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1.
J Biol Chem ; 296: 100345, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33515548

RESUMEN

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.


Asunto(s)
Analgésicos/farmacología , Colestanol/farmacología , Antagonistas del Receptor de Neuroquinina-1/farmacología , Dolor/tratamiento farmacológico , Sustancia P/análogos & derivados , Analgésicos/química , Analgésicos/uso terapéutico , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colestanol/análogos & derivados , Colestanol/uso terapéutico , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Antagonistas del Receptor de Neuroquinina-1/química , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Dolor/metabolismo , Manejo del Dolor , Sustancia P/química , Sustancia P/farmacología , Sustancia P/uso terapéutico
2.
Am J Physiol Gastrointest Liver Physiol ; 322(1): G66-G78, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34755545

RESUMEN

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.


Asunto(s)
Sistema Nervioso Entérico/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , Xantonas/farmacología , Analgésicos Opioides/farmacología , Benzamidas/farmacología , Colon/efectos de los fármacos , Sistema Nervioso Entérico/fisiopatología , Motilidad Gastrointestinal/fisiología , Humanos , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Receptores Opioides mu/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
3.
Clin Exp Pharmacol Physiol ; 46(12): 1201-1215, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31429474

RESUMEN

In patients with breast cancer, metastases of cancer cells to the axial skeleton may cause excruciating pain, particularly in the advanced stages. The current drug treatments available to alleviate this debilitating pain condition often lack efficacy and/or produce undesirable side effects. Preclinical animal models of cancer-induced bone pain are key to studying the mechanisms that cause this pain and for the success of drug discovery programs. In a previous study conducted in our laboratory, we validated and characterised the rat model of Walker 256 cell-induced bone pain, which displayed several key resemblances to the human pain condition. However, gene level changes that occur in the pathophysiology of cancer-induced bone pain in this preclinical model are unknown. Hence, in this study, we performed the transcriptomic characterisation of the Walker 256 cell line cultured in vitro to predict the molecular genetic profile of this cell line. We also performed transcriptomic characterisation of the Walker 256 cell-induced bone pain model in rats using the lumbar spinal cord and lumbar dorsal root ganglia tissues. Here we show that the Walker 256 cell line resembles the basal-B molecular subtype of human breast cancer cell lines. We also identify several genes that may underpin the progression of pain hypersensitivities in this condition, however, this needs further confirmatory studies. These transcriptomic insights have the potential to direct future studies aimed at identifying various mechanisms underpinning pain hypersensitivities in this model that may also assist in discovery of novel pain therapeutics for breast cancer-induced bone pain.


Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Dolor en Cáncer/genética , Carcinoma 256 de Walker/genética , Carcinoma 256 de Walker/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Transcriptoma , Animales , Biomarcadores de Tumor/genética , Neoplasias Óseas/complicaciones , Dolor en Cáncer/etiología , Dolor en Cáncer/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hiperalgesia/etiología , Hiperalgesia/genética , Hiperalgesia/patología , Dolor/etiología , Dolor/genética , Dolor/patología , Ratas , Ratas Wistar , Médula Espinal/metabolismo , Médula Espinal/patología
4.
Biomaterials ; 285: 121536, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35533442

RESUMEN

Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NK1R) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NK1R antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NK1R endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.


Asunto(s)
Dolor Crónico , Antagonistas del Receptor de Neuroquinina-1 , Animales , Aprepitant/farmacología , Aprepitant/uso terapéutico , Dolor Crónico/tratamiento farmacológico , Endosomas , Ratones , Antagonistas del Receptor de Neuroquinina-1/farmacología , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Polímeros/farmacología
5.
Front Mol Neurosci ; 12: 273, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31798411

RESUMEN

Peripheral and central neurons in the pain pathway are well equipped to detect and respond to extracellular stimuli such as pro-inflammatory mediators and neurotransmitters through the cell surface expression of receptors that can mediate rapid intracellular signaling. Following injury or infection, activation of cell surface G protein-coupled receptors (GPCRs) initiates cell signaling processes that lead to the generation of action potentials in neurons or inflammatory responses such as cytokine secretion by immune cells. However, it is now appreciated that cell surface events alone may not be sufficient for all receptors to generate their complete signaling repertoire. Following an initial wave of signaling at the cell surface, active GPCRs can engage with endocytic proteins such as the adaptor protein ß-arrestin (ßArr) to promote clathrin-mediated internalization. Classically, ßArr-mediated internalization of GPCRs was hypothesized to terminate signaling, yet for multiple GPCRs known to contribute to pain, it has been demonstrated that endocytosis can also promote a unique "second wave" of signaling from intracellular membranes, including those of endosomes and the Golgi, that is spatiotemporally distinct from initial cell-surface events. In the context of pain, understanding the cellular and molecular mechanisms that drive spatiotemporal signaling of GPCRs is invaluable for understanding how pain occurs and persists, and how current analgesics achieve efficacy or promote side-effects. This review article discusses the importance of receptor localization for signaling outcomes of pro- and anti-nociceptive GPCRs, and new analgesic opportunities emerging through the development of "location-biased" ligands that favor binding with intracellular GPCR populations.

6.
Nat Nanotechnol ; 14(12): 1150-1159, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31686009

RESUMEN

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK1R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK1R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK1R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK1R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.


Asunto(s)
Aprepitant/administración & dosificación , Dolor Crónico/tratamiento farmacológico , Preparaciones de Acción Retardada/metabolismo , Nanopartículas/metabolismo , Antagonistas del Receptor de Neuroquinina-1/administración & dosificación , Animales , Aprepitant/farmacocinética , Aprepitant/uso terapéutico , Línea Celular , Dolor Crónico/metabolismo , Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones Endogámicos C57BL , Antagonistas del Receptor de Neuroquinina-1/farmacocinética , Antagonistas del Receptor de Neuroquinina-1/uso terapéutico , Ratas , Receptores de Neuroquinina-1/metabolismo
7.
Front Pharmacol ; 9: 495, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29867498

RESUMEN

In the majority of patients with breast cancer in the advanced stages, skeletal metastases are common, which may cause excruciating pain. Currently available drug treatments for relief of breast cancer-induced bone pain (BCIBP) include non-steroidal anti-inflammatory drugs and strong opioid analgesics along with inhibitors of osteoclast activity such as bisphosphonates and monoclonal antibodies such as denosumab. However, these medications often lack efficacy and/or they may produce serious dose-limiting side effects. In the present study, we show that J-2156, a somatostatin receptor type 4 (SST4 receptor) selective agonist, reverses pain-like behaviors in a rat model of BCIBP induced by unilateral intra-tibial injection of Walker 256 breast cancer cells. Following intraperitoneal administration, the ED50 of J-2156 for the relief of mechanical allodynia and mechanical hyperalgesia in the ipsilateral hindpaws was 3.7 and 8.0 mg/kg, respectively. Importantly, the vast majority of somatosensory neurons in the dorsal root ganglia including small diameter C-fibers and medium-large diameter fibers, that play a crucial role in cancer pain hypersensitivities, expressed the SST4 receptor. J-2156 mediated pain relief in BCIBP-rats was confirmed by observations of a reduction in the levels of phosphorylated extracellular signal-regulated kinase (pERK), a protein essential for central sensitization and persistent pain, in the spinal dorsal horn. Our results demonstrate the potential of the SST4 receptor as a pharmacological target for relief of BCIBP and we anticipate the present work to be a starting point for further mechanism-based studies.

8.
Front Pharmacol ; 8: 442, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28729837

RESUMEN

In the majority of patients with advanced breast cancer, there is metastatic spread to bones resulting in pain. Clinically available drug treatments for alleviation of breast cancer-induced bone pain (BCIBP) often produce inadequate pain relief due to dose-limiting side-effects. A major impediment to the discovery of novel well-tolerated analgesic agents for the relief of pain due to bony metastases is the fact that most cancer-induced bone pain models in rodents relied on the systemic injection of cancer cells, causing widespread formation of cancer metastases and poor general animal health. Herein, we have established an optimized, clinically relevant Wistar Han female rat model of breast cancer induced bone pain which was characterized using behavioral assessments, radiology, histology, immunohistochemistry and pharmacological methods. In this model that is based on unilateral intra-tibial injection (ITI) of Walker 256 carcinoma cells, animals maintained good health for at least 66 days post-ITI. The temporal development of hindpaw hypersensitivity depended on the initial number of Walker 256 cells inoculated in the tibiae. Hindpaw hypersensitivity resolved after approximately 25 days, in the continued presence of bone tumors as evidenced by ex vivo histology, micro-computed tomography scans and immunohistochemical assessments of tibiae. A possible role for the endogenous opioid system as an internal factor mediating the self-resolving nature of BCIBP was identified based upon the observation that naloxone, a non-selective opioid antagonist, caused the re-emergence of hindpaw hypersensitivity. Bolus dose injections of morphine, gabapentin, amitriptyline and meloxicam all alleviated hindpaw hypersensitivity in a dose-dependent manner. This is a first systematic pharmacological profiling of this model by testing standard analgesic drugs from four important diverse classes, which are used to treat cancer induced bone pain in the clinical setting. Our refined rat model more closely mimics the pathophysiology of this condition in humans and hence is well-suited for probing the mechanisms underpinning breast cancer induced bone pain. In addition, the model may be suitable for efficacy profiling of new molecules from drug discovery programs with potential to be developed as novel agents for alleviation of intractable pain associated with disseminated breast cancer induced bony metastases.

9.
Front Pharmacol ; 7: 286, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27630567

RESUMEN

The majority of patients with terminal breast cancer show signs of bone metastasis, the most common cause of pain in cancer. Clinically available drug treatment options for the relief of cancer-associated bone pain are limited due to either inadequate pain relief and/or dose-limiting side-effects. One of the major hurdles in understanding the mechanism by which breast cancer causes pain after metastasis to the bones is the lack of suitable preclinical models. Until the late twentieth century, all animal models of cancer induced bone pain involved systemic injection of cancer cells into animals, which caused severe deterioration of animal health due to widespread metastasis. In this mini-review we have discussed details of a recently developed and highly efficient preclinical model of breast cancer induced bone pain: Walker 256 cancer cell- induced bone pain in rats. The model involves direct localized injection of cancer cells into a single tibia in rats, which avoids widespread metastasis of cancer cells and hence animals maintain good health throughout the experimental period. This model closely mimics the human pathophysiology of breast cancer induced bone pain and has great potential to aid in the process of drug discovery for treating this intractable pain condition.

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