Z-360, a novel therapeutic agent for pancreatic cancer, prevents up-regulation of ephrin B1 gene expression and phosphorylation of NR2B via suppression of interleukin-1 b production in a cancer-induced pain model in mice

Background: Z-360 is an orally active cholecystokinin-2 (CCK2)/gastrin receptor antagonist currently under development as a therapeutic drug for pancreatic cancer. It was previously reported that Z-360 treatment in combination with gemcitabine prolonged the survival period in a lethal pancreatic cancer xenograft model in mice. In a phase Ib/IIa clinical study, Z-360 treatment displayed a trend of reduced pain in patients with advanced pancreatic cancer in combination with gemcitabine including analgesics such as opioids. Here, we investigated the mechanism of analgesic action of Z-360 in a severe cancer-induced pain model in mice, which is considered to be opioid-resistant, by examining ephrin B1 gene expression, N-methyl-D-aspartate receptor NR2B subunit phosphorylation, and interleukin-1b (IL-1b) production. Results: In a mouse model of cancer-induced pain, ephrin B1 gene expression in dorsal root ganglia (DRGs) and the phosphorylation of NR2B in the spinal cord were induced. Z-360 treatment inhibited both ephrin B1 gene expression and the phosphorylation of NR2B. In addition, IL-1b production increased in the cancer-inoculated hind paw of mice, but could be suppressed by treatment with Z-360. Moreover, we observed that the CCK1 receptor antagonist devazepide similarly suppressed up-regulation of ephrin B1 gene expression and IL-1b production, and that the intraperitoneal injection of sulfated CCK-8 induced the production of IL-1b in the cancer-inoculated region. Conclusions: We have identified a novel pain cascade, in which IL-1b production in cancer-inoculated regions induces ephrin B1 gene expression in DRGs and then ephrin B1 enhances the tyrosine phosphorylation of NR2B via Eph B receptor in the spinal cord. Notably, Z-360 relieves cancer-induced pain by preventing this pain cascade through the suppression of IL-1b production, likely via the blockade of CCK1 receptor. The pre-clinical results presented here support the analgesic action of Z-360 in pancreatic cancer patients with severe, opioid-resistant pain. Pre-clinical and clinical results have demonstrated that Z-360 combined with gemcitabine represents a promising pancreatic cancer therapy approach with characteristic analgesic effects in addition to the prolongation of survival. * Correspondence: [email protected] Central Research Laboratories, Zeria Pharmaceutical Co., Ltd., 2512-1 Numagami, Oshikiri, Kumagaya-shi, Saitama, Japan Full list of author information is available at the end of the article Orikawa et al. Molecular Pain 2010, 6:72 http://www.molecularpain.com/content/6/1/72 MOLECULAR PAIN © 2010 Orikawa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Patients with cancer develop diverse symptoms, among which pain is a major factor that can drastically reduce quality of life [1-3]. Pancreatic cancer, in particular, is often accompanied by severe pain, with 90% of patients reporting significant pain [4]. Cancer-induced pain is typically relieved with opioids, such as morphine, nonopioid analgesics, such as acetaminophen, and nonsteroidal anti-inflammatory drugs; however, these drugs are not uniformly effective and undesirable side effects often limit their use [5]. For these reasons, it is therefore desirable to develop novel medical agents with increased efficacy and safety for combating cancer-induced pain. Z-360, calcium bis [(R)-(-)-3-[3-{5-cyclohexyl-1-(3,3dimethyl-2-oxo-butyl)-2-oxo-2,3,4,5-tetrahydro-1Hbenzo[b][1,4]diazepin-3-yl}ureido]benzoate], is an orally active 1,5-benzodiazepine derivative cholecystokinin-2 (CCK2)/gastrin receptor antagonist that is currently under development for the treatment of pancreatic cancer [6]. In pre-clinical studies, Z-360 inhibited pancreatic cancer growth and prolonged survival in combination with gemcitabine, a first line drug for the treatment of pancreatic cancer, in mouse pancreatic cancer xenograft models [7-9]. Z-360 was safe, well tolerated, and showed a trend towards reduced pain in phase Ib/IIa clinical study patients with advanced pancreatic cancer when used in combination with gemcitabine and analgesics, such as opioids [6]. In a previous pre-clinical study, we evaluated the analgesic effect of Z-360 using a mouse model of cancer-induced pain that was generated by the orthotopic transplantation of B16/BL6 melanoma cells into the plantar region of the right hind paw of C57BL/6 mice [10]. This model induced licking behaviour (an indicator of spontaneous pain), and mechanical allodynia and hyperalgesia in the periphery of the melanoma mass were observed from approximately day 10 after transplantation [11,12], with intensities that were a notable feature of this model. The mechanical allodynia and hyperalgesia occurring at a late phase in this model were considered to be resistant to existing analgesics, including opioids and adjuvant analgesics [11-13], which is similar to the resistance displayed by moderate-tosevere pain of cancer patients. Therefore, the effects of Z-360 on cancer-induced pain were examined in this model, and it was found that it displayed an analgesic effect that was attributed to the blockade of CCK1 receptor [10]. Moreover, we observed that the combination of Z-360 and morphine were more effective than when each were administered alone. For these reasons, the analgesic effect of Z-360 on cancer-induced pain model in mice is considered to be associated with the observed clinical benefit of Z-360 for pain relief against opioid-resistant pancreatic cancer pain [6]. Despite the evidence from these studies, the detailed analgesic mechanism of Z-360 is unclear. Recent studies suggest that ephrin B-Eph B receptor signalling in dorsal root ganglia (DRGs) contributes to activation of the N-methyl-D-aspartate (NMDA) receptor [14,15] and plays a critical role in neuropathic pain [16,17] and inflammatory hyperalgesia [14]. We speculate that ephrin B-Eph B receptor signalling in DRGs is also involved in cancer-induced pain, as this type of pain is both neuropathic and inflammatory [12]. Interleukin-1b (IL-1b) is a major inflammatory cytokine that has diverse effects on the immune system and is considered to be relevant to cancer development, owing to high levels of serum IL-1b [18,19] and elevated gene expression [20,21] in cancer patients. Recently, it was reported that IL-1b gene promoter single nucleotide polymorphisms (IL-1b secretory phenotype) are associated with an increased risk for pancreatic cancer [19] and that IL-1b participates in opioid-resistant pain, such as bone cancer and neuropathic pain [22-27]. Zhang et al. also reported that spinal IL-1b facilitated bone cancer pain [24], and the intraneural administration of IL-1b into rat sciatic nerves induced signs of neuropathic pain [22]. Conversely, the administration of IL-1b inhibitors exhibited anti-hypernociceptive action in different models of neuropathic pain [26,27]. Based on these results, IL-1b is considered to be a critical factor in the development of opioid-resistant pain, such as cancer-induced pain. Based on the results of these studies, we therefore hypothesised that ephrin B-Eph B receptor signalling and IL-1b play a role in cancer-induced pain. In this study, we attempted to determine the mechanism by which Z-360 mediates analgesic effects using a mouse model of cancer-induced pain and examining ephrin B-Eph B receptor signalling in DRGs, NMDA receptor NR2B subunit phosphorylation, and IL-1b production.