Introduction

The term opiate, or more appropriately opioids, is commonly referred to as a class of compounds, such as the alkaloids morphine, codeine, and thebaine, derived from the opium poppy (Papaver somniferum), and known and utilized by humankind for millennia for both analgesia and sedation. This class of substances also includes semi-synthetic compounds such as heroin, oxycodone, hydrocodone, and hydromorphone, obtained from these natural molecules as well as fully synthetic compounds, including fentanyl, pethidine, levorphanol, methadone, tramadol, and dextropropoxyphene.

Morphine was first isolated in 1806, followed by the isolation of codeine several years later. Following the development of the hypodermic needle and hollow needle in the 1850s, physicians began to use morphine for various surgical procedures as well for the treatment of chronic pain and postoperative pain.[1] With the discovery of different opioid agonists, antagonists, and partial agonist compounds such as nalorphine, various researchers postulated and later proved that there are multiple stereospecific opioid binding sites in the central nervous system (CNS) through which they exert their physiological effects. Researchers further surmised that these receptors were most likely targets of endogenous opioid compounds, which researchers then began to isolate and study.[2]

In 1975, John Hughes and Hans Kosterlitz reported the first evidence of endogenous opioids in brain extracts that they noted were able to inhibit acetylcholine release from nerves in the guinea pig ileum. Further, they indicated that when treated with the opioid receptor antagonist naloxone, the inhibition was blocked. The compounds that they first isolated were termed enkephalins.

Structurally, the enkephalins are pentapeptides that are distinguished into two subgroups by their carboxy-terminal amino acids, leucine, or methionine. As a consequence, the enkephalins either classify as met-encephalins and leu-encephalins, respectively:

• The met-enkephalins present the amino acid sequence Tyr-Gly-Gly-Phe-Met.
• The leu-enkephalins present the amino acid sequence Tyr-Gly-Gly-Phe-Leu.

The enkephalins are one of the three peptide systems that also include beta-endorphins and dynorphins. Of note, the three classes of endogenous opioid peptides share a common N terminus sequence of Tyr-Gly-Gly-Phe and lack a C terminus amide.[3] In molecular terms, Tyr and Phe bind the receptor, and the glycine pair acts as a spacer.

These peptides act as neurotransmitters and neuromodulators throughout the nervous system and various end-organ targets.[4] Additionally, research has found that met-enkephalin has an essential role in cell proliferation and tissue organization during development. When discussed in this context, met-enkephalin is often referred to as the opioid growth factor (OGF).[5]

Issues of Concern

Tolerance/Dependence

Exogenous opioids continue to be the most potent and effective class of agents for the acute treatment of trauma and surgery-related pain as well as for moderate to severe cancer pain. Tolerance and dependence/addiction are significant issues complicating the long-term medical use of opioid compounds. Tolerance is a phenomenon where increased doses are needed to produce the same physical effects. This opioid-related effect is a long-observed clinical entity. Further, it has been noted that the development of tolerance to specific effects of opioid administration occurs at different rates. For example, it has been observed that tolerance to respiratory depression, sedation, and nausea typically occurs more rapidly, whereas constipation and miosis are not subject to tolerance. Proposed mechanisms of opioid tolerance include opioid receptor-G protein uncoupling, decreased receptor internalization/recycling, and increased sensitivity of the NMDA receptor involved in the transmission of nociceptive signals.[6] 

The use of opioids in the long-term management of chronic non-malignant pain has come into question due to epidemic levels of addiction, overdoses, and mortality associated with their use. The process of opioid addiction appears to be driven by a 3 stage cycle beginning with initial intoxication, abstinence which results in dysphoria and hyperalgesia, followed by a strong compulsion to seek opioid drugs to relieve the negative symptoms of withdrawal.[7] The search for potent and efficacious analgesic compounds that either minimize or eliminate the deleterious side effects that come with the long-term administration of exogenous opioids continues to be an active area of research.  

Cellular Level

Enkephalins are generated through the cleavage of the precursor molecule pro-enkephalin, generating either met-enkephalin or leu-enkephalin. The pro-enkephalin gene is composed of three exons that are separated by two introns. Processing of pro-enkephalin generates six copies of met-enkephalin and one copy of leu-enkephalin in humans and other mammals. Enkephalins have a wide distribution throughout multiple brain regions and the spinal cord, as well as the adrenal medulla. There is speculation that enkephalins detected in the plasma are primarily from the adrenal medulla.[8] Enkephalins undergo biodegradation via hydrolysis, which cleaves the pentapeptide at the Tyr-Gly bond. Enkephalinases and aminopeptidases further degrade the molecules into shorter peptides that are from 2 to 4 amino acids in length.[3]

Development

During the 1980s, researchers discovered that the endogenous opioid system, and met-enkephalin, in particular, has a role in regulating cell proliferation in both normal and neoplastic cell lines. Met-enkephalin, in this context, has the name OGF, and its target receptor is the OGF receptor (OGFr). Notably, OGFr is significantly different in its structure and molecular composition as compared to the classical mu, delta, and kappa opioid receptors. The principal mechanism of action is through the upregulation of p16 and p21 cyclin-dependent kinases, which serve to stall the progression of the cell replication cycle from the G0/G1 phase to the S phase. The system is tonically active, and OGFr protein and gene expression are present in most proliferating cell lines.[9]  

Organ Systems Involved

Nervous System

Enkephalins are known to be distributed extensively throughout the central, peripheral, and autonomic nervous systems in mammals. Both leu-enkephalin and met-enkephalin have been detected in 82% of the brainstem tracts/and nuclei of the squirrel monkey. Met-enkephalin has been detected via immunoassay in the CNS in the globus pallidus, hypothalamus, periaqueductal gray area (PAG), amygdala, and spinal cord. Additionally, PENK gene expression has been noted in the posterior pituitary and the spinal cord.[10] 

Cardiac Systems

Patients with heart failure have increased pro-enkephalin levels proportional to the severity of their condition. Both pressure overload and beta-adrenergic stimulation increase the expression of enkephalin in experimental models. The effects of opioids on the cardiac system are multifactorial and seem to have a shorter-term effect of decreasing heart rate and blood pressure while having a longer-term effect of increasing myocardial contraction.[11]

Respiratory System

Respiratory depression is a well-known and potentially fatal side effect of exogenously administered opioids and appears to be primarily mediated by mu-opioid receptors located in the hypothalamus. Nevertheless, fear of respiratory depression must not limit the use of opioids. For instance, in the context of cancer pain management, experience suggests that opioids can be used safely with a reduced risk of respiratory depression.[12]

Gastrointestinal System

The primary opioid receptors in the gastrointestinal tract are mu and delta-opioid receptors, which are found in the submucosal and myenteric plexus levels, respectively. Activation of mu-opioid receptors both inhibits motility in the colon and increases fluid absorption, which can cause constipation.[13]

Endocrine System

Glucocorticoids have shown to directly upregulate the transcription of pro-enkephalin (PENK) mRNA by binding to a DNA sequence termed the glucocorticoid response element.[14] Researchers have postulated that enkephalins play a role in modulating the intensity and duration of the stress response. Utilizing a mouse gene knockout model, Bilkei-Gorzo et al. found that mice lacking the PENK gene showed an approximately doubled hormonal stress load in response to experimentally induced stress as compared to their wild-type counterparts.[15] 

Immune System

Met-enkephalin has shown to have immunomodulatory effects on various cell types. These include the upregulation of CD8+ T cells, inhibiting regulatory T cell activity, stimulating phagocytosis in macrophages, increasing proliferation of CD4+ T-helper 1 cells, and stimulating the natural killer cell response.[16]

Function

The expression of enkephalins and their target opioid receptors have a wide distribution throughout the central, peripheral, and autonomic nervous systems, multiple organ systems, as well as endocrine tissues and their target organs. The various effects of enkephalins are best understood by considering a small sample of these experimentally studied systems. The extensive literature on enkephalins physiological effects includes but is not limited to its role in analgesia, angiogenesis, blood pressure regulation, embryonic development, feeding, hypoxia, limbic system modulation (emotional conditions), memory processes, neuroprotection, peristalsis, pancreatic secretion, wound repair, respiratory control, and hepatoprotective mechanisms.[3] Again, leu-enkephalin seems to be involved in the control of the gonadal function. The main functions concern analgesia, stress response regulation, and peristalsis modulation.

Analgesia

High concentrations of opioid receptors are present in the PAG, locus coeruleus, the rostral ventral medulla, and the substantia gelatinosa of the dorsal horn. Activation of mu-opioid receptors in the midbrain causes descending inhibition of the PAG and nucleus reticular paragigantocellularis. This inhibitory signal becomes further propagated to 5-hydroxytryptamine and enkephalin-containing neurons that connect with the dorsal horn and can ultimately inhibit or reduce the transmission of nociceptive stimuli to the CNS.[17] 

Stress Response Regulation

In response to stress, organisms release Corticotropin-Releasing Factor (CRF), which, in turn, stimulates the production of catecholamines while simultaneously producing endogenous opioids, including enkephalins and endorphins. The postulate is that the endogenous opioid system serves to regulate the intensity and duration of the stress response via multiple mechanisms. Enkephalin, for instance, has been found to modulate the release of CRF from the paraventricular nucleus of the hypothalamus.[18] 

Peristalsis

Enkephalins and endogenous opioids slow gastrointestinal motility by altering neuronal excitability. The main effect occurs by inhibiting K+ and Ca2+ channels, which hyperpolarizes the cell and ultimately prevents conductance of an action potential and the resulting release of neurotransmitter required for gut motility.[19]

Mechanism

Enkephalins exert their physiological effect through specific opioid receptors, which have a broad distribution in the body. Three major classes of opioid receptors exist and are named mu (mainly expressed in the CNS), delta (equally expressed in the SNC and spinal cord), and kappa (expressed primarily in the spinal cord). The fourth class of opioid receptors called nociceptin was discovered in 1994 but is not considered to be part of the aforementioned tripartite group, which is often referred to as the classical opioid receptors.

Enkephalins have the highest affinity for the delta-opioid receptor, followed by the mu-opioid receptor, and exhibit low affinity for the kappa-opioid receptor.

Opioid receptors are in the family of G-protein coupled receptors recognized by their seven membrane-spanning motifs with approximately 60% sequence homology.[2] Their extracellular domains, which determine their selectivity, show between 34 to 49% when comparing sequence similarity.[3] The significant inhibitory effects of enkephalins are mediated by reducing K+ and Ca2+ influx. Signal transduction begins with ligand binding, which causes dissociation of the Gα and Gβγ subunits. The Gα subunit directly interacts with inward rectifying potassium channels causing cellular hyperpolarization. The Gα subunit also inhibits adenylyl cyclase activity, which decreases the formation of cAMP, thus reducing the cAMP-dependent Ca2+ influx. The Gβγ further reduces calcium influx by directly binding to various classes of Ca2+ channels.[20]

Related Testing

While the dysregulation of enkephalins has implications in the pathophysiology of various disease states, there is no widely available assay for either the diagnosis or prognosis of the associated disease entities which have been studied experimentally in both animal and human subjects. Historically immunoassay was regularly used to measure enkephalin levels in various tissues of interest quantitatively. Mass spectrographic techniques were later successfully developed and coupled to both capillary electrophoresis and liquid chromatography. Ozlap et al. describe the application of liquid chromatography coupled to mass spectroscopy for quantifying both leu-enkephalin and met-enkephalin in human plasma.[21]  While it remains unclear if such testing proves to have clinical utility, this technique may be promising for experimental use and further elucidating the role of enkephalins in disease pathogenesis. 

Pathophysiology

Dysregulation of the endogenous opioid system has implications in a wide variety of disease states reflective of its wide distribution throughout the human nervous system and body.

Pain Dysregulation

Research shows that mu-opioid receptor (MOR) availability to decreases in patients diagnosed with fibromyalgia in addition to having an elevated concentration of endogenous opioid concentrations in their CSF. Further, pain-evoked brain activity, as measured by fMRI, was found to be proportional to MOR binding potential.[22]  

Substance Misuse Disorders

Enkephalins have implications in sensitization to cocaine, where they show strong expression in mesocorticolimbic areas.[23] Downregulation of both pro-dynorphin and pro-enkephalin occurred in the caudate nucleus of alcoholics.[24] 

Feeding Behavior

In a mouse gene knockout model for the pro-enkephalin gene, mice without the pro-enkephalin gene were found to have lower overall body weights as compared to the wild-type mice under similar conditions.[25]

Clinical Significance

A deeper understanding of enkephalins and the related ligands, receptors, and signaling pathways that constitute the endogenous opioid system can hold the potential for significant advances in developing therapies pertinent to the wide variety of physiological processes and organ systems in which these agents play a role. Two particular areas of research that may produce improved treatments in the foreseeable future are the creation of potent analgesic therapies without the associated deleterious side effects of opioidergic agents, and the use of met-enkephalin (MENK) in the treatment of various cancers and immune-related diseases.

Analgesia

Postulates regarding the role of enkephalins in producing analgesia existed before their experimental discovery and successfully demonstrated soon after that in animal models.[26] However, one of the main obstacles to therapeutically utilizing them is their relative instability in-vivo, where they are rapidly broken down by endogenous peptidases. There are two separate approaches to trying to mitigate this issue.

One approach has been to chemically modify enkephalins so that they are more difficult to degrade while retaining their ligand specificity for mu and delta-opioid receptors and thus preserving their analgesic efficacy. Many iterations of this approach have been described to this end, beginning with the work of Pert et al. in 1976 with D-Ala-methionine-enkephalin administered into the cerebral ventricles of rats.[27] More recently, Kropotova et al. described the design of several modified enkephalins that capitalize on their finding that neither of the known catabolic peptidases (carboxypeptidase and aminopeptidase) is capable of breaking peptide bonds formed by beta-alanine.[28]  

An alternative approach has been to target the peptidases themselves to amplify the effect of endogenous opioids that are already present but rapidly catabolized under normal physiological conditions. Early work demonstrated that while blocking either enkephalinase or aminopeptidase on its own produced no significant analgesia in either humans or animals, the additive effect of simultaneously blocking both peptidases did indeed produce potent analgesia. This insight prompted investigators to focus their efforts on compounds aptly dubbed dual enkephalinase inhibitors (DENKIs). Perhaps the most promising finding related to DENKIs, is that, unlike exogenous opioids, the undesirable side effects of tolerance, sedation, respiratory depression, constipation, and dependence have yet to be demonstrated. Researchers postulate this to be due in part to their hyper-local effect. Since enkephalins will only be physiologically released at needed areas, DENKIs will only enhance their impact on those areas where enkephalins are present in sufficient quantity. This model is in contrast to exogenously administered opioids, whose agonistic effects will be induced anywhere in the body where sufficient opioid receptors are present. Further factors explaining these findings are differential effects on enkephalin's ability to stimulate receptor internalization and recycling as compared to exogenous opioids, as well as producing a relatively weaker dopamine response in the brains' reward circuitry.[29]  

Immunoregulation and Cancer Therapy 

Shortly after the experimental discovery of enkephalins, evidence for opioid receptors on the surface of various classes of immune cells began to come to light.[30][31] By the late 1980s, researchers published direct experimental evidence of antitumor activity utilizing the met-enkephalin/opioid growth factor in the literature.[32] Subsequently, a large body of experimental evidence has detailed the specific effects of MENK/OGF on various types of immune cells. Currently, there is a large body of both in vivo and in vitro studies demonstrating MENK/OGF's ability to slow tumor growth in a wide variety of cancer types.[16] The mechanism of action for this effect appears to take place through the upregulation of p16 and p21 cyclin-dependent kinases, which serve to stall the progression of the cell replication cycle from the G0/G1 phase to the S phase. Since it believed that the OGF/OGFr system is tonically active in most if not all replicating cell types, it follows that increased concentrations should have an inhibitory effect on replicating cells that are still expressing the OGF receptor and have intact signaling pathways to halt cell progression. MENK/OGF's broad antitumor activity on a wide variety of cancer types combined with its relatively benign side effect profile makes it a highly promising adjuvant therapy in cancer patients.