Palliation of bone cancer pain by antagonists of platelet-activating factor receptors.

Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients

Effects of blockade of PAF receptors by intrathecal injection of PAF receptor antagonists (A–D) or knockdown of spinal PAF receptors by siRNA (E–H) on tactile allodynia (A, B, E, F), guarding behavior (C, G) and limb-use abnormality (D, H) in FBC mice.

<p>(A–D); Tumor cells were implanted into the intramedulla of left femur bone 13 days before the intrathecal (i.t.) injection of PAF receptor antagonists. TCV-309 (10 pg/mouse), WEB 2086 (10 pg/mouse) or the vehicle were injected intrathecally at time “0”. Data are expressed as the mean ± SEM., n = 8–12 mice per group. *P<0.01 compared with the corresponding control (vehicle treated) values, as determined by analysis of variance followed by Tukey-Kramer test. Control mice received injections with a vehicle: ACSF. The pain-related behaviors that developed after tumor implantation in mice were not affected by the vehicle treatments. (E–H); siRNA or mismatched siRNA of PAF-receptor mRNA were transfected into the spinal cord 13 days after tumor implantation. Data are expressed as the mean ± SEM. n = 8–10 mice per group. †P<0.05, *P<0.01 compared with the corresponding control (mismatched siRNA transfection) values, as determined by analysis of valiance followed by an unpaired Student's <i>t</i>-test. Control mice received injections with mismatched siRNA or a vehicle: HVJ-envelope only. The pain-related behaviors that developed after tumor implantation in mice were not affected by mismatched siRNA or vehicle treatments.</p

The Kaplan-Mayer survival curve of FBC mice and the change of body weight (insert).

<p>For the survival experiments, TCV-309 and saline were given once a day and continued every 4 days until the animals died (n = 17 and 50, respectively). Control mice received saline for 32 days. Days for 50% of mice died after receiving TCV-309 were significantly prolonged compared to the saline-treated control, P<0.001. Statistical analysis was performed by log-rank and Gehan–Breslow–Wilcoxon tests.</p

Enhanced pain reliving effect with a combination of TCV-309 and morphine in FBC mice.

<p>TCV-309, 3 and 10 μg/kg were injected i.v. at 10 days post tumor implantation and morphine 0.1 mg/kg and 0.3 mg/kg were injected s.c. 1 day after the injection of TCV-309 (A). Gabapentine 10 and 30 mg/kg i.v. were injected 11 days post tumor transplantation and morphine, 1 mg/kg was injected at 30 min after gabapentin injection (B). Morphine 0.3–30 mg/kg was injected at 11days post tumor transplantation (C). Pain-like behaviors were evaluated at 20 min after morphine injection. Various doses of TCV-309 were injected i.v. at 11 days post tumor implantation and morphine 0.3 mg/kg were injected s.c. 1 days after the injection of TCV-309 (D). One day after the injection of TCV-309, various doses of morphine were injected (E). Control mice received injections with a vehicle: saline. The pain-related behaviors that developed after tumor implantation in mice were not affected by vehicle treatments. Each group contained 11 mice. ^†P<0.05, *P<0.01 compared with the corresponding control (vehicle treated) values, as determined by analysis of variance followed by Tukey-Kramer test. ^§P<0.01 compared with the corresponding control (vehicle treated without morphine) values, as determined by analysis of variance followed by an unpaired Student's <i>t</i>-test.</p

Enhanced pain reliving effect of TCV-309, WEB 2086, BN 50739 and morphine in FBC mice.

<p>TCV-309, WEB 2086 and BN 50739 were administered at 11 days post tumor implantation. Morphine 0.3 mg/kg s.c. was injected at 8 days after the injection of PAF receptor antagonists. Allodynia (A, B), guarding behavior (C) and limb-use abnormality (D) were evaluated at 20 min after the injection of morphine. Values represent the mean ± SEM. n = 11 mice per group. *P<0.01 compared with the corresponding control values, as determined by analysis of variance followed by an unpaired Student's <i>t</i>-test.</p

Effect of the repeated administration of TCV-309 on the pain-like behaviors in FBC mice.

<p>The administration of TCV-309 0.3 mg/kg i.v. was started 6 hr before the tumor implantation, given once a day and continued every 4 days up to 28 days. Allodynia (A, B), guarding behavior (C) and limb-use abnormality (D) were evaluated at 3 hr and 1, 2, 3 days after TCV-309 injection. Data are expressed as the mean ± SEM. n = 15 mice per group.</p

Changes in lysophosphatidylcholinr acyltransferase 2 (LPCAT2), an inducible PAF synthesis enzyume, in the spinal cords of mice after implantation of tumor cells.

<p>Alteration of spinal LPCAT2 expression 3, 8, 15, and 30 days after the levels of immunoreactivity were normalized to that of β-actin and represented as % induction compared with the values of sham-operated (deaden cells implanted) mice (mean ± SEM., n = 5–7). *P<0.05, ***P<0.001 versus the corresponding values in sham-operated mice (unpaired Student's <i>t</i>-test).</p

Morphine-induced constipation with or without TCV-309.

<p>Normal mice received with various doses of morphine and the accumulated feces on the floor over 60-309 was injected 1 day before morphine administration. Data are expressed as the mean. n = 10 mice per group.</p