Transport Characteristics of 5-Aminosalicylic Acid Derivatives Conjugated with Amino Acids via Human H⁺-Coupled Oligopeptide Transporter PEPT1

Abstract

5-Aminosalicylic acid (5-ASA) is used as first line therapy for symptom remission and maintenance of inflammatory bowel disease (IBD). Because 5-ASA is well absorbed from the small intestine when orally administered, several 5-ASA formulations for selective delivery to the colon have been developed and used in clinical practice. However, its delivery efficiency to local inflamed colonic sites remains low. Intestinal H⁺-coupled oligopeptide transporter 1 (PEPT1) expression in the colon is low, whereas its expression is induced in the colon under chronic inflammation conditions, such as IBD. Therefore, we considered that PEPT1 would be a target transporter to improve 5-ASA delivery efficiency to local colonic lesions. We evaluated the transport characteristics of dipeptide-like 5-ASA derivatives, which were coupling glycine (Gly), lysine, glutamic acid (Glu), valine (Val) and tyrosine to amino or carboxyl group of 5-ASA, in Caco-2 cells. [³H]Glycylsarcosine (Gly-Sar) uptake into Caco-2 cells was inhibited by all 5-ASA derivatives. In addition, 5-ASA derivatives (Gly-ASA, Glu-ASA and Val-ASA), which were coupled by glycine, glutamic acid and valine to amino group of 5-ASA, were taken up in a pH- and concentration-dependent manner and their uptake was inhibited by excess Gly-Sar. Two-electrode voltage-clamp experiment using human PEPT1 expressing Xenopus oocytes showed that Gly-ASA, Glu-ASA and Val-ASA induced marked currents at pH 6.0. Taken together, these results showed that these 5-ASA derivatives are transportable substrates for PEPT1.

INTRODUCTION

5-Aminosalicylic acid (5-ASA) is a conventional therapeutic for inflammatory bowel disease (IBD) and is used as a first line drug for remission induction and maintenance in IBD patients.^1,2) Additionally, a very high 5-ASA dose (1.5–3.0 g/d) is usually required for IBD treatment because intestinal absorption of orally administered conventional 5-ASA is rapid and efficient in the upper small intestine. This absorption of 5-ASA might cause some adverse effects, such as nephrotic syndrome, myopericarditis and fever,^3–5) and could result in a low concentration in the colonic region. Therefore, its oral delivery efficiency to the inflamed local colon is low.⁶⁾ The ideal 5-ASA colonic delivery system should decrease the systemic absorption from the small intestine while increasing delivery efficacy of the drug to the colon. To resolve this issue, several new 5-ASA dosage forms (pH-dependent controlled release capsule and enteric coat) have been developed for the efficient targeting of the colon.⁷⁾

In addition to new dosage formulations, various 5-ASA prodrugs improve its delivery efficacy to the colon. 5-ASA has two interactive functional groups, carboxylate and amine, which easily react with various chemical reagents such as polysaccharides, polymers and amino acids.⁸⁾ Many types of polysaccharides have been used as non-toxic chemical agents for the design of 5-ASA prodrugs. Polysaccharides could attach to the 5-ASA via ester bonds (such as dextran, hydroxypropyl methylcellulose or chondroitin sulfate) or an amide bond (chitosan) based on their functional group. These macromolecular 5-ASA prodrugs could not be absorbed from the small intestine. Furthermore, they might be water soluble and release 5-ASA in media containing rat cecal and colonic content.

Amino acids are among the moieties that have been used to prepare 5-ASA prodrugs. Based on these functional groups, amino acids could attach to the 5-ASA via ester,⁹⁾ amide^10–12) or azo¹³⁾ bonds. In particular, amide bond conjugates of 5-ASA with amino acids are structurally similar to dipeptide. Therefore, these 5-ASA conjugates might be recognized as dipeptides and might be transported into the cells via H⁺-coupled oligopeptide transporter PEPT1.

PEPT1 expressed in the brush border membranes of small intestinal epithelial cells accepts small peptide, which comprises two or three amino acids as substrates. In addition to di/tripeptides, PEPT1 is responsible for the intestinal absorption of peptide-like drugs such as oral β-lactam antibiotics.^14–17) Therefore, PEPT1 has been used as a target transporter to improve the intestinal absorption of poorly absorbed compounds with amino acid modification. In clinical practice, the oral bioavailability of valacyclovir and valganciclovir, amino acid prodrugs of acyclovir and ganciclovir, respectively, has increased because of PEPT1-mediated transport compared with parent drugs.^18,19) Interestingly, while PEPT1 is not detectable or poorly expressed under normal conditions,^14,20) colonic PEPT1 is up-regulated in IBD.^21,22)

Here, we report the transport characteristics of dipeptide-like 5-ASA derivatives conjugated with several amino acids (glycine (Gly), lysine (Lys), glutamic acid (Glu), valine (Val) and tyrosine (Tyr)) via PEPT1 in Caco-2 cells, their electrophysiological properties utilizing heterologous expression of human PEPT1 (hPEPT1) in Xenopus laevis (X. laevis) oocytes. We expect that targeting PEPT1 contribute to the enhancement of the therapeutic effect of 5-ASA and the reduction of the non-specific distribution, and consequently reduce the adverse effects.

MATERIALS AND METHODS

Materials

5-ASA derivatives conjugated with amino acid such as glycine, lysine, glutamic acid, valine and tyrosine into NH₂ or COOH-group of 5-ASA (Fig. 1) were purchased from the Chemical Soft Co., Ltd. development laboratory (Mie, Japan). [³H]Glycylsarcosine (Gly-Sar; specific radioactivity, 29.4 Ci/mmol) was purchased from Moravek Biochemicals Inc. (Brea, CA, U.S.A.). Gly-Sar was purchased from Sigma-Aldrich Japan (Tokyo, Japan). 5-ASA was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 2-(N-Morpholino)ethanesulfonate (Mes), 2-[4-(2-hydroxyethl)-a-piperazinyl]ethanesulfonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) were obtained from Nacalai Tesque (Kyoto, Japan). Culture reagents were obtained from Gibco Life Technologies (Waltham, MA, U.S.A.) or Nacalai Tesque. Other chemicals used were of the highest purity available.

Fig. 1. Chemical Structure of 5-ASA and Its Amino Acid Derivatives

Cell Culture and Uptake Studies

The human colon adenocarcinoma cell line Caco-2 was obtained from DS Pharma Biomedical (Osaka, Japan). Caco-2 cells were routinely cultured with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, 100 U/mL penicillin G and 100 µg/mL.^23,24) Caco-2 cells were subcultured in 24-well plate at a seeding density of 3 × 10⁴ cells/cm². Uptake of [³H]Gly-Sar was measured 19-23 d after seeding at 37°C as described previously.²³⁾ In most experiments, the uptake buffer comprised 25 mM Mes/Tris (pH 6.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄ and 5 mM D-glucose. Uptake was initiated by removing the culture medium from the 24-well plate, washing the cells with 1.0 mL uptake buffer and adding 250 µL of the fresh uptake buffer containing [³H]Gly-Sar (2 µCi; 60 nM) and unlabeled Gly-Sar. After incubation for 15 min, the buffer was removed by aspiration and the cells were washed twice with 2.0 mL ice-cold uptake buffer. The cells were solubilized with 500 µL 0.2 M NaOH/1% sodium dodecyl sulfate (SDS), aliquots (400 µL) of samples were transferred to a counting vial and the radioactivity associated with the cells was measured using liquid scintillation spectrometry (Model LS6000; Beckmann, Fullerton, CA, U.S.A.).

In experiments using 5-ASA and 5-ASA derivatives, extraction solution (water/acetonitrile = 10/90) was added to the cells after the uptake period. After standing for 1 h at room temperature, the solutions were centrifuged (13500 × g for 10 min at 4°C) and the supernatants were evaporated at 60°C. The pellets were resolved with mobile phase for HPLC analysis and were subjected to HPLC determination.

Preparation of Caco-2 Cell Homogenate and Degradation of 5-ASA Derivatives

Caco-2 cells grown for 15–18 d were washed twice with ice-cold Ca²⁺/Mg²⁺-free phosphate buffered saline (PBS(−)), detached by a scraper and homogenized using a Teflon homogenizer (5000–6000 rpm, 20 strokes) at 4°C. The resultant homogenate (1 mg protein/mL) was mixed with an equal volume of 10 µM 5-ASA or 5-ASA derivatives and incubated at 37°C under shaking conditions at 120 rpm. The protein concentration in each homogenate was measured using a BCA protein assay kit (Thermo Fisher Scientific) using bovine serum albumin as a standard. The reaction was terminated by adding 10-excess volume of acetonitrile at appropriate time. The reaction mixtures were centrifuged at 13500 × g for 10 min at 4°C and the resultant supernatants were evaporated at 60°C. The pellets were resolved with mobile phase for HPLC analysis and subjected to HPLC determination.

X. laevis Oocytes Expressing hPEPT1 and Electrophysiology

The procedures for preparation of oocytes from X. laevis, synthesis of human PEPT1 cRNA and microinjection of cRNA into the oocytes have been described previously.^25,26) Surgically, mature oocytes (stage IV or V) from Xenopus laevis were injected with 50 nL RNA solution containing 50 ng human PEPT1 cRNA. All electrophysiological measurements were performed after 3–6 d by incubating oocytes in buffer comprising 88 mM NaCl, 1 mM KCl, 0.82 mM CaCl₂, 0.41 mM MgCl₂, 0.33 mM Ca(NO₃)₂, 2.4 mM NaHCO₃ and 10 mM Mes/Tris at pH 6.5 (modified Barth’s solution). The membrane potential was clamped at −50 mV. The oocyte was superfused with the perfusion buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM Hepes/Tris (pH 7.5)). After the current stabilized, the oocyte was superfused with NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Mes/Tris (pH 6.0)), followed by the same buffer containing Gly-Sar (0.25 mM) and/or 5-ASA derivatives (1 mM). After application of the test compound, the currents were recorded; once the currents reached the maximum and steady state, the test compound was washed out with the perfusion buffer (pH 7.5). The substrate-induced current at a given membrane potential was calculated as the difference between the steady-state currents recorded in the absence and presence of substrate.

HPLC Measurement of 5-ASA Derivatives

5-ASA derivatives were quantified according to a reversed-phase HPLC (Shimadzu, Kyoto, Japan) with a fluorescence detector (RF-10XL) and COOSMOSIL 5C₁₈-AR-II column (3.0 mm i.d. × 150 mm; Nacalai Tesque). The eluent was 20 mM phosphate buffer (pH 2.2) : methanol = 95 : 5 for 5-ASA, ASA-Gly, ASA-Lys, ASA-Glu, Gly-ASA, Lys-ASA and Glu-ASA and 20 mM phosphate buffer (pH 2.2) : methanol = 80 : 20 for ASA-Val, ASA-Tyr, Val-ASA and Tyr-ASA. Fluorescence detection was done at excitation 311 nm and emission 449 nm. The injection volume was 5 µL and total flow rate was 0.4 mL/min.

Data Analysis

For saturation kinetics of Gly-Sar and 5-ASA derivatives transport, the data were fitted to the Michaelis–Menten equation:

  

(1)

where V_max is the maximal velocity of test compound transport, K_m is the Michaelis constant, S is the test compound concentration. To confirm the single transport system of test compound uptake in Caco-2 cells, Eadie-Hofstee transformation was performed:

  

(2)

where V′ is the saturable component of test compound transport.

To calculate the half maximal inhibitory concentration (IC₅₀), the data were fitted to the sigmoidal-logistic 3 parameter equation:

  

(3)

Where A is the transport velocity in the absence of the tested inhibitory compound and n is Hill coefficient.

Linear or non-linear regression analyses were performed using SigmaPlot (version 13.0; Jandel Scientific, San Rafael, CA, U.S.A.). The fitting quality was determined by evaluating the correlation coefficient (r) and standard error of parameter estimations. Results are expressed as mean ± standard deviation (S.D.). Statistical comparisons were performed with unpaired or paired t test and ANOVA, as appropriate.

RESULTS

Degradation Properties of 5-ASA Derivatives in Caco-2 Cell Homogenate

To evaluate the stability of 5-ASA derivatives, we initially assessed the degradation properties of 5-ASA derivatives in Caco-2 cells homogenate. All 5-ASA derivatives were stable in PBS(−) at 37°C. Moreover, 5-ASA derivatives conjugated with amino acid into the carboxyl group of 5-ASA (ASA-amino acid derivatives) were not degraded in Caco-2 cell homogenate, whereas the derivatives conjugated with amino acid into the amino group of 5-ASA (amino acid-ASA derivatives: Gly-ASA, Lys-ASA, Glu-ASA, Val-ASA and Tyr-ASA) were converted to parent drug (5-ASA) in Caco-2 cell homogenate (Supplementary Fig. 1). Their half-lives (t_1/2) were approximately 199, 3.2, 136, 30, and 53 min, respectively (Table 1). Therefore, protease inhibitors cocktail was added to the uptake buffer.

Table 1. Degradation Rate Constants and Half-Lifves of 5-ASA Derivatives in Caco-2 Cell Homogenate

	kdeg (min−1)		kdeg (min−1)	t1/2 (min)
ASA-Gly	No degradation	Gly-ASA	3.56 ± 0.67	199 ± 38.1
ASA-Lys	No degradation	Lys-ASA	211 ± 18.5	3.27 ± 0.02
ASA-Glu	No degradation	Glu-ASA	5.23 ± 1.05	136 ± 25.9
ASA-Val	No degradation	Val-ASA	22.9 ± 1.27	30.4 ± 1.51
ASA-Tyr	No degradation	Tyr-ASA	12.9 ± 0.79	53.8 ± 3.38

The Caco-2 homogenate (1 mg protein/mL) was mixed with an equal volume of 10 µM 5-ASA derivatives, and incubated at 37°C for an appropriate time. The apparent first-order degradation rate constants (k_deg) of 5-ASA derivatives were determined by plotting the logarithm of 5-ASA derivatives remaining as a function time. The degradation half-life was calculated by the following equation: t_1/2 = ln2/k. Results are expressed as mean ± S.D. (n = 4).

Effects of 5-ASA Derivatives on Gly-Sar Uptake in Caco-2 Cells

Uptake of Gly-Sar into Caco-2 cells is driven by an inwardly directed pH gradient and mediated by a single-uptake PEPT1 system.^27,28) We first studied the effect of 5-ASA derivatives on the [³H]Gly-Sar uptake in Caco-2 cells. Both amino acid-ASAs and ASA-amino acids significantly inhibited [³H]Gly-Sar uptake (Table 2), which indicates that dipeptide-like 5-ASA derivatives impact on PEPT1-mediated [³H]Gly-Sar uptake. Interestingly, 5-ASA, which is not a PEPT1 substrate, also inhibited [³H]Gly-Sar uptake in Caco-2 cells.

Table 2. Inhibitory Effects of 5-ASA Derivatives on Gly-Sar Uptake in Caco-2 Cells

Unlabeled compound	[3H]Gly-Sar uptake
	pmol/mg protein/15 min	(%)
None	166 ± 5.62	100
Gly-Sar	15.0 ± 0.50	9
5-ASA	79.1 ± 9.93	48
ASA-Gly	56.3 ± 8.75	34
ASA-Lys	39.1 ± 0.45	23
ASA-Glu	2.85 ± 0.72	2
ASA-Val	21.6 ± 1.60	13
ASA-Tyr	14.2 ± 4.59	9
Gly-ASA	11.5 ± 3.39	7
Lys-ASA	24.1 ± 10.6	14
Glu-ASA	5.01 ± 1.00	3
Val-ASA	3.95 ± 0.27	2
Tyr-ASA	12.1 ± 1.28	7

[³H]Gly-Sar (1 µM) uptake was measured in Caco-2 cells at an extracellular pH of 6.0. Unlabeled compounds were used at a concentration of 10 mM. Results are expressed as mean ± S.D. (n = 3).

Transport Properties of 5-ASA Derivatives in Caco-2 Cells

We compared the influence of extracellular pH on amino acid-ASA and ASA-amino acid derivative uptake (Fig. 2). Because 5-ASA derivatives were partially metabolized to 5-ASA and each amino acid with in Caco-2 cells (Table 1), uptake amounts of 5-ASA derivatives were calculated as the sum of the amounts of the unchanged and degraded form. ASA-amino acid derivative uptake decreased when the pH changed from 5.0 to 8.5 (Fig. 2A). The influence of pH on amino acid-ASA derivative uptake differed greatly from that on ASA-amino acid derivative uptake (Fig. 2B). The process of amino acid-ASA derivative uptake showed a distinct pH optimum around pH 6.0. Increasing the pH from 5.0 to 6.0 stimulated amino acid-ASA derivative uptake, but a further increase above pH 6.5 reduced this uptake.

Fig. 2. Influence of pH on 5-ASA Amino Acid Derivative Uptake in Caco-2 Cells

Uptake of (A) ASA-amino acid derivatives (1 mM) and (B) amino acid-ASA derivatives (1 mM) was measured in Caco-2 cells. The uptake was conducted for 15 min in the uptake buffers of varying pH. Values are mean ± S.D. (n = 4).

We next assessed the concentration-dependent inhibitory effect of Gly-Sar on 5-ASA derivative uptake in Caco-2 cells (Fig. 3A). 5-ASA derivative uptake experiments were performed with a 15-min incubation period because we confirmed that the cellular uptake of 5-ASA was linear for up to 30 min (data not shown). Gly-Sar did not change cellular uptake of ASA-amino acid derivatives. However, cellular uptake of Gly-ASA, Glu-ASA and Val-ASA were decreased by Gly-Sar addition in a concentration dependent manner; their IC₅₀ values were 3.4 ± 0.6 mM, 4.6 ± 1.5 mM and 10.2 ± 2.1 mM, respectively (r² = 0.91, 0.70 and 0.78, respectively; Fig. 3B). Lys-ASA and Tyr-ASA uptake was not affected by Gly-Sar addition even at a concentration of 10 mM. These results suggest that three 5-ASA derivatives, Gly-ASA, Glu-ASA and Val-ASA, are preferentially recognized and transported by PEPT1 as substrates.

Fig. 3. Inhibitory Effect of Gly-Sar on the Uptake of 5-ASA and 5-ASA Amino Acid Derivatives in Caco-2 Cells

Uptake of 1 mM ASA-amino acid derivatives (A) and amino acid-ASA derivatives (B) was measured with a 15-min incubation in the uptake buffer at pH 6.0 over a Gly-Sar concentration range of 0–10 mM. Each point represents mean ± S.D. (n = 4).

Figure 4 shows the uptake of amino acid-ASA derivatives into Caco-2 cells that occurred via a saturable process. The cellular uptake of three compounds, Gly-ASA, Glu-ASA and Val-ASA was saturable with K_m values of 1.3 ± 0.1 mM, 1.0 ± 0.1 mM and 0.59 ± 0.08 mM (r² = 0.99, 0.95 and 0.95), respectively. In addition, the V_max value was 487 ± 13.1 nmol/mg protein/15 min for Gly-ASA, 362 ± 17.5 nmol/mg protein/15 min for Glu-ASA, and 591 ± 21.2 nmol/mg protein/15 min for Val-ASA. However, Lys-ASA and Tyr-ASA uptake into Caco-2 cells showed linear profiles (Fig. 4). Cellular uptake of 5-ASA was also linear (data not shown). When the cellular uptake amount at 0.5 mM was compared, Gly-ASA, Glu-ASA and Val-ASA showed higher uptake than 5-ASA (Table 3). On the other hand, the uptake of Lys-ASA and Tyr-ASA in Caco-2 cells was lower than that of 5-ASA because of their poor affinity for PEPT1.

Fig. 4. Saturation Kinetics of Various Amino Acid-ASA Derivatives Transport in Caco-2 Cells

Uptake of various amino acid-ASA derivatives was measured in Caco-2 cells with a 15-min incubation in the uptake buffer (pH 6.0) over their concentration range of 0.01–10 mM. Each point represents mean ± S.D. (n = 4).

Table 3. Uptake Amounts of 5-ASA and 5-ASA Derivatives in Caco-2 Cells

	Uptake (nmol/mg protein/15 min)
5-ASA	110 ± 3.76
Gly-ASA	126 ± 2.24
Lys-ASA	6.69 ± 0.36
Glu-ASA	122 ± 9.01
Val-ASA	271 ± 32.2
Tyr-ASA	34.2 ± 5.28

Uptake of 5-ASA (0.5 mM) or 5-ASA derivatives (0.5 mM) was measured in Caco-2 cells with a 15-min incubation in the uptake buffer at pH 6.0. Results are expressed as mean ± S.D. (n = 4).

Identification of 5-ASA Derivatives Transport in Caco-2 Cells

Figures 3B and 4 indicate that cellular uptake of Gly-ASA, Glu-ASA and Val-ASA might be mediated by PEPT1. We therefore selected Val-ASA as a representative derivative and investigated the kinetic nature of inhibition of PEPT1-mediated Gly-Sar uptake caused by Val-ASA (Fig. 5). In the absence of Val-ASA, Gly-Sar transport was saturable with a K_m value of 2.1 ± 0.1 mM and a V_max value of 315 ± 2.7 nmol/mg protein/15 min (r² = 0.99). The presence of 0.5 mM Val-ASA further increased the K_m value to 10.5 ± 0.7 mM, but V_max value did not change significantly (404 ± 13.7 nmol/mg protein/15 min; r² = 0.99). Moreover, as shown in Fig. 5B, the Gly-Sar uptake was inhibited by Val-ASA in a concentration-dependent of manner, and the IC₅₀ value was 0.14 ± 0.026 mM (r² = 0.99). These data show that Val-ASA is a competitive inhibitor of PEPT1-mediated Gly-Sar transport and the apparent K_i value of Val-ASA for PEPT1-mediated Gly-Sar transport is 0.14 mM. This value is relatively comparable with the K_m value (0.59 mM) for Val-ASA transport in Caco-2 cells (Fig. 4). These results indicate that the transport system of Val-ASA is identical to that of Gly-Sar in Caco-2 cells which is mediated by PEPT1.

Fig. 5. Inhibition of Gly-Sar Transport by Val-ASA and Kinetics of Val-ASA-Induced Inhibition of Gly-Sar Transport in Caco-2 Cells

Uptake of [³H]Gly-Sar (0.06 µM radiolabeled plus 1 µM unlabeled Gly-Sar) was measured with a 15-min incubation in the uptake buffer (pH 6.0) over Val-ASA concentration range of 0–10 mM (A). The uptake of Gly-Sar was measured with a 15-min incubation in the uptake buffer (pH 6.0) (B). The concentration of Gly-Sar ranged between 0.06–10 mM in the presence (△) or absence (●) of 0.5 mM Val-ASA. Inset: Eadie-Hofstee plot analysis of data. Lines were fitted by using linear-regression analysis. Results are mean ± S.D. (n = 4).

Electrophysiological Characterization of hPEPT1-Mediated Transport of 5-ASA Derivatives

hPEPT1 transports di/tripeptide and orally active β-lactam antibiotics in an electrogenic manner; the transport process is coupled to the transport of H⁺ with a 1 : 1 stoichiometry for H⁺ and substrate.^14,25) Therefore, the transport activity of hPEPT1 for any compound as a potential substrate can be monitored by determining if the compound could induce inward currents in hPEPT1-expressing oocytes. Three compounds of 5-ASA derivatives, Gly-ASA, Glu-ASA and Val-ASA, induced significant inward currents (44 ± 6.2, 56 ± 11 and 59 ± 8.6% compared with normalized Gly-Sar current; Fig. 6A). All examined 5-ASA derivatives inhibited Gly-Sar uptake when uptake was measured using radiolabeled tracer (Table 2). This shows that all 5-ASA derivatives compete with [³H]Gly-Sar in the uptake process, but only Gly-ASA, Glu-ASA and Val-ASA are actually transported across the oocyte membrane and thus could induce inward current. We then characterized the transport of these compounds further using the same electrophysiological approach. As shown in Fig. 6B, the I–V relationship for the induced currents exhibited a slightly positive slope for the above three compounds, which indicates that hyperpolarization enhanced the substrate-induced currents. Conversely, the I–V relationship for the current induced by Lys-ASA exhibited a small but detectable negative slope, which suggests that this particular derivative might inhibit the constitutive inward current of H⁺ through hPEPT1, which might be driven by the intracellular negative membrane potential even in the absence of a transportable substrate.

Fig. 6. Inward Currents Induced Gly-Sar and Amino Acid-ASA Derivatives via hPEPT1

Oocytes were perfused with the NaCl buffer (pH 6.0) followed by the same buffer containing 0.25 mM Gly-Sar or 1 mM amino acid-ASA derivatives. Substrate-induced currents were determined by the difference in the currents in the presence and absence of substrate with membrane potential clamped at −50 mV. (A) Magnitude of substrate-induced current was expressed as the normalized value to 0.25 mM Gly-Sar current. Data represent mean ± S.E. (n = 5). The current induced by 0.25 mM Gly-Sar was 151 ± 31 nA, which was taken as 100%. (B) Membrane potential-current (I–V) relationship for Gly-Sar and amino acid-ASA derivatives; Gly-Sar (〇), Gly-ASA (▲), Lys-ASA (▼), Glu-ASA (■), Val-ASA (◆), and Tyr-ASA (×). (Color figure can be accessed in the online version.)

DISCUSSION

In this study, we investigated the PEPT1-mediated transport of dipeptide-like 5-ASA derivatives in Caco-2 cells. Although the mechanism of 5-ASA efficacy has not been clarified enough, it is considered to inhibit cyclooxygenases and lipoxygenases, scavenge effective radicals, and inhibit nuclear factor-kappaB (NF-κB) activation in the large intestinal inflamed epithelium.^29,30) Therefore, PEPT1-targeted delivery of 5-ASA could not only decrease its non-specific distribution but also improve the therapeutic effect.

The derivatives modified with amino acids in the carboxyl group of 5-ASA were stable in Caco-2 homogenate (Table 1). Conversely, the derivatives modified with amino acids in the amino group of 5-ASA were hydrolyzed in Caco-2 homogenate (Table 1, Supplementary Fig. 1). These results suggest that the type of amino acid and the introduced position of 5-ASA might be determining factors for recognition by hydrolytic enzymes. The degradation of the compounds with dipeptide-like structures seems to be mediated by metabolic enzymes such as aminopeptidases and carboxylpeptidases. Various aminopeptidases are functionally expressed in Caco-2 cells and each aminopeptidase has a different affinity to small peptide structure, whereas the enzymatic activity of carboxylpeptidases in Caco-2 cells is very low or none.^31,32) In our previous study, chemical modification of phenylalanylglycine (Phe-Gly) with fatty acids to the N-terminal portion was more stable than the native Phe-Gly.^33,34) However, Phe-Gly chemically modified with fatty acid to the C-terminal portion exhibited no significant increase in stability compared with the native Phe-Gly. These indicated that the introduction of fatty acid to the N-terminal portion might reduce Phe-Gly degradation against aminopeptidases, which are abundantly expressed in the brush border of the intestinal epithelium. Therefore, it is reasonable that amino acid-ASA derivatives are more rapidly degraded than ASA-amino acid derivatives and their degradation rates differ depending on the amino acid. In fact, aminopeptidase activities in human large intestine have been reported to be 10-fold higher than those in Caco-2 cells. Therefore, 5-ASA derivatives would be more rapidly degradation in human large intestinal tract.^32,35)

Uptake of Gly-Sar into Caco-2 cells is driven by an inwardly directed H⁺ gradient and mediated by PEPT1. Gly-Sar was used as a model dipeptide because it is highly resistant to both plasma membrane and intracellular dipeptidases. We therefore investigated the effect of a high concentration (10 mM) of 5-ASA and various 5-ASA derivatives on [³H]Gly-Sar uptake in Caco-2 cells (Table 2). All compounds inhibited Gly-Sar uptake. However, uptake of 5-ASA and ASA-amino acid derivatives into Caco-2 cells was not affected by increasing Gly-Sar concentration (Fig. 3A), which indicates that these compounds were not accepted by PEPT1. We assume that these compounds may bind to the substrate-binding site of PEPT1 and subsequently inhibit Gly-Sar uptake, but they would not be transported via PEPT1. However, amino acid-ASA derivatives, especially Gly-ASA, Glu-ASA and Val-ASA, inhibited Gly-Sar uptake in a concentration-dependent manner (Fig. 3B) and their cellular uptake was saturable with K_m values of 1.3 ± 0.1, 1.0 ± 0.1 and 0.59 ± 0.08 mM, respectively (Fig. 4). These results indicate that Gly-ASA, Glu-ASA and Val-ASA might be substrates for PEPT1. In addition, comparing the cellular uptake amount at 0.5 mM, Gly-ASA, Glu-ASA and Val-ASA showed higher uptake than 5-ASA (Table 3). Since these 5-ASA derivatives are predominantly transported into Caco-2 cells by PEPT1, these derivatives would be transported into the inflamed epithelium more efficiently and selectively than 5-ASA.

In addition to the cellular uptake study, we directly investigated the charge transfer with PEPT1-mediated transport of amino acid-ASAs in Xenopus oocytes expressing PEPT1. At pH 6.0, Gly-ASA, Glu-ASA and Val-ASA evoked marked inward currents (Fig. 6). Conversely, Lys-ASA and Tyr-ASA elicited a weak signal. These results agree very well with data obtained in Caco-2 cells. Interestingly, in Caco-2 cells, the uptake of Gly-Sar (1 mM) is lower than those of 5-ASA derivatives (1 mM) (Figs. 4, 5A), whereas in Xenopus oocytes, the induced current by Gly-Sar (0.25 mM) was higher than those of 5-ASA derivatives (1 mM). At present, the reason for this discrepancy is not known. We hypothesize that posttranslational modifications may play a role. When cloned PEPT1 was expressed heterologously in Xenopus oocytes, transport activity was measured as inwardly current. Posttranslational modifications of PEPT1 such as glycosylation in Xenopus oocytes might be different from those in well-differentiated Caco-2 cells, and therefore posttranslational modifications of the transporter protein are likely to occur these conditions.

PEPT1 has broad recognition of substrates and can transport the compounds with not only di/tripeptide structure³⁶⁾ but also amino acid ester.^1,18,37,38) Furthermore, Börner et al.³⁹⁾ screened transport of alanine (Ala) amide derivatives in Caco-2 cells and Xenopus oocytes expressing PEPT1 and demonstrated that PEPT1 accepts alanine aryl amides as substrates. Taken together with the above reports and our present results, the conjugation position of amino acid might play a key role in the interaction of 5-ASA derivatives with the substrate-binding site of PEPT1 and transport in intact form across the plasma membrane. PEPT1 is reported to be most abundantly expressed in the small intestine, with the highest expression in the duodenum and lower levels toward the ileum, and failed to detect its expression in colon.^14,40) However, controversial reports suggested the presence of PEPT1 in healthy colons of mice, rabbits and humans^41–44) and expression in the colonic cells that is upregulated under intestinal inflammatory conditions. Furthermore, the mucosal pH in colon tends to decrease in inflammatory conditions such as IBD²¹⁾ because of the reduced mucosal bicarbonate secretion and increased mucosal and bacterial lactate and short-chain fatty acid (acetate, propionate and butyrate) production. Nugent et al. reported that the pH in the large intestine is 5.3–6.8 at the onset of IBD.⁴⁵⁾ Therefore, upregulated PEPT1 in the IBD colon would properly function and transport amino acid 5-ASA derivatives to the inflamed lesion. Furthermore, increased expression of PEPT1 is reported to be involved in the induction and progression of IBD by transporting the inflammatory peptide, such as N-formylmethionyl-leucyl-phenylalanine (fMLP), muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP), produced from microbiota in colon.⁴⁶⁾ Therefore, we assume that 5-ASA derivatives could inhibit the transport of inflammatory small peptides into the large intestinal epithelium via PEPT1 and this may enhance the therapeutic effect of the derivatives.

Because PEPT1 is highly expressed in the small intestine compared with the large intestine, Gly-ASA, Glu-ASA and Val-ASA are more rapidly absorbed than a parent drug (5-ASA) in the small intestine. Therefore, colon selective delivery systems should be used to avoid absorption from the small intestine. We have already reported 5-ASA colon-specific delivery system using chitosan capsules and azopolymer.^47–49) We are preparing chitosan-microcapsules incorporating 5-ASA derivatives and evaluating their therapeutic effect in rat colitis. Using this formulation, 5-ASA derivatives might reach the large intestine efficiently and might be selective accumulated in inflamed regions.

Acknowledgments

We would like to thank Ms. Hitomi Yamashima and Mr. Takahiro Nishi (Ritsumeikan University) for technical assistance. This study was supported in part by a Grant from the Strategic Research Foundation at Private Universities and a Grant-in-Aid for Scientific Research (C) (Grant Number: 17K08430) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ritsumeikan Global Innovation Research Organization (R-GIRO) Project at Ritsumeikan University and the Sasagawa Scientific Research Grant from The Japan Science Society.

Author Contributions

Participated in research design: Yuri, Kono, Terada, Fujita. Conducted experiments: Yuri, Okada, Miyauchi. Contributed reagents or analytic tools: Yuri, Kono, Miyauchi, Fujita. Performed data analysis: Yuri, Kono, Miyauchi, Fujita. Wrote or contributed to the writing of the manuscript: Yuri, Kono, Fujita.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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