Search

This study aimed to analyze the contributions of multiple transport mechanisms to the intestinal uptake of serotonin (5-HT) by employing a variety of in vitro experimental techniques, focusing on organic cation transporters expressed in the gastrointestinal (GI) tract, such as SERT, PMAT, THTR2, OCT3, and OCTN2. Analysis of the concentration dependence of 5-HT uptake by Caco-2 cells revealed multi-affinity kinetics with high-affinity and low-affinity components, suggesting that multiple transporters are involved in the intestinal 5-HT uptake. Comparative analysis of transporters using K m values obtained in Xenopus oocyte expression systems suggested that SERT is responsible for the high-affinity transport, while PMAT, THTR2, and OCT3 contribute to the low-affinity transport. Further analysis indicated that the relative contributions of SERT and PMAT to the intestinal 5-HT uptake (0.01 µM) are approximately 94.9% and 1.1%, respectively. Interestingly, at the concentration of 10 µM, the reported steady-state concentration of 5-HT in the human colon, the contributions of SERT, PMAT, THTR2, and OCT3 were estimated to be approximately 37.0%, 1.0%, 18.2%, and 20.5%, respectively. In conclusion, the present study indicated that the contributions of multiple transporters to 5-HT uptake in the GI tract are dependent upon the colon luminal concentration of 5-HT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Chicoric acid (CA), an active phenolic acid of Echinacea purpurea (Linn.) Moench, has been demonstrated to exhibit antioxidative, antiviral and immunological activities. A prior study showed that CA is a water-soluble compound with low bioavailability. The current study was performed to study the intestinal absorption mechanism of CA and improve its bioavailability using natural biodegradable chitosan. A Caco-2 monolayer cell model was established to characterise the mechanisms involved in the intestinal absorption of CA. The bioavailability improvement of CA was studied in Sprague–Dawley rats after oral (20 mg/kg) administration of 0.5% chitosan. In vitro, the results showed that the absorption transport of CA was fairly poor, with Papp values of 8.2 × 10−8 to 2.1 × 10−7 cm/s in the absorption direction and 1.5 × 10−7 to 2.6 × 10−7 cm/s in the secretory direction. The permeability was increased by EDTA and chitosan in both directions. Moreover, the transport through the intestinal monolayer was H+ dependent, and P-glycoprotein and OATP2B1 transporters were involved in the intestinal transport of CA. In vivo, the absorption of CA was increased and accelerated with chitosan in rats because the bioavailability was 1.74-fold that of the prototype drug. The above mentioned results indicated that CA was a poor absorption drug and that paracellular and carrier-mediated trancellular transport both participated in its transport route. Chitosan is an excellent absorption enhancer for CA. The transport characteristics uncovered in this study lay the groundwork for further studies directed toward the development and utilisation of its new formulations.

Background:: Environmental enteropathy (EE) contributes to growth failure in millions of children worldwide, but its relationship to clinical malnutrition has not been elucidated. We used RNA sequencing to compare duodenal biopsies from adults and children with EE, and from children with severe acute malnutrition (SAM), to define key features of these malnutrition-related enteropathies. Methods:: RNA was extracted and sequenced from biopsies of children with SAM in hospital (n=27), children with non-responsive stunting in the community (n=30), and adults living in the same community (n=37) using an identical sequencing and analysis pipeline. Two biopsies each were profiled and differentially expressed genes (DEGs) were computed from the comparisons of the three groups. DEG lists from these comparisons were then subjected to analysis with CompBio software to assemble a holistic view of the biological landscape and IPA software to interrogate canonical pathways. Findings:: Dysregulation was identified in goblet cell/mucin production and xenobiotic metabolism/detoxification for both cohorts of children, versus adults. Within the SAM cohort, substantially greater induction of immune response and barrier function, including NADPH oxidases was noted, concordant with broadly reduced expression of genes associated with the brush border and intestinal structure/transport/absorption. Interestingly, down regulation of genes associated with the hypothalamic-pituitary-adrenal axis was selectively observed within the cohort of children with stunting. Interpretation:: Gene expression profiles in environmental enteropathy and severe acute malnutrition have similarities, but SAM has several distinct transcriptional features. The intestinal capacity to metabolise drugs and toxins in malnourished children requires further study. Funding:: Bill & Melinda Gates Foundation (OPP1066118)

Lipophilic compounds constitute a majority of therapeutics in the pipeline of drug discovery. Despite possessing enhanced efficacy and permeability, some of these drugs suffer poor solubility necessitating the need of a suitable drug delivery system. Nanoemulsion is a drug delivery system that provides enhanced solubility for poorly soluble drugs in an attempt to improve the oral bioavailability. The purpose of this study is to develop a nanoemulsion system using ibuprofen as a model drug in order to investigate the potential of this colloidal system to enhance the absorption of poorly water-soluble drugs. Ibuprofen loaded-nanoemulsion with different drug concentrations (1.5, 3 and 6% w/w) were formulated from olive oil, sucrose ester L-1695 and glycerol using D-phase emulsification technique. A pseudoternary phase diagram was utilised to identify the optimal excipient composition to formulate the nanoemulsion system. In vitro diffusion chamber studies using rodent intestinal linings highlighted improved absorption profile when ibuprofen was delivered as nanoemulsion in comparison to microemulsions and drug-in-oil systems. This was further corroborated by in vivo studies using rat model that highlighted a two-fold increase in ibuprofen absorption when the drug was administered as a nanoemulsion relative to drug-in-oil system. On the other hand, when ibuprofen was administered as microemulsions, only a 1.5-fold increase in absorption was observed relative to drug-in-oil system. Thus, this study highlights the potential of using nanoemulsion as a drug delivery system to enhance the oral bioavailability of hydrophobic drugs.

The development of carrier-based delivery systems for oral administration of retinoic acid (RA), that provides its release and absorption at intestinal level, is of major relevance in the treatment of acute promyelocytic leukemia. The aim of this work was to evaluate RA bioaccessibility and intestinal transport on ethyl cellulose (EC)- and EC + polyethylene glycol (ECP)-based microparticles and to understand the impact of meal co-ingestion by applying in vitro assays. RA-loaded microparticles were produced by spray-drying with an encapsulation efficiency higher than 90 % for both formulations. The gastric bioaccessibility of RA (after in vitro static digestion of RA-loaded particles) was lower than 3 % for both types of microparticles, with and without meal co-ingestion. Whereas after intestinal digestion, RA bioaccessibility was significantly higher and affected by the type of microparticles and the presence of meal. The digestion of EC- and ECP-based microparticles without diet enabled a significantly higher bioaccessibility of RA when compared to the one recorded for the co-digestion of these microparticles with diet. Herein, RA bioaccessibility decreased from 84 ± 1 to 24 ± 6 % (p < 0.0001) for microparticles EC and 54 ± 4 to 25 ± 5 % (p < 0.001) for microparticles ECP. Moreover, comparing both types of microparticles, RA bioaccessibility was significantly higher for EC-based microparticles digested without diet (p < 0.0001). At last, the bioaccessibility of RA was similar among EC- and ECP-based microparticles when co-digested with diet. Intestinal transport experiments performed in Caco-2 monolayers evidenced that after 2 h of transport the amount of RA retained in the apical compartment was higher than the amount that reached the basolateral compartment evidencing a slow transport at intestinal level that was higher when RA is spiked in the blank of digestion and the meal digestion samples compared to RA dissolved in HBSS (44 ± 6 (p < 0.01) and 38 ± 1 (p < 0.05) vs 26 ± 2 %, respectively)., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2023. Published by Elsevier B.V.)

Angiotensin converting enzyme (ACE) inhibitory peptides have been extensively studied as an alternative to synthetic drugs for the treatment of hypertension. Recent studies have shown that dry-cured ham is an important source of naturally generated bioactive peptides, especially showing ACE inhibitory activity. However, due to their excessive degradation by digestive and brush-border enzymes, it is not clear whether these peptides resist intestinal absorption and reach the blood stream where they may exert their antihypertensive effect. Therefore, dry-cured ham extracts and specific pure peptides naturally generated during the dry-curing process, showing ACE inhibitory activity, have been studied for their stability during transepithelial transport in a Caco-2 cell monolayer. The ACE inhibitory activity of transport samples was assayed, showing the highest values in apical samples at 15 min of incubation. In concentrated basal solutions, ACE inhibition values increased during transport for purified peptides, reaching values close to 70% for AAPLAP and KPVAAP at 60 min of cellular transport. Fragments generated by cellular activity were detected by using tandem mass spectrometry techniques, showing that AAATP, AAPLAP, and KPVAAP were hydrolysed during the transport, although KPVAAP was also absorbed intact. This study highlights the potential of intact dry-cured ham peptides as well as their fragments to be absorbed across the intestinal epithelium and reach the blood stream to exert an antihypertensive action.

Background: The purpose of this study was to investigate the effect of hydroxypropyl methyl cellulose phthalate (HPMCP) coating on the digestive stability and intestinal transport of green tea catechins (GTCs). Methods: Two types of HPMCP coating were prepared: one type with size smaller than 500 μm (S-HPMCP) and the other with size larger than 500 μm (L-HPMCP). An in vitro gastrointestinal model system coupled with Caco-2 cells was used for estimating the bioavailability of GTCs. Ultraperformance liquid chromatography with a photodiode array detector was performed to analyze GTCs. Results: The digestive stability of GTCs was enhanced up to 33.73% and 35.28% for S-HPMCP and L-HPMCP, respectively. Intestinal transport of the GTCs was increased to 22.98% and 23.23% for S-HPMCP and L-HPMCP, respectively. Overall, the bioavailability of GTCs increased by 4.08 and 11.71 times for S-HPMCP and L-HPMCP, respectively. Conclusion: The results of this study confirm that coating with HPMCP could be a way to improve the digestive stability and intestinal transport of GTCs.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride products such as feed piping, packing bag, and medical consumable. Our previous studies have demonstrated that DEHP exposure reduced the concentration of nicotinamide adenine dinucleotide (NAD + ) in pregnant mice serum, which cuts off the source of NAD + to placenta and results fetal growth restriction. However, the mechanism of serum NAD + depletion by DEHP remains elusive. This study investigated the intestinal mechanism of NAD + shortage-induced by DEHP in pregnant mice. The transcriptome results implicated that the mRNA level of oxidative response genes Cyp1a1, Gsto2, Trpv1 and Trpv3 were upregulated in colon. These changes induced intestinal inflammation. Transmission Electron Microscopy results displayed that DEHP destroyed the tight junctions and cell polarity of colonic epithelial cells. These dysfunctions diminished the expression of NAD + precursor transporters SLC12A8, SLC5A8, SLC7A5, and the NAD + biosynthetic key enzymes NAMPT, NMNAT1-3, and TDO2 in colonic epithelial cells. Analysis of the gut microbiota showed that DEHP led to the dysbiosis of gut microbiota, reducing the relative abundance of Prevotella copri which possesses the VB3 biosynthetic pathway. Therefore, maternal DEHP exposure during pregnancy decreased the transportation of NAD + precursors from enteric cavity to colonic epithelial cells, and inhibited the synthesis of NAD + in colonic epithelial cells. Meanwhile, DEHP reduced the NAD + precursors provided by gut microbiota. Eventually, serum NAD + content was lowered. Taken together, our findings provide a new insight for understanding the intestinal mechanisms by which DEHP affects serum NAD + levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

In the drug development process, it is important to assess the contributions of drug-metabolizing enzymes and/or drug transporters to the intestinal pharmacokinetics of candidate compounds. For such assessments, chemical inhibitors are often used in in vitro systems. However, this practice poses two problems: one is the low expression levels of pharmacokinetic-related genes in conventional in vitro systems, such as Caco-2 cells, and the other is the off-target and less-efficient effects of their inhibitors. Here, as a model, we have established human biopsy-derived enteroids deficient in MDR1, a key efflux transporter. The expression levels and activities of other pharmacokinetic-related genes, such as CYP3A4, in the MDR1-knockout (KO) enteroid-derived monolayers were maintained at levels as high as those in the WT enteroid-derived monolayers. The contribution of MDR1 to the cytotoxicity of vinblastine, which CYP3A4 metabolized, was accurately evaluated by using the MDR1-KO enteroid-derived monolayers. In contrast, it could not be evaluated in the WT enteroid-derived monolayers treated by verapamil, a widely used MDR1 inhibitor, due to the off-target effect of verapamil, which also inhibits CYP3A4. The combination of human enteroid-derived monolayers and genome editing technology would be a powerful tool to evaluate the contributions of specific pharmacokinetic-related molecules., (Copyright © 2022 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar K m(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Breast cancer resistance protein transporter (ABCG2/BCRP) is highly expressed on the intestinal epithelial membrane and has a significant impact on the oral absorption of topotecan. In this study, we examined 6 pharmaceutical excipients including BL-9EX, Brij97, Cremophor EL, Labrasol, Pluronic F68, and Tween 20 for their BCRP inhibitory effects. A bidirectional transport study using Caco-2 cells demonstrated that Tween 20 and Cremophor EL significantly increased the absorptive transport of topotecan, while simultaneously decreasing secretory transport. Interestingly, Labrasol selectively increased absorptive transport, whereas Pluronic F68 selectively decreased the secretory transport, of topotecan. Further investigation using an in situ closed loop experiment showed that 0.05% (w/v) Tween 20 and Cremophor EL significantly increased the intestinal absorption of topotecan in rats. An LDH assay demonstrated that 0.05% (w/v) Tween 20 and Cremophor EL did not cause significant damage to intestinal epithelial membranes. Furthermore, we examined the absorption-enhancing mechanisms of these excipients and found that Cremophor EL, Tween 20, and Labrasol increased the membrane fluidity of the inner lipid bilayers of the intestine. Therefore, this might be one of the most important mechanisms for inhibition of BCRP function by these excipients in the intestine., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Our previous study introduced a barley protein microparticle for encapsulation of hydrophobic drug/nutraceutical, which could release nanoparticles upon gastric digestion and deliver encapsulated compound to a simulated intestinal environment intact. This work focused on evaluating the potential of liberated nanoparticles to improve the absorption of encapsulated compounds ( e.g ., β -carotene) using in vitro Caco-2 cell and ex vivo small intestine models. Nanoparticles obtained from gastric digestion of barley protein microparticles had a spherical shape and an average size of 351 nm. Nanoparticles showed low cytotoxicity in Caco-2 cells and their cellular uptake was dependent on time, concentration and temperature. In a Caco-2 cell monolayer model, significantly greater uptake and transport of β -carotene were observed when it was delivered by nanoparticles (15%), compared to free β -carotene suspension (2.6%). In an ex vivo rat jejunum model, nanoparticles showed the capacity to retain in small intestinal tissue. Approximately 2.24 and 6.04 μg nanoparticle were able to permeate through each cm 2 intestinal tissue and translocate to the serosal side after 60 and 90 min, respectively. Results from this study demonstrated the absorption improving effect of the barley protein nanoparticles and suggested their potential as vehicles for hydrophobic compounds.

Breast cancer resistance protein (BCRP) transporter is an efflux transporter that utilizes energy from adenosine triphosphate hydrolysis to push its substrates, regardless of the concentration gradient. Its presence on the apical membrane of the intestinal mucosa is a major obstacle for the intestinal absorption of its substrates. In this study, we examined the effects of various pharmaceutical excipients on the intestinal transport and absorption of sulfasalazine, a BCRP substrate. Four excipients, including 0.05% and 0.075% BL-9EX, 0.01% and 0.05% Brij 97, 0.075% Labrasol, and 0.05% and 0.1% Tween 20 decreased the secretory transport of sulfasalazine in an in vitro diffusion chamber. Further investigation in an in situ closed loop experiment in rats showed that 0.05% and 0.1% BL-9EX and 0.1% Brij 97 effectively enhanced the intestinal absorption of sulfasalazine while maintaining minimal toxicity to the intestinal mucosa. However, 0.1% Brij 97 also increased the intestinal absorption of 5(6)-carboxyfluorescein, a paracellular marker compound. These findings suggest that BL-9EX might effectively inhibit the BCRP-mediated efflux of sulfasalazine in vivo, indicating that BL-9EX could improve the intestinal absorption of sulfasalazine and other BCRP substrates., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Nanomaterials have been widely utilized in the food industry in production, packaging, sensors, nutrient delivery systems, and food additives. However, research on the interactions between food-grade nanoparticles and biomolecules as well as their potential toxicity is limited. In the present study, the in vivo solubility, oral absorption, tissue distribution, and excretion kinetics of one of the most extensively used food additives, silica (SiO 2 ) were evaluated with respect to particle size (nano vs bulk) following single-dose oral administration to rats. Intestinal transport mechanism was investigated using a 3D culture system, in vitro model of human intestinal follicle-associated epithelium (FAE). The effect of the presence of food components, such as sugar and protein, on the oral absorption of nanoparticles was also evaluated with focus on their interactions. The results obtained demonstrated that the oral absorption of nanoparticles (3.94±0.38%) was greater than that of bulk materials (2.95±0.37%), possibly due to intestinal transport by microfold (M) cells. On the other hand, particle size was found to have no significant effect on in vivo dissolution property, biodistribution, or excretion kinetics. Oral absorption profile of silica nanoparticles was highly dependent on the presence of sugar or protein, showing rapid absorption rate in glucose, presumably due to their surface interaction on nanoparticles. These findings will be useful for predicting the potential toxicity of food-grade nanoparticles and for understanding biological interactions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Myricetin is a flavonoid that has recently been suggested to induce sustained inhibition of proton-coupled folate transporter (PCFT/SLC46A1), which operates for intestinal folate uptake. The present study was conducted to characterize the inhibitory effect in more detail, using human PCFT stably expressed in Madin-Darby canine kidney II cells, to gain information to cope with problems potentially arising from that. The kinetics of saturable folate transport was first assessed in the absence of myricetin in the cells pretreated with the flavonoid for 60 min. The pretreatment induced PCFT inhibition in a manner dependent on the concentration of myricetin, where the maximum transport rate was reduced by 35.5% and 83.1%, respectively, at its concentrations of 20 μM and 50 μM. The inhibitory effect was, however, less extensive at lower folate concentrations, because the Michaelis constant was also reduced similarly in a manner dependent on myricetin concentration. The inhibition was induced depending on the time of pretreatment and, after removal of myricetin (50 μM) upon the manifestation of an extensive inhibition at 60 min, reversed almost completely in 90 min. This rather short time required for recovery may suggest that the sustained inhibition of PCFT is of a reversible type., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Accumulating evidences have shown that diabetes is often accompanied with depression, thus it is possible that oral antidiabetic agent glyburide and antidepressive agent paroxetine are co-administered in diabetic patients. The aim of this study was to assess interactions between glyburide and paroxetine in rats. Effect of paroxetine on pharmacokinetics of orally administered glyburide was investigated. Effect of naringin (NAR), an inhibitor of rat intestinal organic anion transporting polypeptides 1a5 (Oatp1a5), on pharmacokinetics of glyburide was also studied. The results showed that co-administration of paroxetine markedly reduced plasma exposure and prolonged Tmax of glyburide, accompanied by significant increase in fecal excretion of glyburide. Co-administration of naringin also significantly decreased plasma exposure of glyburide. Data from intestinal perfusion experiments showed that both paroxetine and naringin significantly inhibited intestinal absorption of glyburide. Caco-2 cells were used to investigate whether paroxetine and naringin affected intestinal transport of glyburide and fexofenadine (a substrate of Oatp1a5). The results showed that both paroxetine and naringin greatly inhibited absorption of glyburide and fexofenadine. All results gave a conclusion that co-administration of paroxetine decreased plasma exposure of glyburide in rats via inhibiting intestinal absorption of glyburide, which may partly be attributed to the inhibition of intestinal Oatp1a5 activity., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Epirubicin (EPI) is an anthracycline antineoplastic agent, commercially available for intravenous administration only and its oral ingestion continues to remain a challenge. Present investigation is aimed at the development of poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) for oral bioavailability enhancement of epirubicin. Developed formulation revealed particle size, 235.3±15.12 nm, zeta potential, -27.5±0.7 mV and drug content (39.12±2.13 μg/mg), with spherical shape and smooth surface. Cytotoxicity studies conducted on human breast adenocarcinoma cell lines (MCF-7) confirmed the superiority of epirubicin loaded poly-lactic-co-glycolic acid nanoparticles (EPI-NPs) over free epirubicin solution (EPI-S). Further, flow cytometric analysis demonstrated improved drug uptake through EPI-NPs and elucidated the dominance of caveolae mediated endocytosis for nanoparticles uptake. Transport study accomplished on human colon adenocarcinoma cell line (Caco-2) showed 2.76 fold improvement in permeability for EPI-NPs as compared to EPI-S (p<0.001) whereas a 4.49 fold higher transport was observed on rat ileum; a 1.8 fold higher (p<0.01) in comparison to Caco-2 cell lines which confirms the significant role of Peyer's patches in absorption enhancement. Furthermore, in vivo pharmacokinetic studies also revealed 3.9 fold improvement in oral bioavailability of EPI through EPI-NPs. Henceforth, EPI-NPs is a promising approach to replace pre-existing intravenous therapy thus providing "patient care at home"., (Copyright © 2015. Published by Elsevier B.V.)

Background: The purpose of this study was to investigate the effect of hydroxypropyl methyl cellulose phthalate (HPMCP) coating on the digestive stability and intestinal transport of green tea catechins (GTCs)., Methods: Two types of HPMCP coating were prepared: one type with size smaller than 500 μm (S-HPMCP) and the other with size larger than 500 μm (L-HPMCP). An in vitro gastrointestinal model system coupled with Caco-2 cells was used for estimating the bioavailability of GTCs. Ultraperformance liquid chromatography with a photodiode array detector was performed to analyze GTCs., Results: The digestive stability of GTCs was enhanced up to 33.73% and 35.28% for S-HPMCP and L-HPMCP, respectively. Intestinal transport of the GTCs was increased to 22.98% and 23.23% for S-HPMCP and L-HPMCP, respectively. Overall, the bioavailability of GTCs increased by 4.08 and 11.71 times for S-HPMCP and L-HPMCP, respectively., Conclusion: The results of this study confirm that coating with HPMCP could be a way to improve the digestive stability and intestinal transport of GTCs.