Methionine enkephalin suppresses lung cancer metastasis by regulating the polarization of tumor-associated macrophages and the distribution of myeloid-derived suppressor cells in the tumor microenvironment and inhibiting epithelial-mesenchymal transition.

Metastasis is one of the most difficult challenges for clinical lung cancer treatment. Epithelial-mesenchymal transition (EMT) is the crucial step of tumor metastasis. Immune cells in the tumor microenvironment (TME), such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), promote cancer cell EMT. In this study, we explored the effect of methionine enkephalin (MENK) on the EMT process in vitro and in vivo, and its influence on TAMs, MDSCs, and associated cytokines in vivo. The results showed that MENK suppressed growth, migration, and invasion of lung cancer cells and inhibited the EMT process by interacting with opioid growth factor receptor. MENK reduced the number of M2 macrophages and MDSC infiltration, and downregulated the expression of interleukin-10 and transforming growth factor-ß1 in both primary and metastatic tumors of nude mice. The present findings suggest that MENK is a potential target for suppressing metastasis in lung cancer treatment.

NaV1.7 Channel Blocker [Ala5, Phe6, Leu26, Arg28]GpTx-1 Attenuates CFA-induced Inflammatory Hypersensitivity in Rats via Endogenous Enkephalin Mechanism.

Venom-derived NaV1.7 channel blockers have promising prospects in pain management. The 34-residue tarantula peptide GpTx-1 is a potent NaV1.7 channel blocker. Its powerful analog [Ala5, Phe6, Leu26, Arg28]GpTx-1 (GpTx-1-71) displayed excellent NaV1.7 selectivity and analgesic properties in mice. The current study aimed to elucidate the anti-hyperalgesic activities of GpTx-1-71 in inflammatory pain and reveal the underlying mechanisms. Our results demonstrated that intrathecal and intraplantar injections of GpTx-1-71 dose-dependently attenuated CFA-induced inflammatory hypersensitivity in rats. Moreover, GpTx-1-71-induced anti-hyperalgesia was significantly reduced by opioid receptor antagonists and the enkephalin antibody and diminished in proenkephalin (Penk) gene knockout animals. Consistently, GpTx-1-71 treatment increased the enkephalin level in the spinal dorsal horn and promoted the Penk transcription and enkephalin release in primary dorsal root ganglion (DRG) neurons, wherein sodium played a crucial role in these processes. Mass spectrometry analysis revealed that GpTx-1-71 mainly promoted the secretion of Met-enkephalin but not Leu-enkephalin from DRG neurons. In addition, the combination of subtherapeutic Met-enkephalin and GpTx-1-71 produced synergistic anti-hyperalgesia in CFA-induced inflammatory hypersensitivity. These findings suggest that the endogenous enkephalin pathway is essential for GpTx-1-71-induced spinal and peripheral analgesia in inflammatory pain. PERSPECTIVE: This article presents a possible pharmacological mechanism underlying NaV1.7 blocker-induced analgesia in inflammatory pain, which helps us to better understand and develop venom-based painkillers for incurable pain.

Crotalphine Modulates Microglia M1/M2 Phenotypes and Induces Spinal Analgesia Mediated by Opioid-Cannabinoid Systems.

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for ß-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

Dopamine D2 receptors bidirectionally regulate striatal enkephalin expression: Implications for cocaine reward.

Low dopamine D2 receptor (D2R) availability in the striatum can predispose for cocaine abuse; though how low striatal D2Rs facilitate cocaine reward is unclear. Overexpression of D2Rs in striatal neurons or activation of D2Rs by acute cocaine suppresses striatal Penk mRNA. Conversely, low D2Rs in D2-striatal neurons increases striatal Penk mRNA and enkephalin peptide tone, an endogenous mu-opioid agonist. In brain slices, met-enkephalin and inhibition of enkephalin catabolism suppresses intra-striatal GABA transmission. Pairing cocaine with intra-accumbens met-enkephalin during place conditioning facilitates acquisition of preference, while mu-opioid receptor antagonist blocks preference in wild-type mice. We propose that heightened striatal enkephalin potentiates cocaine reward by suppressing intra-striatal GABA to enhance striatal output. Surprisingly, a mu-opioid receptor antagonist does not block cocaine preference in mice with low striatal D2Rs, implicating other opioid receptors. The bidirectional regulation of enkephalin by D2R activity and cocaine offers insights into mechanisms underlying the vulnerability for cocaine abuse.

Methionine enkephalin inhibited cervical carcinoma via apoptosis promotion and reduction of myeloid derived suppressor cell infiltrated in tumor.

Immunotherapy for cervical carcinoma is becoming increasingly important recently. In these studies methionine enkephalin (menk) is shown to inhibit cervical tumor cell proliferation in vitro in association with an increase in the expression of apoptosis markers and mediators, including an increase in fas, caspase 8, and caspase 3 expression and intrinsic expression of the signaling pathway mediator bax. In vivo, tumor growth was restrained in mice xenotransplant model with typical pathological features of apoptosis. Furthermore, myeloid derived suppressor cells (MDSCs) had a significant decrease in circulation and in tumor site. In brief, these findings showed menk could inhibit tumor growth in vitro and in vivo, providing direction of further research and clinical application prospect.

CIRBP-OGFR axis safeguards against cardiomyocyte apoptosis and cardiotoxicity induced by chemotherapy.

Cold-inducible RNA-binding protein (CIRBP) is documented to be required for maintaining cardiac function, however, its role in chemotherapy-induced cardiotoxicity remains obscured. Herein, we report that CIRBP decreases cardiomyocyte apoptosis and attenuates cardiotoxicity through disrupting OGF-OGFR signal. CIRBP deficiency is involved in diverse chemotherapeutic agents induced cardiomyocyte apoptosis. Delivery of exogenous CIRBP to the mouse myocardium significantly mitigated doxorubicin-induced cardiac apoptosis and dysfunction. Specifically, OGFR was identified as a downstream core effector responsible for chemotherapy-induced cardiomyocyte apoptosis. CIRBP was shown to interact with OGFR mRNA and to repress OGFR expression by reducing mRNA stability. CIRBP-mediated cytoprotection against doxorubicin-induced cardiac apoptosis was demonstrated to largely involve OGFR repression by CIRBP. NTX as a potent antagonist of OGFR successfully rescued CIRBP ablation-rendered susceptibility to cardiac dyshomeostasis upon exposure to doxorubicin, whereas another antagonist ALV acting only on opioid receptors did not. Taken together, our results demonstrate that CIRBP confers myocardium resistance to chemotherapy-induced cardiac apoptosis and dysfunction by dampening OGF/OGFR axis, shedding new light on the mechanisms of chemo-induced cardiotoxicity and providing insights into the development of an efficacious cardioprotective strategy for cancer patients.

Angiotensin-converting enzyme gates brain circuit-specific plasticity via an endogenous opioid.

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin I to produce angiotensin II. In the brain, ACE is especially abundant in striatal tissue, but the function of ACE in striatal circuits remains poorly understood. We found that ACE degrades an unconventional enkephalin heptapeptide, Met-enkephalin-Arg-Phe, in the nucleus accumbens of mice. ACE inhibition enhanced µ-opioid receptor activation by Met-enkephalin-Arg-Phe, causing a cell type-specific long-term depression of glutamate release onto medium spiny projection neurons expressing the Drd1 dopamine receptor. Systemic ACE inhibition was not intrinsically rewarding, but it led to a decrease in conditioned place preference caused by fentanyl administration and an enhancement of reciprocal social interaction. Our results raise the enticing prospect that central ACE inhibition can boost endogenous opioid signaling for clinical benefit while mitigating the risk of addiction.

Research progress of opioid growth factor in immune-related diseases and cancer diseases.

Methionine enkephalin (MENK) has an important role in both neuroendocrine and immune systems. MENK was known as an opioid growth factor (OGF) for its growth regulatory characteristics. OGF interacts with the OGF receptor (OGFr) to inhibit DNA synthesis by upregulating p16 and/or p21, which delays the cell cycle transition from G0/G1 to S phase, and inhibits cell proliferation. In addition, OGF combines with OGFr in immune cells to exert its immunomodulatory activity and regulate immune function. OGF has been studied as an immunomodulator in a variety of autoimmune diseases, including multiple sclerosis, inflammatory bowel disease, diabetes and viral infections, and has been proven to relieve symptoms of certain diseases in animal and in vitro experiments. Also, OGF and OGFr have various anti-tumor molecular mechanisms. OGF can be used as the primary therapy alone or combined with other drugs to treat tumors. This article summarizes the research progress of OGF in immune-related diseases and cancer diseases.

Regulatory role of methionine enkephalin in myeloid-derived suppressor cells and macrophages in human cutaneous squamous cell carcinoma.

The antitumor effects of methionine enkephalin (MENK), also known as opioid growth factor (OGF), including its inhibitory effects on cutaneous squamous cell carcinoma (CSCC), have been established. In this study, we determined the precise mechanism by which MENK suppresses CSCC cell growth. In particular, MENK induced G0/G1 cell cycle arrest and promoted apoptosis in CSCC cells via the Bcl-2/Bax/Caspase-3 signaling pathway. Moreover, MENK reduced immunosuppression by downregulating the number of myeloid-derived suppressor cells (MDSCs) and regulating the polarization of tumor-associated macrophages from M2 to M1 in vivo. Furthermore, JAK2/STAT3, an important tumor-promotion and immunosuppression signaling pathway that is involved in MDSC expansion in tumors and macrophage polarization, was inhibited. These findings highlight the potential of the JAK2/STAT3 signaling pathway as a therapeutic target and suggest the clinical application of MENK for CSCC.

A novel mechanism of lung cancer inhibition by methionine enkephalin through remodeling the immune status of the tumor microenvironment.

This study examined the antitumor effect of methionine enkephalin (MENK) against lung cancer in vivo and in vitro and explored the underlying mechanisms. Changes in the immune status of the tumor microenvironment (TME) in response to MENK administration were examined in mice. MENK significantly inhibited the proliferation of lung cancer cells in vivo and in vitro by regulating the Wnt/ß-catenin pathway and causing cell cycle arrest at the G0/G1 phase. Knockdown of opioid growth factor receptor abolished the effect of MENK on lung cancer cells. The immune status of the TME of mice differed between the MENK and control groups. MENK increased the infiltration of M1-type macrophages, natural killer cells, CD8+ T cells, CD4+ T cells, and dendritic cells into the TME, and decreased the proportion of myeloid inhibitory cells and M2-type macrophages. Immunohistochemical analysis of the expression of cytokines in the TME showed that MENK upregulated IL-15, IL-21, IFN-γ, and granzyme B and downregulated IL-10 and TGF-ß1 in mice. Taken together, these finding indicate that MENK may be a potential agent for lung cancer treatment in the future, especially for overcoming immune escape and immune resistance.

Neuroanatomical distribution of the enkephalinergic and tachykininergic systems in the alpaca brainstem: an immunohistochemical study.

INTRODUCTION: A recent study has shown a close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon. In this study, our aim is to show this relationship in the alpaca brainstem. MATERIAL AND METHODS: Using an immunohistochemical technique, the distribution of immunoreactive (Ir) fibers and cell bodies containing substance P (SP) or methionine-enkephalin (MET) has been studied in the alpaca brainstem. Five adult males were used; brain tissue was fixed and processed by standard methods. RESULTS: SP- and MET-Ir fibers showed a widespread and similar distribution in the mesencephalon, pons and medulla oblongata. The co-localization of fibers containing SP or MET was found in most of the nuclei/tracts of the alpaca brainstem. This close neuroanatomical relationship suggests multiple physiological interactions between both neuropeptides. The distribution of the cell bodies containing SP was very restricted (cell bodies were only observed in a few nuclei located in the mesencephalon and medulla oblongata), whereas MET-Ir perikarya showed a moderately widespread distribution in the mesencephalon, pons and medulla oblongata. CONCLUSIONS: This study increases the knowledge on the neuroanatomical distribution/relationship of the tachykininergic (SP) and enkephalinergic (MET) systems in the alpaca central nervous system.

Methionine enkephalin (MENK) suppresses lung cancer by regulating the Bcl-2/Bax/caspase-3 signaling pathway and enhancing natural killer cell-driven tumor immunity.

The aim of this study was to investigate how methionine enkephalin (MENK) regulates the biological behavior of lung cancer cells and to further explore its anti-lung cancer mechanisms in vitro and in vivo. The results showed that MENK enhanced the expression of opioid receptor (OGFr) and induced apoptosis of lung cancer cells by activating the Bcl-1/Bax/caspase-3 signaling pathway in vitro and in vivo. However, the regulatory effects of MENK disappeared after blockade of the OGFr. This confirmed that a prerequisite for the anti-tumor action of MENK is binding to OGFr. Additionally, we observed that MENK treatment enhanced the immunogenicity of lung cancer by enhancing the exposure of calreticulin and high mobility group box 1, and increasing the expression of NKG2D ligands. Further studies showed that MENK treatment increased the expression of natural killer (NK) cell-related cytokines such as granzyme B and interferon-γ and NK cell activation. Thus, we concluded that MENK might inhibit the proliferation of lung cancer cells by activating the Bcl-2/Bax/caspase-3 signaling pathway and enhancing immunogenicity and NK cell-driven tumor immunity.

Neuroanatomical organization of methionine-enkephalinergic system in the brain of the Mozambique tilapia Oreochromis mossambicus.

Enkephalins are a class of opioid peptides implicated in several physiological and neuroendocrine responses in vertebrates. In this study, using immunocytochemical or immunofluorescence technique, we examined the neuroanatomical distribution of methionine enkephalin (M-ENK) immunoreactivity in the central nervous system (CNS) of the cichlid fish Oreochromis mossambicus. In the telencephalon, no M-ENK-like-immunoreactive (M-ENK-L-ir) perikarya, but sparsely distributed fibres were detected in the glomerular layer and the granule cell layer of the olfactory bulb. Although intensely labeled M-ENK-L-ir cells and fibres were found in the pallium, no M-ENK immunoreactivity was observed in the subpallium. The preoptic area showed a few M-ENK-L-ir somata and dense innervations of fibres. In the hypothalamic area, M-ENK-L-ir cells and fibres were located in magnocellular and parvocellular subdivisions of the nucleus preopticus, and medial and lateral subdivisions of the nucleus lateralis tuberis. Surrounding the recessus lateralis of the third ventricle, several intensely stained and packed M-ENK-L-ir cells and fibres were seen in dorsal, lateral and ventral subdivisions of the nucleus recessus lateralis. In the diencephalon, M-ENK immunoreactivity was restricted to the habenula, the thalamus, the pretectal area and the nucleus posterior tuberis. Dense aggregations of M-ENK-L-ir fibres were seen in the mesencephalic subdivisions, the optic tectum and the torus semicircularis, whereas a few fusiform M-ENK-L-ir cells and fibres were scattered in the midbrain tegmentum. In the rhombencephalon, different populations of ovoid or spindle shaped M-ENK-L-ir cells were observed in the secondary gustatory nucleus, the sensory trigeminal nerve nucleus, the nucleus reticularis medialis and the vagal motor nucleus, whereas bands of fibres were seen in the rostral spinal cord. Collectively, the widespread distribution of M-ENK immunoreactivity in the CNS suggests a role for this opioid peptide in regulation of neuroendocrine control of reproduction and modulation of sensorimotor functions in fish.

Contribution of the µ opioid receptor and enkephalin to the antinociceptive actions of endomorphin-1 analogs with unnatural amino acid modifications in the spinal cord.

Endomorphin analogs containing unnatural amino acids have demonstrated potent analgesic effects in our previous studies. In the present study, the differences in antinociception and the mechanisms thereof for analogs 1-3 administered intracerebroventricularly and intrathecally were explored. All analogs at different routes of administration produced potent analgesia compared to the parent peptide endomorphin-1. Multiple antagonists and antibodies were used to explore the mechanisms of action of these analogs, and it was inferred that analogs 1-3 stimulated the µ opioid receptor to induce antinociception. Moreover, the antibody data suggested that analog 2 may induce the release of immunoreactive [Leu5]-enkephaline and [Met5]-enkephaline to produce a secondary component of antinociception at the spinal level and analog 3 may stimulate the the release of immunoreactive [Met5]-enkephaline at the spinal level. Finally, analogs 2 and 3 produced no acute tolerance in the spinal cord. We hypothesize that the unique characteristics of the endomorphin analogs result from their capacities to stimulate the release of endogenous antinociceptive substances.

Blockade of OGFr delays the onset and reduces the severity of diabetic ocular surface complications.

The opioid growth factor (OGF)-OGF receptor (OGFr) pathway is present in the ocular surface and functions to maintain homeostasis of the epithelium. The OGF-OGFr pathway has been reported to be dysregulated in diabetic individuals and animal models, and is reflected in elevations of the inhibitory growth factor, OGF, chemically termed [Met5]-enkephalin. Recently, our laboratory reported elevated levels of OGF and OGFr in the serum and corneal epithelium of type 1 diabetic rats, suggesting that dysregulation of the OGF-OGFr axis may lead to dry eye, abnormal corneal surface sensitivity, and delayed re-epithelialization. Blockade of OGF-OGFr pathway using naltrexone, a potent opioid receptor antagonist, reverses dry eye symptoms and restores corneal surface sensitivity in diabetic rats when used as a therapy. Based on the evidence that both OGF and OGFr are elevated in type 1 diabetic rats, this study examined whether systemic or topical naltrexone treatment initiated at the time of induction of hyperglycemia could protect against the development of diabetic ocular surface complications. Diabetic male Sprague-Dawley rats treated systemically or topically with naltrexone had a delayed onset of dry eye and altered corneal surface sensitivity, and an improved healing rate for corneal wounds, that were comparable to non-diabetic rats. Serum levels of OGF were normal for rats receiving systemic naltrexone, and OGF tissue levels were normal for type 1 diabetic rats receiving twice daily naltrexone drops. OGFr levels remained elevated. These data support the role of the OGF-OGFr axis in regulation of ocular surface complications, and suggest that naltrexone therapy may be beneficial for pre-diabetic and early diabetic individuals.

Effects of botulinum toxin A on endometriosis­associated pain and its related mechanism.

Endometriosis (EMS) is a common disease in women aged 25­45 years, and pain is the main clinical symptom. The primary clinical treatment is surgical excision and drug therapy targeting the ectopic lesions, but these have not been very effective. Botulinum neurotoxin serotype A (BTX­A) has been reported to be useful in the treatment of pain in a variety of diseases. Based on this, the aim of the present study was to explore the therapeutic effect and mechanism of BTX­A on EMS. A model of nerve injury induced by oxygen glucose deprivation (OGD) was constructed in PC12 cells and EMS mice. Model cells and mice were treated with different concentrations of BTX­A to observe the changes in pain behavior, to detect cell viability and the secretion of norepinephrine (NE) and methionine enkephalin (M­EK) in cells and the spinal cord, and to evaluate the expression of apoptosis­related molecules in spinal cord nerves. The results revealed that BTX­A significantly reduced the amount of writhing in model mice, enhanced the activity of PC12 OGD cells, increased the secretion of NE and M­EK in model cells and the spinal cord of mice, and decreased the apoptosis of neural cells in the spinal cord of the model mice. Therefore, it was hypothesized that BTX­A may alleviate the pain induced by EMS by increasing the secretion of analgesic substances and promoting the repair of nerve injury. The present study provided a theoretical basis for the treatment of pain induced by EMS.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

A close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon.

INTRODUCTION: In the alpaca diencephalon, the distribution of immunoreactive cell bodies and fibers containing methionine-enkephalin (MET) or substance P (SP) has been studied. MATERIAL AND METHODS: The immunohistochemical study was performed by standard method on the diencephalon of four male alpacas that lived at sea level. RESULTS: Nerve fibers containing MET or SP were widely distributed in the thalamus and hypothalamus. METand SP-immunoreactive fibers showed a similar distribution in the whole diencephalon. Immunoreactive cell bodies containing MET or SP were only observed in the hypothalamus. The distribution of MET-immunoreactive cell bodies was more widespread than that observed for cell bodies containing SP. CONCLUSIONS: A close neuroanatomical relationship between the tachykininergic (SP) and enkephalinergic (MET) systems was observed in the whole diencephalon suggestive of the existence of multiple physiological interactions between both systems.

Prospective oncotarget for gynecological cancer: Opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis.

The standard treatments for neoplasia include surgery, chemotherapy, hormone antagonists and radiotherapy, which can prolong survival, but rarely cure the tumors of gynecological cancer patients. OGF - OGFr expression, in various gynecologic cells and tissues, is an intersection point between cell development, neuroendocrine function and immune modulation. It has been identified that OGF and OGFr expression differs between gynecological tumor and normal cells. Further, exogenous or endogenous OGF and OGFr antagonists have been known to have a role in regulating cell viability and apoptosis. Moreover, the expression of proteins in the OGF - OGFr axis modulate differentiation and membrane expression of immune cells, which can enhance the immune response. In vivo and in vitro assays have shown that OGF and OGFr antagonists inhibit mitosis as well as induce apoptosis in gynecologic cancer cells. Although immune augmentation combination therapies can intensify cytotoxic activity, OGF or OGFr antagonists do not increase toxicities associated with dual-immune regulation. In conclusion, the OGF - OGFr axis provides significant strategies for antitumor efficiency in gynecological cancer.

1-Substituted sialorphin analogues-synthesis, molecular modelling and in vitro effect on enkephalins degradation by NEP.

Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from L to D in almost all peptides, except D-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the L and D-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why D-arginine is so unique among all D residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.