Timing of treatment with an endogenous opioid alters immune response and spinal cord pathology in female mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a progressive disease of the central nervous system (CNS) that primarily affects women during the second or third decade of life. The mechanism is hypothesized to involve unregulated peripheral inflammation resulting in blood-brain barrier damage, and eventual axonal damage and demyelination. Based on this understanding, the animal model of MS, experimental autoimmune encephalomyelitis (EAE), often is utilized to study lymphocyte activation. Therapeutic paradigms of exogenous opioid growth factor (OGF) or low-dose naltrexone (LDN) treatment can modulate EAE, but little is reported regarding OGF or LDN effects on peripheral inflammation, microglia activation, and/or macrophage proliferation. Moreover, little is known about differential responses to LDN or OGF relative to the duration and timing of treatment. Utilizing a female mouse model of EAE, two treatment regimens were established to investigate differences between prophylactic treatment and traditional therapy initiated at the time of disease presentation. Prophylactic OGF or LDN treatment delayed the onset of behavior, suppressed neutrophil replication, and curtailed lymphocyte proliferation which ultimately improved behavioral outcome. Traditional therapy with OGF or LDN reversed behavioral deficits, restored OGF and IL-17 serum levels, and inhibited microglial activation within 8 days. Reduced serum OGF levels in untreated EAE mice correlated with increased microglia activation within lumbar spinal cords. Both treatment regimens of OGF or LDN reduced activated microglia, whereas only prophylactic treatment prevented CNS macrophage aggregation. These data demonstrate that the timing of LDN or OGF treatment initiation alters outcomes and can prevent or reverse behavioral deficits, cytokine activation, and spinal cord pathology.

Endogenous Opioid Peptides and Alternatively Spliced Mu Opioid Receptor Seven Transmembrane Carboxyl-Terminal Variants.

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and ß-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of ß-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants.

Design, Synthesis, and Antitumor Evaluation of an Opioid Growth Factor Bioconjugate Targeting Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge with high lethality and limited effective drug treatments. Its heightened metastatic potential further complicates the prognosis. Owing to the significant toxicity of current chemotherapeutics, compounds like [Met5]-enkephalin, known as opioid growth factor (OGF), have emerged in oncology clinical trials. OGF, an endogenous peptide interacting with the OGF receptor (OGFr), plays a crucial role in inhibiting cell proliferation across various cancer types. This in vitro study explores the potential anticancer efficacy of a newly synthesized OGF bioconjugate in synergy with the classic chemotherapeutic agent, gemcitabine (OGF-Gem). The study delves into assessing the impact of the OGF-Gem conjugate on cell proliferation inhibition, cell cycle regulation, the induction of cellular senescence, and apoptosis. Furthermore, the antimetastatic potential of the OGF-Gem conjugate was demonstrated through evaluations using blood platelets and AsPC-1 cells with a light aggregometer. In summary, this article demonstrates the cytotoxic impact of the innovative OGF-Gem conjugate on pancreatic cancer cells in both 2D and 3D models. We highlight the potential of both the OGF-Gem conjugate and OGF alone in effectively inhibiting the ex vivo pancreatic tumor cell-induced platelet aggregation (TCIPA) process, a phenomenon not observed with Gem alone. Furthermore, the confirmed hemocompatibility of OGF-Gem with platelets reinforces its promising potential. We anticipate that this conjugation strategy will open avenues for the development of potent anticancer agents.

Ocular surface complications in diabetes: The interrelationship between insulin and enkephalin.

Diabetes is a multi-faceted disorder with increasing prevalence and rising healthcare costs. The burden of diabetes is increased because of associated complications affecting nearly all organs including the eye. The underlying pathophysiology for the onset of these ocular surface disorders is not well known. Enkephalins are endogenous opioids that originate in the brain and have numerous actions in the human body. Opioid growth factor (OGF), chemically termed [Met5]-enkephalin, binds to a novel, nuclear-associated receptor and mediates cellular homeostasis. Serum OGF levels are elevated in diabetic individuals and rodent models of diabetes. Sustained blockade of the OGF receptor (OGFr) with opioid receptor antagonists, such as naltrexone (NTX), reverses many complications of diabetes in the animal model, including delayed cutaneous wound healing, dry eye, altered corneal surface sensitivity, and keratopathy. The increased enkephalin levels observed in diabetes suggest a relationship between endogenous opioid peptides and the pathophysiology of diabetes. It is common for diabetic patients to undergo insulin therapy to restore normal blood glucose levels. However, this restoration does not alter OGF serum levels nor ameliorate ocular surface complications in the animal model of diabetes. Moreover, sex differences in the prevalence of diabetes, response to insulin therapy, and abnormalities in the OGF-OGFr axis have been reported. This review highlights current knowledge on the dysregulation of the OGF-OGFr pathway and possible relationships of insulin and enkephalins to the development of ocular surface defects in diabetes. It proposes that this dysregulation is a fundamental mechanism for the pathobiology of diabetic complications.

[Met5]-enkephalin preserves diffusion metrics in EAE mice.

Multiple sclerosis is a chronic progressive neurological disorder that has few distinctive biomarkers associated with disease progression or response to therapy. This research investigated whether non-invasive imaging correlated with animal behavior and morphological indicators of disease in response to serum levels of [Met5]-enkephalin. Using the experimental autoimmune encephalomyelitis (EAE) model, adult female C57BL/6 J mice were randomized to receive daily injections of 0.1 mg/kg naltrexone (NTX) (= low dose naltrexone, LDN), 10 mg/kg Opioid Growth Factor (OGF) (chemically termed [Met5]-enkephalin) or saline beginning at the time of disease induction. Daily composite behavior scores were recorded over a 30-day period based on tail tone, gait, righting reflex, and limb strength. Prior to disease onset (day 7), and at peak disease (day 18), mice were imaged and tissues (blood and spinal cord) collected at day 30 for serum analyses of OGF and morphology. Serum OGF levels of EAE mice treated with saline were significantly reduced from baseline and from normal mice. Longitudinal cohort data demonstrated an increase in fractional anisotropy in all cohorts by day 18. There was a significant decrease in radial diffusivity in the saline group seen at day 18 whereas the axial diffusivity was not altered amongst treatment groups. Treatment with OGF or LDN resulted in mean diffusivity rates that were comparable to baseline (normal) levels at days 7 and 18. Luxol fast blue staining of the lumbar spinal cords demonstrated a 16 % reduction in myelin staining in saline treated EAE animals when compared to OGF and LDN treated EAE mice. Immunohistochemistry with Olig2 (pan-oligodendrocyte marker) and myelin basic protein (MBP) revealed that OGF and LDN treatment restored the area (%) of MBP and number of oligodendrocytes to that of normal spinal cord (∼75 %). Saline treated EAE mice had more demyelination and fewer oligodendrocytes than normal mice. Collectively, these data suggest that a panel of biomarkers including imaging, serum biomarker levels, and behavior correlate with progression of disease, and may begin to validate use of specific non-invasive markers for MS.

Featured Article: Serum [Met5]-enkephalin levels are reduced in multiple sclerosis and restored by low-dose naltrexone.

Low-dose naltrexone is a widely used off-label therapeutic prescribed for a variety of immune-related disorders. The mechanism underlying low-dose naltrexone's efficacy for fatigue, Crohn's disease, fibromyalgia, and multiple sclerosis is, in part, intermittent blockade of opioid receptors followed by upregulation of endogenous opioids. Short, intermittent blockade by naltrexone specifically blocks the opioid growth factor receptor resulting in biofeedback events that increase production of the endogenous opioid growth factor (OGF) (chemically termed [Met5]-enkephalin) facilitating interactions between opioid growth factor and opioid growth factor receptor that ultimately, result in inhibited cell proliferation. Preclinical studies have reported that enkephalin levels are deficient in animal models of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our hypothesis is that serum enkephalin levels are diminished in humans with multiple sclerosis and experimental autoimmune encephalomyelitis mice, and that change in serum opioid growth factor levels may serve as a reasonable candidate biomarker for the onset of experimental autoimmune encephalomyelitis and response to therapy. To address this, we designed a two-part study to measure endogenous opioids in multiple sclerosis patients, and to investigate the temporal pattern of decline in serum enkephalin concentrations in mice with chronic progressive experimental autoimmune encephalomyelitis and treated with low-dose naltrexone. For comparison, we investigated whether low-dose naltrexone exposure in normal mice also resulted in altered enkephalin levels. In both animal models, we monitored tactile and heat sensitivity, as well as differential white blood cell counts as indicators of inflammation. Serum [Met5]-enkephalin levels were lower in humans with multiple sclerosis relative to non-multiple sclerosis patients, and low-dose naltrexone restored their levels. In experimental autoimmune encephalomyelitis mice, [Met5]-enkephalin levels were depressed prior to the appearance of clinical disease, and were restored with low-dose naltrexone treatment. Low-dose naltrexone therapy had no effect on serum [Met5]-enkephalin or ß-endorphin in normal mice. Thus, [Met5]-enkephalin (i.e. opioid growth factor) may be a reasonable candidate biomarker for multiple sclerosis, and may signal new pathways for treatment of autoimmune disorders. Impact statement This report presents human and animal data identifying a novel biomarker for the onset and progression of multiple sclerosis (MS). Humans diagnosed with MS have reduced serum levels of OGF (i.e. [Met5]-enkephalin) relative to non-MS neurologic patients, and low-dose naltrexone (LDN) therapy restored their enkephalin levels. Serum OGF levels were reduced in mice immunized with MOG35-55 prior to any clinical behavioral sign of experimental autoimmune encephalomyelitis, and LDN therapy restored their serum OGF levels. ß-endorphin concentrations were not altered by LDN in humans or mice. Thus, blood levels of OGF may serve as a new, selective biomarker for the progression of MS, as well as response to therapy.

Antagonism of the Met5-enkephalin-opioid growth factor receptor-signaling axis promotes MSC to differentiate into osteoblasts.

Chronic opioid therapy is associated with bone loss. This led us to hypothesize that the opioid antagonists, that include naloxone, would stimulate bone formation by regulating MSC differentiation. The opioid growth factor receptor (OGFR) is a non-canonical opioid receptor that binds naloxone with high affinity whereas the native opioid growth factor, met5-enkephalin (met5), binds both the OGFR and the canonical delta opioid receptor (OPRD). Naloxone and an shRNA OGFR lentivirus were employed to disrupt the OGFR-signaling axis in cultured MSC. In parallel, naloxone was administered to bone marrow using a mouse unicortical defect model. OPRD, OGFR, and the met5-ligand were highly expressed in MSC and osteoblasts. A pulse-dose of naloxone increased mineral formation in MSC cultures in contrast to MSC treated with continuous naloxone or OGFR deficient MSC. Importantly, SMAD1 and SMAD8/9 expression increased after a pulse dose of naloxone whereas SMAD1, SMAD7, and ID1 were increased in the OGFR deficient MSC. Inhibited OGFR signaling decreased proliferation and increased p21 expression. The addition of naloxone to the unicortical defect resulted in increased bone formation within the defect. Our data suggest that novel mechanism through which signaling through the OGFR regulates osteogenesis via negative regulation of SMAD1 and p21. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1195-1205, 2016.

The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas.

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

Targeting the opioid growth factor: opioid growth factor receptor axis for treatment of human ovarian cancer.

The opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis is a biological pathway that is present in human ovarian cancer cells and tissues. OGF, chemically termed [Met(5)]-enkephalin, is an endogenous opioid peptide that interfaces with OGFr to delay cells moving through the cell cycle by upregulation of cyclin-dependent inhibitory kinase pathways. OGF inhibitory activity is dose dependent, receptor mediated, reversible, protein and RNA dependent, but not related to apoptosis or necrosis. The OGF-OGFr axis can be targeted for treatment of human ovarian cancer by (i) administration of exogenous OGF, (ii) genetic manipulation to over-express OGFr and (iii) use of low dosages of naltrexone, an opioid antagonist, which stimulates production of OGF and OGFr for subsequent interaction following blockade of the receptor. The OGF-OGFr axis may be a feasible target for treatment of cancer of the ovary (i) in a prophylactic fashion, (ii) following cytoreduction or (iii) in conjunction with standard chemotherapy for additive effectiveness. In summary, preclinical data support the transition of these novel therapies for treatment of human ovarian cancer from the bench to bedside to provide additional targets for treatment of this devastating disease.

Opioid growth factor - opioid growth factor receptor axis inhibits proliferation of triple negative breast cancer.

Triple negative breast cancer (TNBC) represents approximately 15% of the newly diagnosed cancers worldwide and is characterized by tissue lacking in estrogen, progesterone and human epidermal growth factor receptors. TNBC disproportionately affects younger women and women of colour, and new treatments are needed. The opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis is a determinant of cell proliferation in neoplasia, and OGF is an endogenously produced pentapeptide that inhibits cell replication by interacting with OGFr and upregulating cyclin-dependent inhibitory kinase pathways thus reducing DNA synthesis. In these studies we investigated the presence and function of the OGF-OGFr axis in two human TNBC cell lines, as well as in breast cancer cell lines containing hormonal receptors. TNBC cell lines MDA-MD-231 and BT-20, as well as human breast cancer cells SK-BR-3 and MCF-7, were examined for the presence of pentapeptide and receptors, as well as their response to OGF. Specificity of peptide and receptor was confirmed by antibody neutralization and molecular studies to knockdown classical receptor protein. The requirement for protein transcription and translation and RNA transcription were investigated. Growth of TNBC cells in the presence of OGF and standard of care chemotherapeutic agent paclitaxel was evaluated to determine both efficacy and protective effects against toxicity. OGF treatment inhibited TNBC cells in a dosage related, receptor mediated, and reversible manner. OGF was the specific endogenous opioid to inhibit cell proliferation, and this was mediated by p21 cyclin dependent inhibitory kinase pathways, and required protein and RNA synthesis. OGFr was the specific receptor involved; both peptide and receptor were detected in all four cell lines. OGF treatment inhibited growth of all cancer cell lines evaluated, and reduced cell death in cultures exposed to paclitaxel. The OGF-OGFr axis is present and functioning in TNBC cell lines, and provides a novel biological pathway as potential therapy.