Synthesis and Characterization of a New Peptide Prodrug of Glucosamine with Enhanced Gut Permeability

The aim of this study was to synthesize a peptide prodrug of glucosamine (GlcN) with increased gut permeability through the gut peptide transporter 1 (PepT1). Glycine-Valine ester derivative of GlcN (GVG) was synthesised using solid phase synthesis followed by characterization and evaluation of its physicochemical and intestinal stability. In addition, GVG was evaluated for its ability to be biotransformed to GlcN in the liver homogenate. In vitro absorption of the new prodrug through everted rat gut was also assessed. GVG demonstrated significant and meaningful increased gut permeability as compared with GlcN. It showed favorable stability in the gut and a quick cleavage to GlcN after exposure to the liver homogenate. In conclusion, a novel prodrug of glucosamine with superior gut permeability compared to GlcN was developed and successfully tested in vitro

Synthetic scheme for solid-phase synthesis of GVG.

<p>Synthetic scheme for solid-phase synthesis of GVG.</p

Percent average ratio of GlcN concentration (+SD) inside/outside the jejunum sacks (<i>n</i> = 6) after 60 min incubation with GVG or GlcN.

<p>* GVG significantly increased the ratio as compared to GlcN.</p

Characterization of the Gly-Val-COO-GlcN (GVG) ester derivative using 1H NMR spectroscopy.

<p>1H NMR [CD3OD, 400 MHz]: [D2O, 400 MHz]: δ 5.40 (d, 1H, J = 2.9 Hz, H1α), 4.95 (d, 1H, J = 7.5 Hz, H1β), 4.30–4.65 (m, 3H, H5, H6a and H7), 4.06–4.14 (m, 1H, H6b), 3.90 (s, 2H, H10), 3.66–3.85 (m, 1H, H4), 3.45–3.65(m, 1H, H3). 3.00 and 3.30 (m, 1H, H2), 2.20–2.40 (m, 1H, H8), 0.99 (d, 6H, J = 8.6 Hz, H9).</p

Percent average ratio of GlcN concentration (+SD) inside/outside the jejunum sacks after 60 min incubation with GlcN and GVG in the presence and absence of Gly-Sar (Inhibitor).

<p>*GVG permeability was significantly inhibited in the presence of Gly-Sar (<i>P</i> <0.05).</p

Mass spectra of the Gly-Val-COO-GlcN (GVG) conjugate before (A) and after (B) purification using <i>Diaion HP</i>-<i>20</i> resin (MW = 335.357).

<p>Mass spectra of the Gly-Val-COO-GlcN (GVG) conjugate before (A) and after (B) purification using <i>Diaion HP</i>-<i>20</i> resin (MW = 335.357).</p

Synthetic scheme for solid-phase synthesis of GVG.

<p>Synthetic scheme for solid-phase synthesis of GVG.</p

Characterization of the Gly-Val-COO-GlcN (GVG) ester derivative using 13C NMR spectroscopy.

<p>13C NMR [D2O, 150 MHz]: δ 172.8 (C12), 167.2 (C7), 92.7 and 89.1 (C1α and C1β), 73.5, 71.7 and 69.5 (C3, C4 and C5), 63.8 (C6), 58.5 (C8), 56.5 (C2), 40.2 (C13), 30.0 (C9), 18.1 and 17.1 (C10 and C11).</p

Chemical structure of GVG.

<p>Chemical structure of GVG.</p

Cumulative amount of GlcN (μg) inside the jejunum sacks (serosal site) (<i>n</i> = 6) at different time points, after incubation with 1.5 μM of GlcN or GVG.

<p>Data are shown as mean (±SD). Different superscript letters denote significant differences between means in a column (<i>P</i> < 0.05).</p><p>Cumulative amount of GlcN (μg) inside the jejunum sacks (serosal site) (<i>n</i> = 6) at different time points, after incubation with 1.5 μM of GlcN or GVG.</p