Duration of opioid receptor blockade determines biotherapeutic response

Accepted Manuscript Title: Duration of Opioid Receptor Blockade Determines Biotherapeutic Response Author: Patricia J. McLaughlin Ian S. Zagon PII: DOI: Reference:

S0006-2952(15)00332-9 http://dx.doi.org/doi:10.1016/j.bcp.2015.06.016 BCP 12273

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BCP

Received date: Accepted date:

30-4-2015 17-6-2015

Please cite this article as: McLaughlin PJ, Zagon IS, Duration of Opioid Receptor Blockade Determines Biotherapeutic Response, Biochemical Pharmacology (2015), http://dx.doi.org/10.1016/j.bcp.2015.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Revision/Rebuttal Notes

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Duration of Opioid Receptor Blockade Determines Biotherapeutic Response

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Patricia J. McLaughlin ([email protected]) and Ian S. Zagon ([email protected]) Department of Neural and Behavioral Sciences

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Pennsylvania State University College of Medicine

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Hershey, PA 17033 USA

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Corresponding author:

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Dr. Patricia J. McLaughlin

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Department of Neural & Behavioral Sciences, MC H109 Penn State University College of Medicine 500 University Drive

Hershey, PA 17033 USA 717-531-6414 (phone) 717-531-5003 (fax)

Email: [email protected]

Running title: Duration of Receptor Blockade Determines Response

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Abbreviations: OGF, opioid growth factor; OGFr, opioid growth factor receptor, NTX, naltrexone, LDN, low dose naltrexone; EAE, experimental autoimmune encephalomyelitis; DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin; DPDPE, d-Pen2, d-Pen5-enkephalin; CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; VEGF, vascular endothelial growth factor; FGF-2, fibroblast growth factor -2; α-SMA, alpha smooth muscle actin; DMSO, dimethyl sulfoxide; MOG, myelin oligodendrocytic

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glycoprotein; imp, importin; Kd, dissociation constant; Bmax, maximal binding indicating total concentration of

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receptors; NLS, nuclear localization signal

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Number of words: 10,774

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Abstract

Historically, studies on endogenous and exogenous opioids and their receptors focused on the mediation of

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pain, with excess opiate consumption leading to addiction. Opioid antagonists such as naloxone and naltrexone blocked these interactions, and still are widely used to reverse drug and alcohol overdose. Although

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specific opioid antagonists have been designed for mu, delta, and kappa opioid receptors, the general

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antagonists remain the most effective. With the discovery of the opioid growth factor (OGF)-OGF receptor

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(OGFr) axis as a novel biological pathway involved in homeostasis of replicating cells and tissues, the role of opioid receptor antagonists was expanded. An intermittent OGFr blockade by low dosages of naltrexone resulted in depressed cell replication, whereas high (or sustained) dosages of naltrexone that conferred a continuous OGFr blockade resulted in enhanced growth. More than 3 decades of research have confirmed that the duration of opioid receptor blockade, not specifically the dosage, by general opioid antagonists determines the biotherapeutic outcome. Dysregulation of the OGF-OGFr pathway is apparent in a number of human disorders including diabetes, multiple sclerosis, and cancer, and thus opioid antagonist disruption of interaction prevails as a therapeutic intervention. We review evidence that the duration of opioid receptor blockade is correlated with the magnitude and direction of response, and discuss the potential therapeutic effectiveness of continuous receptor blockade for treatment of diabetic complications such as corneal defects and skin wounds, and of intermittent receptor blockade by low dosages of naltrexone for treatment of autoimmune diseases and cancer. 2

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Keywords: Naltrexone, multiple sclerosis, dry eye, diabetic wound healing, LDN 1. Introduction Opioid receptors were identified first followed by the discovery of endogenous opioids that acted as ligands. Concomitantly, pharmacologists began designing opioid antagonists that blocked the neurotransmitter function

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of these receptors in brain and gut. Nearly five decades have elapsed since the initial identification of opioid

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receptors, and two of the original antagonists, naloxone and naltrexone, remain on the forefront of treatment for cancer pain, addiction, drug overdose, alcoholism, and other psychosomatic disorders. The discovery of

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the non-classical opioid receptor, opioid growth factor receptor (OGFr), that shares several pharmacological properties with mu, delta, and kappa opioid receptors, has led to research that broadens the usefulness of

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opioid antagonists. Depending on the duration of opioid receptor blockade, opioid antagonists such as naloxone and naltrexone are effective therapies for cancer, autoimmune diseases, and complications

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associated with diabetes. Understanding the dysregulation of the OGF-OGFr axis in each disease dictates

proliferation is warranted.

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1.1. Opioid receptors

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whether continuous blockade to enhance cellular proliferation or intermittent blockade to inhibit cellular

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1.1.1. Classical G-protein coupled opioid receptors Research to discover opioid receptors commenced in the early 1970s when biochemical studies reported that certain drugs interacted with specific molecules within different regions in the central nervous system [1-3]. Radiolabeled binding of exogenous opiate agonists such as levorphanol were used to locate and isolate specific binding proteins [4]. Pert and Snyder published a seminal paper on the identification of the binding site for radiolabeled naloxone [1], and eventually identified the mu opioid receptor. Many of the investigations involved nervous tissue, and in rapid succession, mu, delta, and kappa opioid receptors were identified and characterized in the brain or enteric nervous system [2,3]. Two decades later, the molecular structure of these classical opioid receptors was revealed [5-7]. Cloning of the mu, delta, and kappa opioid receptors illustrated that all three receptors are G protein-coupled transmembrane proteins that are members of the subfamily of rhodopsin receptors [7]. The receptors share 60% identity with more than 70% identity in the transmembrane domains and intracellular looping regions. The N terminus is least similar among the 3 receptors, but all have 3

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an extracellular domain in the N terminus with glycosylation sites and intracellular loops with multiple amphiphatic α-helixes. All three classical opioid receptors stimulate cAMP accumulation and are blocked by pertussis toxin [8]. 1.1.2. Non-classical nuclear membrane-associated opioid receptor A non-classical opioid receptor, OGFr, was first recognized in the 1980s, and subsequently characterized in

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both murine neural cancer cells [9] and normal rodent brain tissue [10,11]. The isolated protein was originally termed zeta (ζ) to maintain consistent naming with the Greek symbols of mu, delta, and kappa, and was

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appropriately called “zeta” for the Greek word zoe”, loosely defined as “growth”. Concomitantly, other studies

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were conducted to determine the endogenous opioid involved with this binding protein, and the ligand [Met5]enkephalin was identified to have inhibitory growth properties when binding to this receptor. The endogenous

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peptide was termed opioid growth factor (OGF), to distinguish the neurotransmitter function from that of being an inhibitory growth factor, and the zeta receptor was renamed OGFr. The cDNA for the rat OGFr was cloned

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by searching expression libraries [12], and subsequently the sequence was identified in human and mouse [13]. Based on extensive biochemical characterization, and cloning, the similarities of classical mu, delta, and

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kappa opioid receptors with OGFr were in the pharmacology, and not at the molecular level. The open reading

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frame for human OGFr is 697 amino acids with 8 imperfect repeats of 20 amino acids each at the C terminus.

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The human OGFr is located on chromosome 20q13.3 [13]. Thus, the molecular and protein structure of OGFr has no resemblance to classical opioid receptors. Based on NMR studies as well as confirmation from websites such as FoldIndex [14], OGFr is an intrinsically unstructured protein with approximately 78% amino acid identity between mouse, rat, and human. In studies on subcellular localization of OGFr using COS-7 African green monkey kidney cells, it has been documented that the receptor has three nuclear localization signals (NLS) within its sequence, two mono-partite NLS383-386 and NLS456-460, and one bi-partite NLS267-296 [15]. Studies utilizing site directed mutagenesis demonstrated when NLS383-386 and NLS456-469 were both mutated the nuclear localization was decreased by 80%, and the regulatory effects of OGF were diminished indicating that the OGF-OGFr action on proliferation is dependent on the ability of OGFr to translocate into the nucleus requiring the presence of NLS, karyopherin β and Ran [15]. Transport of fluorescein-labeled naltrexone was not temperature dependent, and was observed in the nucleus for 48 hr (Figure 1) [15]. Export of OGFr from the nucleus is CRM-1 dependent. 4

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Subcellular fractionation studies using developing rat brain and cerebellum revealed that OGFr binding is associated with the nucleus [9,11,13]. These biochemical studies were confirmed by confocal microscopy studies in the rat cornea that demonstrated immunogold labeling of OGFr in the paranuclear cytoplasm, within the nucleus, and adjacent to heterochromatin in corneal epithelial cells [16]. Colocalized immunogld labeling of OGFr and OGF was detected on the outer nuclear envelope and inside the nucleus [16]. Collectively, these

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inside the nucleus with its cargo, the endogenous [Met5]-enkephalin ligand.

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data suggest that the receptor is located on or near the outer nuclear envelope and functions by translocating

The gene and protein for OGFr have been identified in cells and tissues arising from all 3 dermal

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derivatives [13]. Gene expression for OGFr has been documented in human fetal tissues including brain, liver, lung, and kidney as well as in adult heart, brain, liver, skeletal muscle, kidney, and pancreas [13]. Binding

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assays on adult and fetal rat brain have quantitated OGFr binding [17], and studies conducted in adult mice demonstrated RNA levels in brain, heart, lung, liver, kidney and skeletal muscle. Additionally, OGFr has been

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detected in neoplasia, as well as in cell lines derived from human cancers [18-20]. 1.2. Opioid receptor antagonists

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Opioid antagonists are compounds that competitively bind to opioid receptors with affinity greater than

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that of specific agonists. However, antagonists have no function other than to block this interfacing. In the

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case of opioid receptors, agonists are both exogenous compounds such as morphine, codeine, congeners of morphine, and endogenous molecules such as endorphins and enkephalins. The general antagonists were synthesized first to block exogenous opiate interactions, and later were instrumental in research on the isolation of opioid receptors [2].

1.2.1. General opioid receptor antagonists Opioid receptor antagonists are either general and bind to all classical opioid receptors, or are specific and selective. The two most widely studied opioid receptor antagonists, naloxone and naltrexone, are general antagonists. Both compounds were discovered more than a half century ago, and remain the most promising pharmaceuticals to reverse opiate overdose and treat drug and alcohol addiction [21,22]. The identification [23] and characterization [24] of naloxone, also termed Narcan, occurred in the 1960s. Interest in Narcan has reemerged with the heightened incidence of heroin addiction and the need to prevent overdose. The primary use of naloxone remains as a medication to reverse opioid overdose and reduce respiratory depression [22]. 5

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Naltrexone hydrochloride is a synthetic congener of oxymorphone, but lacks opioid agonist action, and is trademarked as Trexan, Revia, or the extended release form Vivitrol. Naltrexone and naloxone share similar structures; naloxone is n-allynoroxymorphone [23,24], whereas naltrexone is morphinan-6-1,17(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-, hydrochloride [21]. Both compounds block all classical opioid receptors, as well as OGFr, by competitive binding between the antagonist and their respective exogenous or

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endogenous ligand. Both antagonists can be absorbed orally, with approximately 5-40% oral bioavailability.

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With regards to naltrexone, the parent compound and 6-β-naltrexol metabolites are active, and excreted by the kidney. Peak levels of absorption may occur as quickly as 1 hour, with the half-life for naltrexone being 4

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hours, and 13 hours for its metabolite, 6-β-naltrexol [21]. In comparison, enkephalins that are ligands for µ and δ opioid receptors are degraded within minutes.

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Naltrexone has been shown to provide a complete blockade of exogenous opioid congeners, but is not effective against cocaine or other non-opioid drugs of abuse, thus demonstrating specificity for opioid

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receptors. In some studies, naltrexone has been shown to have a partial inverse agonist effect – such that lowdose naltrexone can reverse the altered homeostasis resulting from long-term abuse of opioid agonist drugs

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[21]. Naloxone has no partial agonist effect, but can work as an inverse agonist at mu receptors – making it

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preferred for reversal of drug overdose [22]. A third general antagonist is the methylated version,

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methylnaltrexone bromide, also called Relistor; this compound does not pass the blood brain barrier, making it useful for treatment of opioid-induced constipation [20,21]. 1.2.2. Selective opioid receptor antagonists Despite classical opioid receptors sharing significant structural homology, selective antagonists have been synthesized and shown to preferentially bind one of the 3 opioid receptors. CTOP, CTAP and cyprodime are selective antagonists for mu opioid receptors, whereas naltrindole is selective for delta, and norbinaltrophimine for kappa receptors. Research is continuing to identify selective antagonists to classical opioid receptors, but at this time, no specific antagonist for OGFr has been identified. 2. Opioid receptor blockade The focus of this commentary is on the duration of opioid receptor blockade by opioid antagonists. In this context, it is appropriate to define “duration of opioid receptor blockade”. Duration is the length of time elapsed between 2 events – e.g., extent of active binding to the receptor. A search of scientific publication 6

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databases (e.g., PUBMED, SCOPUS) revealed that in the 21st century, few laboratories are studying the duration of opioid receptor blockade and biotherapeutic response. Much of the work on the fundamental pharmacological principle first observed in the 1970s was refined during the next two decades. 2.1. Mechanisms of opioid receptor blockade Receptor antagonists have different affinities to each opioid receptor, and binding of the antagonist

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disrupts the interaction between the agonist (or inverse agonist) and the receptor. Pharmacologically,

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antagonists mediate their effects by binding to allosteric or orthosteric sites. The antagonism can be reversible if the agonist concentration exceeds the affinity of the antagonist for the receptor. Because the interactions

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can be reversible depending on the longevity of the antagonist-receptor complex, it is often the duration of opioid receptor blockade that confers the action. For example, at comparable biochemical concentrations,

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naloxone is short–acting and naltrexone is longer-acting. The interplay between two proteins that invoke a reaction has been identified by Tummino and Copeland as the concept of residence time of receptor and

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ligand complexes [25]. This concept implies that all biochemical activities involving a ligand and receptor depend on binary complexes that can be regulated. These authors presented quantitative definitions to

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measure the initial moment of contact, duration of action, and amplitude for every ligand-receptor interaction

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[25]. The pharmacology associated with each, and the duration of receptor blockade, rather than dosage, is

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the subject of the remaining discussion.

2.1.1. Mechanisms of action: Naltrexone Biphasic responses of naltrexone in blockade of opioid receptors were first reported in the 1990s [26]. Radiolabeled naltrexone had high affinity binding for the mu opioid receptor, whereas agonists for delta opioid receptors competed with low affinity binding of naltrexone. Studies on pharmacokinetics of radiolabeled ligands, classical opioid receptors, and antagonist competition documented that radiolabeled naltrexone reached equilibrium in a two-site model within 30 min at room temperature using rat brain homogenates [26]. Competition with mu receptor agonists abolished high-affinity binding of naltrexone, whereas delta agonists (e.g., DPDPE, ICI174,864) were concentration dependent and had less competitive binding [26]. Naltrexone and its active metabolite 6β-naltrexol have affinities for the mu and kappa opioid receptors that approach 0.08 nM and 0.5 nM, respectively; the affinity for the delta opioid receptor is approximately 8 nM [26]. Other studies suggested that 3H-naltrexone had no biphasic binding activity in brain tissue [26]. Radiolabeled naltrexone 7

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competitively inhibited binding of mu receptors at 1 µM concentrations or less, but required ≥ 2 µM concentrations to displace delta opioid receptor agonists such as DPDPE or ICI-174,864 [see table in 26]. Competition between cold and radiolabeled naltrexone was measured in bovine hippocampus, and shown to have a one-site binding with a Kd of 2.2 nM in comparison to competition between radiolabeled naltrexone and delta opioid receptor agonists that demonstrated a Kd of 29 nM [27].

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Dichotomous biological responses following different durations of opioid receptor blockade were

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initially reported in 1983 when low and high dosages of naltrexone were administered to nude mice inoculated with neuroblastoma cells [28]. The low dosages of naltrexone inhibited the growth of the tumors, but the high

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dosages resulted in responses that did not correlate with dosage. Rather than inhibiting growth to a greater degree than low dosages in a normal dose-response manner, the high dosages of naltrexone resulted in larger

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tumors and faster rates of death [28]. This observation was completely unexpected and required further study to understand that it was the duration of opioid receptor blockade that was driving the end result, not the

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dosage of antagonist [28].

Studies in normal rodent body and brain development replicated these observations [29, 30]. Not only

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did the dosages of naltrexone differ between mice [28, 29] and rats [30], but both studies demonstrated

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biphasic responses. Low dosages of naltrexone resulted in inhibitory growth, whereas higher dosages of

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naltrexone resulted in accelerated tumor growth and somatic development. The biochemical pharmacology underlying these observations revealed that the duration of opioid receptor blockade, not the dosage, determined the physiological outcome.

2.1.2. Duration of opioid receptor blockade: Pharmacology studies Similar observations were reported by other investigators. Landymore and Wilkinson examined opioid receptor blockade using naloxone injected subcutaneously at 6 hr intervals to neonatal rats on the timing of puberty. These authors suggested that “…the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effects”, [p. 447, 31]. Other investigators utilized radiolabeled carfentanil binding in normal volunteers and tracked naltrexone blockade of mu-opioid receptors with a positron radiation detection system [32]. The half-time blockade of naltrexone ranged between 3 days and 108 hr, a time significantly longer than the reported plasma clearance rate of approximately 8-12 hr. These data justified the selection of a 50 mg dosage currently recommended for reversal of heroin overdose and were based on the fact that 8

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plasma clearance half-time rate is not necessarily reflective of the duration of receptor blockade. Research to synthesize new antagonists is ongoing, but to date, naltrexone remains the choice to block mu opioid receptors. 2.1.3. Duration of opioid receptor occupancy: Biological response A second avenue of research supporting differential effects for the same ligand at different dosages

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came from studies on the up- and down-regulation of a given receptor. At face value, up- and down-regulation

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can mean both increased and decreased numbers of receptors, or it can mean greater (or lesser) sensitivity and/or activity by the same number of receptors. Using in vitro cell culture of primary brain cells, it was

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reported that agonists invoked down-regulation of receptors in brain cells from forebrain but not hindbrain [33]. Reporting on NG-108 cells subjected to naloxone or naltrexone, Coscia and colleagues revealed a

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transient down-regulation in delta receptors, specifically δ2 [34]. Receptor binding number (Bmax), but not binding affinity (Kd) was reduced following treatment with 10 mg/kg naltrexone. Baker and Meert [35] reported

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that the delta-receptor antagonist naltriben produced both potentiation and attenuation of effects of U50, 488Hinduced hypothermia by way of the kappa opioid receptor. Using the outcome of hypothermia, mice were

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injected with agonists alone and in combination with selective antagonists (e.g., naltrindole, naltriben), as well

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as methyl-naltrexone, a compound with peripheral, rather than centrally-mediated activity. These authors

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concluded that high doses of agonists mediate activity at more than one receptor site, whereas selective antagonists are limited to one receptor [35].

Recent studies on the κ-opioid receptors as a mediator of biological responses to pain, stress, anxiety, and depression have researched the role of naloxone, and the duration of action of selective kappa opioid antagonists and JNK1 activation [36]. Naloxone displayed a short duration of activity at the opioid receptor and did not increase phospho-JNK activation.

2.2. Stereospecificity of opioid receptor blockade The stereospecificity of opioid receptor antagonists, and the duration of blockade, were reported in one investigation that showed (-) naloxone administration in dosages as high as 60 mg/kg resulted in decreased body weights, suggesting that even at this dosage, the opioid receptors associated with growth were transiently blocked [37]. Naloxone administered systemically at 100 mg/kg had no effect on growth which is counter to naltrexone which at dosages equal to or greater than 20 mg/kg accelerates growth. Early work 9

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using the murine neuroblastoma model tested opioid antagonist stereospecificity by injection of (+) and (-) isomers of naloxone; only the (-) isomer was effective at blocking the receptor. Thus, the growth related properties of opioid receptor antagonists were stereospecific and dose-related, but not dose-dependent [37]. Moreover, multiple low dosages of naltrexone could be administered throughout the 24 hr period and invoke a continuous blockade, again confirming that the duration of receptor blockade is important.

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3. Duration of opioid receptor blockade determines response

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Observations that the duration of opioid receptor blockade determined the direction and magnitude of response led to the discovery of the OGF-OGFr regulatory pathway (Figure 2). Duration, not drug dosage, determines response

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3.1.

The Zagon laboratory had interests in both developmental biology and cancer (i.e., neuroblastoma),

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and also explored treatments for narcotic addiction [38-40]. Prompted to investigate brain development in rodent models of opiate addiction, it was noted that opiate treatment of pregnant rats was deleterious for

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offspring, as was prenatal exposure to methadone. Concomitantly, the field of addiction research began to focus on general opioid antagonists as treatments. Transitioning these studies to cancer using a model of

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A/Jax mice inoculated with neuroblastoma, animals were injected daily with 0.1 mg/kg naltrexone. The results

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revealed that tumor development was delayed, and only 33% of the mice developed a tumor in contrast to

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mice receiving saline. It was reasoned that if 0.1 mg/kg naltrexone was effective at retarding tumor growth, increased doses of the antagonist should have more efficacy. Surprisingly, the dose response experiments using 1 mg/kg and 10 mg/kg naltrexone had contradictory results. Instead of tumors being “non-existent” as was expected with higher dosages, tumors were larger than those in mice receiving saline [28], and survival time was decreased. These studies were extended to a mouse model of metastatic neuroblastoma and data revealed that low dosages of naltrexone inhibited tumor take (by 69%), delayed tumor appearance (by 70%), and increased median survival, in comparison to controls; higher dosages of naltrexone exacerbated tumor growth and shortened survival [28,20]. In each study, the duration of receptor blockade determined the biological response. With regard to normal somatic and brain development, naloxone administration to newborn rat offspring had little or no effect. However, treatment with either 1 or 50 mg/kg naltrexone on a daily basis to newborn rat offspring revealed biphasic results. The length of receptor blockade was tested in young rats using hot plate 10

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responsiveness following morphine injections. The concept was that morphine would bind avidly to mu opioid receptors and the animal would not respond to the thermal stimulus. Following naltrexone administration that preferentially blocked mu opioid receptors, the rat was subjected to thermal sensation and responded by licking its paws or jumping off the hot plate [40]. This measure of mu receptor blockade was the original premise for the “duration” studies that followed. Dosages that enabled the animal to remain 30 seconds on the

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hot plate after 12 hours were considered to have induced “continuous opioid receptor blockade”, whereas dosages that allowed the rodent to sense heat within 6-8 hours were considered to produce an “intermittent

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opioid receptor blockade”.

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Treatment of neonatal rats during weaning with 50 mg/kg naltrexone resulted in enhanced body, brain, and cerebellar weights relative to controls [30,38]. The cerebellum was larger in every aspect of neurobiology.

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Each layer of cells had a full complement of appropriate cell types (e.g., granule, Purkinje). Morphometric studies revealed up to 70% more glial cells in the cerebella of weanling rats treated postnatally with 50 mg/kg

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naltrexone relative to saline-injected controls. Neurons that are derived prenatally, and thus not subjected to naltrexone, were not altered in number.

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A thorough exploration of body and organ weights, appearance of physical characteristics, and brain

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development followed [39]. A full dose-response curve of 0.1, 1, 10, 20, 50 or 100 mg/kg naltrexone

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administered throughout the 3-week weaning period was conducted with end points of body weights and brain development. Duration of opioid receptor blockade was measured as well. Dosages of 0.1, 1, and 10 mg/kg naltrexone, that blocked the opioid receptors from morphine analgesia, for less than 12 hr/day (4-8 hr for the 0.1 and 1 mg/kg dosages), resulted in decreased growth. Dosages of 30 – 100 mg/kg increased body and brain weights. It was later determined that no additional enhancement of brain growth occurred when the dosage of 50 mg/kg was increased to 100 mg/kg [38]. To demonstrate that it was the “duration” and not the “dosage” that conferred the outcome, 3 dosages of 3 mg/kg which blocked the receptor for an entire 24 hour period resulted in enhanced body and brain weights relative to controls. The cumulative dosage of 9 mg/kg given once daily diminished growth [36,38]. 3.2. Duration of opioid receptor blockade: normal somatic development Multiple investigations were pursued to determine the extent of somatic growth regulated by blockade of opioid receptors [39]. The effects of naltrexone on organ growth were assessed in rats injected with either a 11

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short-duration blockade (1 mg/kg naltrexone) or complete-blockade (50 mg/kg naltrexone) [39]. Naltrexone altered the wet and dry weights of 10 organs in a dosage and sex dependent manner [39]. Concomitant experiments pursuing the underlying regulatory pathways for these observations led to the discovery and characterization of a new opioid receptor, termed zeta and later renamed OGFr (see review in section 1.1.2)

3.3. Duration of opioid receptor blockade: behavioral development

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[11,13,20].

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The duration of opioid receptor blockade by naltrexone also conferred changes in behavior. Animals exposed to continuous receptor blockade beginning at the time of birth demonstrated early acquisition of

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physical characteristics such as eye opening and hair growth, along with precocious timing in spontaneous motor and reflexive behaviors such as ability to roll over, crawling, bar hanging, and walking in comparison to

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control rats [40]. Ambulation measured by distance traveled, emotionality (fecal pellet deposition), and nociception were not altered by naltrexone.

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3.4. Duration of opioid receptor blockade during gestation: postnatal effects Other investigations on opioid antagonist exposure during pregnancy revealed a variety of effects on

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postnatal behavior [41,42]. Some studies did not completely disrupt peptide-receptor interaction continuously

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from fertilization through pregnancy, and many did not examine whether the dosage of opioid antagonist was

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sufficient to block opioid receptors completely throughout the day resulting in partial blockade and thus enhanced endogenous peptide-receptor interaction [41]. Low dosages of naloxone (1 mg/kg) during pregnancy resulted in cross-fostered offspring with altered behavior [42]. Prenatal naloxone exposure produced changes in body weight development, pain sensitivity, and motor behavior in the offspring. Rats treated prenatally with 1 mg/kg naloxone habituated more rapidly in the open field, and showed less activity as they matured. Bar pressing rates were reduced in male rats exposed to 10 mg/kg naloxone leading the authors to hypothesize that low dosages of naloxone with short receptor blockade may increase opiate [endogenous opioid] function in offspring [42]. Interpretation of maternal treatment and postnatal effects is always difficult, but paramount to the reliability of the studies is the question whether naltrexone passes through the placenta? Reverse phase highperformance liquid chromatography with ultraviolet detection measured a single 50 mg/kg dose of naltrexone administered intraperitoneally to a pregnant rat on day 20 of gestation in brain, heart and liver of fetuses 1 hr 12

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after injection. Thus, maternally administered naltrexone passes through the placenta [43] and could be detected in neonatal pups [44], but not in pups after 2 days of age. 3.5. Duration of opioid receptor blockade: brain development Opioids and their receptors were first discovered in neural tissue, and the brain remains an active area of research on endogenous opioids such as endorphins and enkephalins. Naltrexone‟s effects on the brain

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were studied in a series of investigations of cerebellum, cerebral cortex, and hippocampus [45-47]. Histological

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and morphometric studies of the cerebellum of rats exposed daily to either a continuous blockade by naltrexone (50 mg/kg) or an intermittent blockade (1 mg/kg) revealed that high dosages of naltrexone

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stimulated cerebellar development, whereas the short-receptor blockade inhibited growth [45]. The temporal course of development was consistent with normal development for both dosages; however, the magnitude of

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enhancement seen in morphometric analyses (i.e., cerebellar areal measurements, number of cerebellar internal granule neurons) was greater than the level of reductions.

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Intermittent receptor blockade by 1 mg/kg naltrexone altered cerebral cortex development at both cellular and tissue differentiation levels [46]. Brain tissue from rats injected with 1 mg/kg naltrexone displayed

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a substantial increase in packing density of neural cells, possibly reflecting reduced dendritic arborization and

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synapse connectivity. Hippocampal development was impacted more by the intermittent receptor blockade

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than by continuous blockade, suggesting that the boundaries of accelerated growth may restrict uncontrolled cellular proliferation.

Changes in cerebral development were extended into the ultramicroscopic area of spine formation and dendritic arborization [47]. Meticulous studies revealed that the duration of opioid receptor blockade early in postnatal life conferred long-term effects on brain maturation. Examination of Purkinje cells from the cerebellum and pyramidal cells from the cerebral cortex from rats injected daily with 50 mg/kg naltrexone for only 10 days revealed that both cell types had substantial increases in dendrite and/or spine elaboration compared to controls. In rats receiving intermittent receptor blockade with 1 mg/kg naltrexone, dendritic development was subnormal, with fewer spiny processes and dendritic arborization evident at 21 days of age. Whether these changes in brain development can be translated into learning or behavioral deficits is unknown. 3.6. Duration of opioid receptor blockade: heart development

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Whereas it may be expected that endogenous opioids would modulate growth of neural tissues, the influence of endogenous peptides and receptors on cardiovascular tissue was novel. The heart and vasculature develop prenatally, function during gestation, and essentially undergo hypertrophy throughout development. The presence of endogenous opioid activity in non-neural systems was investigated by examination of heart development [48]. A single injection of 50 mg/kg naltrexone to 1 day old rat pups

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increased DNA synthesis indexes of ventricular and atrial myocardial and epicardial cells. Chronic exposure to

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50 mg/kg naltrexone resulted in increased heart weights and areal measurements of ventricles and atria; the naltrexone effects were not mediated through the sympathetic nervous system or by way of increased thyroid

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hormone production.

A complete opioid receptor blockade during gestation significantly altered the cardiovascular system of

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infant rats [49]. Prenatal naltrexone treatment (50 mg/kg to pregnant rats) resulted in offspring with hearts that weighed more, and had more DNA and protein content in both the ventricles and atria, relative to cardiac

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tissue from saline-treated controls. Morphometric analyses revealed that the myocardium and chamber

term ramifications on cardiac biology.

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volumes were increased, thus suggesting that early exposure to high dosages of naltrexone may have long-

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4. Duration of opioid receptor blockade determines therapeutic outcome

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Our understanding of the biochemistry and pharmacology of opioid receptor blockade has prompted research into the usages of these widely available, non-toxic, drugs for treatment of drug addiction and alcoholism. However, the pharmacological properties of naloxone and naltrexone, and specifically, the ability to modulate growth by altering the duration of opioid receptor blockade, has made these general antagonists very useful therapies for cancer, autoimmune diseases, and complications associated with diabetes. The opposing effects reported following continuous or intermittent opioid receptor blockade are related to the underlying regulatory mechanisms of the OGF-OGFr pathway. Concomitant with opioid receptor blockade is a compensatory upregulation of endogenous opioids and receptors that can interact when the antagonist is no longer present. Thus, receptors are available for heightened interaction following intermittent antagonist blockade, whereas continuous blockade suppresses receptor availability. These mechanisms were delineated in a tissue culture model of human ovarian cancer cells [50]. The paradoxical effects of low and high dosages of naltrexone were demonstrated in vitro using the same drug dosage but different exposure times. Thus, short-term and long14

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term exposure to naltrexone resulted in reduced or accelerated cell growth, respectively. Receptor number and enkephalin levels were measured periodically by western blotting and radioimmunoassay, respectively, confirming the autocrine loop of endogenous peptide and receptor interaction during the period of time that opioid receptors are not longer blocked. With regard to OGFr, continuous opioid receptor blockade with high dosages of naltrexone, or continuous infusion of lower dosages of naltrexone, or multiple injections of

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naloxone, establishes constant „prevention‟ of inhibitory action by endogenous opioids (i.e., OGF). Hence,

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continuous blockade is applicable for treatment of conditions requiring rapid or enhanced cell proliferation such as corneal surface defects [e.g., 51], dry eye [e.g., 55], and closure of cutaneous wounds [57-60]. On the

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contrary, high levels of the inhibitory peptide OGF following intermittent blockade by low dosages of naltrexone (LDN) are biotherapeutic for autoimmune diseases and cancer [e.g., 20, 50] (Figure 2).

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4.1. Continuous OGFr blockade: Enhanced cell proliferation

Type 1 and type 2 diabetes are associated with many complications resulting from delayed

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epithelialization (i.e., keratopathy), lack of cellular function (ocular surface sensitivity, dry eye), and impaired healing (delayed repair of full-thickness cutaneous wounds). Naltrexone, systemically or topically administered,

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at high dosages can reverse and restore these defects [e.g., 55, 57-64].

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OGFr, and not mu, delta, or kappa opioid receptors, specifically mediates the effects of naltrexone in

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wound closure [65]. Specific antagonists for each classical receptor were added to NIH 3T3 cells, and cell number measured over several days. CTOP, naltrindole, and nalmefene, selective for mu, delta, and kappa opioid receptors, respectively, did not accelerate cell replication, whereas naltrexone enhanced growth. Moreover, addition of agonists with high affinity to the classical receptors (i.e., DAMGO, DPDPE, ethylketocyclazocine) did not alter growth, suggesting that these receptors were not pivotal in mediating cell replication, and that continuous blockade of the classical receptors were not involved. Further studies knocking down RNA for each classical receptor using siRNA transfection did not eliminate the increased fibroblast cell number reported following naltrexone. However, addition of OGFr siRNA, nullified the accelerated growth in the presence of naltrexone. In the animal model, the selective blockade of the OGFOGFr pathway by naltrexone was confirmed as only naltrexone, and not selective opioid receptor antagonists, enhanced wound closure [65].

15

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Topical naltrexone also has been shown to be effective at restoring dry eye, repairing corneal surface wounds, and enhancing closure of full-thickness skin wounds in normal and diabetic animals [e.g., 57-60, 63]. Because naltrexone diffuses passively into cells, it is amenable to topical application in a variety of carriers [57], and because epithelial tissues lack vascularity, naltrexone is metabolized slowly. Thus, topical administration can invoke sufficient OGFr blockade to allow for localized, accelerated DNA synthesis of cells

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and tissues, and remain safe and non-apoptotic [e.g., 55, 61].

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4.1.1. Continuous OGFr blockade: Ocular surface wounds and dry eye

A comprehensive series of investigations have been conducted on the role of the OGF-OGFr axis to

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repair ocular surface abnormalities [51-56, 61-64]. Corneal surface integrity maintains the required barrier to enable vision. Loss of sensitivity, dryness, or damage, leads to significant medical discomfort and even

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blindness. The cornea has a well-defined epithelium supported by stroma and peripherally-located renewing limbal cells [50]. Continuous blockade of OGFr and re-epithelialization of the cornea was first studied in rat

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using systemic injections of 50 mg/kg naltrexone [51]. Twice daily injections were required to maintain continuous receptor blockade in the relatively un-vascularized ocular surface [61]. However, within 8 hr of

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inducing a 4 mm diameter surface abrasion, systemic naltrexone increased DNA synthesis and cell replication.

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Topical administration of naltrexone was also effective at diminishing wound size 2.8 fold greater than in

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controls. This was the first indication that naltrexone retained its properties of receptor blockade when administered topically, suggesting that the passive diffusion of naltrexone did not breakdown any of the biochemical/pharmacology characteristics required to prevent endogenous peptide interaction at a nuclearlocated receptor [51,61]. Human donor corneas placed in culture, abraded, and subjected to 10-6 M naltrexone, healed faster than wounded corneas in media alone. The rate of closure, as well as the DNA labeling index was accelerated, in corneas placed in naltrexone [52]. Topical, rather than systemic, naltrexone became an obvious choice for treatment of ocular disorders in diabetic animals that experience delayed epithelialization. Continuous blockade using 10-5 M naltrexone rapidly restored the abraded cornea of chemically – induced, type 1 diabetic rats. Complete blockade of the OGFr by naltrexone also enhanced corneal surface wound repair in alloxan-induced type 1 diabetic rabbits [64], and genetically hyperglycemic type 2 diabetic mice [63]. A concern of whether chronic exposure to naltrexone was detrimental leading to scarring or exuberant granulation tissue in the corneal stroma was 16

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addressed [54,63], and the safety of topical naltrexone was demonstrated in several animal models [51, 52, 64]. It appears that naltrexone accelerates cellular proliferation, but other intrinsic factors provide the “brakes” for enhanced replication following continuous opioid receptor blockade. High dosages of naltrexone are also effective treatment of abnormalities related to corneal sensitivity and tear production in normal [56] and diabetic [55, 64] rodents. Normal rodents experience episodic dry eye;

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Schirmer test scores occur in a biomodal distribution of dryness {~6 mm or less) or wetness (≥ 7 mm). Topical

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naltrexone (10-5 M) dissolved in eyedrops restored dry eye within 1 hour of treatment, and had no effect on rats with normal tear production [56]. Type 1 diabetes is associated with prolonged periods of dry eye and corneal

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surface insensitivity. Topical application of high dosages of naltrexone restored sensitivity and tear production in both type 1 and type 2 diabetic animals [55,63] The reversal of dry eye following a single application lasted 2

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to 3 days, and the restitution of sensitivity lasted 4 to 7 days [55].

4.1.2. Continuous OGFr blockade: Full-thickness cutaneous wounds

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The efficacy of naltrexone for ocular-related complications of diabetes was novel, and studies were extended to another serious medical concern for diabetics – delayed wound repair [57-59]. An animal model of

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type 1 diabetes was established by injections of streptozotocin, and full thickness cutaneous wounds were

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created on the dorsum. Topical treatment of naltrexone in a variety of dosages as well as several carriers

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including DMSO, buffer, and creams, revealed that naltrexone (10-5 M) in moisturizing cream was effective at enhancing wound closure with both cell replication and contracture [57]. Further studies showed that continuous opioid receptor blockade stimulated angiogenesis as measured by FGF-2, VEGF, and α-SMA expression in capillaries [58], accelerated skin remodeling [59], and reinforced the integrity of the skin as monitored by tensile strength measurements [60]. Thus, naltrexone-enhanced wound repair resulted in skin that was strong and intact [60].

4.2. Intermittent OGFr blockade: Treatment of autoimmune disorders Autoimmune disorders such as fibromyalgia, rheumatoid arthritis, Crohn‟s, and multiple sclerosis are difficult to diagnose and are often detected only after multiple clinical visits. Many patients present with decreased enkephalins and endorphins, exacerbating their inflammation, pain, and other immune-mediated metabolic deficits. The concept that low dosages of naltrexone (LDN) have positive effects in individuals with autoimmune diseases preceded most basic science reports. Websites such as LDNNow provide an excellent 17

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summary of the history, uses, and potential for LDN [66,67]. As early as the 1980s, it was noted that LDN inhibited cancer growth [19,20]. Only recently has the potential biofeedback leading to elevated endogenous opioids been evaluated in light of biotherapies for autoimmune diseases. The mechanistic pathways of LDN are unclear. Some investigations suggest that LDN works directly as an immunomodulating agent [68,69], whereas other studies on T and B cell proliferation in vitro [70, 71] and in vivo [72] have demonstrated that the

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activation of peripheral lymphocytes and resultant cytokine production.

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modulation of the immune system most likely is a direct response to increased or decreased proliferation and

4.2.1. Multiple sclerosis

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As mentioned, the clinical use of LDN for treatment of autoimmune disorders outpaced rigorous scientific research. Even today, many internet-originated rumors exist that warrant clarification. For example,

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“more is not better”. The fallacy in this statement is obvious; LDN patients should not be encouraged to increase their dosage or take more than one tablet daily. At present, the timing of administration of LDN is a

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patient preference, and there are no basic science studies that conclude morning or evening consumption is either harmful or better.

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Recently, animal studies on intermittent or continuous blockade of OGFr using naltrexone for treatment

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of experimental autoimmune encephalomyelitis (EAE) were initiated. EAE can be induced in a variety of ways

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to establish a more progressive form using myelin-oligodendrocytic glycoprotein (MOG)35-55 as the antigen, or relapse-remitting form using proteolipid protein (PLP)139-151 [73-79]. In a series of investigations with progressive EAE, high dosages of naltrexone initiated at the time of disease induction had no effect on the behavioral sequelae associated with EAE. LDN however actually prevented 33% of the mice from developing clinical signs of the disease, and the severity and disease index of LDN-treated mice was reduced from that in animals receiving saline. Neuropathology corroborated the behavioral studies showing that LDN treated mice had fewer activated astrocytes, less demyelination and neuronal damage than mice receiving saline or high dosages of naltrexone [73]. Treatment of mice with chronic EAE using OGF appears to be more effective than LDN therapy [73,74]. Using animal models with treatment beginning after established disease, OGF arrested the progression of chronic, MOG-induced EAE and reduced the associated spinal cord pathology [75]. In theory, the behavioral signs appeared several days after the initial immune response, and yet OGF was capable of controlling 18

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autoimmune flairs. Indepth pathology studies indicated that astrocyte proliferation and activation were controlled by OGF. Astrocyte activation in disease states is an important corollary to disease progression and recovery. OGF therapy inhibited the proliferation of astrocytes in vivo, and in vitro [76], and did so by way of OGFr; siRNA technology and diminished protein expression of mu, delta, and kappa receptors in vitro demonstrated the specificity and requirement for OGFr in primary cultures of mouse cerebral astrocytes.

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Relapse-remitting multiple sclerosis is the most common form of multiple sclerosis; the mouse model of relapse-remitting EAE is established by immunization of SJL mice with PLP [77-79]. OGF [78] and LDN [79]

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treatment initiated after clinical disease was observed – establishing a very clinically relevant model – resulted

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in reduced behavioral abnormalities and neuropathological defects. Animals that responded to the treatments were often without sign of relapses, and had sustained remissions over a 40-day period of observation.

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Translation of these observations to the clinic is warranted. Small trials and case reports have documented that LDN usage by MS patients is safe, without side effect, and reduces associated fatigue [80]. Clinical trials have

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concurred that LDN is safe and probably effective – all recommending that larger, randomized, double-blind trials be established [81].

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4.2.2. Inflammatory bowel disease and fibromyalgia

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Other autoimmune diseases that respond to partial opioid receptor blockade include the family of

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inflammatory bowel disorders (Crohn‟s and colitis) [82-85]. Although few basic science reports have been published, patient studies are provocative. In a murine model of inflammatory bowel disease, low dosages of naltrexone were shown to reduce the weight loss and lessen the disease activity, normally associated with progression of the disorder [85]. The short term opioid receptor blockade reversed the physical symptoms, pathology, and molecular markers of inflammation associated with colitis. Fibromyalgia is another chronic, debilitating disorder that incurs sufficient pain, suggesting that opioids and their receptors may be dysregulated. A definitive cause for this disorder that afflicts up to 5% of all women is unknown. Because endorphins are involved in mediating mood, and to some extent pain mediation, studies have explored the role of low dose naltrexone as a biotherapy for this disorder. Studies by Younger and Mackey [86] reported that low dose naltrexone reduced symptoms of fibromyalgia. Other investigators recognized that low dosages of naltrexone would increase endogenous peptides and suppress pain on a

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continual, yet safe, regimen [87]; both groups encouraged larger clinical trials on the use of LDN as a biotherapy for fibromyalgia. 4.3. Intermittent OGFr blockade: treatment of cancer The role for endogenous opioids in cancer therapy commenced more than 30 years ago with our early studies on opioid receptor blockade and proliferation of murine neuroblastoma [28,29]. White mice

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transplanted with S20Y murine neuroblastoma cells were subjected to intermittent receptor blockade following injections of 0.1 mg/kg naltrexone, or complete blockade following 10 mg/kg naltrexone. Plasma levels of β-

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endorphin were not altered by either opioid receptor blockade, whereas tumor tissue levels of both [Met5-

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enkephalin] and β-endorphin were significantly upregulated [88]. Receptor binding sites measured by radioactive-DADLE were significantly upregulated following both intermittent and continuous receptor

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blockade, although putative κ opioid receptors, measured by tritiated ethylketocyclazocine were downregulated. Labeling indexes (thymidine incorporation) and mitotic indexes (microscopic analyses of

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metaphase, anaphase, and telophase in hematoxylin-stained tumor sections) were elevated during the period of opioid receptor blockade, but decreased during the time subsequent to blockade (i.e., post 8 hr for 0.1

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mg/kg naltrexone). Treatment with endogenous opioids, specifically OGF, inhibited proliferation in a receptor-

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mediated, dose-related (not dose-dependent) manner [88]. Tissue culture studies on the regulation of cancer

[89].

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cell proliferation by continuous opioid receptor blockade an all or none response, not a dose-dependent curve

The observation that the duration of opioid receptor blockade mediated the biotherapeutic recourse for cancer [28, 29, 50] stimulated the discovery of the OGF-OGFr regulatory pathway. Although not clinically relevant, studies on continuous receptor blockade in cancer models has provided important insights into the role of the OGF-OGFr axis in cancer biotherapy. Intermittent blockade with LDN, or OGF, are logical biotherapeutic approaches, and each treatment has advantages. LDN is available as an oral tablet, and can be pharmaceutically prepared with a physician‟s prescription. For LDN to be effective however, it requires that the consumer of LDN have an intact OGF-OGFr axis that is fully functioning and able to produce and secrete OGF in sufficient quantity to bind to OGFr located on the targeted tissue. The indirect biofeedback loop presumably requires more time for activation, and is also more susceptible to dysregulation than direct OGF. On the other hand, OGF is a small peptide that acts directly on the cell cycle [90] to inhibit DNA synthesis and 20

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replication. However, OGF is easily degraded (enkephalinases are ubiquitous), not FDA-approved, and currently must be injected either intravenously or subcutaneously. LDN is usually taken once daily, or even every 2nd or 3rd day, whereas OGF has been infused weekly or monthly in the few clinical trials that have been published [91]. Numerous studies on the efficacy of OGF and LDN have been conducted on human cancer cells grown in vitro or transplanted into nude mice [50, 92]. To date, no human cancer cell has been shown to

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be refractive to biotherapy with either OGF or LDN; OGFr is also required to mediate endogenous or

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exogenous inhibition [19]. The mediation of human ovarian cancer [50,92] and human triple negative breast cancer [19] by modulation of the OGF-OGFr axis provides a reasonable portrayal of OGF efficacy. The

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duration of receptor blockade determined the cell proliferative response in human ovarian, pancreatic, colorectal, and squamous cell carcinoma of the head and neck neoplasias. Naltrexone blockade was shown to

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upregulate both peptide and OGFr at the translational but not at the transcriptional level. Naltrexone did not alter cell survival (apoptosis or necrosis), and short-term blockade required p16 and/or p21 cyclin-dependent

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inhibitory kinases to be functional. In culture, sequential administration of short-term naltrexone followed by

support further clinical research.

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OGF was more effective at inhibiting cancer growth than either biotherapeutic regimen alone. These studies

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4.4. Intermittent opioid receptor blockade: Addictive behavior

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The pathways associated with naltrexone‟s blockade of alcoholism, self-injurious behavior and compulsive disorders are not fully known [18]. Recent publications have reported that with regard to alcoholism, NTX therapy at 50 mg once daily was effective at doubling abstention rates 51% vs 23% placebo, reducing (cut in half) the number of drinking days, craving, and relapses. In a second trial of 865 patients, NTX was shown to have no serious side effects [93,94]. Mannelli et al. reported that very low dosages of naltrexone, 0.1 to 0.2 mg/kg daily, resulted in reduced withdrawal and a lower rate of treatment noncompliance in heavy drinkers who were in opioid detoxification programs [95]. In addition to the obvious function as a drug for reversal of drug overdose, naloxone and naltrexone have slowly found their way into a long list of medical uses. In addition to addiction, naltrexone has been recommended to prevent alcohol dependence [94], and even tobacco dependence [96]. All 3 of these events utilize a single 50 mg naltrexone treatment daily. A few studies have shown that long-acting naltrexone can ameliorate self-injurious behavior in autism and other compulsive disorders [97,98]. Thus, low dosages of naltrexone may confer a partial blockade to receptors 21

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craving illicit drugs, attempting to quit smoking, or cease self-injurious behaviors, and even gambling [93-98]. The role of OGFr in mediating the low dose naltrexone is currently under investigation. 5. Conclusions and future perspectives The duration of opioid receptor blockade by antagonists, such as naltrexone, has impacted more biological pathways than addiction pathways, which largely remain undefined. With the widespread use of low

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dosages of naltrexone, the impact of this opioid antagonist biotherapy is broad-based. Approximately 52

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million individuals in the US may benefit from either low dosages of naltrexone or complete receptor blockade for treatment of cancer, multiple sclerosis, inflammatory bowel disorders, or complications associated with

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diabetes. Given that nearly 86 million individuals are pre-diabetic, the need to develop new biotherapies that are non-toxic, and target the underlying pathophysiology of these diseases is an urgent, unmet medical need.

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Worldwide, the potential audience that could benefit from biotherapeutics related to modulation of the OGFOGFr axis approaches more than 350 million. It is anticipated that drug discovery researchers will identify

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specific and selective receptor antagonists that confer no biological activity and can be used as safe,

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Acknowledgements

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inexpensive treatment of these disorders.

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The authors express their gratitude to Nancy Kren who designed the graphic models presented in Figures 1 and 2. This research was supported by grants from NIH, American Diabetes Association, and The Shockey Family Foundation.

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58. McLaughlin PJ, Immonen JA, Zagon IS. Topical naltrexone accelerates full-thickness wound closure in Type 1 diabetic rats by stimulating angiogenesis. Exp. Biol. Med. 238:733-743, 2013. PMID: 23788174.

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59. Immonen JA, Zagon IS, Lewis, GS, McLaughlin PJ. Topical treatment with the opioid antagonist naltrexone accelerates the remodeling phase of full-thickness wound healing in Type 1 diabetic rats. Exp. Biol. Med.

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238:1127-1135, 2013. PMID:23986225

60. Immonen JA, Zagon IS, McLaughlin PJ. Topical naltrexone as treatment for type 2 diabetic cutaneous

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wounds. Advances Wound Care 3: 419-427, 2014. PMID:24940556

61. Zagon IS, Jenkins JB, Lang CM, Sassani JW, Wylie JD, Ruth TB, Fry JL, McLaughlin PJ. Naltrexone, an

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PMID: 12351447

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opioid antagonist, facilitates re-epithelialization of the cornea in diabetic rat. Diabetes 51:3055-3062, 2002.

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62. Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Topically applied naltrexone restores corneal reepithelialization in diabetic rats. J Ocular Pharmacol Ther 23:89-102, 2007. PMID: 17444796 63. Zagon IS, Sassani JW, Immonen JA, McLaughlin PJ. Ocular surface abnormalities related to Type 2 diabetes are reversed by the opioid antagonist naltrexone. Clin Exp Ophthalmol 42:159-168, 2014. PMID: 23777539

64. Zagon IS, Sassani JW, Carroll, MS, McLaughlin PJ. Topical application of naltrexone facilitates reepithelialization of the cornea in diabetic rabbits. Brain Res. Bull. 81:248-255, 2010. PMCID: PMC2815253 PMID: 19853924 65. Immonen JA, Zagon IS, McLaughlin PJ. Selective blockade of the OGF-OGFr pathway by naltrexone accelerates fibroblast proliferation and wound healing. Exp Biol Med 239:1300-1309, 2014. 66. How LDN Works. http://www.ldnnow.co.uk/ 67. LDN Awareness http://www.ldnresearchtrust.org/ 27

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68. Wang Q, Gao X, Yuan Z, Wang Z, Meng Y, Cao Y, Plotnikoff NP, Griffin N, Shan F. Methionine enkephalin (MENT) improves lymphocytes subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs). Hum Vaccin Immunother 10:1836-1849, 2014 69. Li W, Chen W, Herberman RB, Plotnikoff NP, Youkilis G, Griffin N, Wang E, Lu C, Shan F. Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK). Cancer

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Lett 344:212-222, 2014.

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70. Zagon IS, Donahue RN, Bonneau RH, McLaughlin PJ. B lymphocyte proliferation is suppressed by the opioid growth factor-opioid growth factor receptor axis: Implication of the treatment of autoimmune diseases.

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Immunobiology 216:173-183, 2011. PMID: 20598772

71. Zagon IS, Donahue RN, Bonneau RH, McLaughlin PJ. T lymphocyte proliferation is suppressed by the

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opioid growth factor ([Met5]-enkephalin)-opioid growth factor receptor axis: Implication for the treatment of autoimmune diseases. Immunobiology 216:579-590, 2011. PMID: 20965606

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72. McLaughlin PJ, McHugh DP, Magister MJ, Zagon IS. Endogenous opioid inhibition of proliferation of T and B cell subpopulations in response to immunization for experimental autoimmune encephalomyelitis. BMC

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Immunology, 2015, in press.

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73. Zagon IS, Rahn KA, Turel AP, McLaughlin PJ. Endogenous opioids regulate expression of experimental

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autoimmune encephalomyelitis: A new paradigm for the treatment of multiple sclerosis. Exp Biol Med 234:1383-1392, 2009. PMID: 19855075

74. Rahn KA, McLaughlin PJ, Zagon IS. Prevention and diminished expression of experimental autoimmune encephalomyelitis by low dose naltrexone (LDN) or opioid growth factor (OGF) for an extended period: Therapeutic implications for multiple sclerosis. Brain Res 1381:243-253, 2011. PMID: 21256121 75. Campbell, A.M., I.S. Zagon, and P.J. McLaughlin. 2012. Opioid growth factor arrests the progression of clinical disease and spinal cord pathology in established experimental autoimmune encephalomyelitis. Brain Res. 1472:138-148. PMID 22820301 76. Campbell, A.M., I.S. Zagon, and P.J. McLaughlin. 2013. Astrocyte proliferation is regulated by the OGFOGFr axis in vitro and in experimental autoimmune encephalomyelitis. Brain Res Bull 90:43-51. 77. Hammer, L.A., I.S. Zagon, and P.J. McLaughlin. 2013. Treatment of a relapse-remitting model of multiple sclerosis with opioid growth factor. Brain Res Bull 98:122-131. PMID: 23973432 28

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78. Hammer LS, Zagon IS, McLaughlin PJ. Improved clinical behavior of established relapse-remitting experimental autoimmune-encephalomyelitis following treatment with endogenous opioids: Implications for the treatment of multiple sclerosis. Brain Res Bull, 2015, in press. 79. Hammer LA, Zagon IS, McLaughlin PJ. Low dose naltrexone treatment of established relapsing-remitting experimental autoimmune encephalomyelitis. J Mult Scler, 2015, in press.

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80. Turel AP, Oh KH, Zagon IS, McLaughlin PJ. Low dose naltrexone (LDN) for treatment of multiple sclerosis:

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A retrospective chart review of safety and tolerability. J Clin Psychopharmacol, 2015, in press.

81. Cree BA, Komyeyeva E, Goodin DS. Pilot trial of low-dose naltrexone and quality of life in multiple

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sclerosis. Ann Neurol 68:145-150, 2010.

82. Smith JP, Field D, Bingaman SI, Evans R, Mauger DT. Safety and tolerability of low-dose naltrexone

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therapy in children with moderate to serve Crohn‟s disease: a pilot study. J Clin Gastroenterol 47:339-345, 2013.

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83. MacDonald SD, Chande N. Low dose naltrexone for induction of remission in Crohn‟s disease (Review). The Cochrane Collaboration. The Cochrane Library, issue 2, 2014. http://www.thecochranelibrary.com

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84. Smith JP, Bingaman SI, Ruggiero F, Mauger DT, Mukherjee A, McGovern CO, Zagon IS. Therapy with the

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opioid antagonist naltrexone promotes mucosal healing in active Crohn‟s disease: a randomized placebo-

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controlled trial. Dig Dis Sci 56:2088-2097, 2011

85. Matters GL, Harms JF, McGovern C, Fitzpatrick L, Parkh A, Nito N, Smith JP. The opioid antagonist naltrexone improves murine inflammatory bowel disease. J Immunotoxicol 5:179-187, 2008 86. Younger JW, Mackey S. Fibromyalgia symptoms are reduced by low-dose naltrexone: a pilot study. Pain Med 10:663-672, 2009.

87. Ramanathan S, Panksepp J, Johnson B. Is fibromyalgia an endocrine/endorphin deficit disorder? Is low dose naltrexone anew treatment option? Psychosomatics 53:591-594, 2012. 88. Zagon IS, McLaughlin PJ. Stereospecific modulation of tumorigenicity by opioid antagonists. Eur J Pharmacol 113:115-120, 1985. 89. Zagon IS, McLaughlin PJ. Opioid antagonist (naltrexone) stimulation of cell proliferation in human and animal neuroblastoma and human fibrosarcoma cells in culture. Neuroscience 37:223-226, 1990.

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90. Cheng F, Zagon IS, Verderame MF, McLaughlin PJ. The opioid growth factor (OGF)-OGF receptor axis uses the p16 pathway to inhibit head and neck cancer. Cancer Res 67:10511-10518, 2007. PMID: 17974995 91. Smith JP, Bingaman SI, Mauger DT, Harvey HH, Demers LM, Zagon IS. Opioid growth factor improves clinical benefit and survival in patients with advanced pancreatic cancer. Open Access J Clin Trials 2010:3748, 2010.

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92. Donahue, R.N., P.J. McLaughlin, and I.S. Zagon. 2011. The opioid growth factor (OGF) and low dose

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naltrexone (LDN) suppress human ovarian cancer progression in mice. Gynecol Oncol 122:382-388. PMID:21531450

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93. Srisurapanont M, Jarusauraisin N, Leucht S, Vecchi S, Srisurapanont M, Soyka M. Opioid antagonists for alcohol dependence The Cochrane Library 12, 2005. PMID: 15674887

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94. Pettinati HM, O‟Brien CP, Rabinowitz AR, Wortman SP, Oslin DW, Kampman KM, Dackis CA. The status of naltrexone in the treatment of alcohol dependence. J Clin Psychopharmacol 26:610-625, 2006. PMID

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95. Mannelli P, Wu, LT, Peindl KS, Swartz MS, Woody GE. Extended release naltrexone injection is

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performed in the majority of opioid dependent patients receiving outpatient induction: A very low dose

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naltrexone and buprenorphine open label trial. Drug Alcohol Depend 138:83-88, 2014.

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96. King A, deWit, H, Riley R, Cao D, Niaura R, Hatsukam D. Efficacy of naltrexone in smoking cessation: A preliminary study and an examination of sex differences. Nicotine Tobacco Res 8:671-682, 2006. 97. Smith SG, Gupta KK, Smith SH. Effects of naltrexone on self-injury, stereotypy, and social behavior of adults with developmental disabilities. J Dev Physical Disabilities 7:137-146, 1995. 98. Suck WK, Grant JE, Adson DE, Shin YC. Double-bline naltrexone and placebo comparison study in the treatment of pathological gambling. Biological Psychiatry 49:914-921, 2001.

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Figure Legends Fig 1. Nuclear interactions of naltrexone with OGFr in the cytoplasm as well as within the nucleus. Naltrexone binds to OGFr, trafficks into the nucleus and blocks OGF interactions that upregulate p21 and p16 cyclindependent inhibitory kinases. Impα = importin α; impβ – importin β; NTX = naltrexone. Fig 2. Duration of OGFr blockade at the cellular level showing distinction between cytoplasmic receptors and

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nuclear-associated receptors. Impα = importin α; impβ – importin β; NTX = naltrexone.

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Revised manuscript Click here to view linked References

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1 2 3 Duration of Opioid Receptor Blockade Determines Biotherapeutic Response 4 5 6 7 8 9 10 11 12 13 14 Patricia J. McLaughlin ([email protected]) and Ian S. Zagon ([email protected]) 15 16 Department of Neural and Behavioral Sciences 17 18 19 Pennsylvania State University College of Medicine 20 21 Hershey, PA 17033 USA 22 23 24 25 26 27 28 29 30 31 32 Corresponding author: 33 34 Dr. Patricia J. McLaughlin 35 36 Department of Neural & Behavioral Sciences, MC H109 37 38 39 Penn State University College of Medicine 40 41 500 University Drive 42 43 Hershey, PA 17033 USA 44 45 717-531-6414 (phone) 46 47 48 717-531-5003 (fax) 49 50 Email: [email protected] 51 52 53 54 55 56 57 58 59 Running title: Duration of Receptor Blockade Determines Response 60 61 62 63 64 1 65

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Abbreviations: OGF, opioid growth factor; OGFr, opioid growth factor receptor, NTX, naltrexone, LDN, low dose naltrexone; EAE, experimental autoimmune encephalomyelitis; DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin; DPDPE, d-Pen2, d-Pen5-enkephalin; CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; VEGF, vascular endothelial growth factor; FGF-2, fibroblast growth factor -2; α-SMA, alpha smooth muscle actin; DMSO, dimethyl sulfoxide; MOG, myelin oligodendrocytic

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indicating total concentration of receptors; NLS, nuclear localization signal

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glycoprotein; PLP, proteolipid protein; imp, importin; Kd, dissociation constant; Bmax, maximal binding

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Number of words: 10,778

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Abstract

Historically, studies on endogenous and exogenous opioids and their receptors focused on the mediation of

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pain, with excess opiate consumption leading to addiction. Opioid antagonists such as naloxone and naltrexone blocked these interactions, and still are widely used to reverse drug and alcohol overdose. Although

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specific opioid antagonists have been designed for mu, delta, and kappa opioid receptors, the general

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antagonists remain the most effective. With the discovery of the opioid growth factor (OGF)-OGF receptor (OGFr) axis as a novel biological pathway involved in homeostasis of replicating cells and tissues, the role of

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opioid receptor antagonists was expanded. An intermittent OGFr blockade by low dosages of naltrexone resulted in depressed cell replication, whereas high (or sustained) dosages of naltrexone that conferred a continuous OGFr blockade resulted in enhanced growth. More than 3 decades of research have confirmed that the duration of opioid receptor blockade, not specifically the dosage, by general opioid antagonists determines the biotherapeutic outcome. Dysregulation of the OGF-OGFr pathway is apparent in a number of human disorders including diabetes, multiple sclerosis, and cancer, and thus opioid antagonist disruption of interaction prevails as a therapeutic intervention. We review evidence that the duration of opioid receptor blockade is correlated with the magnitude and direction of response, and discuss the potential therapeutic effectiveness of continuous receptor blockade for treatment of diabetic complications such as corneal defects and skin wounds, and of intermittent receptor blockade by low dosages of naltrexone for treatment of autoimmune diseases and cancer. 2

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Keywords: Naltrexone, multiple sclerosis, dry eye, diabetic wound healing, LDN 1. Introduction Opioid receptors were identified first followed by the discovery of endogenous opioids that acted as ligands. Concomitantly, pharmacologists began designing opioid antagonists that blocked the neurotransmitter function

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of these receptors in brain and gut. Nearly five decades have elapsed since the initial identification of opioid

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receptors, and two of the original antagonists, naloxone and naltrexone, remain on the forefront of treatment for cancer pain, addiction, drug overdose, alcoholism, and other psychosomatic disorders. The discovery of

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the non-classical opioid receptor, opioid growth factor receptor (OGFr), that shares several pharmacological properties with mu, delta, and kappa opioid receptors, has led to research that broadens the usefulness of

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opioid antagonists. Depending on the duration of opioid receptor blockade, opioid antagonists such as naloxone and naltrexone are effective therapies for cancer, autoimmune diseases, and complications

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associated with diabetes. Understanding the dysregulation of the OGF-OGFr axis in each disease dictates

proliferation is warranted.

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1.1. Opioid receptors

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whether continuous blockade to enhance cellular proliferation or intermittent blockade to inhibit cellular

1.1.1. Classical G-protein coupled opioid receptors

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Research to discover opioid receptors commenced in the early 1970s when biochemical studies reported that certain drugs interacted with specific molecules within different regions in the central nervous system [1-3]. Radiolabeled binding of exogenous opiate agonists such as levorphanol were used to locate and isolate specific binding proteins [4]. Pert and Snyder published a seminal paper on the identification of the binding site for radiolabeled naloxone [1], and eventually identified the mu opioid receptor. Many of the investigations involved nervous tissue, and in rapid succession, mu, delta, and kappa opioid receptors were identified and characterized in the brain or enteric nervous system [2,3]. Two decades later, the molecular structure of these classical opioid receptors was revealed [5-7]. Cloning of the mu, delta, and kappa opioid receptors illustrated that all three receptors are G protein-coupled transmembrane proteins that are members of the subfamily of rhodopsin receptors [7]. The receptors share 60% identity with more than 70% identity in the transmembrane domains and intracellular looping regions. The N terminus is least similar among the 3 receptors, but all have 3

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an extracellular domain in the N terminus with glycosylation sites and intracellular loops with multiple amphiphatic α-helixes. All three classical opioid receptors stimulate cAMP accumulation and are blocked by pertussis toxin [8]. 1.1.2. Non-classical nuclear membrane-associated opioid receptor A non-classical opioid receptor, OGFr, was first recognized in the 1980s, and subsequently characterized in

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both murine neural cancer cells [9] and normal rodent brain tissue [10,11]. The isolated protein was originally termed zeta (ζ) to maintain consistent naming with the Greek symbols of mu, delta, and kappa, and was

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appropriately called “zeta” for the Greek word zoe”, loosely defined as “growth”. Concomitantly, other studies

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were conducted to determine the endogenous opioid involved with this binding protein, and the ligand [Met5]enkephalin was identified to have inhibitory growth properties when binding to this receptor. The endogenous

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peptide was termed opioid growth factor (OGF), to distinguish the neurotransmitter function from that of being an inhibitory growth factor, and the zeta receptor was renamed OGFr. The cDNA for the rat OGFr was cloned

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by searching expression libraries [12], and subsequently the sequence was identified in human and mouse [13]. Based on extensive biochemical characterization, and cloning, the similarities of classical mu, delta, and

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kappa opioid receptors with OGFr were in the pharmacology, and not at the molecular level. The open reading

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frame for human OGFr is 697 amino acids with 8 imperfect repeats of 20 amino acids each at the C terminus. The human OGFr is located on chromosome 20q13.3 [13]. Thus, the molecular and protein structure of OGFr

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has no resemblance to classical opioid receptors. Based on NMR studies as well as confirmation from websites such as FoldIndex [14], OGFr is an intrinsically unstructured protein with approximately 78% amino acid identity between mouse, rat, and human. In studies on subcellular localization of OGFr using COS-7 African green monkey kidney cells, it has been documented that the receptor has three nuclear localization signals (NLS) within its sequence, two mono-partite NLS383-386 and NLS456-460, and one bi-partite NLS267-296 [15]. Studies utilizing site directed mutagenesis demonstrated when NLS383-386 and NLS456-469 were both mutated the nuclear localization was decreased by 80%, and the regulatory effects of OGF were diminished indicating that the OGF-OGFr action on proliferation is dependent on the ability of OGFr to translocate into the nucleus requiring the presence of NLS, karyopherin β and Ran [15]. Transport of fluorescein-labeled naltrexone was not temperature dependent, and was observed in the nucleus for 48 hr (Figure 1) [15]. Export of OGFr from the nucleus is CRM-1 dependent. 4

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Subcellular fractionation studies using developing rat brain and cerebellum revealed that OGFr binding is associated with the nucleus [9,11,13]. These biochemical studies were confirmed by confocal microscopy studies in the rat cornea that demonstrated immunogold labeling of OGFr in the paranuclear cytoplasm, within the nucleus, and adjacent to heterochromatin in corneal epithelial cells [16]. Colocalized immunogld labeling of OGFr and OGF was detected on the outer nuclear envelope and inside the nucleus [16]. Collectively, these

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inside the nucleus with its cargo, the endogenous [Met5]-enkephalin ligand.

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data suggest that the receptor is located on or near the outer nuclear envelope and functions by translocating

The gene and protein for OGFr have been identified in cells and tissues arising from all 3 dermal

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derivatives [13]. Gene expression for OGFr has been documented in human fetal tissues including brain, liver, lung, and kidney as well as in adult heart, brain, liver, skeletal muscle, kidney, and pancreas [13]. Binding

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assays on adult and fetal rat brain have quantitated OGFr binding [17], and studies conducted in adult mice demonstrated RNA levels in brain, heart, lung, liver, kidney and skeletal muscle. Additionally, OGFr has been

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detected in neoplasia, as well as in cell lines derived from human cancers [18-20]. 1.2. Opioid receptor antagonists

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Opioid antagonists are compounds that competitively bind to opioid receptors with affinity greater than

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that of specific agonists. However, antagonists have no function other than to block this interfacing. In the case of opioid receptors, agonists are both exogenous compounds such as morphine, codeine, congeners of

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morphine, and endogenous molecules such as endorphins and enkephalins. The general antagonists were synthesized first to block exogenous opiate interactions, and later were instrumental in research on the isolation of opioid receptors [2].

1.2.1. General opioid receptor antagonists Opioid receptor antagonists are either general and bind to all classical opioid receptors, or are specific and selective. The two most widely studied opioid receptor antagonists, naloxone and naltrexone, are general antagonists. Both compounds were discovered more than a half century ago, and remain the most promising pharmaceuticals to reverse opiate overdose and treat drug and alcohol addiction [21,22]. The identification [23] and characterization [24] of naloxone, also termed Narcan, occurred in the 1960s. Interest in Narcan has reemerged with the heightened incidence of heroin addiction and the need to prevent overdose. The primary use of naloxone remains as a medication to reverse opioid overdose and reduce respiratory depression [22]. 5

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Naltrexone hydrochloride is a synthetic congener of oxymorphone, but lacks opioid agonist action, and is trademarked as Trexan, Revia, or the extended release form Vivitrol. Naltrexone and naloxone share similar structures; naloxone is n-allynoroxymorphone [23,24], whereas naltrexone is morphinan-6-1,17(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxy-, hydrochloride [21]. Both compounds block all classical opioid receptors, as well as OGFr, by competitive binding between the antagonist and their respective exogenous or

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endogenous ligand. Both antagonists can be absorbed orally, with approximately 5-40% oral bioavailability.

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With regards to naltrexone, the parent compound and 6-β-naltrexol metabolites are active, and excreted by the kidney. Peak levels of absorption may occur as quickly as 1 hour, with the half-life for naltrexone being 4

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hours, and 13 hours for its metabolite, 6-β-naltrexol [21]. In comparison, enkephalins that are ligands for µ and δ opioid receptors are degraded within minutes.

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Naltrexone has been shown to provide a complete blockade of exogenous opioid congeners, but is not effective against cocaine or other non-opioid drugs of abuse, thus demonstrating specificity for opioid

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receptors. In some studies, naltrexone has been shown to have a partial inverse agonist effect – such that lowdose naltrexone can reverse the altered homeostasis resulting from long-term abuse of opioid agonist drugs

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[21]. Naloxone has no partial agonist effect, but can work as an inverse agonist at mu receptors – making it

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preferred for reversal of drug overdose [22]. A third general antagonist is the methylated version, methylnaltrexone bromide, also called Relistor; this compound does not pass the blood brain barrier, making it

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useful for treatment of opioid-induced constipation [20,21]. 1.2.2. Selective opioid receptor antagonists Despite classical opioid receptors sharing significant structural homology, selective antagonists have been synthesized and shown to preferentially bind one of the 3 opioid receptors. CTOP, CTAP and cyprodime are selective antagonists for mu opioid receptors, whereas naltrindole is selective for delta, and norbinaltrophimine for kappa receptors. Research is continuing to identify selective antagonists to classical opioid receptors, but at this time, no specific antagonist for OGFr has been identified. 2. Opioid receptor blockade The focus of this commentary is on the duration of opioid receptor blockade by opioid antagonists. In this context, it is appropriate to define “duration of opioid receptor blockade”. Duration is the length of time elapsed between 2 events – e.g., extent of active binding to the receptor. A search of scientific publication 6

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databases (e.g., PUBMED, SCOPUS) revealed that in the 21st century, few laboratories are studying the duration of opioid receptor blockade and biotherapeutic response. Much of the work on the fundamental pharmacological principle first observed in the 1970s was refined during the next two decades. 2.1. Mechanisms of opioid receptor blockade Receptor antagonists have different affinities to each opioid receptor, and binding of the antagonist

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disrupts the interaction between the agonist (or inverse agonist) and the receptor. Pharmacologically,

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antagonists mediate their effects by binding to allosteric or orthosteric sites. The antagonism can be reversible if the agonist concentration exceeds the affinity of the antagonist for the receptor. Because the interactions

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can be reversible depending on the longevity of the antagonist-receptor complex, it is often the duration of opioid receptor blockade that confers the action. For example, at comparable biochemical concentrations,

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naloxone is short–acting and naltrexone is longer-acting. The interplay between two proteins that invoke a reaction has been identified by Tummino and Copeland as the concept of residence time of receptor and

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ligand complexes [25]. This concept implies that all biochemical activities involving a ligand and receptor depend on binary complexes that can be regulated. These authors presented quantitative definitions to

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measure the initial moment of contact, duration of action, and amplitude for every ligand-receptor interaction

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[25]. The pharmacology associated with each, and the duration of receptor blockade, rather than dosage, is the subject of the remaining discussion.

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2.1.1. Mechanisms of action: Naltrexone Biphasic responses of naltrexone in blockade of opioid receptors were first reported in the 1990s [26]. Radiolabeled naltrexone had high affinity binding for the mu opioid receptor, whereas agonists for delta opioid receptors competed with low affinity binding of naltrexone. Studies on pharmacokinetics of radiolabeled ligands, classical opioid receptors, and antagonist competition documented that radiolabeled naltrexone reached equilibrium in a two-site model within 30 min at room temperature using rat brain homogenates [26]. Competition with mu receptor agonists abolished high-affinity binding of naltrexone, whereas delta agonists (e.g., DPDPE, ICI174,864) were concentration dependent and had less competitive binding [26]. Naltrexone and its active metabolite 6β-naltrexol have affinities for the mu and kappa opioid receptors that approach 0.08 nM and 0.5 nM, respectively; the affinity for the delta opioid receptor is approximately 8 nM [26]. Other studies suggested that 3H-naltrexone had no biphasic binding activity in brain tissue [26]. Radiolabeled naltrexone 7

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competitively inhibited binding of mu receptors at 1 µM concentrations or less, but required ≥ 2 µM concentrations to displace delta opioid receptor agonists such as DPDPE or ICI-174,864 [see table in 26]. Competition between cold and radiolabeled naltrexone was measured in bovine hippocampus, and shown to have a one-site binding with a Kd of 2.2 nM in comparison to competition between radiolabeled naltrexone and delta opioid receptor agonists that demonstrated a Kd of 29 nM [27].

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Dichotomous biological responses following different durations of opioid receptor blockade were

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initially reported in 1983 when low and high dosages of naltrexone were administered to nude mice inoculated with neuroblastoma cells [28]. The low dosages of naltrexone inhibited the growth of the tumors, but the high

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dosages resulted in responses that did not correlate with dosage. Rather than inhibiting growth to a greater degree than low dosages in a normal dose-response manner, the high dosages of naltrexone resulted in larger

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tumors and faster rates of death [28]. This observation was completely unexpected and required further study to understand that it was the duration of opioid receptor blockade that was driving the end result, not the

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dosage of antagonist [28].

Studies in normal rodent body and brain development replicated these observations [29, 30]. Not only

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did the dosages of naltrexone differ between mice [28, 29] and rats [30], but both studies demonstrated

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biphasic responses. Low dosages of naltrexone resulted in inhibitory growth, whereas higher dosages of naltrexone resulted in accelerated tumor growth and somatic development. The biochemical pharmacology

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underlying these observations revealed that the duration of opioid receptor blockade, not the dosage, determined the physiological outcome.

2.1.2. Duration of opioid receptor blockade: Pharmacology studies Similar observations were reported by other investigators. Landymore and Wilkinson examined opioid receptor blockade using naloxone injected subcutaneously at 6 hr intervals to neonatal rats on the timing of puberty. These authors suggested that “…the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effects”, [p. 447, 31]. Other investigators utilized radiolabeled carfentanil binding in normal volunteers and tracked naltrexone blockade of mu-opioid receptors with a positron radiation detection system [32]. The half-time blockade of naltrexone ranged between 3 days and 108 hr, a time significantly longer than the reported plasma clearance rate of approximately 8-12 hr. These data justified the selection of a 50 mg dosage currently recommended for reversal of heroin overdose and were based on the fact that 8

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plasma clearance half-time rate is not necessarily reflective of the duration of receptor blockade. Research to synthesize new antagonists is ongoing, but to date, naltrexone remains the choice to block mu opioid receptors. 2.1.3. Duration of opioid receptor occupancy: Biological response A second avenue of research supporting differential effects for the same ligand at different dosages

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came from studies on the up- and down-regulation of a given receptor. At face value, up- and down-regulation

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can mean both increased and decreased numbers of receptors, or it can mean greater (or lesser) sensitivity and/or activity by the same number of receptors. Using in vitro cell culture of primary brain cells, it was

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reported that agonists invoked down-regulation of receptors in brain cells from forebrain but not hindbrain [33]. Reporting on NG-108 cells subjected to naloxone or naltrexone, Coscia and colleagues revealed a

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transient down-regulation in delta receptors, specifically δ2 [34]. Receptor binding number (Bmax), but not binding affinity (Kd) was reduced following treatment with 10 mg/kg naltrexone. Baker and Meert [35] reported

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that the delta-receptor antagonist naltriben produced both potentiation and attenuation of effects of U50, 488Hinduced hypothermia by way of the kappa opioid receptor. Using the outcome of hypothermia, mice were

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injected with agonists alone and in combination with selective antagonists (e.g., naltrindole, naltriben), as well

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as methyl-naltrexone, a compound with peripheral, rather than centrally-mediated activity. These authors concluded that high doses of agonists mediate activity at more than one receptor site, whereas selective

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antagonists are limited to one receptor [35].

Recent studies on the κ-opioid receptors as a mediator of biological responses to pain, stress, anxiety, and depression have researched the role of naloxone, and the duration of action of selective kappa opioid antagonists and JNK1 activation [36]. Naloxone displayed a short duration of activity at the opioid receptor and did not increase phospho-JNK activation.

2.2. Stereospecificity of opioid receptor blockade The stereospecificity of opioid receptor antagonists, and the duration of blockade, were reported in one investigation that showed (-) naloxone administration in dosages as high as 60 mg/kg resulted in decreased body weights, suggesting that even at this dosage, the opioid receptors associated with growth were transiently blocked [37]. Naloxone administered systemically at 100 mg/kg had no effect on growth which is counter to naltrexone which at dosages equal to or greater than 20 mg/kg accelerates growth. Early work 9

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using the murine neuroblastoma model tested opioid antagonist stereospecificity by injection of (+) and (-) isomers of naloxone; only the (-) isomer was effective at blocking the receptor. Thus, the growth related properties of opioid receptor antagonists were stereospecific and dose-related, but not dose-dependent [37]. Moreover, multiple low dosages of naltrexone could be administered throughout the 24 hr period and invoke a continuous blockade, again confirming that the duration of receptor blockade is important.

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3. Duration of opioid receptor blockade determines response

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Observations that the duration of opioid receptor blockade determined the direction and magnitude of response led to the discovery of the OGF-OGFr regulatory pathway (Figure 2). Duration, not drug dosage, determines response

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3.1.

The Zagon laboratory had interests in both developmental biology and cancer (i.e., neuroblastoma),

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and also explored treatments for narcotic addiction [38-40]. Prompted to investigate brain development in rodent models of opiate addiction, it was noted that opiate treatment of pregnant rats was deleterious for

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offspring, as was prenatal exposure to methadone. Concomitantly, the field of addiction research began to focus on general opioid antagonists as treatments. Transitioning these studies to cancer using a model of

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A/Jax mice inoculated with neuroblastoma, animals were injected daily with 0.1 mg/kg naltrexone. The results

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revealed that tumor development was delayed, and only 33% of the mice developed a tumor in contrast to mice receiving saline. It was reasoned that if 0.1 mg/kg naltrexone was effective at retarding tumor growth,

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increased doses of the antagonist should have more efficacy. Surprisingly, the dose response experiments using 1 mg/kg and 10 mg/kg naltrexone had contradictory results. Instead of tumors being “non-existent” as was expected with higher dosages, tumors were larger than those in mice receiving saline [28], and survival time was decreased. These studies were extended to a mouse model of metastatic neuroblastoma and data revealed that low dosages of naltrexone inhibited tumor take (by 69%), delayed tumor appearance (by 70%), and increased median survival, in comparison to controls; higher dosages of naltrexone exacerbated tumor growth and shortened survival [28,20]. In each study, the duration of receptor blockade determined the biological response. With regard to normal somatic and brain development, naloxone administration to newborn rat offspring had little or no effect. However, treatment with either 1 or 50 mg/kg naltrexone on a daily basis to newborn rat offspring revealed biphasic results. The length of receptor blockade was tested in young rats using hot plate 10

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responsiveness following morphine injections. The concept was that morphine would bind avidly to mu opioid receptors and the animal would not respond to the thermal stimulus. Following naltrexone administration that preferentially blocked mu opioid receptors, the rat was subjected to thermal sensation and responded by licking its paws or jumping off the hot plate [40]. This measure of mu receptor blockade was the original premise for the “duration” studies that followed. Dosages that enabled the animal to remain 30 seconds on the

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hot plate after 12 hours were considered to have induced “continuous opioid receptor blockade”, whereas dosages that allowed the rodent to sense heat within 6-8 hours were considered to produce an “intermittent

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opioid receptor blockade”.

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Treatment of neonatal rats during weaning with 50 mg/kg naltrexone resulted in enhanced body, brain, and cerebellar weights relative to controls [30,38]. The cerebellum was larger in every aspect of neurobiology.

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Each layer of cells had a full complement of appropriate cell types (e.g., granule, Purkinje). Morphometric studies revealed up to 70% more glial cells in the cerebella of weanling rats treated postnatally with 50 mg/kg

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naltrexone relative to saline-injected controls. Neurons that are derived prenatally, and thus not subjected to naltrexone, were not altered in number.

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A thorough exploration of body and organ weights, appearance of physical characteristics, and brain

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development followed [39]. A full dose-response curve of 0.1, 1, 10, 20, 50 or 100 mg/kg naltrexone administered throughout the 3-week weaning period was conducted with end points of body weights and brain

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development. Duration of opioid receptor blockade was measured as well. Dosages of 0.1, 1, and 10 mg/kg naltrexone, that blocked the opioid receptors from morphine analgesia, for less than 12 hr/day (4-8 hr for the 0.1 and 1 mg/kg dosages), resulted in decreased growth. Dosages of 30 – 100 mg/kg increased body and brain weights. It was later determined that no additional enhancement of brain growth occurred when the dosage of 50 mg/kg was increased to 100 mg/kg [38]. To demonstrate that it was the “duration” and not the “dosage” that conferred the outcome, 3 dosages of 3 mg/kg which blocked the receptor for an entire 24 hour period resulted in enhanced body and brain weights relative to controls. The cumulative dosage of 9 mg/kg given once daily diminished growth [36,38]. 3.2. Duration of opioid receptor blockade: normal somatic development Multiple investigations were pursued to determine the extent of somatic growth regulated by blockade of opioid receptors [39]. The effects of naltrexone on organ growth were assessed in rats injected with either a 11

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short-duration blockade (1 mg/kg naltrexone) or complete-blockade (50 mg/kg naltrexone) [39]. Naltrexone altered the wet and dry weights of 10 organs in a dosage and sex dependent manner [39]. Concomitant experiments pursuing the underlying regulatory pathways for these observations led to the discovery and characterization of a new opioid receptor, termed zeta and later renamed OGFr (see review in section 1.1.2)

3.3. Duration of opioid receptor blockade: behavioral development

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[11,13,20].

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The duration of opioid receptor blockade by naltrexone also conferred changes in behavior. Animals exposed to continuous receptor blockade beginning at the time of birth demonstrated early acquisition of

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physical characteristics such as eye opening and hair growth, along with precocious timing in spontaneous motor and reflexive behaviors such as ability to roll over, crawling, bar hanging, and walking in comparison to

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control rats [40]. Ambulation measured by distance traveled, emotionality (fecal pellet deposition), and nociception were not altered by naltrexone.

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3.4. Duration of opioid receptor blockade during gestation: postnatal effects Other investigations on opioid antagonist exposure during pregnancy revealed a variety of effects on

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postnatal behavior [41,42]. Some studies did not completely disrupt peptide-receptor interaction continuously

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from fertilization through pregnancy, and many did not examine whether the dosage of opioid antagonist was sufficient to block opioid receptors completely throughout the day resulting in partial blockade and thus

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enhanced endogenous peptide-receptor interaction [41]. Low dosages of naloxone (1 mg/kg) during pregnancy resulted in cross-fostered offspring with altered behavior [42]. Prenatal naloxone exposure produced changes in body weight development, pain sensitivity, and motor behavior in the offspring. Rats treated prenatally with 1 mg/kg naloxone habituated more rapidly in the open field, and showed less activity as they matured. Bar pressing rates were reduced in male rats exposed to 10 mg/kg naloxone leading the authors to hypothesize that low dosages of naloxone with short receptor blockade may increase opiate [endogenous opioid] function in offspring [42]. Interpretation of maternal treatment and postnatal effects is always difficult, but paramount to the reliability of the studies is the question whether naltrexone passes through the placenta? Reverse phase highperformance liquid chromatography with ultraviolet detection measured a single 50 mg/kg dose of naltrexone administered intraperitoneally to a pregnant rat on day 20 of gestation in brain, heart and liver of fetuses 1 hr 12

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after injection. Thus, maternally administered naltrexone passes through the placenta [43] and could be detected in neonatal pups [44], but not in pups after 2 days of age. 3.5. Duration of opioid receptor blockade: brain development Opioids and their receptors were first discovered in neural tissue, and the brain remains an active area of research on endogenous opioids such as endorphins and enkephalins. Naltrexone‟s effects on the brain

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were studied in a series of investigations of cerebellum, cerebral cortex, and hippocampus [45-47]. Histological

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and morphometric studies of the cerebellum of rats exposed daily to either a continuous blockade by naltrexone (50 mg/kg) or an intermittent blockade (1 mg/kg) revealed that high dosages of naltrexone

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stimulated cerebellar development, whereas the short-receptor blockade inhibited growth [45]. The temporal course of development was consistent with normal development for both dosages; however, the magnitude of

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enhancement seen in morphometric analyses (i.e., cerebellar areal measurements, number of cerebellar internal granule neurons) was greater than the level of reductions.

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Intermittent receptor blockade by 1 mg/kg naltrexone altered cerebral cortex development at both cellular and tissue differentiation levels [46]. Brain tissue from rats injected with 1 mg/kg naltrexone displayed

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a substantial increase in packing density of neural cells, possibly reflecting reduced dendritic arborization and

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synapse connectivity. Hippocampal development was impacted more by the intermittent receptor blockade than by continuous blockade, suggesting that the boundaries of accelerated growth may restrict uncontrolled

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cellular proliferation.

Changes in cerebral development were extended into the ultramicroscopic area of spine formation and dendritic arborization [47]. Meticulous studies revealed that the duration of opioid receptor blockade early in postnatal life conferred long-term effects on brain maturation. Examination of Purkinje cells from the cerebellum and pyramidal cells from the cerebral cortex from rats injected daily with 50 mg/kg naltrexone for only 10 days revealed that both cell types had substantial increases in dendrite and/or spine elaboration compared to controls. In rats receiving intermittent receptor blockade with 1 mg/kg naltrexone, dendritic development was subnormal, with fewer spiny processes and dendritic arborization evident at 21 days of age. Whether these changes in brain development can be translated into learning or behavioral deficits is unknown. 3.6. Duration of opioid receptor blockade: heart development

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Whereas it may be expected that endogenous opioids would modulate growth of neural tissues, the influence of endogenous peptides and receptors on cardiovascular tissue was novel. The heart and vasculature develop prenatally, function during gestation, and essentially undergo hypertrophy throughout development. The presence of endogenous opioid activity in non-neural systems was investigated by examination of heart development [48]. A single injection of 50 mg/kg naltrexone to 1 day old rat pups

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increased DNA synthesis indexes of ventricular and atrial myocardial and epicardial cells. Chronic exposure to

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50 mg/kg naltrexone resulted in increased heart weights and areal measurements of ventricles and atria; the naltrexone effects were not mediated through the sympathetic nervous system or by way of increased thyroid

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hormone production.

A complete opioid receptor blockade during gestation significantly altered the cardiovascular system of

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infant rats [49]. Prenatal naltrexone treatment (50 mg/kg to pregnant rats) resulted in offspring with hearts that weighed more, and had more DNA and protein content in both the ventricles and atria, relative to cardiac

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tissue from saline-treated controls. Morphometric analyses revealed that the myocardium and chamber

term ramifications on cardiac biology.

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volumes were increased, thus suggesting that early exposure to high dosages of naltrexone may have long-

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4. Duration of opioid receptor blockade determines therapeutic outcome Our understanding of the biochemistry and pharmacology of opioid receptor blockade has prompted research

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into the usages of these widely available, non-toxic, drugs for treatment of drug addiction and alcoholism. However, the pharmacological properties of naloxone and naltrexone, and specifically, the ability to modulate growth by altering the duration of opioid receptor blockade, has made these general antagonists very useful therapies for cancer, autoimmune diseases, and complications associated with diabetes. The opposing effects reported following continuous or intermittent opioid receptor blockade are related to the underlying regulatory mechanisms of the OGF-OGFr pathway. Concomitant with opioid receptor blockade is a compensatory upregulation of endogenous opioids and receptors that can interact when the antagonist is no longer present. Thus, receptors are available for heightened interaction following intermittent antagonist blockade, whereas continuous blockade suppresses receptor availability. These mechanisms were delineated in a tissue culture model of human ovarian cancer cells [50]. The paradoxical effects of low and high dosages of naltrexone were demonstrated in vitro using the same drug dosage but different exposure times. Thus, short-term and long14

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term exposure to naltrexone resulted in reduced or accelerated cell growth, respectively. Receptor number and enkephalin levels were measured periodically by western blotting and radioimmunoassay, respectively, confirming the autocrine loop of endogenous peptide and receptor interaction during the period of time that opioid receptors are not longer blocked. With regard to OGFr, continuous opioid receptor blockade with high dosages of naltrexone, or continuous infusion of lower dosages of naltrexone, or multiple injections of

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naloxone, establishes constant „prevention‟ of inhibitory action by endogenous opioids (i.e., OGF). Hence,

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continuous blockade is applicable for treatment of conditions requiring rapid or enhanced cell proliferation such as corneal surface defects [e.g., 51], dry eye [e.g., 55], and closure of cutaneous wounds [57-60]. On the

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contrary, high levels of the inhibitory peptide OGF following intermittent blockade by low dosages of naltrexone (LDN) are biotherapeutic for autoimmune diseases and cancer [e.g., 20, 50] (Figure 2).

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4.1. Continuous OGFr blockade: Enhanced cell proliferation

Type 1 and type 2 diabetes are associated with many complications resulting from delayed

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epithelialization (i.e., keratopathy), lack of cellular function (ocular surface sensitivity, dry eye), and impaired healing (delayed repair of full-thickness cutaneous wounds). Naltrexone, systemically or topically administered,

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at high dosages can reverse and restore these defects [e.g., 55, 57-64].

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OGFr, and not mu, delta, or kappa opioid receptors, specifically mediates the effects of naltrexone in wound closure [65]. Specific antagonists for each classical receptor were added to NIH 3T3 cells, and cell

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number measured over several days. CTOP, naltrindole, and nalmefene, selective for mu, delta, and kappa opioid receptors, respectively, did not accelerate cell replication, whereas naltrexone enhanced growth. Moreover, addition of agonists with high affinity to the classical receptors (i.e., DAMGO, DPDPE, ethylketocyclazocine) did not alter growth, suggesting that these receptors were not pivotal in mediating cell replication, and that continuous blockade of the classical receptors were not involved. Further studies knocking down RNA for each classical receptor using siRNA transfection did not eliminate the increased fibroblast cell number reported following naltrexone. However, addition of OGFr siRNA, nullified the accelerated growth in the presence of naltrexone. In the animal model, the selective blockade of the OGFOGFr pathway by naltrexone was confirmed as only naltrexone, and not selective opioid receptor antagonists, enhanced wound closure [65].

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Topical naltrexone also has been shown to be effective at restoring dry eye, repairing corneal surface wounds, and enhancing closure of full-thickness skin wounds in normal and diabetic animals [e.g., 57-60, 63]. Because naltrexone diffuses passively into cells, it is amenable to topical application in a variety of carriers [57], and because epithelial tissues lack vascularity, naltrexone is metabolized slowly. Thus, topical administration can invoke sufficient OGFr blockade to allow for localized, accelerated DNA synthesis of cells

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and tissues, and remain safe and non-apoptotic [e.g., 55, 61].

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4.1.1. Continuous OGFr blockade: Ocular surface wounds and dry eye

A comprehensive series of investigations have been conducted on the role of the OGF-OGFr axis to

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repair ocular surface abnormalities [51-56, 61-64]. Corneal surface integrity maintains the required barrier to enable vision. Loss of sensitivity, dryness, or damage, leads to significant medical discomfort and even

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blindness. The cornea has a well-defined epithelium supported by stroma and peripherally-located renewing limbal cells [50]. Continuous blockade of OGFr and re-epithelialization of the cornea was first studied in rat

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using systemic injections of 50 mg/kg naltrexone [51]. Twice daily injections were required to maintain continuous receptor blockade in the relatively un-vascularized ocular surface [61]. However, within 8 hr of

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inducing a 4 mm diameter surface abrasion, systemic naltrexone increased DNA synthesis and cell replication.

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Topical administration of naltrexone was also effective at diminishing wound size 2.8 fold greater than in controls. This was the first indication that naltrexone retained its properties of receptor blockade when

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administered topically, suggesting that the passive diffusion of naltrexone did not breakdown any of the biochemical/pharmacology characteristics required to prevent endogenous peptide interaction at a nuclearlocated receptor [51,61]. Human donor corneas placed in culture, abraded, and subjected to 10-6 M naltrexone, healed faster than wounded corneas in media alone. The rate of closure, as well as the DNA labeling index was accelerated, in corneas placed in naltrexone [52]. Topical, rather than systemic, naltrexone became an obvious choice for treatment of ocular disorders in diabetic animals that experience delayed epithelialization. Continuous blockade using 10-5 M naltrexone rapidly restored the abraded cornea of chemically – induced, type 1 diabetic rats. Complete blockade of the OGFr by naltrexone also enhanced corneal surface wound repair in alloxan-induced type 1 diabetic rabbits [64], and genetically hyperglycemic type 2 diabetic mice [63]. A concern of whether chronic exposure to naltrexone was detrimental leading to scarring or exuberant granulation tissue in the corneal stroma was 16

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addressed [54,63], and the safety of topical naltrexone was demonstrated in several animal models [51, 52, 64]. It appears that naltrexone accelerates cellular proliferation, but other intrinsic factors provide the “brakes” for enhanced replication following continuous opioid receptor blockade. High dosages of naltrexone are also effective treatment of abnormalities related to corneal sensitivity and tear production in normal [56] and diabetic [55, 64] rodents. Normal rodents experience episodic dry eye;

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Schirmer test scores occur in a biomodal distribution of dryness {~6 mm or less) or wetness (≥ 7 mm). Topical

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naltrexone (10-5 M) dissolved in eyedrops restored dry eye within 1 hour of treatment, and had no effect on rats with normal tear production [56]. Type 1 diabetes is associated with prolonged periods of dry eye and corneal

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surface insensitivity. Topical application of high dosages of naltrexone restored sensitivity and tear production in both type 1 and type 2 diabetic animals [55,63] The reversal of dry eye following a single application lasted 2

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to 3 days, and the restitution of sensitivity lasted 4 to 7 days [55].

4.1.2. Continuous OGFr blockade: Full-thickness cutaneous wounds

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The efficacy of naltrexone for ocular-related complications of diabetes was novel, and studies were extended to another serious medical concern for diabetics – delayed wound repair [57-59]. An animal model of

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type 1 diabetes was established by injections of streptozotocin, and full thickness cutaneous wounds were

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created on the dorsum. Topical treatment of naltrexone in a variety of dosages as well as several carriers including DMSO, buffer, and creams, revealed that naltrexone (10-5 M) in moisturizing cream was effective at

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enhancing wound closure with both cell replication and contracture [57]. Further studies showed that continuous opioid receptor blockade stimulated angiogenesis as measured by FGF-2, VEGF, and α-SMA expression in capillaries [58], accelerated skin remodeling [59], and reinforced the integrity of the skin as monitored by tensile strength measurements [60]. Thus, naltrexone-enhanced wound repair resulted in skin that was strong and intact [60].

4.2. Intermittent OGFr blockade: Treatment of autoimmune disorders Autoimmune disorders such as fibromyalgia, rheumatoid arthritis, Crohn‟s, and multiple sclerosis are difficult to diagnose and are often detected only after multiple clinical visits. Many patients present with decreased enkephalins and endorphins, exacerbating their inflammation, pain, and other immune-mediated metabolic deficits. The concept that low dosages of naltrexone (LDN) have positive effects in individuals with autoimmune diseases preceded most basic science reports. Websites such as LDNNow provide an excellent 17

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summary of the history, uses, and potential for LDN [66,67]. As early as the 1980s, it was noted that LDN inhibited cancer growth [19,20]. Only recently has the potential biofeedback leading to elevated endogenous opioids been evaluated in light of biotherapies for autoimmune diseases. The mechanistic pathways of LDN are unclear. Some investigations suggest that LDN works directly as an immunomodulating agent [68,69], whereas other studies on T and B cell proliferation in vitro [70, 71] and in vivo [72] have demonstrated that the

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activation of peripheral lymphocytes and resultant cytokine production.

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modulation of the immune system most likely is a direct response to increased or decreased proliferation and

4.2.1. Multiple sclerosis

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As mentioned, the clinical use of LDN for treatment of autoimmune disorders outpaced rigorous scientific research. Even today, many internet-originated rumors exist that warrant clarification. For example,

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“more is not better”. The fallacy in this statement is obvious; LDN patients should not be encouraged to increase their dosage or take more than one tablet daily. At present, the timing of administration of LDN is a

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patient preference, and there are no basic science studies that conclude morning or evening consumption is either harmful or better.

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Recently, animal studies on intermittent or continuous blockade of OGFr using naltrexone for treatment

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of experimental autoimmune encephalomyelitis (EAE) were initiated. EAE can be induced in a variety of ways to establish a more progressive form using myelin-oligodendrocytic glycoprotein (MOG)35-55 as the antigen, or

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relapse-remitting form using proteolipid protein (PLP)139-151 [73-79]. In a series of investigations with progressive EAE, high dosages of naltrexone initiated at the time of disease induction had no effect on the behavioral sequelae associated with EAE. LDN however actually prevented 33% of the mice from developing clinical signs of the disease, and the severity and disease index of LDN-treated mice was reduced from that in animals receiving saline. Neuropathology corroborated the behavioral studies showing that LDN treated mice had fewer activated astrocytes, less demyelination and neuronal damage than mice receiving saline or high dosages of naltrexone [73]. Treatment of mice with chronic EAE using OGF appears to be more effective than LDN therapy [73,74]. Using animal models with treatment beginning after established disease, OGF arrested the progression of chronic, MOG-induced EAE and reduced the associated spinal cord pathology [75]. In theory, the behavioral signs appeared several days after the initial immune response, and yet OGF was capable of controlling 18

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autoimmune flairs. Indepth pathology studies indicated that astrocyte proliferation and activation were controlled by OGF. Astrocyte activation in disease states is an important corollary to disease progression and recovery. OGF therapy inhibited the proliferation of astrocytes in vivo, and in vitro [76], and did so by way of OGFr; siRNA technology and diminished protein expression of mu, delta, and kappa receptors in vitro demonstrated the specificity and requirement for OGFr in primary cultures of mouse cerebral astrocytes.

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Relapse-remitting multiple sclerosis is the most common form of multiple sclerosis; the mouse model of relapse-remitting EAE is established by immunization of SJL mice with PLP [77-79]. OGF [78] and LDN [79]

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treatment initiated after clinical disease was observed – establishing a very clinically relevant model – resulted

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in reduced behavioral abnormalities and neuropathological defects. Animals that responded to the treatments were often without sign of relapses, and had sustained remissions over a 40-day period of observation.

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Translation of these observations to the clinic is warranted. Small trials and case reports have documented that LDN usage by MS patients is safe, without side effect, and reduces associated fatigue [80]. Clinical trials have

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concurred that LDN is safe and probably effective – all recommending that larger, randomized, double-blind trials be established [81].

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4.2.2. Inflammatory bowel disease and fibromyalgia

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Other autoimmune diseases that respond to partial opioid receptor blockade include the family of inflammatory bowel disorders (Crohn‟s and colitis) [82-85]. Although few basic science reports have been

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published, patient studies are provocative. In a murine model of inflammatory bowel disease, low dosages of naltrexone were shown to reduce the weight loss and lessen the disease activity, normally associated with progression of the disorder [85]. The short term opioid receptor blockade reversed the physical symptoms, pathology, and molecular markers of inflammation associated with colitis. Fibromyalgia is another chronic, debilitating disorder that incurs sufficient pain, suggesting that opioids and their receptors may be dysregulated. A definitive cause for this disorder that afflicts up to 5% of all women is unknown. Because endorphins are involved in mediating mood, and to some extent pain mediation, studies have explored the role of low dose naltrexone as a biotherapy for this disorder. Studies by Younger and Mackey [86] reported that low dose naltrexone reduced symptoms of fibromyalgia. Other investigators recognized that low dosages of naltrexone would increase endogenous peptides and suppress pain on a

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continual, yet safe, regimen [87]; both groups encouraged larger clinical trials on the use of LDN as a biotherapy for fibromyalgia. 4.3. Intermittent OGFr blockade: treatment of cancer The role for endogenous opioids in cancer therapy commenced more than 30 years ago with our early studies on opioid receptor blockade and proliferation of murine neuroblastoma [28,29]. White mice

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transplanted with S20Y murine neuroblastoma cells were subjected to intermittent receptor blockade following injections of 0.1 mg/kg naltrexone, or complete blockade following 10 mg/kg naltrexone. Plasma levels of β-

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endorphin were not altered by either opioid receptor blockade, whereas tumor tissue levels of both [Met5-

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enkephalin] and β-endorphin were significantly upregulated [88]. Receptor binding sites measured by radioactive-DADLE were significantly upregulated following both intermittent and continuous receptor

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blockade, although putative κ opioid receptors, measured by tritiated ethylketocyclazocine were downregulated. Labeling indexes (thymidine incorporation) and mitotic indexes (microscopic analyses of

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metaphase, anaphase, and telophase in hematoxylin-stained tumor sections) were elevated during the period of opioid receptor blockade, but decreased during the time subsequent to blockade (i.e., post 8 hr for 0.1

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mg/kg naltrexone). Treatment with endogenous opioids, specifically OGF, inhibited proliferation in a receptor-

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mediated, dose-related (not dose-dependent) manner [88]. Tissue culture studies on the regulation of cancer cell proliferation by continuous opioid receptor blockade an all or none response, not a dose-dependent curve [89].

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The observation that the duration of opioid receptor blockade mediated the biotherapeutic recourse for cancer [28, 29, 50] stimulated the discovery of the OGF-OGFr regulatory pathway. Although not clinically relevant, studies on continuous receptor blockade in cancer models has provided important insights into the role of the OGF-OGFr axis in cancer biotherapy. Intermittent blockade with LDN, or OGF, are logical biotherapeutic approaches, and each treatment has advantages. LDN is available as an oral tablet, and can be pharmaceutically prepared with a physician‟s prescription. For LDN to be effective however, it requires that the consumer of LDN have an intact OGF-OGFr axis that is fully functioning and able to produce and secrete OGF in sufficient quantity to bind to OGFr located on the targeted tissue. The indirect biofeedback loop presumably requires more time for activation, and is also more susceptible to dysregulation than direct OGF. On the other hand, OGF is a small peptide that acts directly on the cell cycle [90] to inhibit DNA synthesis and 20

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replication. However, OGF is easily degraded (enkephalinases are ubiquitous), not FDA-approved, and currently must be injected either intravenously or subcutaneously. LDN is usually taken once daily, or even every 2nd or 3rd day, whereas OGF has been infused weekly or monthly in the few clinical trials that have been published [91]. Numerous studies on the efficacy of OGF and LDN have been conducted on human cancer cells grown in vitro or transplanted into nude mice [50, 92]. To date, no human cancer cell has been shown to

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be refractive to biotherapy with either OGF or LDN; OGFr is also required to mediate endogenous or

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exogenous inhibition [19]. The mediation of human ovarian cancer [50,92] and human triple negative breast cancer [19] by modulation of the OGF-OGFr axis provides a reasonable portrayal of OGF efficacy. The

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duration of receptor blockade determined the cell proliferative response in human ovarian, pancreatic, colorectal, and squamous cell carcinoma of the head and neck neoplasias. Naltrexone blockade was shown to

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upregulate both peptide and OGFr at the translational but not at the transcriptional level. Naltrexone did not alter cell survival (apoptosis or necrosis), and short-term blockade required p16 and/or p21 cyclin-dependent

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inhibitory kinases to be functional. In culture, sequential administration of short-term naltrexone followed by

support further clinical research.

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OGF was more effective at inhibiting cancer growth than either biotherapeutic regimen alone. These studies

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4.4. Intermittent opioid receptor blockade: Addictive behavior The pathways associated with naltrexone‟s blockade of alcoholism, self-injurious behavior and

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compulsive disorders are not fully known [18]. Recent publications have reported that with regard to alcoholism, NTX therapy at 50 mg once daily was effective at doubling abstention rates 51% vs 23% placebo, reducing (cut in half) the number of drinking days, craving, and relapses. In a second trial of 865 patients, NTX was shown to have no serious side effects [93,94]. Mannelli et al. reported that very low dosages of naltrexone, 0.1 to 0.2 mg/kg daily, resulted in reduced withdrawal and a lower rate of treatment noncompliance in heavy drinkers who were in opioid detoxification programs [95]. In addition to the obvious function as a drug for reversal of drug overdose, naloxone and naltrexone have slowly found their way into a long list of medical uses. In addition to addiction, naltrexone has been recommended to prevent alcohol dependence [94], and even tobacco dependence [96]. All 3 of these events utilize a single 50 mg naltrexone treatment daily. A few studies have shown that long-acting naltrexone can ameliorate self-injurious behavior in autism and other compulsive disorders [97,98]. Thus, low dosages of naltrexone may confer a partial blockade to receptors 21

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craving illicit drugs, attempting to quit smoking, or cease self-injurious behaviors, and even gambling [93-98]. The role of OGFr in mediating the low dose naltrexone is currently under investigation. 5. Conclusions and future perspectives The duration of opioid receptor blockade by antagonists, such as naltrexone, has impacted more biological pathways than addiction pathways, which largely remain undefined. With the widespread use of low

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dosages of naltrexone, the impact of this opioid antagonist biotherapy is broad-based. Approximately 52

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million individuals in the US may benefit from either low dosages of naltrexone or complete receptor blockade for treatment of cancer, multiple sclerosis, inflammatory bowel disorders, or complications associated with

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diabetes. Given that nearly 86 million individuals are pre-diabetic, the need to develop new biotherapies that are non-toxic, and target the underlying pathophysiology of these diseases is an urgent, unmet medical need.

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Worldwide, the potential audience that could benefit from biotherapeutics related to modulation of the OGFOGFr axis approaches more than 350 million. It is anticipated that drug discovery researchers will identify

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specific and selective receptor antagonists that confer no biological activity and can be used as safe,

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Acknowledgements

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inexpensive treatment of these disorders.

The authors express their gratitude to Nancy Kren who designed the graphic models presented in Figures 1

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and 2. This research was supported by grants from NIH, American Diabetes Association, and The Shockey Family Foundation.

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preliminary study and an examination of sex differences. Nicotine Tobacco Res 8:671-682, 2006. 97. Smith SG, Gupta KK, Smith SH. Effects of naltrexone on self-injury, stereotypy, and social behavior of adults with developmental disabilities. J Dev Physical Disabilities 7:137-146, 1995. 98. Suck WK, Grant JE, Adson DE, Shin YC. Double-bline naltrexone and placebo comparison study in the treatment of pathological gambling. Biological Psychiatry 49:914-921, 2001.

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Figure Legends Fig 1. Nuclear interactions of naltrexone with OGFr in the cytoplasm as well as within the nucleus. Naltrexone binds to OGFr, trafficks into the nucleus and blocks OGF interactions that upregulate p21 and p16 cyclindependent inhibitory kinases. Impα = importin α; impβ – importin β; NTX = naltrexone. Fig 2. Duration of OGFr blockade at the cellular level showing distinction between cytoplasmic receptors and

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nuclear-associated receptors. Impα = importin α; impβ – importin β; NTX = naltrexone.

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*Graphical Abstract (for review)

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OGF – OGFr Axis – – – –

OGFr OGFr OGFr OGFr

Cell proliferation, Treatment of wound healing

LDN

OGF OGF OGF OGF

– – – –

OGFr OGFr OGFr OGFr

Cell proliferation; treatment of autoimmune disorders, cancer

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HDN

OGF OGF OGF OGF

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