Opioid Activity Research Articles

A Comprehensive Analysis of Fibromyalgia and the Role of the Endogenous Opioid System

Fibromyalgia represents a chronic pain disorder characterized by musculoskeletal pain, fatigue, and cognitive impairments. The exact mechanisms underlying fibromyalgia remain undefined; as a result, diagnosis and treatment present considerable challenges. On the other hand, the endogenous opioid system is believed to regulate pain intensity and emotional responses; hence, it might be expected to play a key role in the enhanced sensitivity experienced by fibromyalgia patients. One explanation for the emergence of disrupted pain modulation in individuals with fibromyalgia is a significant reduction in opioid receptor activity or an imbalance in the levels of endogenous opioid peptides. Further research is essential to clarify the complex details of the mechanisms underlying this abnormality. This complexity arises from the notion that an improved understanding could contribute to the development of innovative therapeutic strategies aimed at targeting the endogenous opioid system in the context of fibromyalgia. Although progress is being made, a complete understanding of these complexities remains a significant challenge. This paradigm has the potential to revolutionize the complex management of fibromyalgia, although its implementation may experience challenges. The effectiveness of this approach depends on multiple factors, but the implications could be profound. Despite the challenges involved in this transformation, the potential for improving patient care is considerable, as this condition has long been inadequately treated.

MODULATION OF MORPHINE ANTINOCICEPTIVE AND REWARDING EFFECT BY MIRTAZAPINE IN AN ANIMAL MODEL OF OSTEOARTHRITIC PAIN

People with chronic pain mitigate their suffering by the action of opioids. Adverse reactions aside, opioids are not exempt from potential complications like addiction and abuse, which have posed a global public health problem lately. Finding new therapeutic strategies to improve analgesia and to reduce opioid side effects has become a priority. In this regard, the association of different adjuvant therapies has been postulated. Despite preclinical and clinical evidence supporting the use of antidepressants as analgesics, it is not clear whether they could help reduce the risk of addiction in combination with opioids. To further explore this idea a model of chronic osteoarthritis pain was employed, and a combination of mirtazapine and morphine was used in a chronic regimen in male and female rats. The effects on the development of tactile allodynia, movement-evoked pain, reward, analgesic tolerance, naloxone-induced withdrawal symptoms, impaired locomotor activity and anxiety were evaluated under a 25-day experimental protocol. Additionally, protein expression of μ-opioid receptors and clusterin (related with substance abuse) was evaluated in plasma and in brain structures of the reward system. Chronic morphine caused analgesic tolerance, reward and naloxone-induced withdrawal symptoms. The combination reduced the analgesic tolerance and prevented the development of withdrawal symptoms but showed sex-based differences regarding reward. Increased levels of clusterin in plasma were observed in females treated with morphine, but not with combined therapy. The combination of mirtazapine and morphine could be a promising strategy to improve the management of long-term opioid treatment and its risk of abuse, especially in females.

Modeling Insights into Potential Mechanisms of Opioid-Induced Respiratory Depression within Medullary and Pontine Networks.

The opioid epidemic is a pervasive health issue and continues to have a drastic impact on the United States. This is primarily because opioids cause respiratory suppression and the leading cause of death in opioid overdose is respiratory failure (i.e., opioid-induced respiratory depression, OIRD). Opioid administration can affect the frequency and magnitude of inspiratory motor drive by activating μ-opioid receptors that are located throughout the respiratory control network in the brainstem. This can significantly affect ventilation and blunt CO2 responsiveness, but the precise neural mechanisms that suppress breathing are not fully understood. Previous research has suggested that opioids affect medullary and pontine inspiratory neuron activity by disrupting upstream elements within this circuit. Inspiratory neurons within this network exhibit synchrony consistent with shared excitation from other neuron populations and recurrent mechanisms. One possible target for opioid suppression of inspiratory drive are excitatory synapses. Reduced excitability of these synaptic elements may result in disfacilitation and reduced synchrony among inspiratory neurons. Downstream effects of disfacilitation may result in abnormal output from phrenic motoneurons resulting in distressed breathing. We tested the plausibility of this hypothesis with a computational model of the respiratory network by targeting the synaptic excitability in fictive medullary and pontine populations. The synaptic conductances were systematically decreased while monitoring the overall respiratory motor pattern and aggregate firing rates of subsets of cell populations. Simulations suggest that highly selective, rather than generalized, actions of opioids on synapses within the inspiratory network may account for different observed breathing mechanics.

G protein Inactivation as a Mechanism for Addiction Treatment.

The endogenous dynorphin/kappa opioid receptor (KOR) system in the brain mediates the dysphoric effects of stress, and KOR antagonists may have therapeutic potential for the treatment of drug addiction, depression, and psychosis. One class of KOR antagonists, the long-acting norBNI-like antagonists, have been suggested to act by causing KOR inactivation through a cJun-kinase mechanism rather than by competitive inhibition. In this study, we screened for other opioid ligands that might produce norBNI-like KOR inactivation and found that nalfurafine (a G-biased KOR agonist) and nalmefene (a KOR partial agonist) also produce long-lasting KOR inactivation. Neither nalfurafine nor nalmefene are completely selective KOR ligands, but KOR inactivation was observed at doses 10-100 fold lower than necessary for mu opioid receptor actions. Daily microdosing with nalfurafine or nalmefene blocked KORs responsible for antinociceptive effects, blocked KORs mediating stress-induced aversion, and mitigated the aversion during acute and protracted withdrawal in fentanyl-dependent mice. Both nalfurafine and nalmefene have long histories of safety and use in humans and could potentially be repurposed for the treatment of dynorphin-mediated stress disorders.

Influence of synthetic neuropeptide of opioid action on functions of 3D cultures in cold stress

influence of synthetic neuropeptide of opioid action on functions of 3D cultures in cold stress

Structure-function relationship of dynorphin B variants using naturally occurring amino acid substitutions.

Dynorphins (Dyn) represent the subset of endogenous opioid peptides with the highest binding affinity to kappa opioid receptors (KOPrs). Activation of the G-protein-coupled pathway of KOPrs has strong anticonvulsant effects. Dyn also bind to mu (MOPrs) and delta opioid receptors (DOPrs) with lower affinity and can activate the β-arrestin pathway. To fully exploit the therapeutic potential of dynorphins and reduce potential unwanted effects, increased selectivity for KOPrs combined with reduced activation of the mTOR complex would be favorable. Therefore, we investigated a series of dynorphin B (DynB) variants, substituted in one or two positions with naturally occurring amino acids for differential opioid receptor activation, applying competitive radio binding assays, GTPγS assays, PRESTO-Tango, and Western blotting on single-opioid receptor-expressing cells. Seven DynB derivatives displayed at least 10-fold increased selectivity for KOPrs over either MOPrs or DOPrs. The highest selectivity for KOPrs over MOPrs was obtained with DynB_G3M/Q8H, and the highest selectivity for KOPrs over DOPrs was obtained with DynB_L5S. Increased selectivity for KOPr over MOPr and DOPr was based on a loss of affinity or potency at MOPr and DOPr rather than a higher affinity or potency at KOPr. This suggests that the investigated amino acid exchanges in positions 3, 5, and 8 are of higher importance for binding and activation of MOPr or DOPr than of KOPr. In tests for signal transduction using the GTPγS assay, none of the DynB derivatives displayed increased potency. The three tested variants with substitutions of glycine to methionine in position 3 displayed reduced efficacy and are, therefore, considered partial agonists. The two most promising activating candidates were further investigated for functional selectivity between the G-protein and the β-arrestin pathway, as well as for activation of mTOR. No difference was detected in the respective read-outs, compared to wild-type DynB. Our data indicate that the assessment of affinity to KOPr alone is not sufficient to predict either potency or efficacy of peptidergic agonists on KOPr. Further assessment of downstream pathways is required to allow more reliable predictions of in vivo effects.

Preclinical evaluation of abuse potential of the peripherally-restricted kappa opioid receptor agonist HSK21542

HSK21542 is a peripherally-restricted kappa opioid receptor (KOR) agonist developed for pain treatment. Because of the CNS pharmacological concern of opioid receptor activation, such as physical dependence and addiction potential, an assessment of abuse potential of HSK21542 was required prior to marketing approval. The preclinical abuse potential assessments for HSK21542 included the following studies: 1) intravenous self-administration study to explore the relative reinforcing efficacy in rats self-administering remifentanil; 2) rat drug discrimination study to examine the pharmacological similarity of the interoceptive or subjective effects of HSK21542 in rats discriminating pentazocine; 3) rat conditioned place preference (CPP) paradigm to test the rewarding effects; 4) rat natural physical dependence-spontaneous withdrawal study in rats chronically treated with HSK21542; 5) naloxone-precipitated withdrawal assay following chronic HSK21542 exposure to evaluate its physical dependence potential. The results showed that HSK21542 was devoid of behavioral evidence of positive reinforcing effect and did not share similar discriminative stimulus effects with pentazocine. HSK21542 also did not produce CPP in rats. In addition, HSK21542 did not produce spontaneous withdrawal or naloxone-precipitated withdrawal in rats with chronic treatments. Collectively, these preclinical findings suggest that HSK21542 has no abuse potential in animals, which demonstrate low abuse potential in humans.

Exogenous Opioids and the Human Endocrine System: An Endocrine Society Scientific Statement.

The use and misuse of opioids are a growing global problem. Although the effects of these drugs on the human endocrine system have been studied for decades, attention on their related clinical consequences, particularly on the hypothalamic-pituitary system and bone health, has intensified over recent years. This Statement appraises research data related to the impact of opioids on the gonadal and adrenal function. Whereas hypogonadism is well recognized as a side effect of opioids, the significance of their inhibitory actions on the hypothalamic-pituitary-adrenal system and the occurrence of clinically relevant adrenal insufficiency is not fully elucidated. The often-inconsistent results of studies investigating how opioids affect the secretion of GH, prolactin, arginine vasopressin, and oxytocin are assessed. The accumulating evidence of opioid actions on bone metabolism and their negative sequelae on bone mineral density and risk of fracture are also reviewed. In each section, available data on diagnostic and management approaches for opioid endocrine sequelae are described. This Statement highlights a plethora of gaps in research associated with the effects and clinical consequences of opioids on the endocrine system. It is anticipated that addressing these gaps will improve the care of people using or misusing opioids worldwide. The Statement is not intended to serve as a guideline or dictate treatment decisions.

Fentanyl enhances immune cell response through TLR4/MD-2 complex.

Opioids have been shown to induce neuroinflammation and immune cell activation, that might contribute to some of the opioid side effects, such as opioid-induced tolerance and paradoxical hyperalgesia. In this context, TLR4/MD-2 complex has been proposed as an off-target site for opioid action. This study was aimed at investigating the effect of fentanyl on lipopolysaccharide (LPS)-induced TLR4/MD-2 activation in rat primary microglia and human monocyte-derived macrophages (MDM). The effect of fentanyl was first explored by measuring the expression and release of different proinflammatory mediators in primary rat microglia and human MDM by real-time PCR and ELISA. Then, the involvement of TLR4/MD-2 signaling was investigated studying NF-κB activation in HEK293 cells stably transfected with human TLR4, MD-2, and CD14 genes (HEK-Blue hTLR4 cells) and in human MDM. Fentanyl increased mRNA levels, as well as the LPS-induced secretion of proinflammatory mediators in primary microglia and MDM. Two inhibitors of TLR4/MD-2 signaling, namely the oxazoline derivative of N-palmitoylethanolamine (PEA-OXA) and CLI-095, blocked the production and release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4/MD-2 could be the target of the proinflammatory activity of fentanyl. Finally, we showed that fentanyl in combination with LPS activated NF-κB signaling in human MDM and in HEK-Blue hTLR4 cells and this effect was blocked by inhibitors of TLR4/MD-2 complex. These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4/MD-2 signaling. Our findings might facilitate the development of novel inhibitors of TLR4/MD-2 signaling to combine with opioid-based analgesics for effective and safe pain management.

Identification of pharmacological inducers of a reversible hypometabolic state for whole organ preservation

Drugs that induce reversible slowing of metabolic and physiological processes would have great value for organ preservation, especially for organs with high susceptibility to hypoxia-reperfusion injury, such as the heart. Using whole-organism screening of metabolism, mobility, and development in Xenopus, we identified an existing drug, SNC80, that rapidly and reversibly slows biochemical and metabolic activities while preserving cell and tissue viability. Although SNC80 was developed as a delta opioid receptor activator, we discovered that its ability to slow metabolism is independent of its opioid modulating activity as a novel SNC80 analog (WB3) with almost 1000 times less delta opioid receptor binding activity is equally active. Metabolic suppression was also achieved using SNC80 in microfluidic human organs-on-chips, as well as in explanted whole porcine hearts and limbs, demonstrating the cross-species relevance of this approach and potential clinical relevance for surgical transplantation. Pharmacological induction of physiological slowing in combination with organ perfusion transport systems may offer a new therapeutic approach for tissue and organ preservation for transplantation, trauma management, and enhancing patient survival in remote and low-resource locations.

Identification of pharmacological inducers of a reversible hypometabolic state for whole organ preservation.

Drugs that induce reversible slowing of metabolic and physiological processes would have great value for organ preservation, especially for organs with high susceptibility to hypoxia-reperfusion injury, such as the heart. Using whole-organism screening of metabolism, mobility, and development in Xenopus, we identified an existing drug, SNC80, that rapidly and reversibly slows biochemical and metabolic activities while preserving cell and tissue viability. Although SNC80 was developed as a delta opioid receptor activator, we discovered that its ability to slow metabolism is independent of its opioid modulating activity as a novel SNC80 analog (WB3) with almost 1000 times less delta opioid receptor binding activity is equally active. Metabolic suppression was also achieved using SNC80 in microfluidic human organs-on-chips, as well as in explanted whole porcine hearts and limbs, demonstrating the cross-species relevance of this approach and potential clinical relevance for surgical transplantation. Pharmacological induction of physiological slowing in combination with organ perfusion transport systems may offer a new therapeutic approach for tissue and organ preservation for transplantation, trauma management, and enhancing patient survival in remote and low-resource locations.

Kappa Opioid Receptor Activation Induces Epigenetic Silencing of Brain-Derived Neurotropic Factor via HDAC5 in Depression.

Treatment-resistant depression (TRD) occurs in almost 50% of the depressed patients. Central kappa opioid receptor (KOR) agonism has been demonstrated to induce depression and anxiety, while KOR antagonism alleviates depression-like symptoms in rodent models and TRD in clinical studies. Previously, we have shown that sustained KOR activation leads to a TRD-like phenotype in mice, and modulation of brain-derived neurotrophic factor (BDNF) expression in the prefrontal cortex (PFC) appears to be one of the molecular determinants of the antidepressant response. In the present study, we observed that sustained KOR activation by a selective agonist, U50488, selectively reduced the levels of Bdnf transcripts II, IV, and Bdnf CDS (protein-coding Exon IX) in the PFC and cultured primary cortical neurons, which was blocked by selective KOR antagonist, norbinaltorphimine. Considering the crucial role of epigenetic pathways in BDNF expression, we further investigated the role of various epigenetic markers in KOR-induced BDNF downregulation in mice. We observed that treatment with U50488 resulted in selective and specific downregulation of acetylation at the ninth lysine residue of the histone H3 protein (H3K9ac) and upregulation of histone deacetylase 5 (HDAC5) expression in the PFC. Further, using anti-H3K9ac and anti-HDAC5 antibodies in the chromatin immune precipitation assay, we detected decreased enrichment of H3K9ac and increased HDAC5 binding at Bdnf II and IV transcripts after U50488 treatment, which were blocked by a selective KOR antagonist, norbinaltorphimine. Further mechanistic studies using HDAC5 selective inhibitor, LMK235, in primary cortical neurons and adeno-associated viral shRNA-mediated HDAC5-knockdown in the PFC of mice demonstrated an essential role of HDAC5 in KOR-mediated reduction of Bdnf expression in the PFC and in depression-like symptoms in mice. These results suggest that KOR engages multiple pathways to induce depression-like symptoms in mice and provide novel insights into the mechanisms by which activation of KOR regulates major depressive disorders.

Detection of N-desethyl etonitazene in a drug checking sample: Chemical analysis and pharmacological characterization of a recent member of the 2-benzylbenzimidazole “nitazene” class

The emergence of 2-benzylbenzimidazole “nitazene” opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ‑opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6–9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the β-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

Baicalein isolated from Oroxylum indicum acts as a potent µ- and κ-opioid receptor antagonist agent via the reversal of agonist-mediated cAMP inhibition

The opioid receptors play a pivotal role in the treatment of several neuropsychiatric and neurological disorders. Oroxylum indicum (L.) Kurtz is a very important medicinal plant with several therapeutic applications. It is a main constituent of the Ayurvedic formulation ‘Dashmool’ used for multifaceted disorders by the Indians. However, the constituents of this plant in neurological conditions have not been well studied. Here, we performed activity-guided isolation of compounds for opioid receptor modulator activity. In the study, we found that the isolated compound baicalein (3) has shown the most potent and competitive antagonistic activity at 20 mg/kg dose in vivo experiments. The acute dose of 3 (20 mg/kg) and pan opioid receptor antagonist naloxone (20 mg/kg) block the morphine-induced antinociception and the paw withdrawal latency decreases up to 8.3 s and 9.6 s, respectively. The in silico studies also support our in vitro data that compound 3 binds with MOR and KOR.

Direct detection of endogenous Gαi activity in cells with a sensitive conformational biosensor.

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is not only a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane, but also the target for a large fraction of clinical drugs. However, approaches typically used to assess this signaling mechanism by directly measuring G-protein activity, like optical biosensors, suffer from limitations. On one hand, many of these biosensors require expression of exogenous GPCRs and/or G-proteins, compromising readout fidelity. On the other hand, biosensors that measure endogenous signaling may still interfere with the signaling process under investigation or suffer from having a small dynamic range of detection, hindering broad applicability. Here, we developed an optical biosensor that detects the endogenous G-protein active species Gαi-GTP upon stimulation of endogenous GPCRs more robustly than current state-of-the-art sensors for the same purpose. Its design is based on the principle of bystander Bioluminescence Resonance Energy Transfer (BRET) and leverages the Gαi-binding protein named GINIP as a high affinity and specific detector module of the GTP-bound conformation of Gαi. We optimized this design to prevent interference with Gi-dependent signaling (cAMP inhibition) and to enable implementation in different experimental systems with endogenous GPCRs, including neurotransmitter receptors in primary astroglial cells or opioid receptors in cell lines, which revealed opioid neuropeptide-mediated activation profiles different from those observed with other biosensors involving exogenous GPCRs and G-proteins. Overall, we introduce a biosensor that directly and sensitively detects endogenous activation of G-proteins by GPCRs across different experimental settings without interfering with the subsequent propagation of signaling.

Regulation of Cannabinoid and Opioid Receptor Levels by Endogenous and Pharmacological Chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), δ opioid receptor (DOR), and CB1R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

Bioactive Peptides Derived from Whey Proteins for Health and Functional Beverages

Milk serves as a crucial source of natural bioactive compounds essential for human nutrition and health. The increased production of high-protein dairy products is a source of whey—a valuable secondary product that, along with other biologically valuable substances, contains significant amounts of whey proteins and is often irrationally used or not utilized at all. Acid whey, containing almost all whey proteins and approximately one-quarter of casein, presents a valuable raw material for generating peptides with potential health benefits. These peptides exhibit properties such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, opioid, mineral-binding, and growth-stimulating activities, contributing to improved human immunity and the treatment of chronic diseases. Bioactive peptides can be produced by enzymatic hydrolysis using a variety of proteolytic enzymes, plant extracts, and microbial fermentation. With the participation of plant enzymes, peptides that inhibit angiotensin-converting enzyme are most often obtained. The use of enzymatic hydrolysis and microbial fermentation by lactic acid bacteria (LAB) produces more diverse peptides from different whey proteins with α-lactalbumin and β-lactoglobulin as the main targets. The resulting peptides of varying lengths often have antimicrobial, antioxidant, antihypertensive, and antidiabetic characteristics. Peptides produced by LAB are promising for use in medicine and the food industry as antioxidants and biopreservatives. Other beneficial properties of LAB-produced, whey-derived peptides have not yet been fully explored and remain to be studied. The development of whey drinks rich in bioactive peptides and based on the LAB proteolytic activity is underway. The strain specificity of LAB proteases opens up broad prospects for combining microorganisms to obtain products with the widest range of beneficial properties.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Susceptibility to Pentylenetetrazole-Induced Seizures in Mice with Distinct Activity of the Endogenous Opioid System.

Currently, pharmacotherapy provides successful seizure control in around 70% of patients with epilepsy; however, around 30% of cases are still resistant to available treatment. Therefore, effective anti-epileptic therapy still remains a challenge. In our study, we utilized two mouse lines selected for low (LA) and high (HA) endogenous opioid system activity to investigate the relationship between down- or upregulation of the opioid system and susceptibility to seizures. Pentylenetetrazole (PTZ) is a compound commonly used for kindling of generalized tonic-clonic convulsions in animal models. Our experiments revealed that in the LA mice, PTZ produced seizures of greater intensity and shorter latency than in HA mice. This observation suggests that proper opioid system tone is crucial for preventing the onset of generalized tonic-clonic seizures. Moreover, a combination of an opioid receptor antagonist-naloxone-and a GABA receptor agonist-diazepam (DZP)-facilitates a significant DZP-sparing effect. This is particularly important for the pharmacotherapy of neurological patients, since benzodiazepines display high addiction risk. In conclusion, our study shows a meaningful, protective role of the endogenous opioid system in the prevention of epileptic seizures and that disturbances in that balance may facilitate seizure occurrence.

Significance of CD10 for Mucosal Immunomodulation by β-Casomorphin-7 in Exacerbation of Ulcerative Colitis.

β-Casomorphin-7 (BCM), a breakdown product of milk β-casein, exhibits opioid activity. Opioids are known to affect the immune system, but the effects of BCM on ulcerative colitis (UC) are not clear. We examined the effects of BCM on mucosal immunity using a mouse dextran sulfate sodium-induced colitis model and an in vitro CD8+ T cell activation model. Human UC patients were examined to reveal the relationship between CD10 and mucosal immunity. Combined treatment of the colitis model with thiorphan (TOP) inhibited BCM degradation by suppressing CD10 in the intestinal mucosa, activating mouse mucosal CD8, and suppressing CD4 and Treg. In the CD8+ T cell in vitro activation assay using mouse splenocytes, BCM inhibited the oxidative phosphorylation (OXPHOS) of CD8+ T cells and induced the glycolytic pathway, promoting their activation. Conversely, in a culture system, BCM suppressed OXPHOS and decreased defensin α production in IEC6 mouse intestinal epithelial cells. In the mouse model, BCM reduced defensin α and butyrate levels in the colonic mucosa. During the active phase of human ulcerative colitis, the downward regulation of ileal CD10 expression by CpG methylation of the gene promoter was observed, resulting in increased CD8 activation and decreased defensin α and butyrate levels. BCM is a potential aggravating factor for UC and should be considered in the design of dietary therapy. In addition, decreased CD10 expression may serve as an indicator of UC activity and recurrence, but further clinical studies are needed.