WO2011139525A1 - Stereoisomer of naloxone and potential therapeutic action of opioid drugs to reverse clinical tolerance to these agents - Google Patents

Abstract

The present invention relates generally to methods of using (+) naloxone for managing feeding disorders and acute debilitating disorders such as pain, Also encompassed by the invention is the use of (+) naloxone to manage any disease, disorder or condition associated with mu opioid receptors (MOPR).

Description

TITLE OF THE INVENTION

Stereoisomer of Naloxone and Potential Therapeutic Action of Opioid Drugs to

Reverse Clinical Tolerance to These Agents BACKGROUND OF THE INVENTION

Opioids are a family of peptides that differentially interact with mainly three G-protein coupled receptor classes (mu, delta and kappa) (Glass, et al., 1999, Neuropeptides 33(5):360-8). All three endogenous opioid families are heavily invested in systems that regulate different physiological responses including nociception, mood, thermoregulation, stress and neuroendocrine function (Akil, et al., 1984, Annu Rev Neurosci. 7:223-55; Daws et al., 1999, Addition Biology 4:391 -397). Moreover, initial data demonstrated that mu opioid receptors (MOPR) in the brain mediate physiological processes that increase feeding (Bakshi et al., 1993, J

Pharmacol Exp Ther 265: 1253-1260; Giraudo, et al., 1998, Brain Res 782(1 -2): 1 8- 23; Mann et al., 1988, Neuropharmacology 27(4):349-55; Ragnauth et al., 2000,

Brain Res 876( i -2)76-87; Zhang et al., 2000, Neuroscience 99(2):267-77; Kotz et al., 1997, Am J Physiol 272(4 Pt 2):R 1028-32; Glass, et al., 1 99, Neuropeptides 33(5):360-8; Bodnar, 2004, Peptides 25:697-725; Kelley et al., 2005, J Comp Neurol 493( l ):723-85 ; Levine et al„ 2006, Physiol Behav 89( l):92-6). For example, the parabrachial nucleus (PBN), enriched with MOPRs, is a region known to be involved in the regulation of feeding among other autonomic functions (Carr et al,, 1996, Neurochem Res 21 (1 1): 1455-67). The PBN receives afferent input from gustatory and visceral-sensory processes, mainly through the nucleus of the solitary tract (NTS). At the same time, the PBN has bidirectional communication with other brain regions related to homeostatic and non-homeostatic feeding (such as the hypothalamus or the nucleus accumbens), Carr et al. (Carr et al., 1991, Brain Res 545(l -2):283-286) reported that infusing the relatively nonselective MOPR antagonist naloxone into the lateral subregion of the PBN (LPBN) increased the threshold for electrical stimulation of the lateral hypothalamus to elicit feeding in rats. These results implicated the involvement of the parabrachial MOPRs in the regulation of feeding behavior.

Previous studies showed that acute infusion of the selective MOPR agonist DAMGO into the LPBN increased food consumption, regardless of the hedonic properties of the diet. This action was blocked by the nonselective MOPR antagonist (-)-naloxone [(-)-NLX], as welt as by the selective, reversible MOPR antagonist CTAP and by the selective, irreversible MOPR antagonist β- funaltrexamine (β-FNA; Wilson et al,_; 2003, Am J Physiol Regul Tntegr Comp Physio! 285(5):R 1055-65; Ward et al._s 2006, Psychopharmacology (Berl) 187:435- 446). These studies also showed that infusion of β-FNA into the LPBN prevented the action of DAMGO to stimulate G-protein coupling, ex vivo (Ward et a!., 2006, Psychopharmacology (Berl) 1 87:435-446).

For at least 30 years, the field of opioid pharmacology has studied drugs that differentially characterize opioid receptor stereospecificity and function. In particular, naloxone (the first opioid antagonist identified), has been used as a "universal" compound to identify opioid actions (Zimmerman et al., 1990, J Med Chem 33(3):895-902). Although naloxone is a nonselective opioid antagonist, its highest affinity is for MOPRs (preference of about 10-fold for μ over and greater preference for μ over δ) (Lord et al., 1977, Nature 267(56 1 1 ):495-9; Goldstein et al., 1989, Mol Pharmacol 36(2):265-72). Two different naloxone stereoisomers exist: (-)- naloxone [(-)-NLX], which is the enantiomer that blocks opioid receptor activation; and (+)-naloxone [(+)-NLX)], shown to be inactive as a narcotic antagonist and to present 10,000-fold less affinity for opioid receptors than (-)-NLX (iijima et ai., 1978, J Med Chem 21(4):398-400; Aceto et ai., 1979, DA Res Monogr. 27:330-350). As opioid receptors display a very strong stereospecificity in their interactions with ligands (Goldstein et ai., 1989, Mol Pharmacol 36(2):265-72), many studies have traditionally used (+)-NLX as a useful control for the detection of opioid receptor mediated effects by (-)-NLX (Rice, KC In The Chemistry and Biology of Isoquinoiine of alkaloids; Phill ipson JD, Roberts MF., Zenk MH, Eds.; Springer-Verlag, New York 1985, pp. 191 -203; Pert et l, 1 84, FEBS Lett 177(2)281 -6). Recent findings however, have shown that the classically described nonselective opioid receptor antagonist (-)-NLX, in cotreatment with mu opioid receptor agonists, may exert actions other than opioid antagonism alone. For example, clinical reports have observed unexpectedly that administration of low doses of (-)-NLX enhances, rather than attenuates, the analgesic effects of morphine or other mu opioid receptor agonists (Buschsbaum et al., 1977,_, Nature 270(5638):620-622; Levine et al., 1979, Nature 278(5706):740- l ; Levine et al., 1986, Pain 27( 1 ):45-9; Levine et al., 1988, J Clin Invest 82(5): 1 574-7; Gillman et al., 1981 , J R Soc Med 74(12)943-4; Gillman et al„ 1985, Neurol Res 7(3): 106- 19; Gillman et al., 1989, Int J Neurosci 48(3-4):321 -4; Schmidt et al., 1985, Anaesthesia 40(6)583-6; Taiwo et al„ 1989, J Pharmacol Exp Ther 249( 1);97-100; La Vincente et al, 2008, Clin Pharmacol Ther 83(1): 144-52; Hamann et al., 2008, J Opioid Manag 4(4):251-4; Cheung et al., 2007, Intensive Care Med 33(1): 190- 194; Tsai et al., 2009, Neiiroscience 164(2):435-43). These enhancing properties have been attributed to blockade by naloxone of a transient activation of the MOPR's excitatory signaling in place of the usual inhibitory (Gi) signaling (Shen et al., 1989, Brain Res 491 :227-242; Crain et al., 1990, Trends Pharmacol Sci 1 1 :77- 81 ; Crain et al, 2001 , Brain Res 888:75-82). Excitatory signaling of opioid receptors was observed initially in an electrophysiological study using dorsal root ganglion cultures (Shen et al., 1989, Brain Res 491 :227-242) that paralleled facilitation rather than an inhibition of neuronal function in preparations of enteric ganglia (Gintzler, et al, 1987, Proc Natl Acad Sci USA 84(8):2537-9). One proposal to explain the signaling mechanism for these phenomena suggested that the opioid-induced excitatory signaling was mediated by an initial and transient switch in G-protein coupling from Gi/o to Gs proteins, which led to the stimulation of adenylate cyclase and increase of cAMP production (Crain et al, 1990, Trends Pharmacol Sci 1 1 :77-81 ; Crain et al, 2000, Pain 84: 121-131 ; Crain et al, 1990, Trends Pharmacol Sci 1 1 :77- 81 ; Wang et al., 2005, Neiiroscience 135(1 ):247-61 ; Chakrabarti, et al, 2005, Brain Res 135 ( 1 -2) :217-224) . Supporting this idea, Wang and Burns (Wang et al, 2009, PLoS One 4( l ):e4282) recently showed in organotypic striatal slice cultures that acute stimulation of MOPRs by morphine causes an initial but transient MOPR-Gs coupling, which subsequently switched to Go coupling after 15 to 20min. They further demonstrated that co-treatment of the tissue with an ultra-low dose of (-)-NLX or with the inactive form (+)-NLX prevented Gs coupling, and thus only the MOPR- Go coupling was observed.

Although many reports have shown that the transitional excitatory stimulation of MOPRs can be blocked by ultra-low doses of (-)-NLX or (+)-NLX, in vitro (Crain et al, 1995, Proc Natl Acad Sci USA 92: 10540- 10544; Crain et al, 2000, Pain 84: 121 - 13 1 ; Wang et al, 2008 PLoS One 3(2):e l 554), it is still unclear if specific concentrations of either of these naloxone enantiomers can actually enhance functional effects of MOPR-induced activation in tissue from a specific brain site and link that enhancement to actions in vivo. The present invention addresses this unmet need in the art. SUMMARY OF THE INVENTION

The invention provides a method of treating pain in a mammal. In one embodiment, the method comprises administering to a mammal in need thereof a therapeutically effective amount of (+)-naloxone.

In one embodiment, the therapeutically effective amount of (+)- naloxone exhibits a positive therapeutic benefit in the absence of an antagonistic property.

The invention also provides a method of treating an eating disorder in a mammal. In one embodiment, the method comprises administering to a mammal in need thereof a therapeutically effective amount of (+)-naIoxone. Preferably, the therapeutically effective amount of (+)-naloxone exhibits a positive therapeutic benefit in the absence of an antagonistic property.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, It should be understood, however, that the invention is not limited to the precise arrangements and

instrumentalities of the embodiments shown in the drawings.

Figure 1 is a schematic of the PBN as an integrator)' relay center. The parabrachial nucleus (PBN) receives afferent inputs from gustatory (nerves VII, VIII and VLX) and visceral-sensory processes (vagus nerve), mainly through the nucleus of the solitary tract (NTS). At the same time, the PBN possesses bidirectional comnninication with other brain regions related with homeostatic and nonhomeostatic feeding, such as the hypothalamus (HYPO), nucleus acc mbens (NAC) and the amygdala (AMY). Through the NAC the PBN also intercommunicates with the ventral tegmental area (VTA) (Norgren et al., 1971 , Science 173(2): 1 136-9; Norgren et al., 1973, J Comp Neurol 150(2):217-37; Saper et al., 1976, Brain Res 1 17(2):305- 12).

Figure 2 is a schematic depicting location and anatomy of the parabrachial nucleus. Location of PBN in dorsal pons (left panel) and subregions sampled for autoradiography atid immunohistochemistry (middle upper panel). The drawing shown in left panel is at the coronal level, 9.8 mm caudal to bregma according to the Paxinos and Watson atlas ( 1 98). The upper middle panel shows the inferior aspect of the lateral inferior PBN (LPBNi), which includes two centra! (C I , C2) and one external subregion of this nucleus (LE). The medial PBN (MPBN) includes a central (MC) and external subregion (ME). Boxes represent the anatomical subregions of the PBN. These subregions can be used for the purpose of creating uniform regions of interest for quantification of autoradiographic and

immiinocytochemical data.

Figure 3 is an image depicting immunoreactivity of cells expressing ran opioid receptors (MOPRs) in the parabrachial nucleus. This region is located in the brainstem, surrounding the brachium conjunctivum (also named superior cerebelar peduncule, scp). Image shows that the iaterai region of the PBN expresses the densest population of MOPRs (as compared to the medial portion of the PBN, MPBN). The figure is modified from Nicklous and Simansky (Nicklous et al., 2003, Am J Physiol Regul Integr Comp Physiol 285: 1046-R1054).

Figure 4 is a schematic depicting molecular structure of the opioid receptor antagonist (-)-naloxone [(-)-NLX] and its inactive stereoisomer (+)~naloxone [(+)-NLX], Naloxone was the first pharmacologically pure opioid antagonist identified. The action of an agonist is characterized as opioid receptor mediated only if its effects are (-)-NLX reversible. The (+)-NLX has been shown to have 10000- fold less affinity for opioid receptors than the active form and it is classically used as a control for the detection of opioid receptor mediated effects by naloxone [(-)-NLX structure from Zimmerman and Leander, (Zimmerman et al,, 1990, J Med Chem 33(3):895-902); (+)-NLX structure from Iijima et al., (lijima et al., 1 78, J Med Chem 21(4):398-400)).

Figure 5 is a graph demonstrating that repetitive infusion of DAMGO did not enhance feeding behavior. On Day Zero rats were infused with 0.5ul vehicle (vehicle (1)), then with 2nmol/0.5ui DAMGO (Day One, DAMGO ( 1 )), infused again with 0.5u) vehicle on Day Five (vehicle (2)) and again with 2nmol/0.5ul DAMGO on Day Six (DAMGO (2)). Rats did not receive any infusions on Days Three or Four (N=7). Cumulative pelleted chow intakes were measured 4hr after infusion from each day. Values are presented as mean±SEM (g). (*) represents different from base line (BL) values, p<0.05, Figure 6 is a graph demonstrating that DAMGO stimulates MOPR G- protein coupling in a concentration dependent manner. [³⁵S]~GTPYS autoradiography on rat tissue sections representing the LPBN. Sections were treated with different DAMGO concentrations (0. 1 - μΜ) and incubated for 120min (Basal 162±1 9 fmoi/g, N=10). Data siiown as GTPyS incorporation (means±SEM). *Different from basa! (p<0.05).

Figure 7 is a graph demonstrating that moderate concentrations of (-)- naloxone [(-)-NLX] inhibit DAMGO-induced G-protein coupling in a concentration dependent manner. Tissue sections containing the LPBN were co-treated with hiM DAMGO and indicated concentrations of (-)-NLX for 120min. In absence of (-)- NLX, DAMGO increased G-protein coupling to 387±21 fmof/g. Basal values:

189±18fmo!/g. Data shown as GTPyS incorporation (means±SEM). *Different from DAMGO alone p<0.05, N=8.

Figure 8 is a graph demonstrating that the stereisomer (-)-naloxone did not affect MOPR-stinuilation. [³⁵S]-GTPyS autoradiography on rat tissue sections representing the LPBN. Sections were treated with either 1 μΜ DAMGO or I nM (-)- naloxone [(-)-NLX], and incubated for I 20min (Basai 1 10±8 fmol/g, N=5). Data shown as GTPyS incorporation (meansiSEM). ^Different from basal (p<0.05).

Figure 9 is a graph demonstrating that the isomer (+)-naloxone [(+)- NLX] did not inhibit DAMGO-induced G-protein coupling. [³⁵S]-GTPyS

Autoradiography on rat tissue sections representing the LPBN. Sections were treated with Ι μΜ DAMGO and indicated concentrations of (+)-na!oxone [(+)-NLX], and incubated for 120min (Basal 96±12 fmol/g, N=8). In absence of (+)-NLX, DAMGO increased G-protein coupl ing to 282±13 fmol/g, Data shown as GTPyS incorporation (meansiSEM). ^Different from basal (p<0.05).

Figure 10 is a graph demonstrating that DAMGO stimulated MOPR G- protein coupling in a dose dependent manner, after 15min incubation, Tissue sections representing the LPBN were treated with 0. 1 , 0.3, 1, 3 and 1 OuM DAMGO for 1 5min (Basal 70±6fmol/g, N=6). Data shown as GTPyS incorporation (means±SE ).

*Different from basal (p<0.05).

Figure 1 1 is a graph demonstrating that ultra-low concentrations of (-)- naloxone enhances DAMGO-induced G-protein coupling, after 15niin incubation. Tissue sections containing the LPBN were co-treated with 1 uM DAMGO and indicated concentrations of (-)-naloxone [(-)-NLX] for 1 5min. The ultra-low concentration of (-)-NLX (0.001 nM) enhanced DAMGO-sti mutated G-protein coupling (1 19±5 fmol/g), as compared to DAMGO alone (94.8±6 fmo!/g). Basal values were 50.4±6 fmol/g. ^Different from DAMGO alone. p<0,05, N==7.

Figure 12 is a series of images demonstrating that tissue sections representing the LPBE were treated with different concentrations of DAMGO in presence or absence of O.OOlnM (-)-NLX, Data shown as stimulation above Basal (40.1±5fmo]/g and 73.4±6 fmol/g, respectively). ^Different from basal p<0,05, ^Difference between DAMGO+NLX and DAMGO alone, p<0.05. Right panel shows a representative autoradiogram demonstrating the increased GTPyS incorporation by 1 μΜ DAMGO co-treated with NLX 0.001 nM.

Figure 1 3 is a graph demonstrating that the stereoisomer (+)-naloxone enhanced DAMGO-stimulated G-protein coupling, after 15m in incubation. [³⁵S]- GTPyS Autoradiography on rat tissue sections representing the LPBN. Sections were treated with ΙμΜ DAMGO and different concentrations of (+)-naloxone (0.001 , 0.01 , 0, 1 , 1 and Ι ΟηΜ), and incubated for 15min (Basal 28±7 fmol/g, N=7). In absence of (+)-NLX, DAMGO stimulated G-protein coupling to 76±5 fmol/g Data shown as GTPyS incorporation (means±SEM), ^Different from basal (p<0,05).

Figure 14 is a graph depicting [³⁵S]-GTPyS Autoradiography on rat tissue sections representing the LPBN. Sections were treated with either Ι Μ

DAMGO or (+)-naloxone [(+)-NLX, O.O lnM], and incubated for 15min (Basal

44.2±3 fmol/g, N=5). Data shown as GTPyS incorporation (means±SEM). *Different from basal (p<0.05).

Figure 15 is a graph demonstrating that infusion of (+)-NLX enhanced DAMGO-induced feeding behavior. All animals were probed with DAMGO

(2^ηηιο!/0.5μ Ι) and, two days after, were treated with VEHICLE (0.5μΙ). Rats were then divided in groups. Each group received either (-)-NLX ( 1 ηιηο1/0.5μ1) +

DAMGO (N=5), (+)-NLX (1 ηηιοϊ/0.5μΙ) + DAMGO (N=7), or (+)-NLX

() 0η(ΏθΙ/0.5μ1) + DAMGO (N=6). Cumulative pelleted chow intakes were measured 4hr after infusion. Values are presented as mean±SEM (g). (*) represents different from vehicle, (#) represents different from DAMGO probe, p<0.05.

Figure 16 is a graph demonstrating that tiie enantiomers (-)-NLX or (+)-NLX did not alter feeding when infused on their own. Three days after receiving DAMGO (2nmol/0.5ul), half of the rats received (-)-NLX (I nmol/O.Sul, N=5) and the remaining animals received (+)-NLX (lnmol/0.5ul, N=8). Cumulative pelleted chow intakes were measured 4hr after infusion from each day. Values are presented as meaniSEM (g). Asterisk (*) represents different from vehicle, p<0.05.

DETAILED DESCRIPTION

The present invention relates generally to methods of managing feeding disorders and acute debilitating disorders such as pain. The invention is based partly on the discovery that the dextro or {+) stereoisomer of naloxone enhances effects of opioids that act at mu opioid receptors, while having no antagonistic properties at these sites. The discovery that (+) naloxone exhibits a positive therapeutic benefit as an adjunct to opioid treatment in the absence of antagonistic properties makes this isomer a new, much better candidate than the currently available (-) counterpart in adjunctive opioid therapy of pain and other clinical pathologies.

In one embodiment the (±) stereoisomer of naloxone can be used to potentiate the therapeutic action of opioid drugs and to reverse clinical tolerance to these agents,

In one embodiment, the present invention includes administration to a human or animal patient in need thereof a therapeutically effective amount of (+) naloxone. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section,

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal 's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system.

Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

The term to "treat," as used herei , means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the frequency with which symptoms are experienced.

As used herein, "treating a disease or disorder" means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient, Disease and disorder are used interchangeably herein,

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format, It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, tiie description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention relates to the use of (+) naloxone to potentiate MOPR agonist-induced activation. In this manner, the invention provides a method of using (+) naloxone as a new pharmacological. For example, (+) naloxone can be used as a tool for analyzing non-traditional mechanisms related to this class of opioid receptors. In contrast to (-) naloxone, the (+) isofonn can be used in a wide concentration range and still be inactive as an opioid antagonist.

The present invention relates generally to methods of using (+) naloxone for managing feeding disorders and acute debilitating disorders such as pain. The following discussion relates to treating pain. However, a skilled artisan when armed with the present disclosure would appreciate that the methods of treating pain using (+) naloxone is equally applicable to treating an eating disorder. Moreover, the artisan would recognize that (+) naloxone can be used to manage any disease, disorder or condition associated with MOPR.

The disclosure presented herein demonstrate that that (+) naloxone can enhance the hyperphagic effects induced by the mu opioid receptor agonist DAMGO, and that this effect was observed in a dose that the active isomer (-) naloxone antagonizes food intake. Accordinigy, the invention provides a method of using (+) naloxone in a broad dose range to potentiate opiate therapeutic actions, without the competing antagonistic effects that would occur with the (-) isomer,

(+) Naloxone may be administered to a mammal in need thereof on a daily or as needed basis, with a suitable daily dosage, Exemplary does range being from about 0, 1 m illigrams to about 10.0 milligrams. Multiple daily doses may also be administered in appropriate correspondingly smaller amounts. The most suitable doses are dependent on the type and location of the source of pain, and the physical characteristics of the patient (e.g., age, weight, etc.). Appropriate doses may be determined by utilizing routine experimental techniques, and/or by trial and error within the perimeters provided above.

In accordance with certain embodiments of the invention, (+) naloxone may be administered along with other pharmacological and/or non-pharmacological treatments for patients experiencing pain. Such treatments will vary depending upon a particular patient's diagnosis. However, the typical course of treatment preferably includes some adjunctive treatment (pharmacological and/or non-pharmacological) for the duration of the period (e.g., six months) during which the patient is being treated with (+) naloxone. Examples of adjunctive treatments include antiinflammatory agents, serotonin specific reuptake inhibitors (SSRIs) and/or physical therapy. The methods of treatment of this invention permit patients, as the symptoms of their pain subside, to more easily engage in such non-pharmacological treatments as physical therapy.

Relief of pain occurs in about one hour and is continually enhanced over approximately the next three weeks. At the end of the treatment period

(typically about six months), the patient will be discontinued from (+) naloxone

] 1 admi istrations, with some patients remaining pain free for an extended period of time after disconti uation of treatment with (+) naloxone.

The administration of (+) naloxone to treat pain may be achieved intranasally, intravenously, intramuscularly, by inhalation, transdermal!)', and/or orally (in immediate release, sustained release, or other modified release form).

Relief of pain typically occurs within about one hour for any of the routes of administration. However, the onset of relief is generally hastened by intravenous or intramuscular injection at or near the source of pain. Inhalation and intranasal administration also provide relatively quick relief and has the advantage of greater patient acceptance and compliance as compared with intravenous and/or intramuscular injection.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The experiments discussed herein present research using the parabrachial MOPRs as a model to further analyze the cellular and behavioral responses associated with manipulating the MOPR system. For example, the disclosure presented herein, demonstrate changes in opioid-elicited G-protein coupling and changes in feeding behavior following the pharmacological modulation of these receptors. Experiments were performed to analyze the consequences on cellular mechanisms and behavioral outcomes of activating MOPRs in situ in a stereospecific manner, in a precise brain locus,

Example 1 : Stereoselectivity of antagonism and enhancement of parabrachial MOPR function by enantiomers of naloxone

Opioids produce their effects by activating μ-, δ- and κ-opioid receptors (MOPRs, DOPRs and KOPRs) that are coupled to Gi-proteins. Classically, activation of these receptors inhibits adenylate cyclase activity and decreases cAMP levels, resulting in an inhibition of neuronal activity (Duman et l., 1988 J Pharmacol Exp Tlier 246(3) 1033-9). Interestingly, previous studies proposed that activating opioid receptors can also stimulate adenylate cyclase, and that these stimulatory effects are due to an initial and transient coupling to Gs-protein (Gain et al,, 1990, Trends Pharmacol Sci 1 1 :77-81 ; Crain et al., 2000, Pain 84: 121- 13 1 ; Wang et al., 2005, Neuroscience 135(1):247-61 ; Chakrabarti, et al„ 2005, Brain Res 135(1-2):2 I 7- 224). For example, some studies have shown that the MOPR agonist morphine can exert excitatory receptor-mediated effects (Chakrabarti, et al., 2005, Brain Res 135(1 - 2):217-224; Wang et al., 2008 PLoS One 3(2):e l 554). Recently, Wang and Burns (Wang et al., 2009, PLoS One 4( l ):e4282) showed that treatment with an ultra-low - dose of the nonselective opioid antagonist (-)-naloxone [(-)-NLXJ or its inactive enantiomer (+)-naloxone [(+)-NLX], can block this excitatory effect and enhance Gi- couplhig. Opioids and their receptors are expressed in different brain regions and are known to be involved in a variety of physiological functions. For example, the parabrachial nucleus (PBN) is a pontine brain region that expresses MOPRs and that has been associated with the regulation of ingestive behavior. Our laboratory has reported previously that infusion of the selective MOPR agonist DAMGO into the lateral PBN (LPBN) increases food intake and that pretreatment with (-)-NLX (1 - lOnmot) blocks this effect (Wilson et al., 2003, Am J Physiol Regul Integr Comp Physiol 285(5):R 1055-65).

In the present study, experiments were conducted to assess the functional and behavioral effects of (-)-NLX and (+)-NLX on parabrachial MOPRs. Specifically, experiments were designed to test the hypothesis that manipulating parabrachial MOPRs by using ultra-low doses of (-)-NLX or a wider dose range of (+)-NLX, can enhance DAMGO-elicited G-protein coupling. Further, to analyze the relevance of these enhancing effects in vivo, experiments were conducted to test if (+)-NLX can potentiate DAMGO-mduced feeding. The results presented herein demonstrate that (-)-NLX but not (+)-NLX, is a stereospecific MOPR antagonist in the PBN. The studies further showed that ultra-low-concentrations of (-)-NLX or several concentrations of (+)-NLX enhanced DAMGO-stimulated G-protein coupling. The results demonstrate that (+)-NLX potentiated DAMGO-stimulated feeding behavior. These results implicate (+)-NLX as a novel tool for studying MOPR function. More importantly, the data demonstrate that this agent has potential therapeutic value to enhance analgesic or other beneficial actions of opiates that act at MOPRs. The materials and methods employed in these experiments are now described. Animals

Male Sprague-Dawiey rats (35( 00 g) purchased from Taconic

Farms (Germantown, NY) were housed individually in suspended wire-mesh cages (43 cm Iengthx22 cm widthx 1 8 cm height). The colony room was maintained at 22- 24 °C and at 40-50% humidity under a 12: 12-h light-dark cycle (lights on at 0600). Animals had free access to water and standard, pelleted chow (3.34 kcai/g; Purina, Milts Laboratory Diet, St. Louis, MO) unless otherwise noted. Subjects were experimentally iiaTve and allowed one week of adaptation in the colony before surgery or any other procedure. Experimental protocols were carried out in accordance with the "Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research of the National Research Council" (2003) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Drexel University. r³⁵S1-GTPyS autoradiography

This procedure was based on an established method by Sim et al. (Sim et al, 1995, Proc Natl Acad Sci USA 92:7242-46), and modified according to (Ward et al., 2006, Psychopharmacology (Berl) 187:435-446; Ward H.G, Simansky, K.J. Regional specialization of mu-opoid receptors within the parabrachial nucleus of the pons in modulating eating. Society for Neuroscience Annual Meeting 2006, Atlanta GA). Naive rats were decapitated by guillotine and the brains were removed immediately, frozen on dry ice and stored at -80 °C. Twenty-micron frozen coronal sections were cut throughout the PBN (Leica cryostatmodel CM3O50, Deerfield, IL), thaw-mounted onto slides coated with chrome-alum and stored at -80 °C until use, To initiate this assay, slides were preincubated for 40 mm in Tris-assay buffer (50mM Tris-HCl, 4mM MgCl₂, 0.3mM EGTA, l OOmM NaCl, pH 7.4) at 25 °C, followed by a 20m in incubation in guanosine diphosphate (GDP, 2mMin Tris-assay buffer) at 25 °C. Sections were then incubated for 15min or 120inin at 25 °C in Tris-assay buffer containing [³⁵S]-GTPyS (0.04 nM, 1250 Ci/mmo!; Perkin Elmer, Boston, MA) and 2 mM GDP (Sigma-Aldrich, St. Louis, MO). Additionally, this incubation was divided into the following conditions: no drug (basal condition); drug corresponding to the specific assay; or unlabeled GTPyS (10 μΜ; guanosine-S'-O- - thio)-triphosphate; MP ICN Biomedicais, Irvine, CA) to determine nonspecific binding. After incubation, slides were rinsed twice in cold 50mMTris-HCl buffer (4 °C, pH 7.4, 2min each rinse), rinsed briefly in cold de-ionized water, dried immediately with a cool stream of air and desiccated overnight. Slides were exposed to Kodak Biomax® MS film (Eastman Kodak Company, Rochester, NY) for 24h (for those tissues incubated for I 20min) or 48h (for those tissues incubated for 15mm), along with [¹⁴C] standards (3 1 -883 nCi/g; Amersham Biosciences, Piscataway, NJ). Images from the developed films were scanned and quantified using Image Pro-Plus Version 4.5 software (Media Cybernetics; Newburyport, MA). Densities (optica! density units, OD) of [³⁵S]- GTPyS incorporated were determined in the lateral parabrachial region (LPBN). The values from multiple sections from each rat were averaged and converted to femtomoles per gram of tissue (fmol/g) using nonlinear curves generated from the [^l C] standards. All OD values (including nonspecific binding) were corrected for background of the film, but not for the different path lengths of isotopic emissions from ⁵S (tissue sections) and ¹⁴C (standards). Each anatomical set of sections contained alternating sections for the nonspecific, basal and the different drug conditions through the PBN. Nonspecific binding was subtracted from each of the conditions, including basal.

In all experiments, solutions were prepared immediately before incubation. Data were analyzed statistically as GTPyS incorporation in fmol/g of tissue. For the anatomical analyses of the LPBN we measured and averaged together three regions; CI, C2 and LE (See Figure 2). The LPBN is an area that we have defined arbitrary in the past as LPBNi (Chaijale et al., 2008, Brain Res 1240: 1 1 1 - 1 1 8),

Drugs

(-)-Naloxone hydrochloride [(-)-NLX, M.W.:363.82, Research

Biochemicals Inc., Wayland, MA, USA] or its enantiomer (+)-natoxone [(+)-NLX, from NIDA] were dissolved in de-ionized water. The MOPR agonist [D-Ala2, N-Me- Phe4, Gtycino]5]-Enkepha!in (DAMGO, MW: 513.7, Tocris Bioscience, Eliisvilie, MO, USA) was dissolved in saline solution (0.9% w/v). All solutions were prepared on test days.

Stereospecificity of the antagonistic properties of naloxone at parabrachial MOPRs Before evaluating the actions of naloxone in vitro, the concentration- response function for the ability of the classical MOPR agonist DAMGO to stimulate [³⁵S]-GTPyS incorporation in the LPBN was determined. Rat tissue sections from this brain region were incubated with different concentrations of DAMGO (0, 0.1 , 0,3, 1 , 3 and 10 μΜ), for 120m in (n=6 rats). Further, to study the prediction that (-)-NLX, but not (+ NLX, inhibits DAMGO-stimulated [³⁵S]-GTPyS incorporation in a stereospecific manner, in vitro coupling of MOPRs at different concentrations of (-)- NLX and (+)-NLX (0, 0.001 , 0.01 , 0.1, 1 and l OnM), in presence of 1 μΜ DAMGO was compared. Rat tissue sections representing the LPBN were incubated for 120min (n=7 rats).

As a control study, in vitro coupling of MOPRs in the presence of DAMGO (I μΜ) or (-)-NLX (I nM) alone (n=5 rats) was compared.

Enhancement by (-)-NLX and (+)-NLX of DAMGO-stimulated G-protein coupling in the LPBN

Recent studies have shown that acute stimulation of MOPRs causes a transient (up to l 5-20min) MOPR-Gs coupling, with subsequent switching to Go coupling, and that co-treatment of the tissue with an ultra-low-dose of (-)-NLX or (+)- NLX can completely prevent this transient effect, allowing the observation of only MOPR-Go coupling (Wang et al., 2008 PLoS One 3(2):el554). For this reason, this series of experiments used autoradiographic studies incubating the tissue with the corresponding treatments for 15min (instead of 120min).

The ability of DAMGO to stimulate MOPR [35S]-GTPyS incorporation in rat tissue sections representing the LPBN was first analyzed. Sections were incubated with different concentrations of DAMGO (0, 0.1 , 0.3, 1 , 3, and 10 μΜ), (n=6).

Secondly, to assess if (-)-NLX or (+)-NLX can enhance parabrachial MOPR-Gi coupling, autoradiography was used to compare in vitro coupling of MOPRs at different concentrations of (-)-NLX or (+)-NLX (0, 0.001 , 0.01 , 0.1 , 1 and 10 tiM) in the presence of Ι μΜ DAMGO.

Additionally, to further confirm the effects of (-)-NLX on MOPR-Gi coupling, the DAMGO concentration-response function for stimulation of MOPR coupling in the PBN was determined, in the presence or absence of the one ultra-low dose of (-)-NLX that enhanced coupling in the initial studies. Finally, as a control study, in vitro coupling of MOPRs in the presence of DAMGO (1 μΜ) or (+)-NLX (0.0 InM) alone (n=5) was compared.

Enhancement by (+)-NLX of DAMGO-induced feeding

The objective of this behavioral experiment was to determine if pretreatment with an infusion of (+)-NLX into the LPBN would enhance the hyperphagic action of DAMGO in the parabrachial nucleus.

Surgeries. Rats were anesthetized with equithesin (3.5 ml/kg i.p.), which was formulated to deliver 36 mg/kg pentobarbital sodium and 160 mg/kg chloral hydrate per dose. Unilateral stainless steel guide cannulas (26-gauge, 3.8 mm center-to-center; Plastics One, Roanoke, VA) were implanted stereotaxically (Kopf Instruments, Tujunga, CA) into the LPBNi with the guide cannula ending at the following coordinates; anteroposterior, - 9.8 mm posterior to bregma; mediolateral, ± 1 .9 mm from the midline; and dorsoventral, - 4.8 mm below the surface of the skull. After surgery, 33-gauge obturators were inserted into the guides, with the tips flush with the end of the guides, to prevent occlusion. Rats were handled daily and allowed at least seven days for recovery before behavioral testing.

Drug infusion. Drug treatment or vehicle was delivered into the LPBN of each animal using a stainless steel 33-gauge microinjector (Plastics One), extending 2.5 mm below the end of the guide cannula. Infusions were made with a microdrive pump {Harvard Apparatus Model 975, South Natick, MA), connected via polyethylene tubing (PE- 10). Drug or vehicle delivery was made in a total volume of 0.5 μ! per side at a rate of 0.33 μΐ/min. Injectors remained in place for a period of 30 s after infusion in order to minimize backflow.

Testing procedure. After surgery and recovery, animals received food (60-70 g of chow; daily ad libitum intake was 28-30 g) and fresh tap water. Food remaining in the cage and the spillage remaining under the cage were weighed 30min, 2hr and 4hr after infusion. Fresh pellets were given at 0900 h daily. The amount of food consumed stabilized within five to seven days and the last three consecutive intakes before infusion day were averaged for each rat. These means were taken as the baselines (BL) for the subsequent studies. On testing days, food was removed from the cage before vehicle or drug infusions. After the infusion, the preweighed food was placed into the cage. As followed for BL measurements, food remaining in the cage and spillage under the cage were weighed 30, 120, and 240 min after infusion.

Actions of f+VNLX on DAMGO-induced feeding

DA GO increases food consumption when infused into the LPBN

(Nickious et al., 2003, Am J Physiol Regui lntegr Comp Physiol 285:R 1046-R 1054; Wilson et al., 2003, Am J Physiol Regul lntegr Comp Physiol 285(5):R 1055-65; Ward et al., 2006, Psychopharmacology (Berl) 1 87:435-446; DiPatritzio et al., 2008, Am J. Physiol Regul lntegr Comp Physiol 295(5):R1409-14). Ail animals were first probed with our standard dose of DAMGO (2nmol/0,5 μΙ, the approximate ED70;

Wilson et al., 2003). The assessments of the actions of (+)-NLX isomer on DAMGO- stimulated eating began 3-4 days later.

On the basis of the response to the initial probe with DAMGO, rats were divided equally into three groups. Ail animals received vehicle + vehicle on the first testing day. On the following day, one group received (-)-NLX ( I ηιηο!/0.5 Ι) before DAMGO infusion (2ηηιο!/0,5μ1, n=5). This dose of (-)-NLX completely blocked the feeding response to DAMGO in a dose-response study previously reported (Wilson et al., 2003). The other two groups were pretreated with either an equivalent dose of (+)-NLX (n=7), or with a dose ten times higher (n=6). Each set of infusions was made 20min apart. The concentration chosen for DAMGO was the same as the one used by Wilson et al. (2003) in which they showed that infusion of 2 nmol/0.5 μΙ DAMGO into the PBN induces hyperphagia.

In separate group of animals, experiments were designed to determine if administration of naloxone by itself would affect intake in rats. For this experiment, a set of 16 rats received vehicle infusion on Day One, followed by DAMGO

(2ηηιο1/0.5μ1) on Day Two. Rats did not receive any treatments on Days Three or Four. Day Five, rats received a second vehicle infusion and were divided in two groups (n=8 per group) on the basis of their response to their initial probe with DAMGO. On Day Six, one group of rats was infused with lnmol (-)-NLX, while the second group received l nmol (+)-NLX. Food intake measurements were followed as described elsewhere herein.

Finally, in order to evaluate further if repeated administrations of DAMGO would either sensitize or desensitize the activation of MOPRs, an experiment in which a set of seven animals underwent the following schedule was performed: vehicle infusion on Day One, DAMGO (2ηιηο1/0.5μΙ) on Day Two, no infusion on Days Three and Four, vehicle on Day Five and DAMGO (2ηιηο1/0.5μ1) on Day Six. This schedule is the same as the one used for I nmol (+)-NLX + DAMGO or lOnmol (+)-NLX + DAMGO studies. Significant responses to PBN infusion of DAMGO were found by 4hr after infusion, but food intake was not different when the two sequential infusions of DAMGO were compared (6,6±l g and 6.6±2g for first and second DAMGO infusions, respectively). Food intake after vehicle infusion on Day One was not different from tiie intake when animals received vehicle on Day Five (Figure 5).

Analysis of cannula placements

Infusion sites from each rat were determined from one to three frozen sections by projecting them onto line-drawn templates with a Camera Lucida (Bausch and Loinb, Rochester, NY, USA). Two observers independently rated the placements without knowledge of the treatments or behavioral results associated with the rat. Animals whose placements were out of the intended targeted area (n=2) or who did not respond to the DAMGO probe (n= l ) were excluded from the experiments.

Statistical analysis

Results from densitometry were analyzed by I -way ANOVA of the actual values (fmol/g), except for the study comparing concentration-response curves for DAMGO in the presence or absence of an ultra-low concentration of (-)-NLX. In that study, densitometry results were analyzed by 2-way ANOVA of the actual values (fmol/g) and after conversion to stimulation above basal. Values from the behavioral experiments comparing the actions of (-)-NLX and (+)-NLX on DAMGO-induced feeding were analyzed by appropriate 2-way ANOVA based upon the actual values. For the treatment effects, the food intake of each rat after administration of DAMGO + NLX was compared to its own responses to vehicle and DAMGO.

For all data analyses, the Student-Newman-Keuis test was used for post-hoc comparisons of means. An alpha level of p<0.05 was the threshold for statistical significance (Sigma Stat version 3.1 , Systat Software inc., Chicago, 1L, USA).

EC50 and 95% confidence intervals from all concentration-response curves were estimated by Prism Graph program. The results of the experiments are now described.

The stereoisomer (-)-naloxone, but not (+)-naloxone, antagonizes DAMGO- stimulated F³⁵SlGTPyS incorporation in the LPBN

In order to select an appropriate concentration of DAMGO for subsequent experiments, the concentration-response curve for DAMGO-stimulated [³⁵S]-GTPyS incorporation in the lateral parabrachial nucleus (LPBN) was initially characterized. Figure 6 shows that DAMGO increased GTPyS incorporation in the LPBN in a concentration-dependent manner. The 0.3, 1 , 3 and 10 μΜ concentrations of DAMGO increased [³⁵S]-GTPyS incorporation between 73-157 % over basal (basal values: 162± 19 fmol/g). The Ι μΜ DAMGO increased incorporation to 123% above basal. The values after the 0.3 μΜ concentration differed from 1 , 3 and Ι ΟμΜ however, the values after the 1- 10 μΜ concentrations did not differ from one another. The 0.1 μΜ concentration was not significantly different from basal (207±24 fmoi/g). The EC₅₀ value of the concentration curve was 0,4 μΜ (0.2 to 0.8μΜ = 95% CI). For subsequent experiments, 1 μΜ DAMGO was chosen being that this is an approximate EC₇₀ concentration.

Figure 7 shows the inhibition by (-)-naloxone [(-)-NLX] of DAMGO- stimulated [³⁵S]-GTPyS incorporation, in the lateral parabrachial nucleus. In the absence of (-)-NLX, Ι μΜ DAMGO increased [³⁵S]-GTPyS incorporation to 387±21 fmol/g (basal value: 1 89±18 fmol/g). In the presence of different concentrations of (-)- NLX, DAMGO-induced GTPyS incorporation was reduced in a concentration- dependent manner. Whereas the 0.1 nM concentration of (-)-NLX was the minimum concentration that significantly reduced DAMGO-stimuiated G-protein coupling (307±31 fmol/g), the l OnM (-)-NLX concentration completely blocked DAMGO- induced GTPyS incorporation (212±15 fmol/g). The value at this concentration was significantly different from any other value after (-)-NLX. Although 0. 111M and 1 nM (-)-NLX reduced significantly DAMGO-stimulated G-protein coupling, the values at these concentrations did not differ from each other.

Finally, it was tested whether (-)-NLX ( InM) alone affected G-protein coupling. The data in Figure 8 showed that whereas 1 μΜ DAMGO stimulated G- protein coupling, I nM (-)-NLX alone did not alter [³⁵S]-GTPyS incorporation (146 ± 12 fmol/g, basal values: 1 10±8 fmol/g). To determine the stereospecificity of (-)-NLX for the functional MOPRs present in the LPBN, the effect of DAMGO-stimulated G-protein coupling in the presence of (+)-NLX was studied (Figure 9). In the absence of (+)-NLX,

DAMGO-stiniulated G-protein coupling (282±13 fmol/g). Cotreatment of the tissue with different concentrations of (+)-NLX did not inhibit DAMGO GTPyS

incorporation, in fact, (+)-NLX enhanced DAMGO-induced G-protein coupling at the Ι ΟηΜ concentration by 29% (332±21 fmol/g). Lower concentrations of (+)-NLX did not alter DAMGO-stimulated GTPyS incorporation (261± 15, 290± 12, 293± 10 and 299±14 fmol/g for 0.001 , 0,01 , 0.1 and l nM concentrations, respectively).

Altogether, these results indicate that only (-)-NLX (see Figure 8) and not (+)-NLX blocks MOPR-stimulated G-protein coupling, demonstrating the stereospecificity of the lateral parabrachial MOPR binding site. Further, these results indicate that the (+)-NLX isomer (inactive as an opioid antagonist), actually enhances DAMGO-stimulated G-protein coupling (see Figure 9).

Further analysis of the potentiation by (-)-naloxone and (+)-naloxone of DAMGO- stimulated G-protein coupling in the LPBN

It was shown that (-)-NLX, in cotreatment with opioid agonists, potentiates agonist-induced effects (Buschsbaum et al., 1977, Nature 270(5638):620- 622; Levine et l., 1979, Nature 278(5706):740- S ; Levine et al., 1986, Pain 27( i ):45- 9; Levine et al, 1988, J Clin Invest 82(5): 1574-7; Gillman et al., 1981 , J R Soc Med 74(12)943-4; Gillman et al., 1985, Neurol Res 7(3): 106- 19; Gillman et al., 1989, Int J Neurosci 48(3-4):321-4; Schmidt et al., 1985, Anaesthesia 40(6)583-6; Taiwo et al.,

1 89, J Pharmacol Exp Ther 249( 1 ):97- 100). One proposal to explain this phenomenon is an opioid-induced excitatory signaling which is mediated by an initial and transient switch in G-protein coupling from Gi/o to Gs proteins (Crain et al.,

1990, Trends Pharmacol Sci 1 1 :77-81 ; Crain et al., 2000, Pain 84: 121 - 13 1 ; Shen et al., 1989, Brain Res 491 :227-242; Wang et al., 2005, Neuroscience 135(1):247-61 ; Chakrabarti, et al., 2005, Brain Res 135( 1 -2):217-224). Wang and Burns (Wang et al., 2009, PLoS One 4( l):e4282) recently showed that the transient MOPR-Gs coupling subsequently switched to Go coupling after 15 to 20min and that cotreatment with an ultra-low-dose of (-)-NLX or (+)~NLX completely prevented Gs coupling (and only the MOPR-Go coupling was observed). Although many reports have shown that the transitional excitatory stimulation of MOPRs can be blocked by ultra-low doses of (-)-NLX or (+)-NLX, in vitro; it is still unclear if specific concentrations of either of these naloxone enantiomers can actually enhance MOPR- induced activation. Thus, by using [³⁵S]-GTPyS autoradiography, experiments were performed to assess if different doses of (-)-NLX or (+)-NLX can potentiate DAMGO-stimulated G- protein coupling after 15min incubation.

Figure 10 shows DAMGO-stimulated [³⁵S]-GTPyS incorporation in the lateral parabrachial nucleus (LPBN), after 15min treatment. DAMGO dose dependency stimulated parabrachial MOPR G-protein coupling at 0.3, 1 , 3 and 10 μΜ. These concentrations increased [³⁵S]-GTPyS incorporation in the range of 55% to 105% above basal (basal values: 73±6 fmol/g). The 1 μΜ DAMGO increased to 83% above basal, The 1, 3 and 10 μΜ concentrations were not significantly different among each other; however, they all differed from the value after 0.3 μΜ ( 1 14±7 fmol/g). The 0. 1 μΜ concentration was not different from basal (85±9 fmol/g). For the subsequent experiments we chose the 1 μΜ DAMGO concentration being that this is an approximate EC70 (Ec₅o value: 0.3 μΜ, 0,2-0.6 at 95% CI).

Figure 1 1 shows the effects by (-)-NLX of DAMGO-stimulated [35S]- GTPyS incorporation in the LPBN, in vitro. In the absence of (-)-NLX, 1 μΜ

DAMGO increased GTPyS incorporation to 94.8±6 fmol/g (basal value: 50.4±6 fmol/g). In these results, unlike the 120min incubation time, only the ! OnM (-)-NLX concentration reduced DAMGO-stimulated G-protein coupling to 70.9±6 fmol/g. Most importantly, 0.00 I nM (-)-NLX enhanced DAMGO-stimulated G-protein coupling ( 1 19±5 fmol/g), indicating that an ultra-low dose of (-)-NLX presents a different pharmacological action than the classical antagonistic effects of this agent.

To confirm the transient enhancing effects of (-)-NLX on MOPR coupling, [ ⁵S]-GTPyS autoradiography was used and different concentrations of DAMGO (0.1 , 0,3, 1 , 3 and 10μΜ) was used in the presence of 0.00 I nM (-)-NLX, following 1 min incubation, The (-)-NLX concentration was chosen according to previous results in which 0.00 I nM enhanced Ι μΜ DAMGO-stimulated G-protein coupling (see figure 1 1). In this study, it was observed that O.OO lnM (-)-NLX enhanced DAMGO-stimulated G-protein coupling at almost all concentrations of

DAMGO (Figure 12). In the absence of (-)-NLX, Ι μΜ DAMGO stimulated G-protein coupling to 60.7±6 fmol/g above basal (basal value of 73.4±6 fmol/g). Tissue sections co-treated with 0,00 I nM (-)-NLX enhanced DAMGO-stimulated G-protein coupling at almost all concentrations of the agonist. The 1 μΜ DAMGO presented the maximal response ( 107.3±6 finol/g above basal; basal value: 40.1±5 fmol/g), whereas 0.1 μΜ DAMGO did not differ between conditions ( 17±7 and 29±9 fmol/g above basal in absence and presence of (-)-NLX, respectively). Although values after 3 and Ι ΟμΜ DAMGO did not differ from one another (90±4 and 94±4 fmol/g above basal), (-)- NLX enhanced the incorporation only after the 10 μΜ DAMGO,

Together, these results indicated that 0.00 I nM (-)-NLX exerts a non traditional effect on activated MOPRs in the LPBN. This ultra-low concentration of (- )-NLX enhances, rather than attenuates, MOPR-stimnlated G-protein coupling.

The data in Figure 9 showed that 10 nM (+)-NLX enhanced GTPyS incorporation at 120min. In the present experiment, the action of (+)-NLX at 1 5min was evaluated. To study if (+)-NLX can enhance parabrachial MOPR-Gi coupling, it fi st needed to be understood the properties of this drug at different concentrations, following I 5min incubation, For this experiment, autoradiography was used to compare in vitro coupling of MOPRs at different concentrations of (+)-NLX (0, 0.001, 0.01 , 0. 1 , 1 and l OnM) in presence of l M DAMGO. The results showed that (+)-NLX did not inhibit DAMGO-induced G-protein coupling, further confirming the stereospecificity of naloxone to inhibit parabrachial MOPR activation (Figure 13).

Interestingly, the data also indicated that almost all concentrations of (+)-NLX (0.01 to l OnM) enhanced DAMGO-induced G-protein coupling. While DAMGO alone stimulated parabrachial MOPR GTPyS incorporation by 75.6±5 fmol/g, the 0.01 , 0. 1 , 1 and 10 nM (+)-NLX enhanced DAMGO-induced G-protein coupling to 91 .2±8, 91.1±7, 96.9±7 and 89.2±7 fmol/g, respectively. Thus, in comparison to the (-)-NLX isofo m, (+)-NLX potentiates DAMGO-induced G-protein coupling at a broad concentration range. Moreover, simitar to the results obtained for 120min incubation time, 15min incubation of 10 nM (+)-NLX + DAMGO enhanced DAMGO-induced G-protein coupling by 3 1 %, suggesting that the 10 nM (+)-NLX isomer (inactive as an opioid antagonist), actually enhances DAMGO-stimulated G- protein coupling at any time point.

Finally, to rule out the possibility that naloxone alone has any agonistic effects on parabrachial MOPR G-protein coupling, a separate experiment was performed in which tissue sections representing the LPBN were treated with either DAMGO (1 μΜ) or (+)-NLX (0.0 I nM) alone (Figure 14). Results from this experiment showed that (+)-NLX (5 1 .1±7 fmo!/g) did not affect MOPR GTPyS incorporation as compared to basal levels (44.2±3 fmol/g). Only the tissues treated with Ι Μ DAMGO significantly increased GTPyS incorporation (71±5fmoi/g). These results suggest that (+)-NLX alone does not affect parabrachial MOPR GTPyS incorporation. Modulation of parabrachial MOPRs in vivo

The in vitro results presented herein showed that tissue representing the LPBN treated with DAMGO and different concentrations of the inactive isomer (+)-NLX can enhance MOPR-stimuiated G-protein coupling. Experiments were performed to further analyze the relevance of these findings in vivo. Specifically, it was determined that DAMGO-induced feeding is enhanced by pretreating rats with (+)-NLX (Figure 15). A hyperphagic dose of DAMGO (2nmol/0.5ul, Wilson et al„ 2003) was infused into the LPBN of rats pretreated either with (+)-NLX ( 1 or l Onmoi) or (-)-NLX ( lnmol). The results showed that rats infused with DAMGO increased the 4h food consumption (4.2±0.4g), as compared to baseline or vehicle treatments (2.1±0.2g and 1 ,9±0.3 g, respectively). Moreover, rats that were pretreated with (-)-NLX and later infused with DAMGO and did not differ from vehicle conditions (2.1±0.9g), confirming that this dose of (-)-NLX blocks the hyperphagic response to DAMGO (Wilson, 2003). In contrast, infusion of DAMGO in animals pretreated with either lnmol or l Onmoi (+)-NLX enhanced DAMGO-induced feeding (6,5± l g and 6. l ±0.5g, respectively, p<0.05). It should be noted that sequential infusions of DAMGO alone neither sensitized nor desensitized the hyperphagic response. Thus the increased response to DAMGO in the presence of (+)-NLX was due to this combination of agents.

Finally, to rule out the possibility that (+)-naloxone alone has agonistic effects on parabrachial MOPRs a separate experiment was performed in which animals were infused with either vehicle, DAMGO, (-)-NLX ( l nmol) or (+)-NLX ( l nmol) (Figure 16). Results from this experiment showed that the food intake of rats infused with either (-)-NLX or (+)-NLX alone did not differ from vehicle treated animals

(2.3±0.4g, 2.4±0.6g and 2.1±0.5g, for (-)-NLX, (+)-MLX and vehicle, respectively), Rats infused with DAMGO increased feeding to 4.8±0.4g. These results confirmed that neither (-)-NLX or (+)-NLX alone affect feeding behavior. Thus, without wishing to be bound by any particular)' theory, it is believed that (+)-NLX can potentiate DAMGO-induced feeding behavior, and that these effects are not due to an agonistic effect of naloxone alone. Example 2: (+)-NLX is a novel pharmacological tool for study of the MOPR function, with potential therapeutic relevance

It has been shown that the μ-opioid receptors (MOPR) in the brain are important for the many behavioral and physiological responses to opiate agents including analgesia and compulsive drug self-administration (Daws and White, 1999). Additionally, MOPRs mediate physiological processes that increase feeding (Bakshi et al., 1993, J Pharmacol fixp Ther 265: 1253- 1260; Giraudo, et al., 1998, Brain Res 782(1 -2): 1 8-23; Mann et a!., 1988, Neuropharmacology 27(4):349-55; Ragnauth et al., 2000, Brain Res 876( I -2)76-87; Zhang et al., 2000, Neuroscience 99(2):267-77; Kotz et al., 1997, Am J Physiol 272(4 Pt 2):R 1028-32; Glass, et al ., 1999,

Neuropeptides 33(5):360-8; Bodnar, 2004, Peptides 25:697-725; Kelley et al., 2005, J Comp Neurol 493( 1 ):723-85; Levine et al., 2006, Physiol Behav 89( l ):92-6). One brain region that expresses MOPRs is the parabrachial nucleus (PBN) especially the lateral PBN (LPBN). The parabrachial MOPR system has been implicated in diverse roles including regulation of food intake. Thus, the present research utilized parabrachial opioid-induced changes in feeding behavior as a model system to further analyze the potential pharmacological manipulation of the MOPRs by enhancing its stimulatory effects, In this regard, experiments were performed to take advantage of the properties of two stereoisomers of the opioid receptor antagonist naloxone - the active agent (-)-naloxone [(-)-NLX], and the compound with no antagonistic actions [(+)-naloxone, (+)-NLXJ - , and used these as pharmacological tools to analyze the parabrachial MOPR system.

This study using the PBN in the rat, confirmed the classical view that naloxone is a stereospecific MOPR antagonist in the rat parabrachial nucleus. Only (- )-NLX, and not (+)-NLX, blocked parabrachial MOPR-stimulated G-protein coupling. Moreover, it was demonstrated that these two stereoisomers can be used as pharmacological tools to manipulate the stimulation of parabrachial MOPRs.

Specifically, while normal concentrations of the active opioid antagonist (-)-NLX inhibited DAMGO-induced GTPyS incorporation, an ultra-low concentration of this drug, potentiated MOPR G-protein coupling. Likewise, the inactive enantiomer (+)- NLX also showed an enhanced MOPR-stimulated G-protein coupling, but in this case, (+)-NLX was effective at higher concentrations than (-)-NLX. Furthermore, by studying these effects of (+)-NLX in a model of feeding behavior, a novel mechanism by which infusion of (+)-NLX into the lateral parabrachial nucleus (LPBN) enhanced DAMGO-elicited feeding has been established, it was possible that the larger response to DAMGO in combination with (+)-NLX was simply to sensitization by the first probe with DAMGO alone, However, DAMGO elicited the same responses on two sequential infusions in the absence of (+)-NLX. Thus, the data presented herein revealed a true potentiation by (+)-NLX of the opiodergic stimulation of eating. It was concluded that (+)-NLX potentiated the response induced by the MOPR iigand in vivo.

These interesting findings strongly suggest the use of (+)-NLX as a new pharmacological tool to potentiate MOPR agonist-induced stimulation. In contrast to (-)-NLX, the (+)-isoform can be used in a wide concentration range and still be inactive as an opioid antagonist. Without wishing to be bound by any particular theory, it is believed that (+)-NLX could be a useful pharmacological tool not only to enhance DAMGO-induced feeding, but to potentiate the activation of MOPRs in other physiological responses as well, Using this particular enantiomer gives a practical advantage, being that (+)-NLX doses that would show enhancing MOPR-induced effects are more flexible than the (-)-NLX doses and, unlike (-)-NLX, reasonable doses of (+)-isoform would not present antagonistic effects.

Administration of reasonable doses of (+)-NLX, instead of an ultra-low dose of (-)- NLX, may also enhance the analgesic effects induced by a MOPR agonist, as it was shown with (-)-NLX.

The present study focused on the pharmacological properties of (+)- NLX and demonstrated that (+)-NLX can enhance MOPR-stimulated G-protein coupling as well as enhance stimulated MOPR-induced feeding. What is more important, these results implicate (+)-NLX as a novel pharmacological too! for the study of MOPR function, not only in the PBN, but potentially in any other MOPR system,

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed :

1 . A method of treating pain in a mammal, the method comprising administering to a mammal in need thereof a theiapeiiticaily effective amount of (+)- naloxone.

2. The method of claim 1 , wherein a therapeutically effective amount of (+)-naloxone exliibits a positive therapeutic benefit in the absence of an antagonistic property.

3. A method of treating an eating disorder in a mammal, the method comprising administering to a mammal in need thereof a therapeutically effective amount of (+)-naloxone.

4. The method of claim 3, wherein a therapeutically effective amount of (+)-naloxone exhibits a positive therapeutic benefit in the absence of an antagonistic property.