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Measurements of the levels of mucus glycoproteins (a) and acidic mucus glycoproteins (b) liberated from the intestinal epithelium. Key: (□) control (untreated); (■) DTT at 10 mM (pretreated for 10 min). Results are presented as the mean ± S.D. (n = 3–9) from three independent experiments. *P < 0.05, **P < 0.01 compared with the control condition.
Source publication
Purpose
Mucins are the principal glycoproteins in mucus and have been implicated in the limitation of intestinal drug absorption; however, the contribution of these molecules to intestinal drug absorption remains unclear. In this study, the relationship between the effect of the mucus layer on intestinal drug permeation and mucin distribution in di...
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Griseofulvin is an antifungal antibiotic applying for the treatment of dermatophyte infections. The most common delivery route of this drug is oral. The dosage of griseofulvin varies depending on whether the drug is administered as a microsized or an ultramicrosized preparation. In order to avoid possible incidental systemic toxicity associated wit...
Citations
... These studies have, however, used different methods of quantification and treatment of the mucus samples, making a comparison to the current study difficult. In a recent study on Wistar rats, the expression of mucin subtype Muc5ac was shown to drastically decrease from the duodenum to the jejunum [33], which has also been observed in pigs [6] and dogs [8]. However, how much the mucins in the rat mucus contributed to the total protein concentration in the mucus is unknown, and further studies are needed to assess the concentration of mucin in rat intestinal mucus. ...
The mucus layer in the small intestine is generally regarded as a barrier to drug absorption. However, the mucus layer is a complex system, and presently, only a few studies have been conducted to elucidate its physicochemical properties. The current study hypothesizes that the mucus layer contains solubility-enhancing surfactants and thus might aid the oral absorption of poorly water-soluble drugs. Mucus was sampled from sections of the small intestine of fasted rats to analyze the rheological properties and determine the mucus pH and concentrations of proteins and endogenous surfactants, i.e., bile salts, polar lipids, and neutral lipids. The mucus layer in the two proximal sections of the small intestine exhibited different rheological properties such as higher zero-shear viscosity and lower loss tangent and higher protein concentrations compared to all subsequent sections of the small intestine. The pH of the mucus layer was stable at ~ 6.5 throughout most of the small intestine, but increased to 7.5 in the ileum. The bile salt concentrations increased from the duodenum (16.0 ± 2.2 mM) until the mid jejunum (55.1 ± 9.5 mM), whereas the concentrations of polar lipids and neutral lipids decreased from the duodenum (17.4 ± 2.2 mM and 37.8 ± 1.6 mM, respectively) until the ileum (4.8 ± 0.4 mM and 10.7 ± 1.1 mM, respectively). In conclusion, the mucus layer of the rat small intestine contains endogenous surfactants at levels that might benefit solubilization and absorption of orally administered poorly water-soluble drugs.
Graphical Abstract
... Alcian Blue is a cationic dye widely used in light microscopy to stain sulfated and sialic acids containing acidic type glycoproteins [29]. The method has later been optimised for quantitative measurement of intestinal mucous glycoproteins by a simple biochemical method [30]. In the present study, the same method was used to analyse saliva with a few modifications. ...
... The Alcian Blue method was used to measure the amount of sulfated and sialylated glycoproteins in stimulated saliva. The method has previously been used to measure intestinal mucous glycoproteins [30] and was applied to the saliva with few modifications, as described below. ...
... A solution of 0.1 w/v (weight/volume) Alcian Blue in 0.1 M sodium acetate acid buffer (pH 5.8) containing 25 mM MgCl 2 was used. Chondroitin sulfate was used to obtain a standard curve for the sulfated and sialylated glycoproteins [30]. A five-point concentration gradient with the highest concentration of 0.2 mg/ml chondroitin sulfate was used to create the absorbance-concentration curve. ...
Objectives
Salivary proteins, acidic glycoproteins, and free calcium might take part in oral mucosal defence against inflammation in oral lichen planus (OLP). The study aimed to investigate whether the levels of sulfated and sialylated glycoproteins, total protein, and free calcium in saliva from patients with OLP differ from those of individuals without oral mucosal diseases.
Material and Methods
Patients diagnosed with OLP (n = 25) and two control groups without any oral mucosal disease; age- and gender-matched controls (n = 25, 65.6 ± 2.9 years), and younger controls (n = 25, 41.8 ± 2.5 years) were included. Subjective dry mouth (xerostomia) was assessed by asking a single-item question. Chew-stimulated whole saliva was collected to measure sulfated and sialylated glycoproteins by the Alcian Blue method. The total protein was determined spectrophotometrically, and the free calcium measured using an electrode.
Results
The output of salivary sulfated and sialylated glycoproteins in the OLP group (21.8 ± 2.4 µg/min) was lower than in the age- and gender-matched controls (43.0 ± 2.9 µg/min, p = 0.0002), whereas the total protein and calcium output did not differ between the three groups (p > 0.05). The prevalence of xerostomia was significantly higher in the OLP group compared to both control groups (p = 0.038).
Conclusions
Patients with OLP showed a high prevalence of xerostomia and lower levels of salivary acidic type glycoproteins compared to the individuals without oral mucosa disease.
Clinical relevance
It is relevant to investigate the role of acidic glycoproteins in the pathogenesis of OLP.
... Using an in situ single-pass perfusion technique, Masaoka et al. demonstrated that the intestinal membrane permeability of griseofulvin was significantly increased in jejunum pre-treated with DTT for 30 min [56]. Similarly, when DTT was pre-activated in vitro for 10 min, the intestinal membrane permeability of griseofulvin and antipyrine was increased in the proximal part of the small intestine of the duodenum and jejunum, but not in the distal part of the small intestine [57] (Figure 4). Mucus layer removal increased the drug's mucosal permeability, indicating that the effect varies depending on the compound administered and the gastrointestinal tract site. ...
... * p < 0.05, ** p < 0.01 compared with the control condition. Reproduced with permission from Ref.[57]. Copyright 2019 Springer Nature. ...
Because of the increasing sophistication of formulation technology and the increasing polymerization of compounds directed toward undruggable drug targets, the influence of the mucus layer on gastrointestinal drug absorption has received renewed attention. Therefore, understanding the complex structure of the mucus layer containing highly glycosylated glycoprotein mucins, lipids bound to the mucins, and water held by glycans interacting with each other is critical. Recent advances in cell culture and engineering techniques have led to the development of evaluation systems that closely mimic the ecological environment and have been applied to the evaluation of gastrointestinal drug absorption while considering the mucus layer. This review provides a better understanding of the mucus layer components and the gastrointestinal tract’s biological defense barrier, selects an assessment system for drug absorption in the mucus layer based on evaluation objectives, and discusses the overview and features of each assessment system.
... In addition, because of the peristaltic action of the digestive tract, the UWL was similar to the thickness in the μFlux test. 68 Therefore, the animal study showed a similar tendency to the dissolutionpermeation study with the μFlux system. Orally administered coamorphous salts can pass through the UWL and supply free drugs to the surface of the intestinal membrane. ...
Coamorphous formulation is a useful approach for enhancing the solubility of poorly water-soluble drugs via intermolecular interactions. In this study, a hydrogen-bonding-based coamorphous system was developed to improve drug solubility, but it barely changed the apparent permeability (Papp) of the drug. This study aimed to design a novel coamorphous salt using ionic interactions to improve drug permeability and absorption. Telmisartan (TMS), with an acidic group, was used to form a coamorphous salt with basic amlodipine (AML). Evaluation of the physicochemical properties confirmed the formation of a coamorphous salt via ionic interactions between the amine group of AML and the carboxyl group of TMS at a molar ratio of 1:1. The coamorphous salt of TMS/AML enhanced the partitioning of both drugs into octanol, indicating increased lipophilicity owing to the interaction between TMS and AML. The coamorphous salt dramatically enhanced TMS solubility (99.8 times that of untreated TMS) and decreased AML solubility owing to the interaction between TMS and AML. Although the coamorphous salt showed a decreased Papp in the permeation study in the presence of a thicker unstirred water layer (UWL) without stirring, Papp increased in the presence of a thinner UWL with stirring. The oral absorption of TMS from the coamorphous salt increased by up to 4.1 times compared to that of untreated TMS, whereas that of AML remained unchanged. Although the coamorphous salt with increased lipophilicity has a disadvantage in terms of diffusion through the UWL, the UWL is thin in human/animal bodies owing to the peristaltic action of the digestive tract. Dissociation of the coamorphous salt on the membrane surface could contribute to the partitioning of the neutral form of drugs to the membrane cells compared with untreated drugs. As a result, coamorphous salt formation has the advantage of improving the membrane permeation and oral absorption of TMS, owing to the enhanced solubility and supply of membrane-permeable free TMS on the surface of the membrane.
... Most often, membrane barriers consist of an unstirred water layer (UWL) at the exterior surface of a lipophilic membrane [33]. In the gastrointestinal tract, the UWL is the relatively thick (can be several hundred µm in humans) and viscous mucus layer that covers the lipophilic mucosal epithelium [90,91]. Figure 5 is a schematic presentation of permeation of a lipophilic drug through a simple two-layer biomembrane showing passive drug permeation first through an UWL and then through a lipophilic membrane. ...
Cyclodextrins (CDs) are cyclic oligosaccharides that emerged as industrial excipients in the early 1970s and are currently found in at least 130 marketed pharmaceutical products, in addition to numerous other consumer products. Although CDs have been the subject of close to 100,000 publications since their discovery, and although their structure and properties appear to be trivial, CDs are constantly surprising investigators by their unique physicochemical properties. In aqueous solutions, CDs are solubilizing complexing agents of poorly soluble drugs while they can also act as organic cosolvents like ethanol. CDs and their complexes self-assemble in aqueous solutions to form both nano- and microparticles. The nanoparticles have diameters that are well below the wavelength of visible light; thus, the solutions appear to be clear. However, the nanoparticles can result in erroneous conclusions and misinterpretations of experimental results. CDs can act as penetration enhancers, increasing drug permeation through lipophilic membranes, but they do so without affecting the membrane barrier. This review is an account of some of the unexpected results the authors have encountered during their studies of CDs as pharmaceutical excipients.
... Кроме того, ребамипид усиливает секрецию муцина бокаловидными клетками кишечника и, следовательно, увеличивает толщину слизистого слоя кишечника [48]. Возможность влияния ребамипида на всем протяжении ЖКТ связано с дифференцированным его взаимодействием с определенными типами муцина, в частности MUC 1, который в основном присутствует в желудке, а также MUC 4, экспрессируемом в кишечнике [49]. ...
Syndrome of increased epithelial permeability (SPEP) is considered as one of the universal mechanisms that determine the subsequent development of chronic systemic inflammation of varying severity. Studies carried out in the last 10 years have shown the important role of SPEP in the pathogenesis of many diseases of internal organs, and, first of all, of the gastrointestinal diseases. The article discusses possible ways of correcting impaired epithelial permeability from the point of view of the cytoprotective effects of drugs most often prescribed to patients with gastrontestinal diseases.
... The western blotting analysis was carried out according to previous reports (Miyazaki et al., 2019;Kishimoto et al., 2022). Briefly, the cultured cells were collected from the cell culture dishes by using This article has not been copyedited and formatted. ...
Mucin 1 (MUC1) is aberrantly expressed in various cancers and implicated in cancer progression and chemoresistance. Although the C-terminal cytoplasmic tail of MUC1 is involved in signal transduction, promoting chemoresistance, the role of the extracellular MUC1 domain [N-terminal glycosylated domain (NG)-MUC1] remains unclear. In this study, we generated stable MCF7 cell lines expressing MUC1 and cytoplasmic tail-deficient MUC1 (MUC1ΔCT) and show that NG-MUC1 is involved in drug resistance by modulating the transmembrane permeation of various compounds without cytoplasmic tail signaling. Heterologous expression of MUC1ΔCT increased cell survival in treating anticancer drugs (such as 5-fluorouracil, cisplatin, doxorubicin, and paclitaxel), in particular by causing an approximately 150-fold increase in the IC50 of paclitaxel, a lipophilic drug, compared with the control [5-fluorouracil (7-fold), cisplatin (3-fold), and doxorubicin (18-fold)]. The uptake studies revealed that accumulations of paclitaxel and Hoechst 33342, a membrane-permeable nuclear staining dye, were reduced to 51% and 45%, respectively, in cells expressing MUC1ΔCT via ABCB1/P-gp-independent mechanisms. Such alterations in chemoresistance and cellular accumulation were not observed in MUC13-expressing cells. Furthermore, we found that MUC1 and MUC1ΔCT increased the cell-adhered water volume by 2.6- and 2.7-fold, respectively, suggesting the presence of a water layer on the cell surface created by NG-MUC1. Taken together, these results suggest that NG-MUC1 acts as a hydrophilic barrier element against anticancer drugs and contributes to chemoresistance by limiting the membrane permeation of lipophilic drugs. Our findings could help better the understanding of the molecular basis of drug resistance in cancer chemotherapy. SIGNIFICANCE STATEMENT: Membrane-bound mucin (MUC1), aberrantly expressed in various cancers, is implicated in cancer progression and chemoresistance. Although the MUC1 cytoplasmic tail is involved in proliferation-promoting signal transduction thereby leading to chemoresistance, the significance of the extracellular domain remains unclear. This study clarifies the role of the glycosylated extracellular domain as a hydrophilic barrier element to limit the cellular uptake of lipophilic anticancer drugs. These findings could help better the understanding of the molecular basis of MUC1 and drug resistance in cancer chemotherapy.
... pH) may also influence its porosity and molecular permeability. Consequently, the GI mucosa of the STZ-icv rats may be exposed to a greater concentration of intraluminal toxins and some drugs [55][56][57]. The results obtained from the in vitro experiment designed for the assessment of the ability of reconstituted mucus to prevent the diffusion of caffeine indicate that the STZ-icv mucus has a reduced capacity to bind and/or prevent the diffusion of small molecules (Fig 5). ...
Accumulating evidence supports the involvement of the gastrointestinal (GI) system in Alzheimer's disease (AD), however, it is currently unknown whether GI alterations arise as a consequence of central nervous system (CNS) pathology or play a causal role in the pathogenesis of the disease. The GI mucus system is a possible mediator of GI dyshomeostasis in neurological disorders as CNS controls mucus production and secretion via the efferent arm of the brain-gut axis. The aim was to use a brain-first model of sporadic AD induced by intracerebroventricular streptozotocin (STZ-icv) to dissect the efferent (i.e. brain-to-gut) effects of isolated central neuropathology on the GI mucus system. Quantification and morphometric analysis of goblet cell mucigen granules revealed altered GI mucus secretion in the AD model possibly mediated by the insensitivity of AD goblet cells to neurally-evoked mucosal secretion confirmed by ex vivo cholinergic stimulation of isolated duodenal rings. The dysfunctional efferent control of the GI mucus secretion results in altered biochemical composition of the mucus associated with reduced glycoprotein aggregation and binding capacity in vitro. Finally, functional consequences of the reduced barrier-forming capacity of the AD mucus are demonstrated using the in vitro two-compartment caffeine diffusion interference model. Isolated central AD-like neuropathology results in the loss of efferent control of GI homeostasis via the brain-gut axis characterized by the insensitivity to neurally-evoked mucosal secretion, altered mucus constitution, and reduced barrier-forming capacity potentially increasing the susceptibility of STZ-icv rat model of AD to GI and systemic inflammation induced by intraluminal toxins, microorganisms, and drugs.
... Moreover, due to their complex chemical composition, mucins can also act as a selective physicochemical barrier via binding to their negatively charged O-glycans clustered in the heavily glycosylated central domains and to hydrophobic, non-O-glycosylated cysteine-rich domains 6,7 . These barrier properties of mucins are key factors to be considered in the intestinal absorption of drugs 9 . However, mucus is a dynamic barrier, and its composition (in particular, mucin composition) and biological functions differ depending on the mucosal tissue 7,8 . ...
Cyclic peptides are good candidates for orally delivered therapeutics, however, issues remain in their development due to low intestinal permeability. Although some of the biological factors have been reported that regulate intestinal permeation of cyclic peptides, the influence of the mucus barrier, a major hurdle to epithelial drug delivery, on cyclic peptide bioavailability is unclear. In this study, we show that the lipophilic cyclic peptide, cyclosporin A (CsA), interacted with, and likely induced aggregation, of polymeric, gel-forming mucins (MUC2, MUC5AC and MUC5B) which underpin the mucus gel-networks in the gastrointestinal tract. Under similar conditions, two other cyclic peptides (daptomycin and polymyxin B) did not cause mucin aggregation. Using rate-zonal centrifugation, purified MUC2, MUC5AC and MUC5B mucins sedimented faster in the presence of CsA, with a significant increase in mucins in the pellet fraction. In contrast, mucin sedimentation profiles were largely unaltered after treatment with daptomycin or polymyxin B. CsA increased MUC5B sedimentation was concentration-dependent, and sedimentation studies using recombinant mucin protein domains suggests CsA most likely causes aggregation of the relatively non-O-glycosylated N-terminal and C-terminal regions of MUC5B. Furthermore, the aggregation of the N-terminal region, but not the C-terminal region, was affected by pH. CsA has partially N-methylated amide groups, this unique molecular structure, not present in daptomycin and polymyxin B, may potentially be involved in interaction with gel-forming mucin. Taken together, our results indicate that the interaction of gel-forming mucins with the cyclic peptide CsA is mediated at the N- and C-terminal domains of mucin polymers under physiological conditions. Our findings demonstrate that the mucus barrier is an important physiological factor regulating the intestinal permeation of cyclic peptides in vivo.
... [10][11][12][13][14][15] The mucus has been shown to limit diffusion of drug molecules toward, for example, the intestinal epithelium. 16 The UWL is also referred to as the aqueous diffusion layer, static diffusion layer or stagnant liquid film, and has long been recognized as a part of the total barrier to solute permeation through not only biomembranes but also membranes in general. [17][18][19] It has been shown that hydrophilic complexing agents, such as CDs, can enhance permeation of lipophilic compounds through the UWL 20,21 although CDs have also been shown to hamper drug permeation. ...
Cyclodextrins are hydrophilic oligosaccharides that can increase aqueous solubility of lipophilic drugs through formation of water-soluble drug/cyclodextrin complexes. Although the complexes are hydrophilic, and as such do not permeate biological membranes, the complexes are known to enhance drug permeation through lipophilic membranes and improve drug bioavailability after, for example, oral administration. However, it is not clear how cyclodextrins enhance the permeation. An artificial biomembrane (PermeaPad®) was used to study the effect of donor medium composition on drug permeation. It was observed that in aqueous solutions the hydrophilic cyclodextrins behave not like disperse systems but rather like organic cosolvents such as ethanol, increasing the solubility without having significant effect on the molecular mobility and ability of lipophilic drug molecules to partition into the lipophilic membrane. Also, that partition of dissolved drug molecules from the aqueous exterior into the membrane is at its maximum when their thermodynamic activity is at its maximum. In other words, that drug flux from aqueous cyclodextrin solutions through lipophilic membranes depends on both the concentration and the thermodynamic activity of dissolved drug. Maximum flux is obtained when both the drug concentration and thermodynamic activity of the dissolved drug molecules are at their maximum value.
