Search Results (29)

Background/Objectives: The oral cavity represents a convenient route of administration for drugs that exhibit significant hepatic first-pass extraction. In this study, the mucosal permeation properties of selected active pharmaceutical ingredients (APIs) incorporated into oral cavity drug products that are approved by the U.S. Food and Drug Administration were quantified using the human-derived sublingual HO-1-u-1 and buccal EpiOral™ in vitro tissue models. Methods: Epithelial barrier properties were monitored using propranolol and Lucifer Yellow as prototypic transcellular and paracellular markers. APIs were dissolved in artificial saliva, pH 6.7, and transepithelial flux from the apical to the basolateral compartment was quantified using HPLC. Results: Apparent permeability coefficients (Papp) calculated for these APIs in the sublingual HO-1-u-1 tissue model varied from Papp = 2.72 ± 0.06 × 10⁻⁵ cm/s for asenapine to Papp = 6.21 ± 2.60 × 10⁻⁵ cm/s for naloxone. In contrast, the buccal EpiOral™ tissue model demonstrated greater discrimination power in terms of permeation properties for the same APIs, with values ranging from Papp = 3.31 ± 0.83 × 10⁻⁷ cm/s for acyclovir to Papp = 2.56 ± 0.68 × 10⁻⁵ cm/s for sufentanil. The tissue-associated dose fraction recovered at the end of the transport experiment was significantly increased in the buccal EpiOral™ tissue model, reaching up to 8.5% for sufentanil. Conclusions: Experimental permeation data collected for selected APIs in FDA-approved oral cavity products will serve as a training set to aid the development of predictive computational models for improving algorithms that describe drug absorption from the oral cavity. Following a robust in vitro–in vivo correlation analysis, it is expected that such innovative in silico modeling strategies will the accelerate development of generic oral cavity products by facilitating the utility of model-integrated evidence to support decision making in generic drug development and regulatory approval. Full article

Background/Objectives: The purpose of this study was to create bile acid-containing liposomes to improve methotrexate blood-brain barrier penetration and to assess the liposome transportation mechanism across the blood–brain barrier. Methods: The improvement of liposome penetration was investigated utilizing human brain microvascular endothelial cells in an in vitro blood-brain barrier model. Using confocal laser scanning microscopy (CLSM) and flow cytometry, liposomes were labeled with fluorescent phospholipids to facilitate their passage across the blood–brain barrier. Results: The produced liposomes with bile acid exhibited a negative surface charge and an average particle size of between 30 and 148 nm. According to an in vitro blood-brain barrier penetration study, the methotrexate penetration was increased by liposomes containing 1% glycocholic acid but not by liposomes containing taurocholic acid. For transport pathway evaluation across the blood-brain barrier of these liposomes, CLSM revealed that fluorescent liposomes were present inside cells treated with specific endocytosis inhibitors, indicating that the cellular internalization of the particles was not involved in endocytosis. Conclusions: Liposomes supplemented with 1% glycocholic acid could enhance the penetration of methotrexate across the blood-brain barrier, while taurocholic acid could not. The transport of liposomes with 1% glycocholic acid across the blood-brain barrier occurs via the transcellular pathway through which it penetrates cells. In contrast, the paracellular pathway was a minor pathway. Full article

The tympanic membrane forms an impenetrable barrier between the ear canal and the air-filled middle ear, protecting it from fluid, pathogens, and foreign material entry. We previously screened a phage display library and discovered peptides that mediate transport across the intact membrane. The route by which transport occurs is not certain, but possibilities include paracellular transport through loosened intercellular junctions and transcellular transport through the cells that comprise the various tympanic membrane layers. We used confocal imaging to resolve the phage’s path through the membrane. Phages were observed in puncta within the cytoplasm of tympanic membrane cells, with no evidence of phages within junctions between epithelial cells. This result indicates that transport across the membrane is transcellular and within vesicles, consistent with the transcytosis process. The trans-tympanic peptide phages display a wide range of transport efficiencies for unknown reasons. This could include variation in tympanic membrane binding, entry into the membrane, crossing the membrane, or exiting into the middle ear. To address this, we titered phages recovered from within the membrane for phages with differing transport rates. We found that differences in the transport rate were inversely related to their presence within the tympanic membrane. This suggests that differences in the transport rate primarily reflect the efficiency of an exocytotic exit from the mucosal epithelium rather than entry into, or passage across, the membrane. Full article

It is currently impossible to perform an evidence-based risk assessment for medication use during breastfeeding. The ConcePTION project aims to provide information about the use of medicines during lactation. The study aimed to develop and characterize an in vitro model of the blood–milk barrier to determine the extent of the milk transfer of xenobiotics, relying on either on human mammary epithelial cells (hMECs) or immortalized cell lines derived from breast tissue. The hMECs were cultured and characterized for epithelial markers; further, the ability to form an epithelial barrier was investigated. Drug transporter functionality in the cultured hMECs was analyzed with specific probe substrates. The hMECs showed an epithelial morphology and the expression of epithelial markers and tight junctions. They formed a reproducible tight barrier with a transepithelial electrical resistance greater than 400 Ωcm², unlike immortalized cell lines. Different levels of mRNA expression were detected for 81 genes of membrane transporters. Functional assays showed no evidence for the transporter-mediated secretion of medicines across the hMECs. Nevertheless, the hMEC-based in vitro model covered a 50-fold range of permeability values, differentiating between passive transcellular and paracellular-mediated transport. The cultured hMECs proved to be a promising in vitro model for biorelevance; the wide characterization of hMECs makes them useful for studying medicine partitioning in milk. Full article

Neuromyelitis optica spectrum disorder (NMOSD) is associated with pathological aquaporin-4 immunoglobulin G (AQP4-IgG), which cause brain damage. However, the impact of AQP4-IgG on retinal tissue remains unclear. Additionally, dysregulated complement anaphylatoxins C3a and C5a, known to modulate the endothelial barrier, are implicated in NMOSD. This study evaluates the susceptibility of human brain microvascular endothelial cells (HBMEC) and human retinal endothelial cells (HREC) to C3a- and C5a-mediated stress using real-time cell barrier analysis, immunocytochemical staining, qPCR and IgG transmigration assays. The findings reveal that C3a induced a concentration-dependent paracellular barrier breakdown and increased transcellular permeability in HBMEC, while HREC maintained barrier integrity under the same conditions. C5a attenuated C3a-induced disruption in HBMEC, indicating a protective role. Anaphylatoxin treatment elevated transcript levels of complement component C3 and increased C5 gene and protein expression in HREC, with no changes observed in HBMEC. In HBMEC, C5a treatment led to a transient upregulation of C3a receptor (C3AR) mRNA and an early decrease in C5a receptor 1 (C5AR1) protein detection. Conversely, HREC exhibited a late increase in C5aR1 protein levels. These results indicate that the retinal endothelial barrier is more stable under anaphylatoxin-induced stress compared to the brain, potentially offering better protection against paracellular AQP4-IgG transport. Full article

Brain endothelial cells (ECs) are essential elements of the blood–brain barrier (BBB), maintaining its integrity through both paracellular junctions and transcellular transport systems. Myosin IIA, a multifunctional protein, plays a significant role in various cellular processes, including cytoskeletal maintenance, cell division, and signal transduction. While Myosin IIA has been implicated in bleeding and ischemic stroke, its role in regulating BBB integrity under physiological conditions remains unclear. In this study, we investigated the impact of Myosin IIA deficiency on BBB integrity using intravenous tracer injections and models of epilepsy. Flow cytometry, Western blot, and real-time PCR were employed to isolate brain cells and assess changes in protein and mRNA levels. Additionally, immunofluorescence staining and electron microscopy were used to explore alterations in protein expression and the structure of BBB. Our results demonstrate that endothelial Myosin IIA deficiency increased BBB permeability and exacerbated symptoms in BBB-related diseases. Mechanistically, we found that Myosin IIA modulates β-catenin transcription and protein interactions. The overexpression of β-catenin in brain endothelial Myosin IIA deficiency mice improved BBB integrity and reduced disease severity. This study establishes Myosin IIA as a critical regulator of BBB integrity and suggests new therapeutic targets for vascular diseases. Full article

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most prescribed drugs to treat pain or fever. However, oral administration of NSAIDs is frequently associated with adverse effects due to their inhibitory effect on the constitutively expressed cyclooxygenase enzyme 1 (COX-1) in, for instance, the gastrointestinal tract. A systemic delivery, such as a buccal delivery, of NSAIDs would be beneficial and additionally has the advantage of a non-invasive administration route, especially favourable for children or the elderly. To investigate the transport of NSAIDs across the buccal mucosa and determine their potential for buccal therapeutic usage, celecoxib, diclofenac, ibuprofen and piroxicam were tested using an established oral mucosa Transwell^® model based on human cell line TR146. Carboxyfluorescein and diazepam were applied as internal paracellular and transcellular marker molecule, respectively. Calculated permeability coefficients revealed a transport ranking of ibuprofen > piroxicam > diclofenac > celecoxib. Transporter protein inhibitor verapamil increased the permeability for ibuprofen, piroxicam and celecoxib, whereas probenecid increased the permeability for all tested NSAIDs. Furthermore, influence of local inflammation of the buccal mucosa on the transport of NSAIDs was mimicked by treating cells with a cytokine mixture of TNF-α, IL-1ß and IFN-γ followed by transport studies with ibuprofen (+ probenecid). Cellular response to pro-inflammatory stimuli was confirmed by upregulation of cytokine targets at the mRNA level, increased secreted cytokine levels and a significant decrease in the paracellular barrier. Permeability of ibuprofen was increased across cell layers treated with cytokines, while addition of probenecid increased permeability of ibuprofen in controls, but not across cell layers treated with cytokines. In summary, the suitability of the in vitro oral mucosa model to measure NSAID transport rankings was demonstrated, and the involvement of transporter proteins was confirmed; an inflammation model was established, and increased NSAID transport upon inflammation was measured. Full article

Deciphering the dynamics of water transport across bronchial epithelial cell monolayers is pivotal for unraveling respiratory physiology and pathology. In this study, we employ an advanced microfluidic system to explore bidirectional water transport across 16HBE14σ bronchial epithelial cells. Previous experiments unveiled electroneutral multiple ion transport, with chloride ions utilizing transcellular pathways and sodium ions navigating both paracellular and transcellular routes. Unexpectedly, under isoosmotic conditions, rapid bidirectional movement of Na⁺ and Cl⁻ was observed, leading to the hypothesis of a substantial transport of isoosmotic solution (145 mM NaCl) across cell monolayers. To validate this conjecture, we introduce an innovative microfluidic device, offering a 500-fold sensitivity improvement in quantifying fluid flow. This system enables the direct measurement of minuscule fluid volumes traversing cell monolayers with unprecedented precision. Our results challenge conventional models, indicating a self-regulating mechanism governing water transport that involves the CFTR channel and anion exchangers. In healthy subjects, equilibrium is achieved at an apical potential of Δφ_ap = −30 mV, while subjects with cystic fibrosis exhibit modulation by an anion exchanger, reaching equilibrium at [Cl] = 67 mM in the airway surface liquid. This nuanced electrochemical basis for bidirectional water transport in bronchial epithelia sheds light on physiological intricacies and introduces a novel perspective for understanding respiratory conditions. Full article

The blood-brain barrier (BBB) is a dynamic barrier separating neurocytes and brain tissues from blood that is extremely sealed and strictly regulated by transporters such as aquaporin-4 (AQP-4), glucose transporter (GLUT), and specialized tight junctional complexes (TJCs) including tight junctions (TJs), adherens junctions (AJs), and Zonulae occludens (ZOs). With specifically selective transcellular and paracellular permeability, the BBB maintains a homeostatic microenvironment to protect the central nervous system (CNS). In recent years, increasing attention has been paied to the importance of BBB disruption and dysfunction in the pathology of neurological disorders and diseases, such as Alzheimer’s diseases (AD), Parkinson diseases (PD), stroke and cerebral edema. However, the further research on how the integral structure and function of BBB are altered under the physiological or pathological conditions is still needed. Focusing on the ultrastructural features of the BBB and combining the latest research on associated proteins and transporters, physiological regulation and pathological change of the BBB were elucidated. By summarizing the protective effects of known bioactive compounds derived from marine life on the BBB, this review aims to highlight the BBB as a key to the treatment of several major neurological diseases instead of a normally described obstacle to drug absorption and transport. Overall, the BBB’s morphological characteristics and physiological function and their regulation provide the theoretical basis for the study on the BBB and inspire the diagnosis of and therapy for neurological diseases. Full article

The transport of low-density lipoprotein (LDL) through the endothelium is a key step in the development of atherosclerosis, but it is notorious that phenotypic differences exist between endothelial cells originating from different vascular beds. Endothelial cells forming the blood–brain barrier restrict paracellular and transcellular passage of plasma proteins. Here, we systematically compared brain versus aortic endothelial cells towards their interaction with LDL and the role of proteins known to regulate the uptake of LDL by endothelial cells. Both brain endothelial cells and aortic endothelial cells bind and internalize LDL. However, whereas aortic endothelial cells degrade very small amounts of LDL and transcytose the majority, brain endothelial cells degrade but do not transport LDL. Using RNA interference (siRNA), we found that the LDLR–clathrin pathway leads to LDL degradation in either endothelial cell type. Both loss- and gain-of-function experiments showed that ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which promote LDL uptake and transport into aortic endothelial cells, limit neither binding nor association of LDL to brain endothelial cells. Together, these results indicate distinct LDL trafficking by brain microvascular endothelial cells and aortic endothelial cells. Full article

The blood-brain barrier (BBB) is a selective barrier and a functional gatekeeper for the central nervous system (CNS), essential for maintaining brain homeostasis. The BBB is composed of specialized brain endothelial cells (BECs) lining the brain capillaries. The tight junctions formed by BECs regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps and receptors. Cytokine-induced neuroinflammation, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), appear to play a role in BBB dysfunction and contribute to the progression of Alzheimer’s disease (AD) by contributing to amyloid-β (Aβ) peptide accumulation. Here, we investigated whether TNF-α and IL-1β modulate the permeability of the BBB and alter Aβ peptide transport across BECs. We used a human BBB in vitro model based on the use of brain-like endothelial cells (BLECs) obtained from endothelial cells derived from CD34+ stem cells cocultivated with brain pericytes. We demonstrated that TNF-α and IL-1β differentially induced changes in BLECs’ permeability by inducing alterations in the organization of junctional complexes as well as in transcelluar trafficking. Further, TNF-α and IL-1β act directly on BLECs by decreasing LRP1 and BCRP protein expression as well as the specific efflux of Aβ peptide. These results provide mechanisms by which CNS inflammation might modulate BBB permeability and promote Aβ peptide accumulation. A future therapeutic intervention targeting vascular inflammation at the BBB may have the therapeutic potential to slow down the progression of AD. Full article

Background: Chemotherapy treatment of cancer in children can influence formation of normal tissues, leading to irreversible changes in their structure and function. Tooth formation is susceptible to several types of chemotherapy that induce irreversible changes in the structure of enamel, dentin and dental root morphology. These changes can make the teeth more prone to fracture or to caries when they have erupted. Recent studies report successful treatment of brain tumors with the alkylating drug temozolomide (TMZ) in combination with veliparib (VLP) in a glioblastoma in vivo mouse model. Whether these drugs also affect tooth formation is unknown. Aim: In this study the effect of TMZ/VLP on incisor formation was investigated in tissue sections of jaws from mice and compared with mice not treated with these drugs. Materials and method: The following aspects were studied using immunohistochemistry of specific protein markers including: (1) proliferation (by protein expression of proliferation marker Ki67) (2) a protein involved in paracellular ion transport (expression of tight junction (TJ) protein claudin-1) and (3) in transcellular passage of ions across the dental epithelium (expression of Na+, K+ 2Cl- cotransporter/NKCC1). Results: Chemotherapy with TMZ/VLP strongly reduced immunostaining for claudin-1 in distal parts of maturation ameloblasts. No gross changes were found in the treated mice, either in cell proliferation in the dental epithelium at the cervical loop or in the immunostaining pattern for NKCC1 in (non-ameloblastic) dental epithelium. The salivary glands in the treated mice contained strongly reduced immunostaining for NKCC1 in the basolateral membranes of acinar cells. Discussion/Conclusions: Based on the reduction of claudin-1 immunostaining in ameloblasts, TMZ/VLP may potentially influence forming enamel by changes in the structure of TJs structures in maturation ameloblasts, structures that are crucial for the selective passage of ions through the intercellular space between neighboring ameloblasts. The strongly reduced basolateral NKCC1 staining seen in fully-grown salivary glands of TMZ/VLP-treated mice suggests that TMZ/VLF could also influence ion transport in adult saliva by the salivary gland epithelium. This may cause treated children to be more susceptible to caries. Full article

The purpose of this study was to evaluate mechanisms behind the intestinal permeability of minoxidil, with special emphasis on paracellular transport, and elucidate the suitability of minoxidil to be a reference drug for Biopharmaceutics Classification System (BCS). The permeability of minoxidil (vs. metoprolol) was evaluated in-silico, in-vitro using both the PAMPA assay and across Caco-2 cell monolayers, as well as in-vivo in rats throughout the entire intestine. The permeability was studied in conditions that represent the different segments of the small intestine: upper jejunum (pH 6.5), mid small intestine (pH 7.0), distal ileum (pH 7.5), and colon (pH 6.5). Since we aimed to investigate the paracellular transport of minoxidil, we have also examined its permeability in the presence of quercetin (250 µM), which closes the tight junctions, and sodium decanoate (10 mM), which opens the tight junctions. While metoprolol demonstrated segmental-dependent rat and PAMPA permeability, with higher permeability in higher pH regions, the permeability of minoxidil was pH-independent. Minoxidil PAMPA permeability was significantly lower than its rat permeability, indicating a potential significant role of the paracellular route. In rat intestinal perfusion studies, and across Caco-2 monolayers, tight junction modifiers significantly affected minoxidil permeability; while the presence of quercetin caused decreased permeability, the presence of sodium decanoate caused an increase in minoxidil permeability. In accordance with these in-vitro and in-vivo results, in-silico simulations indicated that approximatelly 15% of minoxidil dose is absorbed paracellularly, mainly in the proximal parts of the intestine. The results of this study indicate that paracellular transport plays a significant role in the intestinal permeability of minoxidil following oral administration. Since this permeation route may lead to higher variability in comparison to transcellular, these findings diminish the suitability of minoxidil to serve as the low/high BSC permeability class benchmark. Full article

Various types of artificial biomimetic barriers are widely utilized as in vitro tools to predict the passive “transcellular” transport of drug compounds. The current study investigated if the sandwich barrier PermeaPad^®, which is composed of tightly packed phospholipid vesicles enclosed between two support sheets, contributes to a transport mechanism that is paracellular-like, representing one of the alternative pathways of passive transport in vivo, primarily of relevance for hydrophilic drug compounds. To this end, we pretreated the commercial PermeaPad^® barrier with NaCl solutions of either high or low osmolality prior to permeation experiments on reversed Franz cell setups with hydrophilic model compounds calcein and acyclovir and hydrophobic model compounds hydrocortisone and celecoxib. Our starting hypothesis was that the liposomes formed in the barrier during the hydration step should either shrink or swell upon contact with test media (drug solutions) due to osmotic pressure difference as compared to the pretreatment solutions. Apparent permeabilities for calcein and acyclovir across the PermeaPad^® barrier were found to increase approximately 2.0 and 1.7 fold, respectively, upon hypo-osmotic pretreatment (soaking in hypotonic medium, while the permeation of hydrocortisone and celecoxib remained unchanged. A control experiment with lipid-free barriers (support sheets) indicated that the permeation of all the compounds was virtually unchanged upon hypo-osmotic pretreatment. In conclusion, soaking PermeaPad^® in a medium of lower osmotic pressure than that used during the permeation study appears to induce the osmotic shrinking of the lipid vesicles in the barrier, leaving wider water channels between the vesicles and, thus, allowing hydrophilic compounds to pass the barrier in a paracellular-like manner. Full article

We explored whether metabolic health is linked to intestinal permeability, using a multi-sugar (MS) permeability test, and whether intestinal permeability is correlated with the leaky gut-related markers (LGM) zonulin, LBP, and sCD14. Metabolically healthy (n = 15) and unhealthy subjects (n = 15) were recruited based on waist circumference, fasting glucose, and high-density lipoprotein cholesterol levels. Participants underwent an MS permeability test that assessed site-specific permeabilities of the gastroduodenum and small and large intestines. The test was performed with/without an acetylsalicylic acid challenge to measure and correlate the gut permeability, LGM, and metabolic health. At baseline, metabolic health showed no correlation with gut permeability. Significant correlations were found between the metabolic health parameters and LGM. In the acetylsalicylic acid challenged MS permeability test, low-density lipoprotein cholesterol was correlated with the sucralose/erythritol ratio, reflecting the whole intestinal permeability. Correlations between most metabolic health parameters and LGM during the acetylsalicylic acid challenge were less pronounced than at baseline. In both MS permeability tests, no significant correlations were found between LGM (plasma and serum) and gut permeability. Thus, correlations between LGM and metabolic health might not be linked with paracellular gut permeability. Transcellular translocation and/or lipoprotein-related transportation is a more likely mechanism underlying the association between LGM and metabolic health. Full article