Galacturonic acid-capsaicin prodrug for prolonged nociceptive-selective nerve blockade

Abstract

There is an urgent clinical need to develop nerve-blocking agents capable of inducing long duration sensory block without muscle weakness or paralysis to treat post-operative and chronic pain conditions. Here, we report a galacturonic acid-capsaicin (GalA-CAP) prodrug as an effective nociceptive-selective axon blocking agent. Capsaicin selectively acts on nociceptive signaling without motor nerve blockade or disruption of proprioception and touch sensation, and the galacturonic acid moiety enhance prodrug permeability across the restrictive peripheral nerve barriers (PNBs) via carrier-mediated transport by the facilitative glucose transporters (GLUTs). In addition, following prodrug transport across PNBs, the inactive prodrug is converted to active capsaicin through linker hydrolysis, leading to sustained drug release. A single injection of GalA-CAP prodrug at the sciatic nerves of rats led to nociceptive-selective nerve blockade lasting for 234 ± 37 h, which is a sufficient duration to address the most intense period of postsurgical pain. Furthermore, the prodrug markedly mitigated capsaicin-associated side effects, leading to a notable decrease in systemic toxicity, benign local tissue reactions, and diminished burning and irritant effects.

Keywords: Capsaicin; Galactose; Glucose transporters; Nociceptive-selective nerve blockade; Prodrug.

Figure 1.

Synthesis route and characterization of the GalA-CAP prodrug. (a) Structure of galactose and galacturonic acid. (b) Synthesis route a of the GalA-CAP prodrug. (c) ¹H NMR (500 MHz) spectrum of GalA-CAP prodrug in methanol-d₄. j’, k’, l’ are assigned to dihydrocapsaicin. (d) Mass spectrum of GalA-CAP prodrug. (e) UPLC chromatography illustrated the GalA-CAP prodrug degradation to capsaicin after 7 days. (f) Hydrolysis kinetics of GalA-CAP prodrug in DI water at 37 °C determined by UPLC. The concentration of GalA-CAP prodrug is 1 mg/mL. The solution was stirred at 200 rpm and kept at 37°C.

Figure 2.

Dose effects of capsaicin and GalA-CAP prodrug on (a) frequency of successful sensory nerve blockade, (b) duration of nociceptive axon blockade, (c) frequency of acute respiratory distress, (d) frequency of severe seizure, (e) frequency of contralateral nociceptive axon blockade, and (f) frequency of irreversible nociceptive axon blockade. n = 4 for all groups except for n = 8 for 3 mg GalA-CAP prodrug in (b). Data are means ± SD.

Figure 3.

Assessment of nociceptive axon blockade (a–h) and motor function impairment (i–p) in rats post-administration with 3 mg of GalA-CAP prodrug formulated with 2.5% (w/v) Tween 20 in 0.3 mL PBS buffer. Data in panels (a–h) are means ± SD.

Figure 4.

Representative histology of dissected sciatic nerves and surrounding muscle tissue. H&E-stained sections of muscle tissue (a) and toluidine blue-stained sections of sciatic nerves (b). Tissues harvested at 4- and 14-days post-injection were injected 2 mg capsaicin and GalA-CAP prodrug, whereas those harvested at 28 days post-injection were injected 3 mg capsaicin and GalA-CAP prodrug. Red arrow denotes perifascicular internalization of nucleus. M: Muscle; N: Nerve region; Inf: Inflammation. Scale bar: 200 μm.

Figure 5.

Representative TEM images of dissected sciatic nerves and the diameter of axons. Red arrows denote unmyelinated fibers. The nerve tissues were harvested at 28 days post-injection, and the nerve from rats without injections were harvested at the same time. Scale bar: 2 μm. n = 200 fibers for diameter measurement. Data shown mean ± SD. *P < 0.05, ns‒not significant, analysis of variance (ANOVA), n = 4.

Figure 6.

Inhibition of the prodrug uptake by glucose and KL-11743. (a) Representative time courses of thermal latency in rats co-injected with 2 mg GalA-CAP prodrug and different doses of glucose. (b) Time courses of thermal latency of the ipsilateral side of rats co-injected with 2 mg GalA-CAP prodrug and different doses of KL-11743. (c) The average duration of nociceptive axon blockade in rats co-injected with 2 mg GalA-CAP prodrug or capsaicin with different doses of inhibitors. (d) Structures of GalA-CAP prodrug and Gly-CAP prodrug. Comparison on frequency of successful axon blockade (e) and duration of nociceptive axon blockade (f) between 3 mg GalA-CAP prodrug and 3 mg Gly-CAP prodrug. Data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant, analysis of variance (ANOVA).

Figure 7.

In vivo nerve penetration of FITC, Gal-FL, Glc-UDP-FL, and PEG₇₅₀-FL. (a) Representative confocal images of sciatic nerves and surrounding tissues 4 h after injecting FITC, Gal-FL, Gal-FL@ 100 μM KL-11743, Glc-UDP-FL, Glc-UDP-FL@ 100 μM KL-11743, and PEG₇₅₀-FL. The image taken from higher laser intensity were inserted on the left top for groups of Gal-FL@ 100 μM KL-11743, Glc-UDP-FL@ 100 μM KL-11743, and PEG₇₅₀-FL, due to poor signals collected using the same camera settings. The contour of nerves was circled in red dash lines. Scale bar: 100 μm. (b) Mean fluorescent intensity in epineurium and perineurium. (c) Mean fluorescent intensity in nerves. (d) Relationship between mean fluorescent intensity and normalized distance from the epineurium/perineurium of the nerve. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are means ± SD.

Figure 8.

Enhanced efficacy and reduced side effects due to sustained release of capsaicin from the less active prodrug. (a) Structures of Gal-CAP prodrug and capsaicin. Comparison on frequency of successful axon blockade (b) and duration of nociceptive axon blockade (c) between 3 mg GalA-CAP prodrug, 3 mg Gly-CAP prodrug, 3 mg Gal-CAP prodrug, and 3 mg capsaicin. Data shown are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, analysis of variance (ANOVA). (d-j) Molecular docking of capsaicin and prodrugs. Molecular docking of capsaicin and prodrugs. (d) Published binding mode of capsaicin with TRPV1 (generated from PDB code 7LPE). The atoms of capsaicin are colored as follows: carbon, green; oxygen, red; nitrogen, blue. The cartoon shows the protein and key residues are colored as follows: carbon, grey; oxygen, red; nitrogen, blue. Yellow dotted lines indicate H-bonds, and the red dotted lines represent π-π stacking interactions. (e, g, i) Proposed binding modes of GalA-CAP, Gly-CAP, and Gal-CAP prodrug with TRPV1. (f, h, j) Superposed docking poses of capsaicin and GalA-CAP, Gly-CAP, or Gal-CAP prodrug. The atoms of prodrugs are colored as follows. GalA-CAP: carbon, cyan; oxygen, red; nitrogen, blue. Gly-CAP: carbon, purple; oxygen, red; nitrogen, blue. Gal-CAP: carbon, pink; oxygen, red; nitrogen, blue.

Figure 9.

Licking frequency (a) and licking duration (b) during the first 5 min after intraplantar application of 0.1% (w/v) capsaicin, 0.05% (w/v) capsaicin, 0.1% (w/v) GalA-CAP prodrug, 0.1% (w/v) Gal-CAP prodrug and 1×PBS buffer. *P < 0.05, ***P < 0.001 analysis of variance (ANOVA), n = 4.

Scheme 1.

Schematic illustration of the sugar-capsaicin prodrug for sensory selective nerve blockade. Sugar-capsaicin prodrugs penetrate the peripheral nerve barrier, mediated by the GLUT transporters. After crossing the peripheral nerve barriers, the inactive prodrug is converted to active capsaicin by hydrolysis of the ester bond. Capsaicin acts selectively on sensory but not motor neurons.