Evidence of D-phenylglycine as delivering tool for improving L-dopa absorption

Abstract

Background: L-dopa has been used for Parkinson's disease management for a long time. However, its wide variety in the rate and the extent of absorption remained challenge in designing suitable therapeutic regime. We report here a design of using D-phenylglycine to guard L-dopa for better absorption in the intestine via intestinal peptide transporter I (PepT1).

Methods: D-phenylglycine was chemically attached on L-dopa to form D-phenylglycine-L-dopa as a dipeptide prodrug of L-dopa. The cross-membrane transport of this dipeptide and L-dopa via PepT1 was compared in brush-boarder membrane vesicle (BBMV) prepared from rat intestine. The intestinal absorption was compared by in situ jejunal perfusion in rats. The pharmacokinetics after i.v. and p.o. administration of both compounds were also compared in Wistar rats. The striatal dopamine released after i.v. administration of D-phenylglycine-L-dopa was collected by brain microdialysis and monitored by HPLC. Anti-Parkinsonism effect was determined by counting the rotation of 6-OHDA-treated unilateral striatal lesioned rats elicited rotation with (+)-methamphetamine (MA).

Results: The BBMV uptake of D-phenylglycine-L-dopa was inhibited by Gly-Pro, Gly-Phe and cephradine, the typical PepT1 substrates, but not by amino acids Phe or L-dopa. The cross-membrane permeability (Pm*) determined in rat jejunal perfusion of D-phenylglycine-L-dopa was higher than that of L-dopa (2.58 ± 0.14 vs. 0.94 ± 0.10). The oral bioavailability of D-phenylglycine-L-dopa was 31.7 times higher than that of L-dopa in rats. A sustained releasing profile of striatal dopamine was demonstrated after i. v. injection of D-phenylglycine-L-dopa (50 mg/kg), indicated that D-phenylglycine-L-dopa might be a prodrug of dopamine. D-phenylglycine-L-dopa was more efficient than L-dopa in lowering the rotation of unilateral striatal lesioned rats (19.1 ± 1.7% vs. 9.9 ± 1.4%).

Conclusion: The BBMV uptake studies indicated that D-phenylglycine facilitated the transport of L-dopa through the intestinal PepT1 transporter. The higher jejunal permeability and the improved systemic bioavailability of D-phenylglycine-L-dopa in comparison to that of l-dopa suggested that D-phenylglycine is an effective delivery tool for improving the oral absorption of drugs like L-dopa with unsatisfactory pharmacokinetics. The gradual release of dopamine in brain striatum rendered this dipeptide as a potential dopamine sustained-releasing prodrug.

Figure 1

The structures of d-phenylglycine-l-dopa and l-dopa.

Figure 2

The uptake of d-phenylglycine-l-dopa in BBMV with or without the presence of l-Phe, l-dopa, l-Gly-l-Pro, l-Gly-l-Phe and cephradine (**: p < 0.01; ***: p < 0.001.). The BBMV was prepared according to material and methods. The BBMV preparation (20 ml containing approximately 20 mg protein/ml) was added into 200 ml of a reaction buffer (composed of 300 mM mannitol, 25 mM HEPES/Tris buffer pH 7.4, (pH was adjusted by adding MES) and the test solution (to 1 - 2 mM of final conc.) was added. After incubation at room temperature for acquired time, an ice-cold stop solution (1.5 ml) containing NaCl (150 mM) and HEPES/Tris (16 mM, pH 7.4) was added and the solution was filtered through a filter paper (Whatman WCN, 0.45 μm pore size, 2.5 cm diameter) under a vacuum. The filter paper was washed twice with 3 ml of the same stop solution. The test compound remained on the filter paper were extracted with 0.5 ml of 0.01 M aqueous HCl solution by virtue of a vortex motion. The solution (100 μl) was injected onto the HPLC column. Test compound bound on the filter paper was determined for correction in different runs using preparations without BBMV added.

Figure 3

Comparison of the stability of d-phenylglycine-l-dopa and l-Gly-l-Phe in rat intestinal mucosa suspension. Each point represents mean ± SE. of 3 experiments. A methanolic solution (100 μl) of the test compound (1 mg/ml) was diluted with an isotonic mannitol buffer solution (pH 6.5, 2.4 ml) as the stock solution. This stock solution (1 ml) was mixed with the freshly prepared mucosal suspension (1 ml). The mixture was incubated in a water bath at 37°C and subjected to sampling at intervals between zero to 90 min of incubation. Each sampled solution (200 μl) was denatured with 0.8 ml of MeOH and centrifuged at 6,600 g for 5 min. Each of the supernatant (20 - 100 μl) was subjected to HPLC assay.

Figure 4

Plasma concentration-time profile of d-phenyglycine-l-dopa (a), (b) and l-dopa (c), (d) after i.v. (a), (c) and oral (b), (d) administration in Wistar rats (n = 6). The aqueous solution of test compound with dose equivalent to 5.97 mg/kg body weight of l-dopa was administered either intravenously from the tail vein or orally by a feeding tube. Blood samples were collected from the carotid artery at time intervals of from 1 to 180 min. Heparin sodium (25 I.U./ml in 0.3 ml of saline) was added to blood samples, and were then centrifuged at 6,600 g for 5 min. Plasma was stored at -78°C until being analyzed. A 200 μl of the plasma sample in a 10 ml test tube was mixed with 500 μl of 1.0 M Tris buffer (pH 8.6, adjusted by EDTA-2Na⁺) and 10 μl of 3,4-dihydroxybenzylamine (DHBA, 2 μg/ml) was added as internal standard. Alumina 100 mg was then added and then shake for 15 sec and the supernatant was decanted. The alumina was washed four times with 5 ml of water, and the adsorbed compounds on the alumina was eluted with 200 μl of an acidic solution (0.9 ml of glacial acetic acid in 4.0 ml of 1.0 M phosphate buffer). A 30 μl of the eluent was then analyzed by HPLC.

Figure 5

Striatal dopamine level after i. v. injection of d-phenylglycine-l-dopa. Values represent the group mean ± s.e.m. (n = 4). Single dose d-phenylglycine-l-dopa (50 mg/kg in 2.5 mL of normal saline) was administered i.v. via femoral vein to anesthetized male Sprague-Dawley rats (280 - 320 g). The dialysates collected from the brain microdialysis probe was subjected to HPLC to measure the striatal dopamine concentration at 10 min interval.