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Biol Pharm Bull
2020 Jan 01;434:697-706. doi: 10.1248/bpb.b19-01048.
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Transport Characteristics of 5-Aminosalicylic Acid Derivatives Conjugated with Amino Acids via Human H+-Coupled Oligopeptide Transporter PEPT1.
Yuri T
,
Kono Y
,
Okada T
,
Terada T
,
Miyauchi S
,
Fujita T
.
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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.
Fig. 1. Chemical Structure of 5-ASA and Its Amino Acid Derivatives
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).
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).
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).
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).
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.)
Supplementary figure 1- HPLC analysis of the degradation property of 5-ASA derivatives in Caco-2 cell homogenate. Chromatograms of 5-ASA (A), Gly-ASA (B,C), Glu-ASA (D,E), and Val-ASA (F,G). The 5-ASA derivatives (10 microM) were incubated with an equal volume of Caco-2 cell homogenate (1 mg protein/mL) for predetermined time (Gly-ASA and Glu-ASA: 60 min, Val-ASA: 15 min) at 37 degrees C under shaking conditions (120 rpm). HPLC analysis of the derivatives was performed before (B,D,F) and after (C,E,G) the incubation. Arrows indicate the peak derived from 5-ASA.
