Blockade and reversal of spinal morphine tolerance by peptide and non-peptide calcitonin gene-related peptide receptor antagonists

Abstract

This study examined the effects of the peptide CGRP receptor antagonist CGRP(8-37) and the newly-developed non-peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 microg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED(50) value in the tailflick and paw pressure tests. Co-administration of CGRP(8-37) (4 microg) or BIBN4096BS (0.05, 0.1 microg) with morphine (15 microg) prevented the decline of antinociceptive effect and increase in ED(50) value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP(8-37) also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP(8-37) (4 microg) with morphine (15 microg) partially restored the antinociceptive effect and ED(50) value of acute morphine, reflecting the reversal of tolerance. Animals tolerant to intrathecal morphine expressed increased CGRP and substance P-like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P-like immunostaining, was blocked by a co-treatment with CGRP(8-37) (4 microg). In animals already tolerant to morphine, the increase in CGRP but not substance P-like immunostaining was partially reversed by CGRP(8-37) (4 microg). These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

Figure 1

Time course of the antinociceptive effects of daily administration of intrathecal morphine (15 μg) alone and in combination with CGRP_8-37 (4 μg) in the (A) tailflick and (B) paw pressure tests. Morphine and the test agent were administered as a single dose. Nociceptive testing was performed 30 min following each injection. The data are expressed as mean±s.e.mean for 5–7 animals. # Significant differences from the action of morphine # (P<0.05); ## (P<0.01).

Figure 2

Time course of the antinociceptive effects of daily administration of intrathecal morphine (15 μg) alone and in combination with BIBN4096BS (0.001, 0.05, 0.1 μg) in the (A) tailflick and (B) paw pressure tests. Morphine and the test agent were administered as a single dose. Nociceptive testing was performed 30 min following each injection. The data are expressed as mean±s.e.mean for 5–7 animals. # Significant differences from the action of morphine # (P<0.05); ## (P<0.01).

Figure 3

The effects of intrathecal CGRP_8-37 on established tolerance to intrathecal morphine in the tailflick test. Tolerance was induced by administration of single morphine (15 μg) injections from days 1–5. CGRP_8-37 (4 μg) was administered with morphine from days 6–10. Morphine and the test agent were given as a single injection followed by nocicpetive testing 30 min after each injection. The data are expressed as mean±s.e.mean for 5–7 animals. # Significant differences from the action of morphine (P<0.001).

Figure 4

The effect of CGRP_8-37 on established tolerance to intrathecal morphine expressed as area under the curve. Area under the curve was calculated for the antinociceptive responses obtained in the tailflick test. # Significant differences from morphine days 6–10 (P<0.001).

Figure 5

Photomicrographs of CGRP immunoreactive axons in the dorsal horn of L4–L5 spinal cords of rats following daily intrathecal injection of (A) saline 7 days, (B) CGRP_8-37 (4 μg) 7 days, (C) morphine (15 μg) 7 days, (D) morphine +CGRP_8-37 7 days, (E) morphine 5 days, then CGRP_8-37 5 days, (F) morphine 5 days, then morphine +CGRP_8-37 5 days. Scale bar 200 um.

Figure 6

Mean optical density of CGRP and substance P immunoreactive axons in the dorsal horn of L4–L5 spinal cords of rats. ^*Significant from saline ^*(P<0.01), ^**(P<0.001). # Significant from morphine (P<0.001).

Figure 7

Photomicrographs of substance P immunoreactive axons in the dorsal horn of L4–L5 spinal cords of rats following daily intrathecal injection of (A) saline 7 days, (B) CGRP_8-37 (4 μg) 7 days, (C) morphine (15 μg) 7 days, (D) morphine +CGRP_8-37 7 days, (E) morphine 5 days, then CGRP_8-37 5 days, (F) morphine 5 days, then morphine +CGRP_8-37 5 days. Scale bar 200 um.