400-fold lower than for intravenous rifampin (0.16 vs. 65 mg/kg, respectively). Microscopy showed that P-gp was located in endothelial cells that line the olfactory bulb and within the olfactory epithelium.
Nasal delivery of rifampin enhanced brain uptake of [3H]-verapamil. The magnitude of transport inhibition was dependent on the dose and route of the inhibitor, the time after administration of the inhibitor, and the specific brain region examined. P-gp is localized to both the olfactory epithelium and the endothelial cells that surround the olfactory bulb.
The purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasal epithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell. Characterization of this cell culture model was achieved by bioelectric measurements and morphological studies. The passages 2--4 of cell monolayers exhibited the TEER value of 1731+/-635 Omega cm(2) after 2 days of seeding and maintained high TEER value for 4--6 days. Morphological study by TEM and SEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugs, albuterol hemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model compounds for transport studies. All the drugs were assayed using reversed-phase HPLC under isocratic conditions. Results indicated that within the logP (apparent 1-octanol/water partition coefficient) range from --1.58 (albuterol) to 3.21 (budesonide), there existed 100-fold difference in the apparent permeability coefficients (P(app)). A log-linear relationship was shown between the drug logP and the P(app) across passaged human nasal epithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was concentration-dependent in the direction of apical to basolateral. The direction dependent transport studies were investigated among all these drugs and no significant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture model may be a useful in vitro model for studying the passive transport processes in nasal drug delivery.
Nasal drug delivery has now been recognized as a very promising route for delivery of therapeutic compounds including biopharmaceuticals. It has been demonstrated that low absorption of drugs can be countered by using absorption enhancers or increasing the drug residence time in the nasal cavity, and that some mucoadhesive polymers can serve both functions. This article reviews the background of nasal mucoadhesive drug delivery with special references to the biological and pharmaceutical considerations for nasal mucoadhesive drug administration. Applications of nasal mucoadhesives for the delivery of small organic molecules, antibiotics, proteins, vaccines and DNA are also discussed. Furthermore, new classes of functionalized mucoadhesive polymers, the characterization and safety aspects of nasal drug products as well as the opportunities presented by nasal drug delivery are extensively discussed.
The objective of this study was to establish a drug transport study using human nasal epithelial (HNE) cell monolayers cultured by the air-liquid interface (ALI) method using serum-free medium (BEGM:DME/F12, 50:50). The cells were developed and characterized in comparison to those that have been previously cultured by the liquid-covered culture (LCC) method. The epithelial cell monolayer cultured by the ALI method resulted in a significantly higher transepithelial electrical resistance value (3,453 +/- 302 ohm x cm(2)) that was maintained (>1,000 ohm x cm(2)) for up to 20 days compared with that cultured by the LCC method. Observation by scanning electron microscopy revealed mature cilia after 2 weeks in the ALI culture, while flatten unhealthy ciliated cells were observed in the LCC method. After 21 days, higher level of MUC5AC and 8 mRNA were expressed in ALI culture which confirmed the secretory differentiation of HNE monolayers in vitro. No significant difference in the permeability coefficients of a model hydrophilic marker ((14)C-mannitol) and a lipophilic drug (budesonide) was observed between the two conditions on day 7. The passage 2-3 of the HNE monolayer using ALI condition retained the morphology and differentiated features of normal epithelium. Thus it would be a suitable model for in vitro nasal drug delivery studies.
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.
Serially passaged normal human bronchial epithelial (NHBE) cell monolayers were established on Transwell inserts via an air-liquid interface (ALI) culture method. NHBE cells were seeded on polyester Transwell inserts, followed by an ALI culture from day 3, which resulted in peak TEER value of 766+/-154 Omegaxcm2 on the 8th day. Morphological characteristics were observed by light microscopy and SEM, while the formation of tight junctions was visualized by actin staining, and confirmed successful formation of a tight monolayer. The transepithelial permeability (Papp) of model drugs significantly increased with the increase of lipophilicity and showed a good linear relationship, which indicated that lipophilicity is an important factor in determining the Papp value. The expression of P-gp transporter in NHBE cell monolayers was confirmed by the significantly higher basolateral to apical permeability of rhodamine123 than that of reverse direction and RT-PCR of MDR1 mRNA. However, the symmetric transport of fexofenadine.HCl in this NHBE cell monolayers study seems to be due to the low expression of P-gp transporter and/or to its saturation with high concentration of fexofenadine.HCl. Thus, the development of tight junction and the expression of P-gp in the NHBE cell monolayers in this study imply that they could be a suitable in vitro model for evaluation of systemic drug absorption via airway delivery, and that they reflect in vivo condition better than P-gp over-expressed cell line models.
The distribution of the P-glycoprotein (P-gp/MDR1) efflux transporter at mucosal barriers has defined it as a functionally important element in limiting drug absorption into the systemic circulation. However, little is known about the distribution and functionality of P-gp/MDR1 in the human lung. Here, the presence of P-gp/MDR1 was investigated immunohistochemically in distal human lung tissue and at mRNA and protein levels in human alveolar epithelial cells (hAEpC) in primary culture. We studied the presence and activity of P-gp/MDR1 in hAEpC monolayers by Western blotting, by immunofluorescence microscopy and by conducting bi-directional transport studies employing a P-gp substrate (rhodamine 123) with and without a P-gp inhibitor (verapamil). The flux of fluorescein sodium was also examined as a paracellular transport marker. Alveolar tissue specimens showed P-gp localised at the luminal membranes of type I pneumocytes. Reverse transcription-polymerase chain reaction revealed the presence of mRNA encoding for P-gp/MDR1 in freshly isolated (i.e. type II) hAEpC and in monolayers of hAEpC cultured for 8 days (i.e. type I-like morphology). At the protein level, P-gp could be detected in hAEpC monolayers after 8 days in culture but not in freshly isolated type II pneumocytes. The flux of rhodamine 123 across hAEpC monolayers on day 8 in culture exhibited net secretion, which disappeared in the presence of verapamil. Fluorescein sodium fluxes showed no distinct directionality. Our findings indicate that P-gp is functionally active in the human alveolar airspace and that hAEpC monolayers might provide a suitable in vitro model for studying P-gp function mechanistically in the distal human lung.
Interest in intranasal (IN) administration as a non-invasive route for drug delivery continues to grow rapidly. The nasal mucosa offers numerous benefits as a target issue for drug delivery, such as a large surface area for delivery, rapid drug onset, potential for central nervous system delivery, and no first-pass metabolism. A wide variety of therapeutic compounds can be delivered IN, including relatively large molecules such as peptides and proteins, particularly in the presence of permeation enhancers. The current review provides an in-depth discussion of therapeutic aspects of IN delivery including consideration of the intended indication, regimen, and patient population, as well as physicochemical properties of the drug itself. Case examples are provided to illustrate the utility of IN dosing. It is anticipated that the present review will prove useful for formulation scientists considering IN delivery as a delivery route.
P-gp expression in HNE cell monolayers
- Hyun-Jong
- Cho
P-gp expression in HNE cell monolayers Hyun-Jong Cho et al.
Nasal absorption and local tissue reaction of insulin nanocomplexes of trimethyl chitosan derivatives in rats
- Jintapattanakit
Brain targeting studies on buspirone hydrochloride after intranasal administration of mucoadhesive formulation in rats
- Khan
P-glycoprotein efflux pump expression and activity in Calu-3 cells
- Hamilton