Search Results (32)

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

Objectives: Ex vivo nasal mucosa models provide physiologically relevant platforms for evaluating nasal drug permeability; however, their application is often limited by high experimental variability and the absence of standardized methodologies. This study aimed to improve experimental design by addressing two major limitations: the confounding effects of mucosal thickness and the questionable reliability of fluorescein sodium (Flu-Na) as an integrity marker for chemically induced mucosal injury. Methods: Permeability experiments were conducted using porcine nasal tissues mounted in Franz diffusion cells, with melatonin and Flu-Na as model compounds. Tissues of varying thickness were collected from both intra- and inter-individual sources, and a numerical simulation-based method was employed to normalize apparent permeability coefficients (Papp) to a standardized mucosal thickness of 0.80 mm. The effects of thickness normalization and chemically induced damage were systematically evaluated. Results: Thickness normalization substantially reduced variability in melatonin Papp, particularly within same-animal comparisons, thereby improving statistical power and data reliability. In contrast, Flu-Na exhibited inconsistent correlations across different pigs and failed to reflect the expected increase in permeability following isopropyl alcohol (IPA)-induced epithelial damage. These results suggest that the relationship between epithelial injury and paracellular transport may be non-linear and not universally applicable under ex vivo conditions, limiting the suitability of Flu-Na as a standalone marker of mucosal integrity. Conclusions: The findings highlight the importance of integrating mucosal thickness correction into standardized experimental protocols and call for a critical reassessment of Flu-Na in nasal drug delivery research. Full article

Background/Objectives: The low permeability of the blood-brain barrier (BBB) represents a significant challenge to effective systemic chemotherapy for primary and metastatic brain cancers. Kinin receptors play a crucial role in modulating BBB permeability, and their agonist analogs have been explored in preclinical animal models to enhance drug delivery to the brain. In this study, we investigated whether des-Arg⁹-bradykinin (DBK), a physiological agonist of kinin B₁ receptor (B1R), acts as a brain drug delivery adjuvant by promoting the transient opening of the BBB. Methods: Human brain microvascular endothelial cells (HBMECs) were treated with DBK in the culture medium and in conditioned media from glioblastoma cell lines, namely T98G (CMT98G) and U87MG (CMU87). Immunofluorescence, RT-qPCR, in-cell Western assay, and proximity ligation assay (PLA) were performed to analyze BBB components, kinin receptors and TLR4, a receptor associated with the kinin pathway and inflammation. The effect of DBK on enhancing paracellular molecule transport was evaluated using Evans blue dye (EB) quantification in a cell culture insert assay and in an in vivo model, where mice with and without brain tumors were treated with DBK. To assess the functional impact of the transient BBB opening induced by DBK, the chemotherapeutic drug doxorubicin (DOX) was administered. Results: Treatment with DBK facilitates the presence of EB in the brain parenchyma by transiently disrupting the BBB, as further evidenced by the increased paracellular passage of the dye in an in vitro assay. B1R activation by DBK induces transient BBB opening lasting less than 48 h, enhancing the bioavailability of the DOX within the brain parenchyma and glioma tumor mass. The interaction between B1R and TLR4 is disrupted by the secreted factors released by glioblastoma cells, as conditioned media from T98G and U87 reduce TLR4 staining in endothelial cells without affecting B1R expression. Conclusions: These results further support the potential of B1R activation as a strategy to enhance targeted drug delivery to the brain. Full article

Background Despite many studies on polymer-incorporated nanocarriers for ophthalmic drug delivery, few have thoroughly explored the relationship between coating composition and performance. This study aimed to evaluate the effects of three commonly used cationic polymers—distearoyl phosphatidylethanolamine-polyethylene glycol 1000-poly(amidoamine) (DSPE-PEG1000-PAMAM), trimethyl chitosan (TMC), and (2,3-dioleoyloxypropyl) trimethylammonium chloride (DOTAP)—on the corneal behaviors and anti-cataract efficacy of diosmetin (DIO)-loaded micelles (D-M-P, D-M-T, and D-M-D, respectively). Methods The DIO-loaded micelles were prepared using the thin-film dispersion method and incorporated with the three polymers through hydrophobic interactions and electrostatic adsorption. Structural characterization was demonstrated by TEM imaging and particle size analyzer. In vitro release behavior was detected by the dialysis method. Cell viability of D-M-P, D-M-T, and D-M-D on L929 cells was detected by CCK-8 assays, with cellular uptake performed using coumarin 6 as the fluorescence indicator. Precorneal retention behaviors of these three vesicles were observed by In Vivo Imaging System. Transcorneal permeability was determined by modified Franz diffusion method and the permeation routes of the vesicles are investigated. Selenite-induced cataract model was established. The anti-cataract effects of three different DIO-loaded micelles were evaluated by the observation of lens opacity and antioxidant enzyme activities. Eye Irritation of the DIO in different preparations was estimated using the Draize test, along with H&E staining of the corneas. Results Structural characterization of DIO-loaded micelles revealed that the vesicles were spherical, with a uniform size distribution of around 28 nm, a similar surface potential of approximately 6.0 mV, and a high DIO entrapment efficiency of about 95%. Compared to the DIO suspension, all three formulations exhibited a significant sustained-release effect. They showed no signs of irritation and demonstrated increased IC50 values in L929 cells, indicating improved biocompatibility. Cellular uptake in human lens epithelial cells (HLECs) was assessed using confocal laser scanning microscopy. C-M-T displayed the highest fluorescence signals, with a cellular internalization 3.2 times greater than that of the solution group. Both C-M-T and C-M-P enhanced vesicle retention on the corneal surface by at least 47.8% compared to the Cou-6 solution. Furthermore, TMC facilitated the paracellular transport of vesicles into the deepest layers of the cornea and delivered DIO across the cornea, with a P_app value 3.11 times and 1.49 times those of D-M-D and D-M-P, respectively. In terms of therapeutic efficacy, D-M-T demonstrated the most significant attenuation of lens opacity, along with enhanced antioxidant enzyme activities and inhibition of lipid peroxidation. Conclusion The modification of micelle vesicles with different cationic polymers significantly influences their performance in ocular drug delivery. Among the tested formulations, D-M-T stands out due to its multiple advantages, including enhanced transcorneal drug delivery, therapeutic efficacy for DIO, and safety, making it the most promising candidate for ophthalmic applications. Full article

ATP-binding cassette (ABC) transporters, particularly those of subfamily B, are involved in cell detoxification, multidrug resistance, drug treatment pharmacodynamics, and also ecological adaptation. In this regard, ABCB transporters may play a decisive role in the co-evolution between plants and herbivores. Cardenolides, toxic steroid glycosides, are secondary plant metabolites that defend plants against herbivores by targeting their sodium–potassium ATPase. Despite their toxicity, several herbivorous insects such as the large milkweed bug (Oncopeltus fasciatus) have evolved adaptations to tolerate cardenolides and sequester them for their own defense. We investigate the role of two ABCB transporters of O. fasciatus for the paracellular transport of cardenolides by docking simulations and ATPase assays. Cardenolide binding of OfABCB1 and OfABCB2 is predicted by docking simulations and calculated binding energies are compared with substrate specificities determined in ATPase assays. Both tested ABCB transporters showed activity upon exposure to cardenolides and Km values that agreed well with the predictions of our docking simulations. We conclude that docking simulations can help identify transporter binding regions and predict substrate specificity, as well as provide deeper insights into the structural basis of ABC transporter function. 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

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

The biological barriers existing in the human body separate the blood circulation from the interstitial fluid in tissues. The blood–brain barrier (BBB) isolates the central nervous system from the bloodstream, presenting a dual role: the protection of the human brain against potentially toxic/harmful substances coming from the blood, while providing nutrients to the brain and removing metabolites. In terms of architectural features, the presence of junctional proteins (that restrict the paracellular transport) and the existence of efflux transporters at the BBB are the two major in vivo characteristics that increase the difficulty in creating an ideal in vitro model for drug permeability studies and neurotoxicity assessments. The purpose of this work is to provide an up-to-date literature review on the current in vitro models used for BBB studies, focusing on the characteristics, advantages, and disadvantages of both primary cultures and immortalized cell lines. An accurate analysis of the more recent and emerging techniques implemented to optimize the in vitro models is also provided, based on the need of recreating as closely as possible the BBB microenvironment. In fact, the acceptance that the BBB phenotype is much more than endothelial cells in a monolayer has led to the shift from single-cell to multicellular models. Thus, in vitro co-culture models have narrowed the gap between recreating as faithfully as possible the human BBB phenotype. This is relevant for permeability and neurotoxicity assays, and for studies related to neurodegenerative diseases. Several studies with these purposes will be also presented and discussed. Full article

Acromegaly is a condition most commonly diagnosed in the fifth decade of life and has numerous treatment options. In this regard, Mycapssa^® is the first FDA-approved oral octreotide capsule for treating acromegaly, combining the efficacy of the somatostatin receptor ligand, octreotide, with the ease of a twice-daily oral capsule. Where surgical treatment is not an option, somatostatin analogs, including octreotide, are the first line of medical treatment for acromegaly, requiring regular subcutaneous or intramuscular injections administered by a patient’s healthcare provider. Octreotide capsules (Mycapssa^®) provide an alternative to these somatostatin receptor ligand injections by combining octreotide with other excipients to produce a transient permeability enhancer technology that improves paracellular transport of octreotide across the gastrointestinal wall into the small intestine. Across multiple trials, including open-label (CH-ACM-01), double-blind placebo-controlled (CHIASMA OPTIMAL), and open-label extension of the trial period (CHIASMA OPTIMAL OLE), Mycapssa^® octreotide capsules maintained a consistent biochemical normalization of IGF-1 and GH levels, safety profiles similar to injected somatostatin receptor ligands, and patient preference to continued treatment with octreotide capsules. While clinical trial data supports the use of octreotide capsules (Mycapssa^®) in the pharmacological management of GH and IGF-1 levels, very little data exist regarding the drug’s efficacy, tolerability, and use in female or pediatric-specific populations. A better understanding of the efficacy, application, and role of oral octreotide capsules in the long-term medical management of acromegaly in a diversity of populations is imperative to best determine the risks/benefits for the clinician. 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

Some pharmaceutical excipients are able to modify intestinal permeability, thus influencing drug absorption and bioavailability. The effect of four polyols (mannitol, maltitol, sorbitol and xylitol) on the permeability of seven active pharmaceutical ingredients (API), representing different BCS classes (furosemide, amiloride, atenolol, ranitidine, nadolol, L-thyroxine and acyclovir), was investigated using the Caco-2 cell permeability model. Analytical methods for the sensitive polyol and API quantification were developed using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ). Apparent permeability coefficients (P_app) were calculated from the measured concentrations in the apical and basolateral compartments. The cell monolayer remained intact throughout the experiment in all trials, neither significant Lucifer Yellow (LY) passage, nor modification of the electrical resistance was detected, demonstrating that no active principle or excipient (or combinations thereof) modulated the paracellular transport. The P_app values for apical to basolateral and basolateral to apical directions of drug + excipient combinations were compared with the Papp values for the drug substance alone. Our results show that mannitol, maltitol, sorbitol and xylitol did not modify the permeability of furosemide, amiloride, atenolol, ranitidine, nadolol, acyclovir and L-thyroxine APIs. Moreover, the presence of polyols did not alter the efflux of the active principle (basolateral to apical). Full article

Modulation of drug transporter activity at mucosal sites of HIV-1 transmission may be exploited to optimize retention of therapeutic antiretroviral drug concentrations at target submucosal CD4+ T cells. Previously, we showed that darunavir was a substrate for the P-glycoprotein efflux drug transporter in colorectal mucosa. Equivalent studies in the cervicovaginal epithelium have not been reported. Here, we describe the development of a physiologically relevant model to investigate the permeability of antiretroviral drugs across the vaginal epithelium. Barrier properties of the HEC-1A human endometrial epithelial cell line were determined, in a dual chamber model, by measurement of transepithelial electrical resistance, immunofluorescent staining of tight junctions and bi-directional paracellular permeability of mannitol. We then applied this model to investigate the permeability of tenofovir, darunavir and dapivirine. Efflux ratios indicated that the permeability of each drug was transporter-independent in this model. Reduction of pH to physiological levels in the apical compartment increased absorptive transfer of darunavir, an effect that was reversed by inhibition of MRP efflux transport via MK571. Thus, low pH may increase the transfer of darunavir across the epithelial barrier via increased MRP transporter activity. In a previous in vivo study in the macaque model, we demonstrated increased MRP2 expression following intravaginal stimulation with darunavir which may further increase drug uptake. Stimulation with inflammatory modulators had no effect on drug permeability across HEC-1A barrier epithelium but, in the VK2/E6E7 vaginal cell line, increased expression of both efflux and uptake drug transporters which may influence darunavir disposition. Full article

A hybrid blood–brain barrier (BBB)-on-chip cell culture device is proposed in this study by integrating microcontact printing and perfusion co-culture to facilitate the study of BBB function under high biological fidelity. This is achieved by crosslinking brain extracellular matrix (ECM) proteins to the transwell membrane at the luminal surface and adapting inlet–outlet perfusion on the porous transwell wall. While investigating the anatomical hallmarks of the BBB, tight junction proteins revealed tortuous zonula occludens (ZO-1), and claudin expressions with increased interdigitation in the presence of astrocytes were recorded. Enhanced adherent junctions were also observed. This junctional phenotype reflects in-vivo-like features related to the jamming of cell borders to prevent paracellular transport. Biochemical regulation of BBB function by astrocytes was noted by the transient intracellular calcium effluxes induced into endothelial cells. Geometry-force control of astrocyte–endothelial cell interactions was studied utilizing traction force microscopy (TFM) with fluorescent beads incorporated into a micropatterned polyacrylamide gel (PAG). We observed the directionality and enhanced magnitude in the traction forces in the presence of astrocytes. In the future, we envisage studying transendothelial electrical resistance (TEER) and the effect of chemomechanical stimulations on drug/ligand permeability and transport. The BBB-on-chip model presented in this proposal should serve as an in vitro surrogate to recapitulate the complexities of the native BBB cellular milieus. 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