The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells.

BACKGROUND: Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood-brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). METHODS: We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. RESULTS: We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min-1·mg protein-1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. CONCLUSIONS: We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.

Neonatal intestinal mucus barrier changes in response to maturity, inflammation, and sodium decanoate supplementation.

The integrity of the intestinal mucus barrier is crucial for human health, as it serves as the body's first line of defense against pathogens. However, postnatal development of the mucus barrier and interactions between maturity and its ability to adapt to external challenges in neonatal infants remain unclear. In this study, we unveil a distinct developmental trajectory of the mucus barrier in preterm piglets, leading to enhanced mucus microstructure and reduced mucus diffusivity compared to term piglets. Notably, we found that necrotizing enterocolitis (NEC) is associated with increased mucus diffusivity of our large pathogen model compound, establishing a direct link between the NEC condition and the mucus barrier. Furthermore, we observed that addition of sodium decanoate had varying effects on mucus diffusivity depending on maturity and health state of the piglets. These findings demonstrate that regulatory mechanisms governing the neonatal mucosal barrier are highly complex and are influenced by age, maturity, and health conditions. Therefore, our results highlight the need for specific therapeutic strategies tailored to each neonatal period to ensure optimal gut health.

Generation of an iPSC-line (BIONi010C-48) with restored P-glycoprotein functionality following transfection with the human MDR1 gene in the AAVS1 locus.

The human MDR1 gene encodes the efflux transporter P-glycoprotein, which plays an important part of the blood-brain barrier function of brain microvascular endothelial cells (BMECs). Here, we report the generation of an iPSC line, where a construct of the human MDR1 gene was inserted into the safe-site locus AAVS1. This iPSC line (BIONi010-C-48) shows functional expression of P-gp and can further be differentiated and cultured into electrically tight BMEC-like monolayers exhibiting polarized expression of P-gp in the apical membrane.

Application of a new MDCKII-MDR1 cell model to measure the extent of drug distribution in vitro at equilibrium for prediction of in vivo unbound brain-to-plasma drug distribution.

INTRO: Reliable estimates of drug uptake from blood to brain parenchyma are crucial in CNS drug discovery and development. While in vivo Kp,uu,brain estimates are the gold standard for investigating brain drug disposition, animal usage is a limitation to high throughput application. This study investigates an in vitro model using P-gp expressing MDCKII-MDR1 cells for predicting in vivo brain drug penetration. METHODS: In vitro equilibrium distribution studies were conducted in apical and basolateral solutions with high protein content to estimate Kp,brain and Kp,uu,brain values. The correlation between in vitro and in vivo Kp,brain values for a set of compounds was examined. RESULTS: We observed a good correlation between in vitro and in vivo Kp,brain values (R2 = 0.69, Slope: 1.6), indicating that the in vitro model could predict in vivo drug brain penetration. The 'unilateral (Uni-L)' in vitro setup correctly classified 5 out of 5 unrestricted compounds and 3 out of 5 restricted compounds. Possible reasons for the observed disparities for some compounds have been discussed, such as difference in transport areas between in vitro and in vivo settings and effect of pH changes. CONCLUSION: The in vitro assay setup developed in this study holds promise for predicting in vivo drug brain penetration in CNS drug discovery. The correlation between in vitro and in vivo Kp,brain values, underscores that the model may have potential for early-stage screening. With minor refinements, this in vitro approach could reduce the reliance on in vivo experiments, accelerating the pace of CNS drug discovery and promoting a more ethical research approach.

Barrier properties of ex vivo porcine intestinal mucus are highly independent of isolation and storage conditions.

Porcine intestinal mucus (PIM) is often utilized as an ex vivo mucus model in mucus interaction studies. However, numerous isolation procedures and storage conditions for PIM are reported, yet their potential impact on preserving the critical properties of PIM remains unknown. This study investigated the effect of isolation procedures (rinsing and anatomical site of mucus isolation) and storage conditions (-20 °C, -80 °C, snap frozen in li-quid nitrogen prior to storage at -80 °C, or freeze-dried followed by storage at room temperature and reconstitution) of PIM in regard to the permeation of fluorescein-isothiocyanate-labelled dextran (FD) macromolecules of 4, 40 and 150 kDa, rheological properties as well as pH, osmolality, protein and water content. Rinsing intestines with tap water or phosphate-buffered saline as well as isolating PIM from different regions of the first five meters of the proximal jejunum did not affect the pH or osmolality of isolated PIM. The permeation of FD4, FD40 and FD150 through stored PIM was similar to permeation through fresh PIM. The rheological properties of stored PIM were similar to properties of fresh PIM. Osmolality, protein and water content were similar in stored and fresh PIM whereas pH decreased with 0.3 unit for all stored PIM. Overall, PIM samples stored at -20 °C, -80 °C, snap frozen or freeze-dried were found to have similar properties to freshly isolated PIM and can all be consi-dered good alternatives to fresh PIM for mucus studies.

Drug Delivery Strategies to Overcome the Blood-Brain Barrier (BBB).

The brain capillary endothelium serves both as an exchange site for gases and solutes between blood and brain and as a protective fence against neurotoxic compounds from the blood. While this "blood-brain barrier" (BBB) function protects the fragile environment in the brain, it also poses a tremendous challenge for the delivery of drug compounds to the brain parenchyma. Paracellular brain uptake of drug compounds is limited by the physical tightness of the endothelium, which is tightly sealed with junction complexes. Transcellular uptake of lipophilic drug compounds is limited by the activity of active efflux pumps in the luminal membrane. As a result, the majority of registered CNS drug compounds are small lipophilic compounds which are not efflux transporter substrates. Small molecule CNS drug development therefore focuses on identifying compounds with CNS target affinity and modifies these in order to optimize lipophilicity and decrease efflux pump interactions. Since efflux pump activity is limiting drug uptake, it has been investigated whether coadministration of drug compounds with efflux pump inhibitors could increase drug uptake. While the concept works to some extent, a lot of challenges have been encountered in terms of obtaining efficient inhibition while avoiding adverse effects.Some CNS drug compounds enter the brain via nutrient transport proteins, an example is the levodopa, a prodrug of Dopamine, which crosses the BBB via the large neutral amino acid transporter LAT1. While carrier-mediated transport of drug compounds may seem attractive, the development of drugs targeting transporters is very challenging, since the compounds should have a good fit to the binding site, while still maintaining their CNS target affinity.Receptor-mediated transport of drug compounds, especially biotherapeutics, conjugated to a receptor-binding ligand has shown some promise, although the amounts transported are rather low. This also holds true for drug-conjugation to cell-penetrating peptides. Due to the low uptake of biotherapeutics, barrier-breaching approaches such as mannitol injections and focused ultrasound have been employed with some success to patient groups with no other treatment options.

Screening novel CNS drug candidates for P-glycoprotein interactions using the cell line iP-gp: In vitro efflux ratios from iP-gp and MDCK-MDR1 monolayers compared to brain distribution data from mice.

Drug efflux by P-glycoprotein (P-gp, ABCB1) is considered as a major obstacle for brain drug delivery for small molecules. P-gp-expressing cell monolayers are used for screening of new drug candidates during early states of drug development. It is, however, uncertain how well the in vitro studies can predict the in vivo P-gp mediated efflux at the blood-brain barrier (BBB). We previously developed a novel cell line of porcine origin, the iP-gp cell line, with high transepithelial resistance and functional expression of human P-gp. The aim of the present study was to evaluate the applicability of the cell line for screening of P-gp interactions of novel drug candidates. For this purpose, bidirectional fluxes of 14 drug candidates were measured in iP-gp cells and in MDCK-MDR1 cells, and compared with pharmacokinetic data obtained in male C57BL/6 mice. The iP-gp cells formed extremely tight monolayers (>15 000 Ω∙cm2) as compared to the MDCK- MDR1 cells (>250 Ω∙cm2) and displayed lower Papp,a-b values. The efflux ratios obtained with iP-gp and MDCK-MDR1 monolayers correlated with Kp,uu,brain values from the in vivo studies, where compounds with the lowest Kp,uu,brain generally displayed the highest efflux ratios. 12 of the tested compounds displayed a poor BBB penetration in mice as judged by Kp,uu less than 1. Of these compounds, nine compounds were categorized as P-gp substrates in the iP-gp screening, whereas analysis of data estimated in MDCK-MDR1 cells indicated four compounds as potential substrates. The results suggest that the iP-gp cell model may be a sensitive and useful screening tool for drug screening purposes to identify possible substrates of human P-glycoprotein.

A new phenothiazine derivate is active against Clostridioides difficile and shows low cytotoxicity.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matters of concern for public health. Thioridazine, a compound belonging to the phenothiazine group, has previous shown antimicrobial activity against C. difficile. The purpose of this present study was to investigate the potential of a novel phenothiazine derivative, JBC 1847, as an oral antimicrobial for treatment of intestinal pathogens and CDIs. The minimal inhibition concentration and the minimum bactericidal concentration of JBC 1847 against C. difficile ATCC 43255 were determined 4 µg/mL and high tolerance after oral administration in mice was observed (up to 100 mg/kg bodyweight). Pharmacokinetic modeling was conducted in silico using GastroPlusTM, predicting low (< 10%) systemic uptake after oral exposure and corresponding low Cmax in plasma. Impact on the intestinal bacterial composition after four days of treatment was determined by 16s rRNA MiSeq sequencing and revealed only minor impact on the microbiota in non-clinically affected mice, and there was no difference between colony-forming unit (CFU)/gram fecal material between JBC 1847 and placebo treated mice. The cytotoxicity of the compound was assessed in Caco-2 cell-line assays, in which indication of toxicity was not observed in concentrations up to seven times the minimal bactericidal concentration. In conclusion, the novel phenothiazine derivative demonstrated high antimicrobial activity against C. difficile, had low predicted gastrointestinal absorption, low intestinal (in vitro) cytotoxicity, and only induced minor changes of the healthy microbiota, altogether supporting that JBC 1847 could represent a novel antimicrobial candidate. The clinical importance hereof calls for future experimental studies in CDI models.

Efficacy of a novel antimicrobial hydrogel for eradication of Staphylococcus epidermidis, Staphylococcus aureus and Cutibacterium acnes from preformed biofilm and treatment performance in an in vivo MRSA wound model.

BACKGROUND: Bacterial biofilm formation is a complicating factor in the antimicrobial treatment of bacterial infections. OBJECTIVES: In this study, we assessed the impact of a novel hydrogel with the active antimicrobial compound JBC 1847 on eradication of preformed biofilms of Staphylococcus epidermidis, Cutibacterium acnes and MRSA in vitro, and evaluated the in vivo efficacy of MRSA wound treatment. METHODS: Biofilms were exposed to JBC 1847 for 24 h and subsequently the treatments were neutralized and surviving biofilm-associated bacteria recovered and enumerated. The efficacy of the hydrogel on post-treatment load of MRSA was determined in a murine model of MRSA wound infection, and skin samples of the infected mice were examined histologically to evaluate the degree of healing. RESULTS: A concentration-dependent eradication of biofilm-embedded bacteria by JBC 1847 was observed for all three pathogens, and the hydrogel caused a greater than four log reduction of cfu in all cases. In the mouse model, treatment with the hydrogel significantly reduced the cfu/mL of MRSA compared with treatment of MRSA-infected wounds with pure hydrogel. Histopathological analysis of the wounds showed that the JBC 1847 treatment group had a lower grade of inflammation, a higher mean score of re-epithelization and higher mean scores of parameters assessing the maturity of the newly formed epidermis, compared with both the fusidic acid 2% and vehicle treatment groups. CONCLUSIONS: The novel hydrogel shows promising results as a candidate for future wound treatment, likely to be highly effective even in the case of biofilm-complicating infected wounds.

Gluten Degradation, Pharmacokinetics, Safety, and Tolerability of TAK-062, an Engineered Enzyme to Treat Celiac Disease.

BACKGROUND AND AIMS: Celiac disease (CeD) is an immune-mediated disorder triggered by the ingestion of gluten. Despite adhering to a gluten-free diet (the only management option available to patients with CeD), many patients continue to experience symptoms and intestinal injury. Degradation of immunogenic fractions of gluten peptides in the stomach has been proposed as an approach to reduce toxicity of ingested gluten; however, no enzymes evaluated to date have demonstrated sufficient gluten degradation in complex meals. TAK-062 is a novel, computationally designed endopeptidase under development for the treatment of patients with CeD. METHODS: Pharmacokinetics, safety, and tolerability of TAK-062 100-900 mg were evaluated in a phase I dose escalation study in healthy participants and patients with CeD. Gluten degradation by TAK-062 was evaluated under simulated gastric conditions in vitro and in healthy participants in the phase I study, with and without pretreatment with a proton pump inhibitor. Residual gluten (collected through gastric aspiration in the phase I study) was quantified using R5 and G12 monoclonal antibody enzyme-linked immunosorbent assays. RESULTS: In vitro, TAK-062 degraded more than 99% of gluten (3 g and 9 g) within 10 minutes. In the phase I study, administration of TAK-062 was well tolerated and resulted in a median gluten degradation ranging from 97% to more than 99% in complex meals containing 1-6 g gluten at 20-65 minutes postdose. CONCLUSIONS: TAK-062 is well tolerated and rapidly and effectively degrades large amounts of gluten, supporting the development of this novel enzyme as an oral therapeutic for patients with CeD. (ClinicalTrials.gov: NCT03701555, https://clinicaltrials.gov/ct2/show/NCT03701555.).

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration.

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847-treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

IPEC-J2 rMdr1a, a New Cell Line with Functional Expression of Rat P-glycoprotein Encoded by Rat Mdr1a for Drug Screening Purposes.

The efflux pump P-glycoprotein (P-gp) affects drug distribution after absorption in humans and animals. P-gp is encoded by the multidrug resistance gene (MDR1) gene in humans, while rodents (the most common preclinical animal model) express the two isoforms Mdr1a and Mdr1b. Differences in substrate selectivity has also been reported. Our aim was to generate an in vitro cell model with tight barrier properties, expressing functional rat Mdr1a P-gp, as an in vitro tool for investigating species differences. The IPEC-J2 cell line forms extremely tight monolayers and was transfected with a plasmid carrying the rat Mdr1a gene sequence. Expression and P-gp localization at the apical membrane was demonstrated with Western blots and immunocytochemistry. Function of P-gp was shown through digoxin transport experiments in the presence and absence of the P-gp inhibitor zosuquidar. Bidirectional transport experiments across monolayers of the IPEC-J2 rMDR1a cell line and the IPEC-J2 MDR1 cell line, expressing human P-gp, showed comparable magnitude of transport in both the absorptive and efflux direction. We conclude that the newly established IPEC-J2 rMdr1a cell line, in combination with our previously established cell line IPEC-J2 MDR1, has the potential to be a strong in vitro tool to compare P-gp substrate profiles of rat and human P-gp.

A Novel Derivative of Thioridazine Shows Low Toxicity and Efficient Activity against Gram-Positive Pathogens.

Thioridazine hydrochloride (HCl) has been suggested as a promising antimicrobial helper compound for the treatment of infections with antimicrobial-resistant bacteria. Unfortunately, the therapeutic concentration of thioridazine HCl is generally higher than what can be tolerated clinically, in part due to its toxic side effects on the central nervous system. Therefore, we aimed to synthesize a less toxic thioridazine derivative that would still retain its properties as a helper compound. This resulted in a compound designated 1-methyl-2-(2-(2-(methylthio)-10H-phenothiazin-10-yl)ethyl)-1-pentylpiperidin-1-ium bromide (abbreviated T5), which exhibited low blood-brain barrier permeability. The lowest minimal inhibitory concentration (MIC) against Staphylococcus aureus exposed to the novel compound was reduced 32-fold compared to thioridazine HCl (from 32 µg/mL to 1 µg/mL). The MIC values for T5 against five Gram-positive pathogens ranged from 1 µg/mL to 8 µg/mL. In contrast to thioridazine HCl, T5 does not act synergistically with oxacillin. In silico predictive structure analysis of T5 suggests that an acceptably low toxicity and lack of induced cytotoxicity was demonstrated by a lactate dehydrogenase assay. Conclusively, T5 is suggested as a novel antimicrobial agent against Gram-positive bacteria. However, future pharmacokinetic and pharmacodynamic studies are needed to clarify the clinical potential of this novel discovery.

The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

SEDDS for intestinal absorption of insulin: Application of Caco-2 and Caco-2/HT29 co-culture monolayers and intra-jejunal instillation in rats.

To face the challenges of oral delivery of peptide and protein (P/P) drugs, self-emulsifying drug delivery systems (SEDDSs) containing monoacyl phosphatidylcholine (MAPC), Labrasol (LAB) and medium-chain (MC) monoglycerides as permeation enhancers (PEs) were evaluated for their effect on intestinal absorption of insulin. In this study, insulin was complexed with phosphatidylcholine (SPC) to form an insulin-SPC complex (ins-SPC) with increased lipophilicity. The following three SEDDSs: MCT(MAPC) (MC triglycerides and MAPC included), MCT(RH40) (MC triglycerides and Kolliphor® RH40 included) and LCT(MAPC) (long-chain triglycerides and MAPC included) were loading with ins-SPC (4% or 8% w/w of SPC). Three SEDDSs generated emulsions with droplet sizes between 50 and 470 nm and with zeta potentials between -5 to -25 mV in a simulated intestinal medium. Mucus-secreting Caco-2/HT29-MTX-E12 co-culture and Caco-2 monolayers were used as in vitro cell transport models to investigate insulin permeability. In comparison to insulin HBSS solution, MCT(MAPC) significantly increased the insulin permeability across co-culture and Caco-2 monolayers (2.0-2.5 × 10-7 cm/s). In an intra-jejunal (i.j.) instillation model in rats, MCT(RH40) significantly decreased the rat blood glucose after 0.5 h by 17.0 ±â€¯2.5% and for MCT(MAPC), it was 23.6 ±â€¯10.6%. Furthermore, a lipase inhibitor orlistat was incorporated into MCT(MAPC) to evaluate the effect of lipid digestion on insulin absorption. Results indicated that the incorporation of orlistat did not significantly alter the in vivo insulin absorption. Overall, the SEDDS MCT(MAPC) composed of natural PEs (MAPC and MC glycerides) and synthetic PE (LAB) significantly increased the intestinal absorption of insulin upon i.j. instillation. Although it is not possible to conclude if a single PE is dominating the intestinal absorption of insulin, MCT(MAPC) seems to have the potential for oral insulin delivery.

Dark chocolate as a stable carrier of microencapsulated Akkermansia muciniphila and Lactobacillus casei.

The viability of probiotics is affected by several factors during manufacturing, storage and gastrointestinal tract passage. Protecting the probiotics from harmful conditions is particularly critical for oxygen sensitive species like Akkermansia muciniphila, a bacterium which recently has been proposed as a next-generation probiotic candidate. Previously, we have developed a protocol for microencapsulating A. muciniphila in a xanthan/gellan gum matrix. Here, we report the enhanced survival during storage and in vitro gastric passage of microencapsulated A. muciniphila embedded in dark chocolate. Lactobacillus casei, as a representative species of traditional probiotics, was included in order to compare its behavior with that of A. muciniphila. For A. muciniphila we observed a 0.63 and 0.87 log CFU g-1 reduction during 60 days storage at 4°C or 15°C, respectively. The viability of L. casei remained stable during the same period. During simulated gastric transit (pH 3), microencapsulated A. muciniphila embedded in chocolate showed 1.80 log CFU mL-1 better survival than naked cells, while for L. casei survival was improved with 0.8 log CFU mL-1. In a hedonic sensory test, dark chocolate containing microcapsules were not significantly different from two commercially available chocolates. The developed protocol constitutes a promising approach for A. muciniphila dosage.

Data demonstrating the challenges of determining the kinetic parameters of P-gp mediated transport of low-water soluble substrates.

The presented data are related to the research article entitled "Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates" by Ozgur et al. (2017) [1]. This data report describes the challenges of investigating the concentration-dependent transport of P-glycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, representing P-gp substrates with a relatively low- and high-aqueous solubility, respectively. We present a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin to demonstrate that the maximal donor concentration, which can be reached in the experimental transport buffer, is too low to yield transport data in the saturable range of the Michaelis-Menten relationship. Furthermore, we present data on the bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 cell line and iP-pg cell line in the presence of the selective P-gp inhibitor, zosuquidar/LY335979.

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates.

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm2), immunocytochemistry and low fluxes of the paracellular flux marker [14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [3H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124µM and 111±16pmol·cm-2·min-1 (n=3, total N=6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01µM (n=3, total N=6) and 0.04±0.01µM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.

A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as Km and Vmax, for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC50) and the disassociation constant for an inhibitor/P-gp complex (Ki) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions.

Apomorphine and its esters: Differences in Caco-2 cell permeability and chylomicron affinity.

Oral delivery of apomorphine via prodrug principle may be a potential treatment for Parkinson's disease. The purpose of this study was to investigate the transport and stability of apomorphine and its esters across Caco-2 cell monolayer and their affinity towards chylomicrons. Apomorphine, monolauroyl apomorphine (MLA) and dilauroyl apomorphine (DLA) were subjected to apical to basolateral (A-B) and basolateral to apical (B-A) transport across Caco-2 cell monolayer. The stability of these compounds was also assessed by incubation at intestinal pH and physiological pH with and without Caco-2 cells. Molecular dynamics (MD) simulations were performed to understand the stability of the esters on a molecular level. The affinity of the compounds towards plasma derived chylomicrons was assessed. The A-B transport of intact DLA was about 150 times lower than the transport of apomorphine. In contrast, MLA was highly unstable in the aqueous media leading to apomorphine appearance basolaterally. MD simulations possibly explained the differences in hydrolysis susceptibilities of DLA and MLA. The affinity of apomorphine diesters towards plasma derived chylomicrons provided an understanding of their potential lymphatic transport. The intact DLA transport is not favorable; therefore, the conversion of DLA to MLA is an important step for intestinal apomorphine absorption.