For many patients, pain is the first sign of cancer and, while pain can be present at any time, the frequency and intensity of pain tend to increase with advancing stages of the disease. Thus, between 75 and 90% of patients with metastatic or advanced-stage cancer will experience significant cancer-induced pain. One major unanswered question is why cancer pain increases and frequently becomes more difficult to fully control with disease progression. To gain insight into this question we used a mouse model of bone cancer pain to demonstrate that as tumor growth progresses within bone, Tropomyosin receptor kinase A (TrkA)-expressing sensory and sympathetic nerve fibers undergo profuse sprouting and form neuroma-like structures. To address what is driving the pathological nerve reorganization we administered an antibody to nerve growth factor (anti-NGF). Early sustained administration of anti-NGF, whose cognate receptor is TrkA, blocks the pathological sprouting of sensory and sympathetic nerve fibers, the formation of neuroma-like structures, and inhibits the development of cancer pain. These results suggest that cancer cells and their associated stromal cells release NGF, which induces a pathological remodeling of sensory and sympathetic nerve fibers. This pathological remodeling of the peripheral nervous system then participates in driving cancer pain. Similar to therapies that target the cancer itself, the data presented here suggest that the earlier that therapies blocking this pathological nerve remodeling are initiated, the more effective the control of cancer pain.

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 (CJL-1-140) possessed a functional profile that was unique from its monovalent counterparts providing evidence of the discrete effects of bivalent ligands. Lead compound 7 (CJL-1-87) significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other non-malignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene related peptide (CGRP+) and neurofilament 200 kDa (NF200+) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also co-express tropomyosin receptor kinase A (TrkA+). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, RT-PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA+ sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state.

Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP + ) sensory nerve fibers. Nearly all CGRP + nerve fibers that undergo sprouting also co-express tropomyosin receptor kinase A (TrkA + ) and growth associated protein-43 (GAP43 + ). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP + / TrkA + / GAP43 + sensory nerve fibers.

Objective Many forms of arthritis are accompanied by significant chronic joint pain. Here we studied whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathological reorganization. Methods A painful arthritic knee joint was produced by injection of complete Freund’s adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry using antibodies raised against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kDa (NF200; sensory nerve fibers), growth associated protein-43 (GAP43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells) or CD68 (monocytes/macrophages). Results In CFA-injected mice, but not vehicle-injected mice, there was a significant increase in the density of CD68+ macrophages, CD31+ blood vessels, CGRP+, NF200+, GAP43+, and TH+ nerve fibers in the synovium as well as joint pain-related behaviors. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31+ blood vessels or CD68+ macrophages. Conclusions Ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.

A closed femur fracture pain model was developed in the C57BL/6J mouse. One day after fracture, a monoclonal antibody raised against nerve growth factor (anti-NGF) was delivered intraperitoneally and resulted in a reduction in fracture pain-related behaviors of ∼50%. Anti-NGF therapy did not interfere with bone healing as assessed by mechanical testing and histomorphometric analysis.Introduction: Current therapies to treat skeletal fracture pain are limited. This is because of the side effect profile of available analgesics and the scarcity of animal models that can be used to understand the mechanisms that drive this pain. Whereas previous studies have shown that mineralized bone, marrow, and periosteum are innervated by sensory and sympathetic fibers, it is not understood how skeletal pain is generated and maintained even in common conditions such as osteoarthritis, low back pain, or fracture. Materials and Methods:In this study, we characterized the pain-related behaviors after a closed femur fracture in the C57BL/6J mouse. Additionally, we assessed the effect of a monoclonal antibody that binds to and sequesters nerve growth factor (anti-NGF) on pain-related behaviors and bone healing (mechanical properties and histomorphometric analysis) after fracture. Results: Administration of anti-NGF therapy (10 mg/kg, days 1, 6, and 11 after fracture) resulted in a reduction of fracture pain-related behaviors of ∼50%. Attenuation of fracture pain was evident as early as 24 h after the initial dosing and remained efficacious throughout the course of fracture pain. Anti-NGF therapy did not modify biomechanical properties of the femur or histomorphometric indices of bone healing. Conclusions: These findings suggest that therapies that target NGF or its cognate receptor(s) may be effective in attenuating nonmalignant fracture pain without interfering with bone healing.

Current therapies to treat skeletal fracture pain are extremely limited. Some non-steroidal anti-inflammatory drugs have been shown to inhibit bone healing and opiates induce cognitive dysfunction and respiratory depression which are especially problematic in the elderly suffering from osteoporotic fractures. In the present report, we developed a closed femur fracture pain model in the mouse where skeletal pain behaviors such as flinching and guarding of the fractured limb are reversed by 10mg/kg morphine. Using this model we showed that the administration of a monoclonal antibody against nerve growth factor (anti-NGF) reduced fracture-induced pain-related behaviors by over 50%. Treatment with anti-NGF reduced c-Fos and dynorphin up-regulation in the spinal cord at day 2 post-fracture. However, anti-NGF treatment did not reduce p-ERK and c-Fos expression at 20 and 90 min, respectively, following fracture. This suggests NGF is involved in maintenance but not the acute generation of fracture pain. Anti-NGF therapy did not inhibit bone healing as measured by callus formation, bridging of the fracture site or mechanical strength of the bone. As the anti-NGF antibody does not appreciably cross the blood-brain barrier, the present data suggest that the anti-hyperalgesic action of anti-NGF therapy results from blockade of activation and/or sensitization of the CGRP/trkA positive fibers that normally constitute the majority of sensory fibers that innervate the bone. These results demonstrate that NGF plays a significant role in driving fracture pain and that NGF sequestering therapies may be efficacious in attenuating this pain.

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.