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Interaction of food additives with intestinal efflux transporters Noora Sjöstedt, Feng Deng, Oskari Rauvala, Tuomas Tepponen, and Heidi Kidron Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00563 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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Molecular Pharmaceutics

1

Interaction of food additives with intestinal efflux transporters

Noora Sjöstedt 1, #, Feng Deng 1, #, Oskari Rauvala 1, Tuomas Tepponen 1, Heidi Kidron 1*

1

Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014

University of Helsinki, Helsinki, Finland

#

Authors contributed equally to this work

* Corresponding author: Dr Heidi Kidron Mailing address: P.O. Box 56 (Viikinkaari 5E), 00014 University of Helsinki, Finland Tel: +358 2941 59518 E-mail: [email protected]

ACS Paragon Plus Environment


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2 GRAPHICAL ABSTRACT

ABSTRACT Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2) and P-glycoprotein (P-gp) are ABC transporters that are expressed in the intestine, where they are involved in the efflux of many drugs from enterocytes back into the intestinal lumen. The inhibition of BCRP, MRP2 and P-gp can result in the enhanced absorption and exposure of substrate drugs. Food additives are widely used by the food industry to improve the stability, flavor and consistency of food products. Although they are considered safe for consumption, their interactions with intestinal transporters are poorly characterized. Therefore, in this study, selected food additives, including preservatives, colorants and sweeteners, were studied in vitro for their inhibitory effects on intestinal ABC transporters. Among the studied compounds, several colorants were able to inhibit BCRP and MRP2, whereas P-gp was fairly insensitive to inhibition. Additionally, one sweetener was identified as a potent inhibitor of BCRP. Dose-response studies revealed that the IC50 values of the inhibitors were lower than the estimated intestinal concentrations after the consumption of beverages containing food colorants. This suggests that there is potential for previously unrecognized transporter-mediated food additive - drug interactions.

KEY WORDS ABC transporter, ABCB1, ABCC2, ABCG2, p-glycoprotein, multidrug-resistance associated protein 2, breast cancer resistance protein, food-drug interaction


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3 ABBREVIATIONS BCRP

breast cancer resistance protein (ABCG2)

CDCF

5(6)-carboxy-2’,7’-dichlorofluorescein

EFSA

European Food Safety Authority

JECFA

Joint Food and Agriculture Organization and World Health Organization (FAO/WHO) Expert Committee on Food Additives

LY

Lucifer yellow

MRP2

multidrug resistance associated protein 2 (ABCC2)

P-gp

P-glycoprotein (ABCB1)

1

INTRODUCTION

Food additives are substances added into foodstuffs to enhance their properties. For instance, food colorants are used to improve the appearance of food, the sweet taste in beverages can be obtained without sugar by adding sweeteners and the shelf life of food products can be extended with antimicrobial preservatives. Additives are present in almost any industrial food products, often in combinations and sometimes in high concentrations. For example, the concentration of some sweeteners in beverages could reach millimolar concentrations if used at the highest permitted levels.1 Like any substances intended for human (or animal) consumption, food additives are required to follow certain safety regulations. In the European Union, the European Food Safety Authority (EFSA) evaluates and approves the safety of food additives. All EU-approved food additives have a so-called E-number, which is required in food labelling. 2 The Joint Food and Agriculture Organization and World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) also provide recommendations on the safety and the maximum use levels of food additives for the harmonization of international food standards. Animal studies are usually needed to ensure the safety of new food additives by determining possible toxic effects and identifying no-observed-adverse-effects levels for any adverse effects. 3

Acceptable daily intake levels are set based on the safety studies and used with exposure data for risk



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4 assessment and these describe the amount of additive that can be consumed daily over a lifetime with no harmful effects.

Although food additives on the market are deemed safe for consumption with few adverse effects, it is possible that they could affect the function of the proteins they encounter during their transit through the body. The intestinal tissue is one of the first barriers for ingested food additives. Embedded in the cell membranes of enterocytes, ATP-binding cassette (ABC) transporters use ATP as energy to extrude xenobiotics, including drugs, from cells. Breast cancer resistance protein (BCRP, ABCG2), multidrug resistance associated protein 2 (MRP2, ABCC2) and P-glycoprotein (P-gp, ABCB1) are the major ABC transporters expressed in the apical membranes of the enterocytes, where they act as gatekeepers for absorption.4-8 In addition to their intestinal expression, BCRP, MRP2 and P-gp are present in other tissues such as blood-brain barrier, liver and kidney and they are involved in the excretion of many endogenous metabolites, as for instance conjugated estrogens,9, 10 bilirubin conjugates11 and uric acid.12 Their presence in these physiological barriers and excretory organs can be of importance to the pharmacokinetics of their drug substrates. These include, for example, mitoxantrone,13 sulfasalazine14 and rosuvastatin15 for BCRP; methotrexate,16 olmesartan17 and pravastatin18 for MRP2 and digoxin,19 loperamide20 and paclitaxel for P-gp.21 Furthermore, numerous drugs and other xenobiotics are able to inhibit these transporters and the inhibitory specificity for these transporters is often overlapping.22, 23 The inhibition of these transporters is of interest, because inhibition can increase the exposure and the potential adverse effects of their substrate drugs.

The significance of BCRP on oral bioavailability is demonstrated by the increased systemic exposure to orally dosed drugs in patients harboring a decreased function genetic variant (p.Q141K) of BCRP.24-27 Although human data is lacking, evidence from animal and in vitro studies suggest that MRP2 could also have a role in the absorption of drugs.28 Data on verified BCRP or MRP2-mediated drug-drug interactions (DDIs) is, however, rare.


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5 As an example, Martin et al.

29

recently reported a near 2-fold increase in rosuvastatin exposure and peak

concentrations following concomitant administration with tyrosine kinase inhibitor fostamatinib, a potent inhibitor of BCRP. Despite the lack of DDIs ascribed to BCRP and MRP2, the large number of BCRP and MRP2 inhibitors identified in vitro suggests that there is the potential for many more DDIs that are yet to be identified. P-gp, however, is involved in many DDIs that have been reported to affect the oral bioavailability of drugs such as digoxin,30 paclitaxel,31 docetaxel,32 tacrolimus33 and talinolol.34

In addition to DDIs, interactions of different foods or food components, such as fruit juice and tea flavonoids, with intestinal ABC transporters have been studied.35-37 There is also evidence that some drug excipients can inhibit the activity of these transporters.38 While some of the previously studied excipients or food components may be used as food additives, food additives in general have received little attention concerning drug transporter inhibition or induction. Nevertheless, a few indications of interaction do exist. For example, polysorbate 20 (Tween 20), used as an excipient and food additive, increased topotecan AUC by 1.8 fold in mice through the inhibition of BCRP39 and digoxin AUC by 1.5 fold in wild-type Sprague Dawley rats.40 However, the most effective doses used in these studies, greatly exceed the acceptable daily intake level of polysorbate 20 recommended by EFSA.41 On the other hand, titanium dioxide nanoparticles used to produce a white color were shown to increase the expression of several ABC transporters, including MRP2 and BCRP, in the Caco-2 intestinal cell model.42

To begin to bridge the gap in the knowledge concerning food additive - transporter interactions, we selected 25 food additives and examined whether they could modulate the transport activity of BCRP, MRP2 and P-gp in vitro. The selected compounds included natural and artificial sweeteners and food colorants as well as preservatives. Using the vesicular transport assay, we show that several food colorants can inhibit BCRP and



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6 MRP2. Based on the relationship between the IC50 values calculated in this study and the concentrations of the food additives reported in beverages, transporter inhibition in vivo cannot be ruled out.

2

MATERIALS AND METHODS

Materials A set of 25 food additives, including 10 coloring agents, 6 sweeteners and 9 preservatives, were obtained from Sigma-Aldrich (USA) and dissolved at 10 mM in DMSO. HyClone SFX Insect medium (Fisher Scientific) and fetal bovine serum (Gibco) for Sf9 cell culture were purchased from Thermo Fisher Scientific (USA). Cholesterol/RAMEB complex, used for the cholesterol loading of BCRP vesicles, was from Cyclolab Ltd (Hungary). Formic acid and acetonitrile for HPLC analysis, as well as all other reagents, were from Sigma-Aldrich if not otherwise specified. All water used was of ultrapure grade. Dr. Douglas Ross (University of Maryland School of Medicine, USA) and Dr. Piet Borst (Netherlands Cancer Institute, Netherlands) kindly provided the human BCRP and MRP2 cDNAs and recombinant baculovirus preparations were constructed from these as described previously23, 43 using the Bac-to-bac expression protocol (Invitrogen, USA). The human P-gp cDNA was a kind gift from Dr. Branimir Sikic (Stanford University School of Medicine, USA). The cDNA was cloned from the pcDNA3-MDR1.4 plasmid by digesting with BamHI and XhoI and the cleaving the 5’UTR sequence with AccII before ligation into the pFastBac1-BamHI(blunt)-XhoI site. The pFastBac-MDR1 plasmid was further used to generate the recombinant baculovirus as with BCRP and MRP2.

Vesicular transport assay Membrane vesicles from Sf9 cells transduced to express either BCRP, MRP2 or P-gp were produced and BCRP and P-gp vesicles loaded with cholesterol as described in Sjostedt et al.

23

. The BCRP and MRP2 vesicular

transport assays were performed as reported previously23 using probe substrates Lucifer yellow (LY) at 50 µM


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7 and 5(6)-carboxy-2,7-dichlorofluorescein (CDCF) at 5 µM, for BCRP and MRP2, respectively. The reaction time of LY and CDCF was 10 and 30 min, respectively. For P-gp, 2 µM N-methyl-quinidine (NMQ)(Solvo Biotechnology, Hungary) was used and the reaction was terminated after 3 min.

The initial testing of the food additives was performed at 50 µM. All assays were carried out in triplicates in a 96-well plate format. Positive inhibition controls were 50 µM sulfasalazine for BCRP, 100 µM benzbromarone for MRP2 and 100 µM verapamil for P-gp. Potential inhibitors, i.e. compounds resulting in more than 50% inhibition at 50 µM, were subjected to further dose-response studies. The accumulation of substrates in the vesicles was analyzed in BCRP and MRP2 experiments by fluorescent detection using a Varioskan Flash plate reader (Thermo Fisher Scientific, Finland). CDCF was detected using excitation and emission wavelengths 510 nm and 535 nm, respectively. The corresponding wavelengths for LY were 430 nm and 538 nm. The accumulation into P-gp vesicles was studied by HPLC after lysis and elution of the vesicle contents using 3:1 methanol:H2O with 0.1% formic acid.

The analysis of NMQ was performed with high-performance liquid chromatography (HPLC) (Agilent 110 series, Agilent Technologies, USA), using a Poroshell 120 EC-C18 column with a size of 4.6 x 100 mm and 2.7 micron particle size. The temperature of the column was kept at 40 ˚C and the flow rate of eluent was 1 ml/min. 0.1 % formic acid was used as eluent A and acetonitrile as eluent B. The following method was used for analysis: 0-1 min (15 % B), 1-3 min (15 % -> 30 % B), 3-4 min (30 % -> 90 % B), 4-6 min (90 % -> 30 % B). The injection volume of samples was 10 µl and the retention time for NMQ was 2.6 minutes. A fluorescence detector was used for detection with the excitation and emission of NMQ at 248 nm and 442 nm, respectively.



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8 Assay interference studies Fluorescence based assays are liable to false results due to the interference of test compounds. Therefore, the intrinsic fluorescence or quenching of probe fluorescence by the food additives was evaluated as described previously.23 Briefly, to mimic the measurement step, food additives (or DMSO in control) were diluted in 0.1 M NaOH (CDCF samples) or a 1:1 mixture of 0.1 M NaOH and 0.1 M HCl (LY samples). The concentration was set to the maximal achievable concentration, assuming all test compound (100%) used in the assay is present in the final elute. CDCF or LY were added to the wells and fluorescence was measured as described above. Fluorescence signals were compared to the DMSO control with CDCF or LY. A subset of potential inhibitory food additives with possible interference were studied at additional lower retained amounts, namely 5% and 20% of the initial assay concentration of 50 µM. To rule out any false inhibition caused by the aggregation of the food additives in the assay, the possible aggregation of identified inhibitors was evaluated in assay conditions (omitting membrane vesicles) with a Nepheloskan Ascent nephelometer (Thermo Fisher Scientific Inc., USA).

Data analysis ATP-dependent transport was calculated by subtracting the transport in the absence of ATP from the measured transport in the presence of ATP. Results are presented as relative transport values (%), which were obtained by comparing the uptake of probe substrate in the presence of food additive to the control containing only the vehicle (DMSO). GraphPad Prism 6.05 (GraphPad Software Inc., USA) was used to calculate the concentrations required for 50% inhibition (IC50) by fitting data from the inhibition studies to the four-parameter logistic curve: = +

− [] 1 + ( )


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9 where Minimum and Maximum correspond to the plateaus of minimal and maximal relative transport (%), [I] is the concentration of inhibitor (i.e. food additive) and h is the Hill slope, which describes the steepness of the slope.

3

RESULTS

The inhibition of BCRP, MRP2 and P-gp transport activity by the food additives was initally tested at 50 µM using the vesicular transport assay (Figure 1, Supporting Information Table S1). None of the tested preservatives inhibited any of the studied transporters. Neohesperidin dihydrochalcone (DC) was the only sweetener identified as a potential inhibitor. It had a clear inhibitory effect on BCRP transport, but almost no effect on P-gp and MRP2. However, the group of food colorants contained several potential inhibitors of BCRP and MRP2. Azo dyes, in particular, were identified as an important group, because all of these tested dyes inhibited at least one of the two transporters. Altogether out of the colorants, allura red AC, brilliant black BN, carmoisine, chlorophyllin sodium copper complex and curcumin inhibited both BCRP and MRP2. Additionally, sunset yellow FCF and tartrazine inhibited BCRP and brilliant blue FCF inhibited MRP2 transport. In the case of P-gp, curcumin was the only additive that exhibited inhibitory activity.

To rule out false inhibition, the fluorescence of the probe substrate in the presence of the food additives was examined. At the initial maximum concentration, eight compounds changed the fluorescence of LY more than 20% and nine compounds quenched CDCF fluorescence more than 20% (Figure 2). Sorbic acid was the only non-colored compound out of the interfering compounds. As the worst-case scenario of total retention is unlikely, fluorescence quenching was further tested at a lower concentration for compounds identified as inhibitors in the transport assay. Assuming lower retained amounts (5% and 20%), the interference of the additives, excluding brilliant black, chlorophyllin sodium copper complex and curcumin, was less than 10%



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10 (Figure 3). Chlorophyllin sodium copper complex quenched the CDCF signal around 20% at both concentrations, whereas the interference of brilliant black and curcumin was only around 10% at the lower retained amount tested. False or indirect inhibition by the aggregation of the potential BCRP, MRP2 and P-gp inhibitors was also tested at assay conditions at 50 µM using nephelopmetry (data not shown). Curcumin and chlorophyllin showed signs of aggregation and were therefore excluded from further studies. Dose-response studies were performed and IC50 values (concentration required for 50% inhibition) were calculated for the rest of the inhibitors identified in the initial studies (Figure 4, Table 1). Brilliant blue was the most potent MRP2 inhibitor with an IC50 of 3.22 (2.31 – 4.49) µM. The strongest inhibitor of BCRP transport was neohesperidin DC (IC50 = 0.86 µM, 95% CI = 0.622 – 1.19 µM), followed by carmoisine and brilliant black with IC50 values of 5.38 (4.50 – 6.42) and 6.06 (5.00 – 7.36) µM, respectively.

Table 1. Calculated IC50 values and 95% confidence intervals (95% CI) for the inhibition of MRP2 and BCRP transport by selected food additives. BCRP

MRP2

IC50 (µM)

95% CI

IC50 (µM)

95% CI

Allura red AC

13.3

11.6 – 15.2

20.0

11.8 – 33.9

Brilliant black BN

6.06

5.00 – 7.36

7.21

4.25 – 12.2

Brilliant blue FCF

N.A.

N.A.

3.22

2.31 – 4.49

Carmoisine

5.38

4.50 – 6.42

29.0

17.9 – 47.0

Neohesperidin DC

0.860

0.622 - 1.19

N.A.

N.A.

Sunset yellow FCF

10.6

8.56 – 13.1

N.A.

N.A.

Tartrazine

23.5

15.5 – 35.5

N.A.

N.A.

N.A. Not applicable

4

DISCUSSION


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11 Out of the 25 food additives that were tested, several compounds showed inhibitory activity against BCRP and MRP2 with low micromolar IC50 values, while only one of the compounds decreased P-gp transport activity more than 50% at 50 µM. The most potent inhibitor was neohesperidin DC, a sweetener, which inhibited BCRP with an IC50 value of 0.86 µM. The preservatives had no inhibitory activity towards any of the transporters, but several BCRP and MRP2 inhibitors were identified among the food colorants. In addition to the synthetic colorants, two naturally derived dyes, curcumin and chlorophyllin sodium copper complex, inhibited both transporters in the initial testing. Curcumin was also the only compound to inhibit P-gp in this study. However, curcumin and chlorophyllin sodium copper complex were excluded from further studies due to potential assay interference. Nevertheless, our observations of inhibition are in line with previous reports, where curcumin was observed to inhibit rhodamine 123 transport in P-gp overexpressing multidrug resistant human cervical carcinoma cells44 and MRP2 in Sf9-MRP2 vesicles.45 In addition, curcumin was reported to increase the AUC of sulfasalazine 3.2 fold in healthy volunteers through the inhibition of intestinal BCRP.46 The inhibition of BCRP by the chlorophyllin sodium copper complex is likely, since pheophorbide A, a catabolic product of chlorophyll, is a BCRP substrate.47 Likewise, MRP2 is known to transport bilirubin conjugates and coproporphyrins, which have a porphyrin structure similar to chlorophyllin.11, 48

The molecular properties of the preservatives can help to explain the lack of BCRP, MRP2 and P-gp inhibition observed for food preservatives and sweeteners in this study (Supporting Information Table S1). The inhibitors of ABC transporters tend to be lipophilic with aromatic properties and are generally larger than noninhibitors,22, 49 whereas the preservatives are small in size (MW < 200 g/mol) and contain a maximum of one ring in their structure. The amount of hydrogen bond donors and acceptors is also much lower than in the identified inhibitors, partly due to the size differences of the molecules. Although LogD7.4 is known to be an important descriptor for inhibitory activity against ABC transporters,22 the calculated LogD7.4 values of the azo dyes identified here as inhibitors are surprisingly low, ranging from - 6.63 to - 0.18. The calculated LogD7.4 of



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12 neohesperidin DC is higher, 2.59. Surprising is also the similarity in inhibition patterns of MRP2 and BCRP. Although the inhibitor specificity is overlapping, the number of compounds inhibiting MRP2 tends to be considerably lower than for other ABC transporters.22, 23, 50 The similarity might, however, be explained by the narrow chemical space of the compounds tested here. Regarding P-gp, it was unexpected that only one P-gp inhibitor was found despite the previously observed considerable overlap observed in P-gp and BCRP inhibitors.22

The interaction of sweeteners with ABC transporters could potentially have a high impact, as the use of lowcalorie sweeteners is ever popular in the age of increasing health burden from obesity and diabetes. Neohesperidin DC, a dihydrochalcone derivative of the flavanone neohesperidin, a flavonoid found in citrus fruits,51 was the only sweetener identified as an inhibitor in this study. The fact that neohesperidin DC inhibited BCRP is not surprising, because a plethora of flavonoids are known to decrease BCRP activity.35, 52 Additionally, several chalcones have shown potential as BCRP inhibitors.53 Since neohesperidin DC is approximately 1500 times sweeter than sucrose, it can be used in small amounts, thus increasing its safety and decreasing interaction potential. However, reported concentrations of neohesperidin DC in hard candies and a soft drink are 11 ± 10 mg/kg and 3.02 mg/l, respectively,54, 55 which are higher than the IC50 of neohesperidin DC determined in this study (0.86 µM ≈ 0.53 mg/l).

The current use of neohesperidin DC is scarce compared to older low-calorie sweeteners such as acesulfame-K and aspartame,54-56 but could increase during the coming years since it is one of the newer sweeteners approved for use. The most commonly used sweeteners are acesulfame-K, aspartame, cyclamate, saccharin and sucralose.56 Although they were not identified as inhibitors at the 50 µM concentration used in this study, testing at higher concentrations could be warranted since concentrations can reach high levels in beverages. At the maximum accepted use level of acesulfame-K (600 mg/kg), the concentration can reach almost 3 mM.57


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13 The levels of aspartame, acesulfame-K and cyclamate detected in beverages are around 50 – 200 mg/l.54, 56 The highest detected amounts correspond to 0.55, 1.23 and 1.16 mM for aspartame, acesulfame K and cyclamate respectively. Furthermore, these sweeteners are often used in combination with each other.

Colorants, especially azo dyes, were the primary class of inhibitors for both transporters. The studied azo dyes vary to some extent in structure and molecular weight, but they are all characterized by the R−N=N−R’ azo bond and contain multiple (aromatic) ring structures and sulfonic acid functional groups. Despite the rather negative general opinion on the use of azo dyes, which is due to their suspected adverse effects such as attention deficiency,58 they are still used in many food products worldwide. The exposure burden of consumers to food additives is a function of the concentrations used in food products and their consumption patterns. The levels at which certain additives are used can vary between countries and food products. For example, confectionaries, ice cream and beverages are typical foods where coloring agents are used to make products more appealing. The burden of food additives may therefore be higher in groups where the consumption of these products is high, such as children and youths. Several studies have been conducted to study the levels of azo dyes present in foods and evaluate the exposure of consumers to these dyes. The risk of local effects of food additives on drug absorption is highest for products that can be consumed in considerable amounts in short periods, such as beverages. In fact, drinks and juices are a major dietary source of azo dyes, constituting up to over 80% of average daily intakes.59 Table 2 compiles the findings concerning the concentrations found in beverages. The most highly consumed colorants in a Korean study were allura red and tartrazine,60 whereas tartrazine and sunset yellow were the most popular used azo dyes in Indian samples.61 The levels of colorants detected in beverages range from below 0.1 mg/l up to over 2000 mg/l (Table 2). However, the JEFCA recommends the maximum use levels of these colorants to be around 100 – 300 mg/kg in beverages, depending on the colorant and the specific product in question.57



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14 Table 2. Reported concentrations (mg/kg or mg/l) of selected food colorants in beverages. Colorant a

Allura red AC IC50, BCRP = 6.60 IC50, MRP2 = 9.93

Brilliant blue FCF IC50, MRP2 = 2.55

Carmoisine IC50, BCRP = 2.70 IC50, MRP2 = 14.6

Sunset yellow FCF IC50, BCRP = 4.80

Tartrazine IC50, BCRP = 12.56

Minimum

Maximum

Mean

Country

5.77 1.6 16.03 − 0.1 − 0.03 − 2.75 0.9 21 4.3 0.63 2.9 11.9 17 − 0.64 0.35 3.3 − 25.1 0.48 0.1 4 3.7 − 0.06 0.2 21 10

7.15 8.5 55.91 63.5 2335 − 0.08 11.2 71.44 73 144 39.1 125 448 603.14 848 − 2.09 2.7 17.4 19 27.33 215.14 270 1000 10 47.4 121.82 633 1558 −

6.46 − −

Iran France Italy Korea Kuwait Turkey Brazil Korea Iran Kuwait India France Greece Kuwait Iran India Turkey Brazil Greece Taiwan Korea Iran Iran Kuwait India Taiwan Korea Iran Kuwait India Iran

17.8 − 65.28 − 2.2 24.85 − − − − − 208.57 198 b 24.26 − − − 6.8 − 25.49 − 172 b − 9.5 24.42 − 239 b −

a

Reference 62 63 64 60 59 65 66 60 62 59 61 63 67 59 62 61 65 66 67 68 60 69 62 59 61 68 60 62 59 61 69

The IC50 values determined in this study (Table 2) are given in mg/l below the colorant name to enable comparison. b

Median concentration


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15 The data in Table 2 shows that the concentrations of colorants in drinks can exceed the IC50 values for BCRP and MRP2 inhibition determined in this study. For instance, the consumption of allura red AC and carmoisine containing beverages could, in extreme cases, lead to intestinal concentrations that are more than 200 times higher than the IC50 values. This greatly exceeds the threshold for in vivo studies set by the International Transporter Consortium (ITC) for new drug molecules that may be P-gp or BCRP inhibitors.70 In vivo drug interaction studies are recommended, if the expected intestinal concentration of the inhibitor exceeds the in vitro IC50 value by more than 10-fold. Applying this criterion, further studies to elucidate the impact of the presented azo dyes on intestinal drug transporters would be justified. It should also be kept in mind that the magnitude of interactions for BCRP and MRP2 dual substrates may be larger than expected, since several of the azo dyes inhibited both of these transporters.

While our results suggest that certain food additives may cause transporter inhibition in vivo, it should be noted that according to the EFSA safety evaluations, the absorption of the studied colorants is generally very low. Therefore, the intracellular concentrations in enterocytes may be considerably lower than the concentration in the intestinal lumen. Although the vesicular transport assay used in this study is a useful tool for studying the effects of different compounds on efflux transporter activity, it has certain limitations. The IC50 value measured in vesicles reflects unbound intracellular concentrations, because the inhibitor has direct access to the intracellular domains of the transporter. In the intestine, the inhibitor has to permeate into the enterocyte to access this site and therefore the vesicle-derived IC50 values are not directly comparable to extracellular concentrations such as luminal or plasma concentrations. Further studies in a whole-cell system are thus needed to verify the effects observed in this study and draw conclusions about the possible in vivo effects. Moreover, it should be kept in mind that food additives may undergo extensive metabolism in the body. For instance, azo dyes are cleaved by azo reductases of intestinal bacteria71 while neohesperidin DC and



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16 stevioside are known to be deglycosylated by fecal bacteria.72, 73 These metabolic products may have better bioavailability and distinct interactions with BCRP, MRP2 and P-gp compared to parent compounds.

In conclusion, the possible involvement of food additives in drug transporter-mediated interactions is to date an overlooked area. Using the vesicular transport assay, we report here the inhibition of intestinal efflux transporters BCRP and MRP2 by selected food additives, namely the sweetener neohesperidin DC and several azo dyes. The food preservatives studied here did not affect transporter activity. By comparing the reported concentrations of azo dyes in beverages to the IC50 values determined here, it is suggested that the concomitant ingestion of BCRP or MRP2 substrate drugs with azo dye containing beverages could result in the increased absorption of the substrates. However, results should be confirmed in cell-based assays, because the cellular permeation of the additives may be low. Additionally, since only a fraction of food additives were studied here (25 compounds in total), further investigations are needed to elucidate the impact of other food additives on intestinal ABC transporters and their consequences for drug pharmacokinetics.

5

ACKNOWLEDGEMENTS

We would like to thank M.Sc. Erkka Järvinen for help with the HPLC analysis and Pedro López-Terradas Mota for technical assistance. We would also like to thank the Academy of Finland, the University of Helsinki Doctoral Program in Drug Research and the Magnus Ehrnrooth Foundation for funding this research. We acknowledge the Drug Discovery and Chemical Biology Network, funded by Biocenter Finland, for providing access to the screening instrumentation.

6

REFERENCES

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Figure 1. The inhibition of BCRP, MRP2 and P-gp transport by food additives. The inhibitory potential of 25 food additives including (A.) colorants, (B.) sweeteners and (C.) preservatives was tested at 50 µM in the vesicular transport assay. BCRP (black bars), MRP2 (grey bars) or P-gp (green bars) expressing vesicles were used with 50 µM LY, 5 µM CDCF and 2 µM NMQ as probe substrates, respectively. Results are presented as a relative transport activity (%), which is the activity in the presence of the food additive normalized to uptake in the presence of vehicle (DMSO) only. Bars show the mean ± SD (n = 3). 80x193mm (300 x 300 DPI)


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Figure 2. Interference of the food additives on probe fluorescence assuming the complete retention of the tested additives in the final elute. The fluorescence of the probe substrates LY (black bars) and CDCF (grey bars) was studied in the presence of the tested food additives to rule out fluorescence quenching that could result in false positives. Fluorescence conditions were set to mimic the measurement step of the vesicular transport assay. For a more detailed description, see Materials and Methods. Results (mean ± SD) are reported as relative fluorescence (%), which is the probe fluorescence in the presence of the food additive normalized to the fluorescence in the presence of vehicle (DMSO) (n = 3). 100x65mm (300 x 300 DPI)



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Figure 3. Interference of the food additives on probe fluorescence assuming that 5% or 20% of the total additive amount used in the assay is retained in the final elute. The fluorescence of the probe substrates LY (top panel) and CDCF (bottom panel) was studied in the presence of the tested food additives at 5% (black bars) and 20% retained (grey bars). Fluorescence conditions were set to mimic the measurement step of the vesicular transport assay. For a more detailed description, see Materials and Methods. Results (mean ± SD) are reported as relative fluorescence (%), which is the probe fluorescence in the presence of the food additive normalized to the fluorescence in the presence of vehicle (DMSO) (n = 3). 72x136mm (300 x 300 DPI)


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Figure 4. Dose-response curves of BCRP and MRP2 inhibition by selected food additives. 50 µM LY and 5 µM CDCF were used as probe substrates for BCRP (closed circles) and MRP2 (open circles), respectively. Where only one curve is shown, the compound only inhibited either of the transporters. Points represent the mean ± SD from two to three separate experiments performed in triplicates and the solid lines show the results from curve fitting using GraphPad Prism 6.05 (GraphPad Software Inc., USA). 207x269mm (300 x 300 DPI)


Interaction of Food Additives with Intestinal Efflux ... - ACS Publications

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