Transport Of Digoxin Research Articles

Digestion of surfactants does not affect their ability to inhibit P-gp-mediated transport in vitro

While various non-ionic surfactants at low concentrations have been shown to increase the transport of P-gp substrates in vitro, in vivo studies in rats have shown that a higher surfactant concentration is needed to increase the oral absorption of e.g. the P-gp substrates digoxin and etoposide. The aim of the present study was to investigate if intestinal digestion of surfactants could be the reason for this deviation between in vitro and in vivo data. Therefore, Kolliphor EL, Brij-L23, Labrasol and polysorbate 20 were investigated for their ability to inhibit P-gp and increase digoxin absorption in vitro. Transport studies were performed in Caco-2 cells, while P-gp inhibition and cell viability assays were performed in MDCKII-MDR1 cells. Polysorbate 20, Kolliphor EL and Brij-L23 increased absorptive transport and decreased secretory digoxin transport in Caco-2 cells, whereas only polysorbate 20 and Brij-L23 showed P-gp inhibiting properties in the MDCKII-MDR1 cells. Polysorbate 20 and Brij-L23 were chosen for in vitro digestion prior to transport- or P-gp inhibiting assays. Brij-L23 was not digestible, whereas polysorbate 20 reached a degree of digestion around 40%. Neither of the two surfactants showed any significant difference in their ability to affect absorptive or secretory transport of digoxin after pre-digestion. Furthermore, the P-gp inhibiting effects of polysorbate 20 were not decreased significantly. In conclusion, the mechanism behind the non-ionic surfactant mediated in vitro P-gp inhibition seemed independent of the intestinal digestion and the results presented here did not suggest it to be the cause of the observed discrepancy between in vitro and in vivo.

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08–0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.

P-glycoprotein-mediated transport in a mucus-supplemented Caco-2 cell model in the presence of different surfactants

The aim of the present study was to investigate if mucus applied to Caco-2 cell monolayers protects cells from high concentrations of surfactants, while still allowing for an identification of the surfactant’s inhibitory effects on P-glycoprotein (P-gp). Two types of porcine mucin and six surfactants (Polysorbate 20 (PS20) and 80 (PS80), Kolliphor EL (Kol. EL) and RH40 (Kol. RH40), Labrafil M 2125 CS (L.fil) and Labrasol (L.sol)) were applied to Caco-2 cells, and TEER, paracellular transport and P-gp mediated digoxin transport was measured. The results showed that 15% porcine mucin type II was incompatible with Caco-2 cell monolayer integrity, resulting in a dramatic drop in monolayer TEER and increased mannitol transport. In contrast, mucin type III was compatible with Caco-2 cell monolayers in the concentration range of 2.5–15% without substantially disturbing barrier properties. The highest concentration of mucin type III impaired the ability of all six surfactants to decrease P-gp mediated digoxin transport. Subsequently lowering the mucin concentration to 5% facilitated adequate protection of cells and enabled e.g., 5% PS20 to inhibit P-gp mediated digoxin transport. Overall, the present work is useful for early-stage permeability investigations on how mucus affects P-gp mediated transport in the presence of formulation excipients.

Selection of an Optimal In Vitro Model to Assess P-gp Inhibition: Comparison of Vesicular and Bidirectional Transcellular Transport Inhibition Assays

The multidrug resistance protein 1 (MDR1) P-glycoprotein (P-gp) is a clinically important transporter. In vitro P-gp inhibition assays have been routinely conducted to predict the potential for clinical drug-drug interactions (DDIs) mediated by P-gp. However, high inter- laboratory and inter-system variability of P-gp IC50 data limits accurate prediction of DDIs using static models and decision criteria recommended by regulatory agencies. In this study, we calibrated two in vitro P-gp inhibition models: vesicular uptake of N-methyl-quinidine (NMQ) in MDR1 vesicles and bidirectional transport (BDT) of digoxin in Lilly Laboratories Cell Porcine Kidney 1 cells overexpressing MDR1 (LLC-MDR1) using a total of 48 P-gp inhibitor and non-inhibitor drugs, and digoxin DDI data from 70 clinical studies. Refined thresholds were derived using receiver operating characteristic (ROC) analysis and their predictive performance was compared with the decision frameworks proposed by regulatory agencies and selected reference. Furthermore, the impact of various IC50 calculation methods and non-specific binding of drugs on DDI prediction was evaluated. Our studies suggest that the concentration of inhibitor based on highest approved dose dissolved in 250 ml divided by IC50(I2/IC50) is sufficient to predict P-gp related intestinal DDIs. IC50 obtained from vesicular inhibition assay with a refined threshold of I2/IC50 {greater than or equal to} 25.9 provides comparable predictive power than those measured by net secretory flux and efflux ratio in LLC-MDR1 cells. We therefore recommend vesicular P-gp inhibition as our preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters than BDT assay. Significance Statement We have conducted comprehensive calibration of two in vitro P-gp inhibition models: uptake in MDR1 vesicles and bidirectional transport in LLC-MDR1 cell monolayers to predict DDIs. Our studies suggest that IC50s obtained from vesicular inhibition with a refined threshold of I2/IC50 ≥ 25.9 provide comparable predictive power than those in LLC-MDR1 cells. We therefore recommend vesicular P-gp inhibition as preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters.

In vitro evaluation of the interaction of the cannabis constituents cannabichromene and cannabichromenic acid with ABCG2 and ABCB1 transporters

Cannabichromene (CBC) and cannabichromenic acid (CBCA) are cannabis constituents currently under evaluation for their therapeutic potential, but their pharmacological properties have not been thoroughly investigated. The most studied ATP-binding cassette (ABC) transporters, ABC subfamily G member 2 (ABCG2) and ABC subfamily B member 1 (ABCB1) limit absorption of substrate drugs in the gut and brain. Moreover, inhibitors of these proteins can lead to clinically significant drug-drug interactions (DDIs). The current study sought to examine whether CBC and CBCA affect ABCB1 and ABCG2 to advance their basic pharmacological characterisation. The plant cannabinoids CBC and CBCA were screened in vitro in a bidirectional transport assay to determine whether they were substrates and/or inhibitors of ABCB1 and ABCG2. Transwell assays with polarized epithelial Madin-Darby Canine Kidney II (MDCK) cells expressing ABCB1 or ABCG2 were used. Samples were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). CBCA was found to be an ABCB1 substrate, but not an ABCG2 substrate. CBC was not a substrate of either transporter. Neither CBCA nor CBC inhibited ABCB1 transport of prazosin or ABCG2 transport of digoxin. In silico molecular docking suggested CBCA binds ABCB1 in the access tunnel and the central binding pocket. CBC, an agent with anticonvulsant, anti-inflammatory and anti-depressant properties, is not a substrate or inhibitor of ABCB1 or ABCG2, which is favourable to its therapeutic development. CBCA is an ABCB1 substrate in vitro which might contribute to its poor absorption. These findings provide important basic pharmacological data to assist the therapeutic development of these cannabis constituents.

Evaluation of the Potency of Anti-HIV and Anti-HCV Drugs to Inhibit P-Glycoprotein Mediated Efflux of Digoxin in Caco-2 Cell Line and Human Precision-Cut Intestinal Slices.

The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug’s ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [3H]-digoxin and compare it with our previous rhodamine123 study. At concentrations of up to 100 µM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug–drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes.

Involvement of P-gp on Reversing Multidrug Resistance Effects of 23-Hydroxybetulinic Acid on Chemotherapeutic Agents.

Betulinic acid (BA) and 23-Hydroxybetulinic acid (23-HBA) are natural products with similar structures, which show a range of biological effects including cytotoxicity activity. The aim of current research was to investigate and evaluate the combinational cytotoxicity of BA and 23-HBA with chemotherapeutic agents in vitro, and to clarify the potential interaction and related mechanism with P-gp. Instead of BA, 23-HBA could increase cytotoxicity of MCF-7/ADR cells to adriamaycin (ADR) and vincristine (VCR). The intracellular accumulation of ADR/VCR in MCF-7/ADR cells was obviously increased in the presence of 23-HBA. Furthermore, 23-HBA could show dose-dependent increase on the transport of VCR and digoxin, which are typical P-gp substrates, in both MDCK-MDR1 and Caco-2 cells. However, the transport of BA and 23-HBA was not influenced by P-gp inhibition in MDCK-MDR1 cells. MDR1 shift assay and molecular docking model suggested that both compounds showed interaction with P-gp, yet the binding affinity and sites are different. In conclusion, 23-HBA could strongly improve the efficacy of anti-tumor agents in multidrug resistance (MDR) cells, which was related to P-gp inhibition. The MDR1 shift assay and molecular docking study further revealed that 23-HBA and BA showed different interaction modes with P-gp.

Differences in P-glycoprotein activity in human and rodent blood\u2013brain barrier assessed by mechanistic modelling

Variation in the efficacy and safety of central nervous system drugs between humans and rodents can be explained by physiological differences between species. An important factor could be P-glycoprotein (Pgp) activity in the blood–brain barrier (BBB), as BBB expression of this drug efflux transporter is reportedly lower in humans compared to mouse and rat and subject to an age-dependent increase. This might complicate animal to human extrapolation of brain drug disposition and toxicity, especially in children. In this study, the potential species-specific effect of BBB Pgp activity on brain drug exposure was investigated. An age-dependent brain PBPK model was used to predict cerebrospinal fluid and brain mass concentrations of Pgp substrate drugs. For digoxin, verapamil and quinidine, in vitro kinetic data on their transport by Pgp were derived from literature and used to scale to in vivo parameters. In addition, age-specific digoxin transport was simulated for children with a postnatal age between 25 and 81 days. BBB Pgp activity in the model was optimized using measured CSF data for the Pgp substrates ivermectin, indinavir, vincristine, docetaxel, paclitaxel, olanzapine and citalopram, as no useful in vitro data were available. Inclusion of Pgp activity in the model resulted in optimized predictions of their brain concentration. Total brain-to-plasma AUC values (Kp,brain) in the simulations without Pgp were divided by the Kp,brain values with Pgp. Kp ratios ranged from 1 to 45 for the substrates investigated. Comparison of human with rodent Kp,brain ratios indicated ≥ twofold lower values in human for digoxin, verapamil, indinavir, paclitaxel and citalopram and ≥ twofold higher values for vincristine. In conclusion, BBB Pgp activity appears species-specific. An age-dependent PBPK model-based approach could be useful to extrapolate animal data to human adult and paediatric predictions by taking into account species-specific and developmental BBB Pgp expression.

S1304 Intestinal Cell Culture Model System to Test the Effect of Natural Products on Drug Transport: Effect of Naringin on the Transport of Digoxin

INTRODUCTION: Certain foods can modify the luminal plasma membrane pumps of intestinal absorptive cells (enterocytes) and significantly change drug absorption. This project aims to create a cell culture model and a series of drug immunoassays to assess the ability of specific natural products to change enterocyte pump activity. Current work focuses upon naringin, a flavonoid that occurs naturally in citrus fruits, especially grapefruit. Both naringin and slow-release naringin microparticles will be assessed for their ability to inhibit the ABC transporter. Inhibition of this pump increases drug absorption in vivo. METHODS: Intestinal enteroids and a cell line derived from a human colonic adenocarcinoma (T84) were obtained from collaborators. A survey of drugs whose absorption was stimulated by ABC pump inhibition was cross-referenced to commercially available antibodies, and digoxin was chosen as the model drug. A digoxin-bovine serum albumin (BSA) conjugate was prepared by NaIO4 oxidation of the glucose moiety in digoxin. The newly generated aldehydes were then reacted with the lysine residues on the BSA to create a Schiff base, which was then reduced to stabilize the covalent bond. Our in-house synthesized conjugate was then compared to commercially available digoxin-BSA conjugate as a capture reagent in a competitive ELISA for digoxin. RESULTS: The competitive ELISA using in-house made digoxin-BSA conjugate was ∼ 10-fold more sensitive than the assay formatted with the commercial conjugate. The limit of detection of the final ELISA was 0.4 ng/mL and the IC50 was 5.0 ng/mL. The assay was not affected by the addition of up 25% of culture media or ethanol (used as a solvent for digoxin). CONCLUSION: An assay to detect the levels of digoxin in culture media has been optimized. The next steps will be to grow intestinal cell monolayers on transwell plates and measure any increase in digoxin reaching the basolateral side in the presence of naringin. This could ultimately lead to a novel model system to test food-drug interactions that affect intestinal transporter pump activity.

One molecule two goals: A selective P-glycoprotein modulator increases drug transport across gastro-intestinal barrier and recovers doxorubicin toxicity in multidrug resistant cancer cells.

In the present study a series of tetrahydroisoquinoline derivatives were synthesized and evaluated for their activity towards three ABC transporters, P-gp, MRP1 and BCRP. The compounds proved to be selective against P-gp. One of them, 8b, displayed activity in the nanomolar range (EC50=94nM). Thus, compound 8b was tested for its ability to restore the cytotoxic activity of a well-known anti-cancer agent and P-gp substrate, doxorubicin, as first proof of concept. Moreover, compound 8b was also tested in an invitro model of competent gastro-intestinal (GI) barrier (Caco-2 cells) for its ability to inhibit P-gp, present on luminal side, and increase the apical-to-basolateral transport of several structurally uncorrelated drugs, belonging to different therapeutic areas but actively excreted by P-gp. Notably the transport of the drugs across the GI barrier was increased by a concentration of 8b devoid of toxicity and of perturbing effects on barrier function. An invitro simulated digestion process was set up: interestingly the effect of 8b on the transport of digoxin was preserved also after the simulated digestion process. This result may suggest 8b as a safe and effective P-gp modulator that can increase the bioavailability of a wide spectrum of drugs administered per os, improving their transport across the GI barrier.

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.

MRP2-mediated transport of etoposide in MDCKII MRP2 cells is unaffected by commonly used non-ionic surfactants.

The aim of the present study was to investigate the ability of non-ionic surfactants to inhibit MRP2-mediated transport in vitro in MDCKII MRP2 cells. Transport studies across MDCKII MRP2 cell monolayers were performed using 3H-etoposide and 3H-digoxin. 19 different non-ionic surfactants, including several polysorbates (PS), cremophor EL, vitamin E-TPGS, and n-nonyl β-D-glucopyranoside (NG), were investigated. Barrier function of the cells was investigated measuring TEER and transport of 14C-glycine. The amount of isotope was quantified using liquid scintillation counting. In MDCKII MRP2 cells a polarized transport of etoposide and digoxin in the secretory (basolateral to apical) direction with efflux ratios of 5.5 ± 0.7 and 18.5 ± 4.2, respectively, was measured. P-gp inhibitors such as valspodar and zosuquidar did not affect etoposide transport, and furthermore PS20 decreased secretory transport of digoxin, but not of etoposide. Transport of etoposide was therefore mainly MRP2-mediated and used as a probe to investigate pharmaceutical excipients. Non-ionic surfactants did not modulate etoposide transport across intact cell monolayers of MRP2 overexpressing MDCKII cells, although preliminary studies suggest that most were able to alter MRP2-mediated efflux of the fluorescent 5-chloromethylfluorescein (CMF). In conclusion, etoposide transport across MDCKII MRP2 cells was modulated by cyclosporin A, an inhibitor of MRP2 and P-gp, but not by specific P-gp inhibitors (valspodar and zosuquidar), which suggests that etoposide transport is primarily influenced by MRP2. In addition, commonly used non-ionic surfactants did not decrease MRP2-mediated etoposide transport in MDCKII MRP2 cells. These results suggest that etoposide transport in MDCKII MRP2 cells is a model system to investigate MRP2 interactions, and that surfactants may not have a large potential for increasing oral bioavailability of drugs through inhibition of MRP2 transport activity.

In Vitro Profiling for Potential Cytochrome P450 and P-Glycoprotein–Mediated Drug–Drug Interaction By Maribavir (Shp620)

Introduction Maribavir is a potent and orally bioavailable antiviral with a novel mechanism of action against cytomegalovirus (CMV). The efficacy and safety of maribavir for treatment of CMV infections are being evaluated in ongoing Phase 3 trials. Characterization of drug–drug interaction (DDI) properties of maribavir is necessary in informing potential DDIs with co-medications. Objective To evaluate the potential cytochrome p450 and p-glycoprotein–mediated drug–drug interaction by maribavir. Methods Cytochrome P450 (CYP) reversible inhibition: Probe substrates of CYPs were incubated with human liver microsomes (HLM) and maribavir. Percent remaining CYP activity was plotted against maribavir concentrations for IC50 determination. Time-dependent inhibition (TDI) for CYPs: After pre-incubation with maribavir for varying durations, percent remaining CYP activity in HLM was measured. The concentration at half-maximal inactivation (KI) and maximum inactivation rate constant (kinact) were calculated. CYP induction: After treating human hepatocytes with maribavir for three days, CYP mRNA expression was plotted against maribavir concentrations to determine half-maximal induction concentration (EC50) and maximal fold induction (Emax). P-glycroprotein (P-gp) inhibition: Bi-directional transport of digoxin in Caco-2 cells with maribavir was measured and IC50 calculated by plotting percent transporter activity against maribavir concentration. Results Maribavir is not a reversible inhibitor of CYP2A6, CYP2B6, CYP2C8, CYP2D6, CYP2E1, or CYP3A4 and is a weak inhibitor of CYP1A2, CYP2C9, and CYP2C19 (IC50 40, 18, and 35 µM). Maribavir is not a TDI of CYP1A2, CYP2C8, CYP2C9, CYP2C19, or CYP2D6 but a weak TDI for CYP3A (KI 41.2 μM, kinact 0.0117 min−1). Maribavir is not an inducer of CYP1A2 or CYP2B6 mRNA but is a weak, possibly donor-dependent inducer of CYP3A4 mRNA. Maribavir is a weak inhibitor of P-gp (IC50 33.7 µM). Conclusions Maribavir is not expected to affect exposures of substrates of major CYPs or P-gp when co-administered at therapeutic dose of 400 mg BID, based on its estimated maximal unbound plasma concentration (Cmax,u) of 0.74 μM in transplant patients with CMV infection.

The inhibitory effects of mitragynine on P-glycoprotein in vitro.

Mitragynine is a major component isolated from Mitragyna speciosa Korth or kratom, a medicinal plant known for its opiate-like and euphoric properties. Multiple toxicity and fatal cases involving mitragynine or kratom have been reported but the underlying causes remain unclear. P-glycoprotein (P-gp) is a multidrug transporter which modulates the pharmacokinetics of xenobiotics and plays a key role in mediating drug-drug interactions. This study investigated the effects of mitragynine on P-gp transport activity, mRNA, and protein expression in Caco-2 cells using molecular docking, bidirectional assay, RT-qPCR, Western blot analysis, and immunocytochemistry techniques, respectively. Molecular docking simulation revealed that mitragynine interacts with important residues at the nucleotide binding domain (NBD) site of the P-gp structure but not with the residues from the substrate binding site. This was consistent with subsequent experimental work as mitragynine exhibited low permeability across the cell monolayer but inhibited digoxin transport at 10μM, similar to quinidine. The reduction of P-gp activity in vitro was further contributed by the downregulation of mRNA and protein expression of P-gp. In summary, mitragynine is likely a P-gp inhibitor in vitro but not a substrate. Hence, concurrent administration of mitragynine-containing kratom products with psychoactive drugs which are P-gp substrates may lead to clinically significant toxicity. Further clinical study to prove this point is needed.

Transport characteristics of Shuxiong prescription across Caco-2 cell monolayer model

Shuxiong prescription (Notoginseng Radix et Rhizoma, Chuanxiong Rhizome and Carthami Flos) has the function of activating blood circulation to dissipate blood stasis, activating meridians to stop pain. This paper was mainly aimed to discuss the transport characteristics of Shuxiong prescription across Caco-2 cell monolayer. Safe concentration range of Shuxiong prescription against Caco-2 cell monolayer model was determined by MTT assay. The mechanism of Shuxiong prescription bidirectional transport was investigated by Caco-2 cell monolayer model. The apparent permeability coefficient Papp of digoxin was determined by high performance liquid chromatography (HPLC). The test results showed that the Papp of extract from Notoginseng Radix et Rhizoma, Chuanxiong Rhizome, Carthami Flos, Chuanxiong Rhizome+Carthami Flos and Shuxiong prescription transport from apical (AP) side to basolateral (BL) side was (3.12±0.73)×10⁻⁶, (2.58±0.41)×10⁻⁶, (4.97±0.64)×10⁻⁶, (4.63±0.57)×10⁻⁶, (5.79±0.68)×10⁻⁶ cm·s⁻¹, respectively, indicating that the transport of digoxin across Caco-2 cell monolayer model was active absorption, and the P-gp protein took part in the process. Chuanxiong Rhizome could significantly decrease the transport of digoxin from BL→AP(P<0.01) and increase its transport from AP→BL(P<0.05) significantiy. After the addition of Shuxiong prescription, the transport of digoxin from BL→AP was significantly inhibited(P<0.01). The results suggested that the extract of safflower had no effect on P-gp transport, nor on the independence diffusion of digoxin. The transport of digoxin could be degraded by the extract of Chuanxiong Rhizome and the extract of Shuxiong prescription from BL→AP(P<0.01), significantly; pseudo-ginseng had no effect on the independence diffusion of digoxin; the extract of safflower+Chuanxiong Rhizome had the same experimental result as Chuanxiong Rhizome extract.

Use of a Caco-2 permeability assay to evaluate the effects of several Kampo medicines on the drug transporter P-glycoprotein

In modern medical care in which Kampo and Western drugs are often combined, it is extremely important to clarify drug–drug interaction (DDI) to ensure safety and efficacy. However, there is little evidence of DDI in Kampo medicines. Therefore, as part of our studies to clarify the DDI risk for Kampo medicines, we evaluated the effects of five Kampo medicines [yokukansan (YKS), rikkunshito (RKT), shakuyakukanzoto (SKT), hangeshashinto (HST), and goshajinkigan (GJG)] that are widely used in Japan, on drug transporter P-glycoprotein (P-gp) using a Caco-2 permeability assay. These Kampo medicines inhibited the P-gp transport of digoxin through a Caco-2 cell monolayer. The IC50 values were 1.94–10.80 mg/ml. Of the five Kampo medicines, YKS showed the strongest inhibition (IC50 = 1.94 mg/ml), which was attributed to Uncariae Uncis Cum Ramulus. Unfortunately, we could not find the active ingredients responsible for its action. Finally, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance issued by the Japanese Ministry of Health, Labor, and Welfare and the US Food and Drug Administration. The Igut/IC50 values for the five Kampo medicines were ≤3.4. As these values were <10, they were evaluated as having a weak P-gp inhibitory effect that does not require further verification in humans, suggesting that the DDI risk due to P-gp inhibition for these Kampo medicines is low. The results should provide useful clinical information on the safety and efficacy of the combined use of Kampo and Western medicines.

Derivation of a System-Independent Ki for P-glycoprotein Mediated Digoxin Transport from System-Dependent IC50 Data.

It has been previously demonstrated that IC50 values for inhibition of digoxin transport across confluent polarized cell monolayers are system-dependent. Digoxin IC50 data from five laboratories participating in the P-glycoprotein (P-gp) IC50 Initiative, using Caco-2, MDCKII-hMDR1 or LLC-PK1-hMDR1 cells, were fitted by the structural mass action kinetic model for P-gp-mediated transport across confluent cell monolayers. We determined their efflux-active P-gp concentration [T(0)], inhibitor elementary dissociation rate constant from P-gp (krQ), digoxin basolateral uptake clearance (kB), and inhibitor binding affinity to the digoxin basolateral uptake transporter (KQB). We also fitted the IC50 data for inhibition of digoxin transport through monolayers of primary human proximal tubule cells (HPTCs). All cell systems kinetically required a basolateral uptake transporter for digoxin, which also bound to all inhibitors. The inhibitor krQ was cell system-independent, thereby allowing calculation of a system-independent Ki. The variability in efflux-active P-gp concentrations and basolateral uptake clearances in the five laboratories was about an order of magnitude. These laboratory-to-laboratory variabilities can explain more than 60% of the IC50 variability found in the principal component analysis plot in a previous study, supporting the hypothesis that the observed IC50 variability is primarily due to differences in expression levels of efflux-active P-gp and the basolateral digoxin uptake transporter. HPTCs had 10- to 100-fold lower efflux-active P-gp concentrations than the overexpressing cell lines, whereas their digoxin basolateral uptake clearances were similar. HPTC basolateral uptake of digoxin was inhibited 50% by 10 μM ouabain, suggesting involvement of OATP4C1.

Natural flavonoids silymarin and quercetin improve the brain distribution of co-administered P-gp substrate drugs.

P-glycoprotein (P-gp), a well known efflux transporter in the blood brain barrier inhibits the uptake of substrate drugs into brain. The main aim of this study is to evaluate the effect of natural product based P-gp inhibitors on brain penetration of various CNS drugs which are P-gp substrates. In this study, we have evaluated the inhibitory effects of natural bioflavonoids (quercetin and silymarin) on P-gp by using digoxin and quinidine as model P-gp model substrate drugs. In vitro inhibitory effects were evaluated in Caco-2 cell lines using digoxin as a model drug and in vivo P-gp inhibiting effect was evaluated in mice model using quinidine as model drug. The accumulation and bidirectional transport of digoxin in Caco-2 cells was determined in presence and absence of quercetin and silymarin. Elacridar was used as standard P-gp inhibitor and used to compare the inhibitory effects of test compounds. The apical to basolateral transport of digoxin was increased where as basolateral to apical transport of digoxin was decreased in concentration dependent manner in the presence of elacridar, quercetin and silymarin. After intravenous administration of P-gp inhibitors, brain levels of quinidine were estimated using LC-MS method. Increased brain uptake was observed with quercetin (2.5-fold) and silymarin (3.5-fold). Though the brain penetration potential of P-gp substrates was lower than that observed in elacridar, both quercetin and silymarin improved plasma quinidine levels. Caco-2 permeability studies and brain uptake indicate that both quercetin and silymarin can inhibit P-gp mediated efflux of drug into brain. Our results suggest that both silymarin and quercetin could potentially increase the brain distribution of co-administered drugs that are P-gp substrates.

Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells

1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells.2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor.3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp.4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

Polysorbate 20 increases oral absorption of digoxin in wild-type Sprague Dawley rats, but not in mdr1a(-/-) Sprague Dawley rats

The aim was to investigate the ability of polysorbate 20 to alter oral digoxin absorption in vitro and drug exposure in vivo via modulation of transporter mediated efflux. Transport studies were performed in MDCKII-MDR1 and Caco-2 cells using 3H-digoxin. Pharmacokinetic studies were performed in wild type and mdr1a deficient Sprague Dawley rats. 3H-digoxin was quantified using liquid scintillation counting. The results showed an increased absorptive transport and a reduced secretory transport in MDCKII-MDR and Caco-2 cells as a function of polysorbate 20 concentrations. The secretory transport (B–A) of digoxin was reduced by 50% at lower polysorbate 20 concentrations than required to increase the absorptive transport (A-B). In vivo, the oral bioavailability of digoxin in wild type animal was increased by 10–25% (w/v) polysorbate 20. In mdr1a deficient Sprague Dawley rats 25% (w/v) polysorbate 20 did not alter the absorption of digoxin after oral administration, but digoxin exposure was significantly different between wild type and mdr1a deficient rats. In conclusion, polysorbate 20 increased absorptive transport across Caco-2 cell monolayers and in vivo in rats in a concentration dependent manner, most likely via inhibition of P-gp rather than through solubilization of digoxin.