Recommended Daily Dose of Sesame Lignans Has No Influence on Oral Absorption of P-Glycoprotein Substrates in Rats

Abstract

Sesamin (SM) and episesamin (ESM) are constituents of sesame seeds, which are used in health foods and considered to have various beneficial effects in the prevention of lifestyle-related diseases. P-Glycoprotein (P-gp) is an ATP-binding cassette transporter involved in drug absorption in the human gastrointestinal tract. A recent report indicated that SM influences P-gp-mediated drug transport. In the present study, we investigated whether SM and ESM inhibit P-gp in vitro, using Caco-2 cells and the typical P-gp substrates rhodamine123 (Rho123) and fexofenadine. SM and ESM showed no effect on accumulation of these compounds, indicating that SM and ESM do not influence P-gp function. In addition, an in vivo study using Rho123 indicated that SM and ESM do not affect absorption of P-gp substrates. Overall, these results suggest that health foods containing SM and ESM are unlikely to interact with P-gp substrates.

Sesamin (SM) is a major constituent of sesame seeds, and episesamin (ESM) is formed by epimerization of SM.¹⁾ These lignans are found in various health foods and are considered to have a number of physiological effects, including anti-oxidative,²⁾ serum cholesterol and lipid-decreasing,^3–5) anti-hypertensive^6,7) and anti-carcinogenic⁸⁾ activity. In addition, in vitro and in vivo experiments have indicated that SM and ESM have synergistic effects, such as an anti-cancer effect with γ-tocotrienol⁹⁾ and a triacylglycerol-lowering effect with soybean phospholipid.¹⁰⁾

P-Glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter consisting of 1280 amino acid residues.^11,12) Initially, P-gp was discovered in various tumor cells^13–15) and its overexpression led to failure of chemotherapy due to increased efflux of anti-cancer drugs from the tumor cells.^16,17) Subsequently, many reports have shown that P-gp is expressed in not only cancer cells, but also normal tissues, such as brain capillaries, renal proximal tubules, and the small and large intestine.^14,18) It is localized in apical plasma membrane and transports various compounds, including anti-cancer drugs,^16,19) cardiovascular drugs²⁰⁾ and natural products¹⁹⁾ from cells. However, the broad substrate recognition of P-gp may result in drug–drug interaction and drug–food interaction.

Although it was reported that SM inhibits drug efflux activity of P-gp in cells overexpressing the transporter,²¹⁾ the effect of SM in vivo remains unclear. In the present study, we investigated whether SM and ESM, at concentrations expected to be present in the gastrointestinal tract after ingestion of health foods, affect the transport function of P-gp in vitro and in vivo.

MATERIALS AND METHODS

Chemicals

SM and ESM were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Nagara Science (Gifu, Japan) respectively. All other chemicals were commercial products of reagent grade.

In Vitro Uptake Experiments

Human colon adenocarcinoma cell line Caco-2 was obtained from American Type Culture Collection (ATC C, Rockville, MD, U.S.A.). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 units/mL penicillin, 0.1 mg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. Caco-2 cells were seeded on 12-well culture plates (Greiner bio-one) at a cell density of 2×10⁵ cells/mL. Cells cultured for two weeks were used for uptake experiments. Caco-2 cells grown on the plates were gently rinsed twice with Hank’s balanced salt solution (HBSS) buffered with 4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES) (pH 7.4) (HBSS-HEPES). Before the uptake experiment, cells were incubated in HBSS-HEPES for 30 min. Then they were treated with 10 μM rhodamine123 (Rho123) or 30 μM fexofenadine (Fex) in the absence or presence of 100 μM SM, 100 μM ESM or 20 µM cyclosporine A (CsA) in HBSS-HEPES at 37°C for 60 min. The buffer was then removed, and the cells were washed three times with ice-cold HBSS-HEPES, and lysed in 1 N NaOH. The lysate was neutralized with 5 N HCl.

In Vivo Inhibition of P-gp in Rats

Eight-week-old male Wistar rats were obtained from SLC Japan (Hamamatsu, Japan). All animal experiments were performed according to the Guidelines for the Care and Use of Animals at Takasaki University of Health and Welfare. Rats weighing 180–200 g were fasted for 18 h with free access to water. Two miligrams per kilogram of Rho123 was orally administered to rats in the absence or presence of 0.35 mg/kg SM or 0.35 mg/kg ESM. The solution volume in all cases was adjusted to 10 mL solution/kg of body weight. Blood samples were collected from the jugular vein using a heparinized syringe at the designated times after administration.

Determination of Drug Concentration

Plasma and intracellular concentrations of Rho123 were measured with a WALLAC multilabel/luminescence counter (PerkinElmer, Inc., Waltham, MA, U.S.A.) at wavelengths of 485 nm (excitation) and 538 nm (emission). Fex samples were prepared as previously described, with some modifications.²²⁾ Briefly, 100 µL of 50% methanol and 100 µL of 20 mM ammonium formate (pH 6.4) were added to 200 µL of neutralized cell lysate. The solution was mixed with 2.5 mL of ethyl acetate using a reciprocal shaker for 20 min and centrifuged at 1120×g for 5 min at 4°C. Then, 1.92 mL of the organic layer was concentrated under nitrogen gas flow and the residue was dissolved in the mobile phase. The resulting solution was analyzed with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system, with an API3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, U.S.A.) using electrospray ionization in the positive mode. The multiple reaction monitor for Fex was set at 502.4–466.3 m/z. Fex samples were injected into an HPLC system (HP1100 system Agilent, Waldbronn, Germany) equipped with a Capcell pak C18 MGII column (50×2.0 mm i.d., 3 µm; Shiseido Company, Tokyo, Japan) using isocratic elution at 0.1 mL/min. The mobile phase consisted of equal parts of 20 mM ammonium formate (pH 6.4) and acetonitrile.

Statistical Analysis

All data are presented as the mean±standard error of the mean (S.E.M.). Statistical analysis was undertaken using the two-sided Tukey’s test. Values of p<0.05 were considered significant.

RESULTS

Effect of SM and ESM on Uptake of P-gp Substrates in Caco-2 Cells

SM and ESM had no effect on intracellular accumulation of Rho123 in Caco-2 cells (118.0±14.2% and 128.4±20.5% of control, respectively). In the presence of CsA, Rho123 accumulation was increased (219.6±40.1%) (Fig. 1). Fex uptake was not affected by SM or ESM (85.6±3.9% and 104.6±8.5% of control, respectively). However, CsA increased the intracellular accumulation of Fex (235.0±15.4%) (Fig. 2).

Fig. 1. Rho123 Uptake in Caco-2 Cells

The data are presented as the mean±S.E.M. (n=3). *p<0.05.

Fig. 2. Fex Uptake in Caco-2 Cells

The data are presented as the mean±S.E.M. (n=3). *p<0.01.

Pharmacokinetic Study of Rho123 in Rats

When Rho123 was administered to rats together with SM or ESM we observed no significant change of its pharmacokinetic parameters. Namely, maximum plasma concentration (C_max) (37.73±4.79 ng/mL control, 31.91±10.30 ng/mL with SM, and 29.03±2.16 ng/mL with ESM), time to C_max (T_max) (0.51±0.26 h control, 1.01±0.29 h with SM, and 0.58±0.08 h with ESM) and the area under the plasma concentration–time curve from 0 to 6 h (AUC_0–6h) (112.37±7.28 ng·h/mL control, 105.24±24.94 ng·h/mL with SM, and 99.34±8.11 ng·h/mL with ESM) (Fig. 3, Table 1).

Fig. 3. Effect of SM and ESM on Pharmacokinetics of Oral Administrated Rho123 in Rats

Plasma concentrations of Rho123 are shown in the absence of SM and ESM (closed circle), in the presence of SM (closed square) and in the presence of ESM (closed triangle). The data are presented as the mean±S.E.M. (n=3).

Table 1. Pharmacokinetic Parameters of Rho123 in Rats

	Cmax (ng/mL)	Tmax (h)	AUC 0–6 h (ng·h/mL)
Control	37.73±4.79	0.51±0.26	112.37±7.28
+SM	31.91±10.30	1.01±0.29	105.24±24.94
+ESM	29.03±2.16	0.58±0.08	99.34±8.11

The data are presented as the mean±S.E.M. (n=3).

DISCUSSION

Our results indicated that SM and ESM are likely to have no effect on drug efflux activity of P-gp in Caco-2 cells at physiologically relevant concentrations. To evaluate a suitable test concentration of SM or ESM for in vitro study, we considered the case of ingestion of a health food containing 10 mg SM, together with 250 mL of water (the volume recommended by the U.S. Food and Drug Administration for administration of a drug product to humans²³⁾), which would give an estimated concentration of 113 µM. However, because of the low solubility of SM and ESM, we rounded this down to 100 µM SM or ESM for the present in vitro experiments. On the other hand, for the in vivo study, we envisioned that commercial health food containing the maximum recommended amounts of SM or ESM (equivalent to approximately 20 mg SM or ESM) would be ingested by a human weighing 60 kg. On this basis, we administered 0.35 mg/kg of SM or ESM with Rho123 to rats. Our finding that SM and ESM have no effect on P-gp is different from that of a prior report using transport experiments in Caco-2 cells²⁴⁾ and P-gp-overexpressing LS-180 V cells.²¹⁾ However, the transport experiments showed that digoxin transport in both the apical-to-basal and basal-to-apical directions was increased in the presence of 50 or 100 µM SM.²⁴⁾ Therefore, their results are not necessarily inconsistent with our present finding. On the other hand, the result of our Rho123 uptake study differs from a previous study using LS-180 V cells.²¹⁾ The difference might be due to various factors, such as different cell type and different cell status (vinblastine-treated LS-180 cells and non-treated Caco-2 cells). We think our in vitro study using Caco-2 cells may better reflect the in vivo kinetics of Rho123. Moreover, Caco-2 cells are widely used as a human gastrointestinal absorption model, and we consider that our results are more likely to reflect the real effect of SM and ESM on P-gp function in the human gastrointestinal tract. This conclusion is supported by our observation that neither SM nor ESM influenced the in vivo kinetics of Rho123. In addition, a previous report indicated that SM is not a substrate of P-gp.²⁵⁾ Overall, it seems reasonable to conclude that neither SM nor ESM is likely to influence absorption of P-gp substrates in vivo. On the other hand, a patent has indicated that 500 mg/kg of SM has a P-gp-inhibitory effect in vivo.²⁴⁾ Although this amount of SM is higher than that contained in commercial health foods, we cannot rule out the possibility that ingestion of large amounts of health foods containing SM might have the potential to enhance intestinal absorption of P-gp substrates.

CONCLUSION

Our results suggest that SM and ESM at physiologically relevant concentrations have no effect on P-gp function or on absorption of a P-gp substrate in vivo. We conclude that a single daily dose of the maximum recommended amount of SM or ESM is unlikely to interact with simultaneously ingested P-gp substrates.

Acknowledgment

This work was partially supported by Grant-in-Aid for Scientific Research from the Minisry of Education, Culture, Sports, Science and Technology of Japan (No. 24590667).

Conflict of Interest

The authors declare no conflict of interest.

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