WO2013056105A2 - INHIBITION OF SPINAL MAMMALIAN TARGET OF RAPAMYCIN (mTOR) REDUCES CANCER PAIN, OPIOID TOLERANCE, AND HYPERALGESIA - Google Patents

Description

INHIBITION OF SPINAL MAMMALIAN TARGET OF RAPAMYCIN (mTOR) REDUCES CANCER PAIN, OPIOID TOLERANCE, AND HYPERALGESIA

BACKGROUND

Cancer, particularly metastatic bone tumor, produces intractable and persistent pain (Mercadante 1997; Bubendorf et al. 2000). Current treatment for cancer pain has had limited success, at least in part, due to an incomplete understanding of the mechanisms that underlie the induction and maintenance of cancer-related pain. Cancer-induced peripheral nerve and tissue damage leads to unique changes of neuronal plasticity in spinal dorsal horn and primary afferent neurons (Schwei et al. 1999; Clohisy and Mantyh 2003; Svendsen et al. 2005). These changes are thought to contribute to the generation and maintenance of cancer pain. Understanding the molecular mechanisms that underlie these changes can lead to the development of novel therapeutic strategies to treat cancer pain.

Opioids (such as morphine) remain the gold standard of pharmacologic treatment of moderate to severe pain in the clinical setting. Many patients, particularly those with advanced cancer and nerve injury, require long-term and high- dose morphine therapy. Achieving clinical efficacy and tolerability of such treatment regimens is often hindered by morphine-induced analgesic tolerance and abnormal pain. Adaptations of complex neuronal circuits in the nervous system in response to chronic morphine exposure are thought to be critical for the development and maintenance of morphine-induced tolerance and abnormal pain.

Recent studies revealed that increased protein translation plays a key role in regulating long-term changes of neuronal circuits. Thus, exploring translational control in chronic morphine tolerance may uncover novel mechanisms and open the door for development of new adjuvant drugs for use with morphine in treating chronic pain.

SUMMARY

In some aspects, the presently disclosed subject matter provides methods for preventing or treating opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain by administering a mammalian Target of Rapamycin (mTOR) inhibitor to a subject in an amount effective to prevent or treat opioid tolerance, opioid- induced hyperalgesia, and/or cancer pain.

In other aspects, the presently disclosed subject matter provides methods for preventing or treating opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain associated with the mu receptor//PI3K/Akt/mTOR signaling pathway, the method comprising administering an mTOR inhibitor to a subject in an amount effective to prevent or treat opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain associated with the mu receptor//PI3K/Akt/mTOR signaling pathway.

In still other aspects, the presently disclosed subject matter provides methods for preventing or treating chronic pain associated with the mu

receptor//PI3K/Akt/mTOR signaling pathway in a subject in need of treatment thereof, the method comprising administering an mTOR inhibitor to a subject in an amount effective to prevent or treat chronic pain associated with the mu

receptor//PI3K/Akt/mTOR signaling pathway.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 shows that intrathecal rapamycin (Rap), but not ascomycin (Asc), significantly and dose-dependently attenuated the decrease in morphine (Mor) antinociceptive effect in the development of morphine tolerance. Rapamycin alone did not affect the baseline morphine analgesic effect. ** P< 0.01 or * P<0.05 vs the corresponding morphine + vehicle (Veh);

FIG. 2 shows that intrathecal rapamycin (Rap), but not ascomycin (Asc), significantly attenuated decreases in paw withdrawal thresholds in response to mechanical stimulation (A) and paw withdrawal latencies in response to thermal stimulation (B) in the development of morphine tolerance. ** P< 0.01 or * P<0.05 vs the corresponding morphine + vehicle (Veh); FIG. 3 shows that intrathecal rapamycin (Rap), but not ascomycin (Asc), attenuated the decreases in morphine (Mor) analgesic effect (A), paw withdrawal threshold in response to mechanical stimuli (B), and paw withdrawal latency in response to thermal stimulation (C) during the maintenance period of chronic morphine tolerance. ** P < 0.01 or * P < 0.05 vs the corresponding baseline. ##P < 0.01 or # P < 0.05 vs the corresponding morphine + vehicle (Veh);

FIG. 4 shows the effect of spinal mTOR knockdown on chronic morphine (Mor) tolerance and abnormal pain: (A) Intrathecal mTOR siRNA knocks down mTOR, but not PSD-95, p70S6K, and 4E-BP1 in spinal cord. Top: an example of Western blot. Bottom: statistical analysis; (B and C) intrathecal mTOR siRNA attenuates the decreases in morphine analgesic effect (B) and paw withdrawal thresholds (C) in chronic morphine tolerance. Scramble (Scram) iRNA and PEI have no effects. ** P <0.01 vs saline group (A), 0.5 h (B), or baseline (C); ##P < 0.01 vs morphine alone;

FIG. 5 shows that intrathecal repeated injection of morphine (Mor), but not saline, increases phosphorylation of mTOR in L5, but not C2, spinal cord (A). This repeated injection time-dependently changes the levels of phosphorylation of mTOR, p70S6K, and 4E-BP1 in dorsal horn (C) but not in the dorsal root ganglion (B). Total expression levels of mTOR, p70S6K, and 4E-BP1 proteins are not altered after intrathecal repeated morphine injection (D);

FIG. 6 shows that: (A) morphine time-dependently induces expression of p- mTOR and p-p70S6K (top) and dose-dependently induces expression of p-mTOR, p- p70S6K, and p-4E-BP l (bottom) in cultured dorsal horn neurons; (B) DAMGO time- dependently induces expression of mTOR (top) and dose-dependently induces expression of p-mTOR and p-p7066K in cultured dorsal horn neurons; (C) The neurons were treated with vehicle (DMSO, 3 μί), 20 μΜ morphine (M), 10 μΜ CTAP (Ct, dissolved in DMSO), and Ct + Μμ; (D) The cells were treated with vehicle (V; saline, 4 μΐ), 20 μΜ DAMGO (D), 10 μΜ Ct, and Ct + D; E: The neurons were treated with vehicle, morphine, 20 μΜ BNI, BNI + morphine, 20 μΜ Nal, or Nal + morphine. N: Naive;

FIG. 7 shows the effect of CTAP (Ct) on chronic morphine tolerance (A) and activation of dorsal horn mTOR, p70S6K, and 4E-BP1 (B) during the development period (S: saline; V: vehicle; M: morphine; N 5-7/group; P < 0.05 vs morphine + vehicle); FIG. 8 shows that PI3K and Akt participate in morphine-and DAMGO- induced activation of mTOR and downstream effectors: (A) the neurons were treated with vehicle (V; 4 μΐ, of 20% DMSO), morphine (M), Ly294002 (Ly), and Ly + M; (B) the neurons were treated with vehicle, M, Akt inhibitor IV (Ak), and Ak + M; (C) the neurons were treated with vehicle, DAMGO (D), Akt, and Ak + M; (D) the neurons were treated with vehicle, DAMGO (D), Ly, and Ly + M (N: Naive);

FIG. 9 shows the involvement of spinal PI3K and Akt in chronic morphine tolerance and activation of dorsal horn mTOR and p70S6K during the development period. The rats received i.th. injections of saline (S) + vehicle (V), morphine (M) + vehicle (V), Ly294002 (Ly) + S, Akt inhibitor IV (Ak) + S, Ly + M, or Ak + M: (A) behavioral test; and (B and C) Western blots. N = 3/group;

FIG. 10 shows that rapamycin does not affect proliferation of splenocytes in response to ConA or LPS. N= 5/group;

FIG. 1 1 shows the establishment of cancer-related pain model in male rats: (A and B) effect of AT-3.1 prostate cancer cells (PCCs; 4.5 x 10⁵ cells/ 15 Hank's solution) or Hank's solution injection into the tibia on paw withdrawal threshold (A) and latency (B) on the ipsilateral (ipsi) and contralateral (con) sides. Mechanical allodynia and thermal hyperalgesia were observed on the ipsilateral, but not contralateral, side after PCC injection. N = 6/group. ** P< 0.01 vs the corresponding Hanks -injected side; (C) Radiographs of tibiae 12 days after PCC or Hank's injection. Arrowhead shows the injection site. Arrows indicate structural destruction of the proximal cortical bone; (D) HE staining of the proximal cortical bones 12 days after PCC or Hank's injection. Note that tumor cells were densely packed in the narrow cavity and induced the destruction of trabeculae. Scale bar: 150 μιη;

FIG. 12 shows the effect of intrathecal administration of rapamycin on

PCC(P)-induced mechanical allodynia and thermal hyperalgesia during development: (A and B) Pre-treatment of rapamycin (Rap), but not of ascomycin (Asc), completely blocked PCC-induced decreases in paw withdrawal threshold (A) and latency (B) on the ipsilateral side. Neither rapamycin nor ascomycin affected basal responses to mechanical (A) and thermal (B) stimuli on the contralateral side in PCC-treated rats or on either side in Hanks (H)-treated rats (A and B). V: vehicle (50% DMSO). N = 5/group;

FIG. 13 shows the dose-dependent effect of intrathecal rapamycin (Rap) on PCC(P)-induced mechanical allodynia and thermal hyperalgesia during development: PCC-induced decreases in paw withdrawal threshold (A) and latency (B) on the ipsilateral side were significantly attenuated by rapamycin at two higher doses (1 and 10 μg), but not at a lower dose (0. 1 μg). N = 5/group. * P < 0.05, ** P < 0.01 vs the corresponding vehicle (V)-treated group;

FIG. 14 shows the effect of intrathecal administration of rapamycin on

PCC(P)-induced mechanical allodynia and thermal hyperalgesia during maintenance: (A and B) Post-treatment of rapamycin ( Rap), but not of ascomycin (Asc), significantly attenuated PCC-induced decreases in paw withdrawal threshold (A) and latency (B) on the ipsilateral side. Neither rapamycin nor ascomycin affected basal responses to mechanical (A) and thermal (B) stimul i on the contralateral side in PCC- treated rats or on either side in Hanks ( H (-treated rats (A and B). V: vehicle (50% DMSO ). N = 6/group;

FIG. 15 shows the dose-dependent effect of intrathecal rapamycin ( Rap) on PCC( P (-induced mechanical al lodynia and thermal hyperalgesia during maintenance. PCC-induced decreases in paw withdrawal threshold (A) and latency ( B ) on the ipsilateral side were significantly attenuated by rapamycin at two higher doses (1 ng and 10 Lig ), but not at a lower dose (0. 1 iig ). N = 6/group. * P < 0.05, ** P < 0.01 vs the corresponding vehicle (V)-treated group;

FIG. 16 shows time-dependent activation of L₄„s dorsal horn mTOR and p70S6K after PCC injection: (A and B) The levels of phosphorylated (p-) mTOR (A) and p-p70S6K (B) on the ipsilateral side were significantly increased on days 3, 5, 7, and 12 post-PCC in jection. The levels of neither p-mTOR (C) nor p-p70S6K ( D ) on the contralateral side were changed after PCC injection. PCC injection did not alter expression of total mTOR (A and C) and total p70S6K (B and D) in L .5 dorsal horns on either side. N = 5/group. * P < 0.05, * * P < 0.01 vs the corresponding na^'ive rats

(0);

FIG. 1 7 shows transient activation of L4.5 DRG mTOR and p70S6K after PCC injection: (A and B) The levels of phosphorylated (p-) mTOR (A) and p-p70S6K (B) on the ipsilateral side were significantly increased only on day 3 post-PCC injection. The levels of neither p-mTOR (C) nor p-p70S6K (D) on the contralateral side were changed after PCC injection. PCC injection did not alter expression of total mTOR (A and C) and total p70S6K ( B and D ) on either side. N = 5/group. * P < 0.05, ** P < 0.01 vs the corresponding na^'ive rats (0); FIG. 18 shows the effect of blocking NMDA receptors on PCC (P)-induced mechanical allodynia, thermal hyperalgesia, and activation of dorsal horn mTOR and P-70S6K: (A to D) Intrathecal infusion of DL-AP5 (AP5) completely blocked PCC- induced decreases in paw withdrawal threshold (A) and latency (C) on the ipsilateral side. DL-AP5 did not affect basal responses to mechanical (B) and thermal (D) stimuli on the contralateral side in PCC-treated rats or on either side in Hanks (Entreated rats (A to D). H: Hanks. S: saline. N = 5/group. E and F: Intrathecal infusion of DL-AP5 significantly repressed PCC-induced increases in the levels of p-mTOR (E) and p-p70S6K (F) in dorsal horn on day 7 post-PCC injection. N = 5/group. ** P < 0.01 vs the corresponding group with Hank's (H) injection and saline (S) infusion. ## P < 0.01 vs the corresponding group treated with PCC (P) injection and saline infusion;

FIG. 19 shows co-localization of NMDA receptor subunit Rl with mTOR and p70S6K in dorsal horn neurons. Immunofluorescent co-localization (arrows) of red reaction products for mTOR and p70S6K and green products for NR 1. Scale bar: 30 μΜ;

FIG. 20 shows that systemic administration of rapamycin attenuated the development (A) and maintenance (B) of chronic morphine tolerance. (A) Mice received subcutaneous injection of 20 mg/kg morphine (M) twice a day and intraperitoneal injection of 2.5 mg/kg rapamycin (Rap) or vehicle (50% DMSO) once a day for 5 days. Tail-flick test was carried out before morphine injection, and 30 min after morphine injection in the morning of day 1, 3 and 5. N = 5/group. ** p < 0.01 vs the morphine and vehicle group at the corresponding time point. (B) Mice received subcutaneous injection of 20 mg/kg twice a day for 10 days and also intraperitoneal injection of 2.5 mg/kg rapamycin or vehicle starting on day 6 post-morphine injection. Tail-flick test was carried out before morphine injection, and 30 min after morphine injection in the morning of day 1, 3, 5, 8, and 10. N = 5/group, **p < 0.01 vs the morphine and vehicle group at the corresponding time points;

FIG. 21 shows that systemic administration of rapamycin attenuated the development (A) and maintenance (B) of morphine-induced cold allodynia. (A) Mice received subcutaneous injection of 20 mg/kg morphine (M) twice a day and intraperitoneal injection of 2.5 mg/kg rapamycin (Rap) or vehicle (50% DMSO) once a day for 5 days. Cold plate test was carried out prior to morphine injection and day 6 after morphine injection. N = 5/group. ** p < 0.01 vs the morphine + vehicle group at the corresponding time point. (B) Mice received subcutaneous injection of 20 mg/kg twice a day for 10 days and also intraperitoneal injection of 2.5 mg/kg rapamycin or vehicle starting on day 6 post-morphine injection. Cold plate test was carried out prior to morphine injection, in the morning of day 6 before morphine injection, and day 11 after morphine injection. N = 5/group, **p < 0.01 vs the morphine + vehicle group at the corresponding time point;

FIG. 22 shows that systemic administration of rapamycin attenuated the development (A) and maintenance (B) of morphine-induced mechanical allodynia. (A) Mice received subcutaneous injection of 20 mg/kg morphine (M) twice a day and intraperitoneal injection of 2.5 mg/kg rapamycin (Rap) or vehicle (50% DMSO) once a day for 5 days. Mechanical test was carried out prior to morphine injection and day 6 after morphine injection. N = 5/group. * p < 0.05 vs the morphine + vehicle group at the corresponding time point. (B) Mice received subcutaneous injection of 20 mg/kg twice a day for 10 days and also intraperitoneal injection of 2.5 mg/kg rapamycin or vehicle starting on day 6 post- morphine injection. Mechanical test was carried out prior to morphine injection, in the morning of day 6 before morphine injection, and day 11 after morphine injection. N = 5/group, *p < 0.05 vs the morphine + vehicle group at the corresponding time point; and

FIG. 23 shows that mu receptor (MOR) mRNA detected by using in situ hybridization histochemistry co-localizes with p-mTOR and mTOR detected by using immunohistochemistry in dorsal horn neurons.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

I. Spinal Mammalian Target of Rapamycin (mTOR) Inhibition

Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase.

Its activation, especially in a complex sensitive to rapamycin (mTOR complex 1), promotes the phosphorylation of mTOR downstream effectors, such as p70 ribosomal S6 protein kinase (p70S6K), and governs mRNA translation (Gingras et al. 1999; Hay and Sonenberg 2004; Costa-Mattioli et al. 2009). mTOR plays an important role in the modulation of long term plasticity, memory processes, and spinal cord central sensitization. Mice with deletion of mTOR downstream effectors exhibit deficits in synaptic plasticity and long-term memory (Banko et al. 2005; Antion et al. 2008; Costa-Mattioli et al. 2009). mTOR and its downstream effectors are expressed in dorsal root ganglion (DRG) and spinal cord dorsal horn, two major pain-related regions (Jimenez-Diaz et al. 2008; Geranton et al. 2009; Xu et al. 2010). Intrathecal (i.th) administration of rapamycin, a specific inhibitor of mTOR, produced anti- nociception in models of nerve injury and inflammation (Price et al. 2007; Jimenez- Diaz et al. 2008; Geranton et al. 2009; Asante et al. 2009; Norsted et al. 2010; Xu et al. 2011). Local perfusion of rapamycin into spinal cord significantly reduced formalin-induced neuronal hyperexcitability in dorsal horn (Asante et al. 2009).

A. mTOR inhibitors

The mammalian target of rapamycin (mTOR), also known as mechanistic target of rapamycin or FRAP 1 , is a serine/threonine protein kinase that affects neuronal plasticity, cell growth, proliferation, motility, and survival. Because it is a protein kinase, it is capable of regulating other proteins by modulating their phosphorylation. In addition, mTOR itself is activated when it is phosphorylated. mTOR also regulates other proteins by governing mRNA translation.

The presently disclosed subject matter provides mTOR inhibitors for use in the methods of the presently disclosed subject matter. Particular embodiments include rapamycin, sirolimus, temsirolimus, everolimus, derivatives of rapamycin, and isomers of rapamycin. Rapamycin is a compound that has been assigned the generic name sirolimus and the trade-mark name Rapamune. Temsirolimus is an intravenous derivative of rapamycin and everolimus an oral derivative of rapamycin. It also has been found herein that siRNA (small interfering RNA, short interfering RNA, or silencing RNA) of mTOR can be used as an mTOR inhibitor. siRNA is a class of short double-stranded RNA molecules that interfere with the expression of specific genes with a complementary nucleotide sequence. Therefore, in some embodiments, the presently disclosed subject matter provides methods that use siRNA of mTOR as an mTOR inhibitor.

In some embodiments, the mTOR that is inhibited is a spinal mTOR. mTOR and its downstream effectors are expressed in dorsal root ganglion (DRG) and spinal cord dorsal horn. The presently disclosed subject matter suggests that spinal mTOR is critical for the maintenance of opioid tolerance, hyperalgesia, and/or cancer pain and is involved in chronic pain.

The method of administering the mTOR inhibitor varies depending on the circumstances. Depending on the specific conditions being treated, such inhibitors may be formulated into liquid or solid dosage forms and administered systemically or locally. In a particular embodiment, administration of the mTOR inhibitor is by intrathecal injection, without any observable side effects. In particular embodiments, the subject that is administered the mTOR inhibitor is a mammalian subject, such as a human subject.

B. Methods to Prevent or Treat Opioid Tolerance, Hyperalgesia, and/or Cancer Pain

Opioids are chemical substances that provide pain relief. Opioid receptors are found principally in the central and peripheral nervous system and the gastrointestinal tract. Examples of opioids include natural opiates, such as morphine and codeine, semi-synthetic opiates, such as buprenorphine, hydrocodone, oxycodone,

hydromorphone, and oxymorphone, and fully synthetic opiates, such as fentanyl, tramadol, and methadone, and derivatives thereof.

Opioids are commonly used for the treatment of chronic pain. Repeated injection of opioids, such as morphine, however, leads to opioid tolerance, and hyperalgesia. Therefore, increasing doses of opioid must be given to treat the pain, which results in increased severe side effects of the opioid to a subject. Repeated opioid injection can lead to neuroadaptive changes of cellular signals in the spinal cord that is thought to contribute to central mechanisms underlying chronic opioid tolerance and opioid-induced hyperalgesia. Such changes are determined by increased protein synthesis. In some embodiments, the presently disclosed subject matter provides methods for preventing or treating opioid tolerance, hyperalgesia, and/or cancer pain by administering an mTOR (mammalian Target of Rapamycin) inhibitor to a subject. The administration of the mTOR inhibitor attenuates the development and maintenance of opioid tolerance, hyperalgesia, and/or cancer pain. In a particular embodiment, the presently disclosed subject matter provides methods to prevent or treat opioid tolerance, hyperalgesia, and/or cancer pain, wherein the opioid is morphine.

Opioid tolerance refers to the physiological state characterized by a decrease in the effects of the opiate with chronic administration. The mTOR inhibitor of the presently disclosed subject matter can be administered along with an opioid, it can be administered prior to administering an opioid, or it can be administered after administering an opioid to a subject. The methods of the presently disclosed subject matter include methods for preventing opioid tolerance, hyperalgesia, and/or cancer pain, such as when an mTOR inhibitor is administered before opioid tolerance, hyperalgesia, and/or cancer pain sets in, or can include methods to treat opioid tolerance, hyperalgesia, and/or cancer pain such as when an mTOR inhibitor is administered after opioid tolerance, hyperalgesia, and/or cancer pain is found in a subject. More than one mTOR inhibitor can be administered simultaneously or sequentially.

It has been found herein that repeated opioid injection increases the levels of phosphorylation of mTOR, but not the total expression levels of the mTOR protein. It also has been found that mTOR inhibitors can decrease the activity of mTOR by decreasing the levels of phosphorylated mTOR after repeated administration of opioid.

Therefore, in some embodiments, the presently disclosed subject matter provides methods for preventing or treating opioid tolerance, hyperalgesia, and/or cancer pain by using mTOR inhibitors that decrease the activity of mTOR. In some embodiments, administering the mTOR inhibitor results in a decrease in the phosphorylation of mTOR. In other embodiments, administering the mTOR inhibitor results in a decrease in the activity of mTOR. In still other embodiments, administering the mTOR inhibitor results in a decrease in mTOR protein kinase activity. Particular embodiments of mTOR inhibitors for use in preventing or treating opioid tolerance, hyperalgesia, and/or cancer pain include rapamycin, sirolimus, temsirolimus, everolimus, derivatives of rapamycin, and isomers of rapamycin.

It also has been found herein that siR A (small interfering R A, short interfering RNA, or silencing RNA) of mTOR can be used as an mTOR inhibitor. siRNA is a class of short double-stranded RNA molecules that interfere with the expression of specific genes with a complementary nucleotide sequence. Therefore, in some embodiments, the presently disclosed subject matter provides methods for preventing or treating opioid tolerance, hyperalgesia, and/or cancer pain by administering an mTOR inhibitor to a subject in an amount effective to prevent or treat opioid tolerance, hyperalgesia, and/or cancer pain, wherein the mTOR inhibitor is an siRNA of mTOR.

mTOR and its downstream effectors are expressed in dorsal root ganglion (DRG) and spinal cord dorsal horn. The presently disclosed subject matter suggests that spinal mTOR is critical for the development and maintenance of opioid tolerance, hyperalgesia and/or cancer pain, particularly morphine. In some embodiments, the mTOR that is inhibited is spinal mTOR. In further embodiments, the mTOR inhibitor prevents or treats opioid tolerance, hyperalgesia, and/or cancer pain in dorsal horn neurons.

In further embodiments, the opioid tolerance, hyperalgesia, and/or cancer pain is associated with the novel signal pathway of mu receptor/PI3K/Akt/mTOR in dorsal horn neurons. Activation of the mu receptors (also called MOR or MOP receptors), a class of opioid receptors with a high affinity for enkephalins and β-endorphin, but low affinity for dynorphins, has been shown to activate the PI3K family of enzymes (phosphatidylinositol 3 -kinases or PI 3 -kinases), as well as Akt (also known as

Protein Kinase B or PKB). Activation of PI3K and Akt has been shown to result in an increased phosphorylation of mTOR and its downstream effectors, p70S6K and 4E- BP1. The increased phosphorylation of mTOR and its downstream effectors, p70S6K and 4E-BP1, increases the activity of PI3K, Akt, p70S6K and 4E-BP1.

Therefore, in some embodiments, the presently disclosed subject matter provides a method for preventing or treating opioid tolerance, hyperalgesia, and/or cancer pain, wherein the opioid tolerance, hyperalgesia, and/or cancer pain is associated with the mu receptor/P13K/Akt/mTOR signaling pathway and blocking the mu receptor/P13K/Akt/mTOR signaling pathway reduces morphine-induced activation of mTOR, p70S6K and 4E-BP 1. Hence, a method is provided wherein administering the mTOR inhibitor results in a decrease in the activity of the mu receptor. Another method is provided wherein administering the mTOR inhibitor results in a decrease in the activity of the P13K protein. In one embodiment, administering the mTOR inhibitor results in a decrease in the activity of the Akt protein. In a further embodiment, administering the mTOR inhibitor results in a decrease in the phosphorylation of p70S6K. In still another embodiment, administering the mTOR inhibitor results in a decrease in the phosphorylation of 4E- BP1. In some embodiments, the mTOR inhibitor prevents or treats opioid tolerance, hyperalgesia, and/or cancer pain in dorsal horn neurons.

In other embodiments, methods are provided to inhibit the development of opioid tolerance, hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment by reducing the activity of spinal PI3K in neurons to reduce activation of spinal mTOR and its downstream effectors the method comprising administering an amount of an inhibitor of PI3K to effectively reduce the activity of PI3K, wherein reduction of the activity of PI3K reduces the activity of spinal mTOR and its downstream effectors and slows development of opioid tolerance,

hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment.

In still other embodiments, methods are provided to inhibit development of opioid tolerance, hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment by reducing the activity of spinal Akt in neurons to reduce activation of spinal mTOR and its downstream effectors comprising the steps of administering an amount of an inhibitor of Akt to effectively reduce the activity of Akt, wherein reduction of the activity of Akt reduces the activity of spinal mTOR and its downstream effectors and slows development of opioid tolerance, hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment.

In further embodiments, methods are provided to inhibit development of opioid tolerance, hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment by reducing the activity of mu receptors in spinal neurons to reduce activation of spinal mTOR and its downstream effectors comprising the steps of administering an amount of a mu receptor antagonist to effectively reduce the activation of spinal mTOR and its downstream effectors, wherein reduction of the activation of mu receptors reduces development of opioid tolerance, hyperalgesia, and/or cancer pain in a subject that is receiving chronic opioid treatment.

In some embodiments, the downstream effectors of spinal mTOR are members of the group consisting ofP70S6K and 4E-BP1.

C. Methods for Preventing or Treating Chronic Pain

Chronic pain is pain that lasts for a long time, such as about three months or longer, six months or longer, or twelve months or longer. Chronic pain also can be described as pain that extends beyond the expected period of healing. Chronic pain can be caused by many different diseases or disorders. In some embodiments of the presently disclosed subject matter, chronic pain is caused by cancer in a subject.

In other embodiments, chronic pain is due to allodynia, pain caused by a stimulus that does not normally provoke pain. For example, the allodynia may be mechanical or tactile allodynia, which is pain in response to a light touch or pressure, or it may be thermal allodynia, which is pain from normally mild skin temperatures in the affected area. In still other embodiments, the chronic pain is caused by hyperalgesia, which is an increased sensitivity to pain. Types of hyperalgesia include, but are not limited to, primary hyperalgesia, which is pain sensitivity that occurs directly in damaged tissues, for example by damage to nociceptors or peripheral nerves, and secondary hyperalgesia, which is pain sensitivity that occurs in surrounding undamaged tissues. Opioid-induced hyperalgesia may develop as a result of long term opioid use in the treatment of chronic pain. In a particular embodiment, the hyperalgesia is thermal hyperalgesia and therefore is in response to heat.

In still other embodiments, pain hypersensitivity is caused by the development and maintenance of cancer pain, nerve injury-induced chronic neuropathic pain, or by chronic inflammatory pain, such as by the development and maintenance of complete Freund's adjuvant-induced chronic inflammatory pain.

As disclosed hereinabove, opioid tolerance is associated with the novel signal pathway of mu receptor/PI3K/Akt/mTOR. It has further been found in Examples described herein below, that the mTOR inhibitors of the presently disclosed subject matter can be used for preventing or treating chronic pain associated with the mu receptor/PI3K/Akt/mTOR signaling pathway. In some embodiments, the presently disclosed subject matter provides methods that can be used as adjuvant drugs to reduce the dose of opioids used in the treatment of chronic neuropathic pain and cancer pain. In some embodiments, the presently disclosed methods comprise

administering an mTOR inhibitor to a subject in an amount effective to prevent or treat chronic pain associated with the mu receptor/PI3K/Akt/mTOR signaling pathway. Similarly to the methods described herein for the use of mTOR inhibitors to attenuate the effects of repeated opioid administration, these methods also can be used to prevent or treat chronic pain associated with mu receptor/PI3K/Akt/mTOR signaling pathway.

In some embodiments, the mTOR inhibitors can be used as adjuvant drugs of an opioid, such as morphine, in the treatment of chronic pain. In other embodiments, the mTOR inhibitors are given to the subject prophylactically before chronic pain occurs, especially for subjects that are expected to have chronic pain due to a disease, disorder, or dysfunction. In some embodiments, the chronic pain is associated with the mu receptor/PI3K/Akt/mTOR signaling pathway in dorsal horn neurons.

The presently disclosed subject matter also provides methods that include the involvement of the NMDA (N-methyl-D-aspartate) receptor, a specific type of ionotropic glutamate receptor. Calcium flux through the NMDA receptor is thought to be critical in synaptic plasticity, a cellular mechanism for learning and memory. The increases in dorsal horn could be abolished by intrathecal infusion of DL-AP5, an NMDA receptor antagonist. Examples provided herein show that a NMDA receptor subunit is co-localized with mTOR and p70S6K. These findings suggest that the activation of the mTOR pathway is mediated by NMDA receptors. As such, methods are provided for preventing or treating chronic pain associated with the mu receptor/PI3K/Akt/mTOR signaling pathway, the methods comprising administering an mTOR inhibitor to a subject in an amount effective to prevent or treat chronic pain associated with the mu receptor/PI3K/Akt/mTOR signaling pathway, wherein there is a decrease in activity of an NMDA receptor.

In some embodiments, the mTOR inhibitor is administered along with an opioid or prior to administration of an opioid to a subject. In other embodiments, the mTOR inhibitor is administered after administration of an opioid to a subject. In some embodiments, the opioid is morphine.

In some embodiments, administration of the mTOR inhibitor is by intrathecal injection. In other embodiments, the mTOR inhibitor prevents or treats chronic pain in dorsal horn neurons. In some embodiments, the subject is human. In some embodiments, the mTOR inhibitor prevents chronic pain due to allodynia, such as mechanical allodynia. In other embodiments, the chronic pain is due to hyperalgesia, such as thermal hyperalgesia. II. Administration of mTOR Inhibitors

The mTOR inhibitors of the presently disclosed subject matter can be administered alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient. The term

"pharmaceutically-acceptable excipient" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration into a subject.

The mTOR inhibitors are effective over a wide dosage range. For example, in treating adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained- low release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20^th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articullar, intra -sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery. For injection, the agents may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate the mTOR inhibitors for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the mTOR inhibitors, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as, saline, preservatives, such as benzyl alcohol, absorption promoters, and fluorocarbons.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium

carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked

polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye- stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler, such as lactose, binders, such as starches, and/or lubricants, such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

As used herein, the terms "treat," treating," "treatment," and the like, are meant to decrease, suppress, attenuate, diminish, arrest, the underlying cause of a disease, disorder, or condition, or to stabilize the development or progression of a disease, disorder, condition, and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease, disorder or condition does not require that the disease, disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition. Thus, in some embodiments, an agent can be administered prophylactically to prevent the onset of a disease, disorder, or condition, or to prevent the recurrence of a disease, disorder, or condition.

The term "therapeutic agent" means a substance that has the potential of affecting the function of an organism. Such an agent may be, for example, a naturally occurring, semi-synthetic, or synthetic agent. For example, the therapeutic agent may be a drug that targets a specific function of an organism. A therapeutic agent also may be an antibiotic or a nutrient. A therapeutic agent may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of disease, disorder, or condition in a host organism. The term "effective amount" of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. More particularly, the term " effective amount" refers to an amount sufficient to produce the desired effect, e.g., to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence,

development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

An effective amount of a compound according to the presently disclosed methods can range from, e.g., about 0.001 mg/kg to about 1000 mg/kg, or in certain embodiments, about 0.01 mg/kg to about 100 mg/kg, or in certain embodiments, about 0.1 mg/kg to about 50 mg/kg. Effective doses also will vary, as recognized by those skilled in the art, depending on the disorder treated, route of administration, excipient usage, the age and sex of the subject, and the possibility of co-usage with other therapeutic treatments, such as use of other agents. It will be appreciated that an amount of a compound required for achieving the desired biological response may be different from the amount of compound effective for another purpose.

By "in combination with" is meant the administration of an mTOR inhibitor with one or more therapeutic agents and/or with another type of mTOR inhibitor either simultaneously, sequentially, or a combination thereof. Therefore, a cell or a subject administered an mTOR inhibitor can receive one or more therapeutic agents and/or other type of mTOR inhibitor at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the cell or the subject. When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as for treating an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs;

lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a subject" includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about," when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term "about" when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

EXAMPLE 1

Inhibition of spinal mTOR attenuates the development of morphine tolerance and mechanical allodynia and thermal hyperalgesia induced by

repeated i.th. morphine injection

To determine whether spinal mTOR is involved in the development of morphine tolerance and abnormal pain induced by repeated intrathecal (i.th.) morphine injection in rats, a polyethylene (PE-10) tube was inserted into the subarachnoid space at the rostral level of the spinal cord lumbar enlargement segment through an incision at the atlanto-occipital membrane. A specific mTOR inhibitor, rapamycin, was given i.th.. Ascomycin was used as a negative control.

The model of chronic tolerance to repeated i.th. morphine in rats was made. Briefly, rapamycin (dissolved in 50% DMSO; LC Laboratories, Woburn, MA), ascomycin (dissolved in 50% DMSO; LC laboratories), and vehicle (50% DMSO in distilled water) were given i.th. once daily (8:00 A.M.) for 7 days, whereas saline (10 μί) and morphine sulfate (10 μg/10 dissolved in saline; Elkins-Sinn, Inc., Cherry Hill, NJ) were injected i.th. twice daily at 12-h intervals (8:30 A.M and 8:30 P.M.) for 7 days. Five groups of rats were used, including (1) vehicle (10 μΚ) + saline, (2) vehicle (10 μί) + morphine, (3) 1 μg/10 μΐ_^ rapamycin + morphine, (4) 5 μg/10 μΐ_^ rapamycin + morphine, (5) 10 μg/10 μΐ_^ rapamycin + morphine, (6) 10 μg/10 μΐ_^ rapamycin + saline, and (7) 10 μg/10 μϊ_^ ascomycin + morphine. The tail flick test for evaluating morphine analgesic effect was performed 1 day before drug injection and at 0.5 h after i.th. injection of morphine in the morning on days 1, 3, 5, and 8. Paw withdrawal thresholds in response to mechanical stimuli and paw withdrawal latencies in response to thermal stimulation were carried out bilaterally before drug injection and 2 h before the tail-flick test on day 8. In addition, locomotor function testing was performed.

Consistent with previous studies, rats that received repeated i.th. morphine injections developed tolerance to the antinociceptive effect of morphine, mechanical allodynia, and thermal hyperalgesia (FIGS. 1-2). It was found that rapamycin, but not ascomysin, dose-dependently attenuated the decreases in morphine antinociceptive effect (FIG. 1), paw withdrawal threshold, and paw withdrawal latency (FIG. 2) during the development period. The rats treated i.th. with saline (10 μΐ, twice daily) plus rapamycin (10 μg/10 μΐ, once daily) or ascomycin (10 μg/10 μΐ, once daily) for 7 days did not show any significant changes in tail-flick latencies, paw withdrawal thresholds, or paw withdrawal latencies compared to baselines (FIGS. 1-2). Neither rapamycin nor ascomycin at the dose used affected motor function (including placing, righting, and grasping reflexes; Table 1). These observations indicate that spinal mTOR might participate in the development of morphine tolerance and associated abnormal pain. Accordingly, intrathecal pre-treatment of mTOR inhibitors delays the development of chronic morphine tolerance.

N= 3/ group; 5 trials

EXAMPLE 2

Inhibition of spinal mTOR prevents the maintenance of chronic morphine tolerance mechanical allodynia. and thermal hyperalgesia induced by

repeated i.th. morphine injection

To determine the role of spinal mTOR in the maintenance of morphine tolerance and abnormal pain induced by repeated i.th. morphine injection, the rats were injected i.th. with morphine sulfate (10 μg /10 μί) twice daily at 12-h intervals (8:30 A.M and 8:30 P.M) for 12 days; rapamycin, ascomycin, or vehicle was given i.th. once daily (8:00 A.M.) for 5 days beginning on day 8 of morphine injection. Three groups of rats were used: (1) vehicle (10 μί) + morphine, (2) 10 μg/10 μΐ_^ rapamycin + morphine, and (3) 10 μg/10 μϊ_^ ascomycin + morphine. The tail-flick test was performed 1 day before i.p. morphine injections were begun and at 0.5 h after i.p. morphine injection on mornings 8, 10, and 13. Paw withdrawal thresholds in response to mechanical stimuli and paw withdrawal latencies in response to thermal stimulation were conducted 1 day before morphine injection and 2 h before the tail- flick test on days 8 and 13.

Consistent with the previous results provided hereinabove, morphine tolerance, mechanical allodynia, and thermal hyperalgesia were significantly induced by day 8 and maintained for at least for 5 days after repeated i.th. morphine injection (FIG. 3). Rapamycin, but not ascomycin, prevented the decreases in morphine antinociceptive effects (FIG. 3), paw withdrawal thresholds, and pain withdrawal latencies (FIG. 3) during the maintenance period. These results suggest that spinal mTOR is critical for the maintenance of morphine tolerance and associated abnormal pain. Accordingly, intratheral post-treatment of mTOR inhibitors could be used as an adiuvant drug to reduce the dose of morphine used in treatment of chronic pain.

EXAMPLE 3

Knockdown of spinal mTOR represses the development

of chronic morphine tolerance

To further confirm the role of spinal mTOR in the development of chronic morphine tolerance, an mTOR siRNA duplex was designed corresponding to bases 2162-2182 from the open reading frame of rat mTOR mRNA 5'- UGAACCCUGCCUUUGUCAUGC-3' (rapamycin binding site; GenBank accession number NM_019906). A mismatch siRNA with scrambled sequence and no known homology to a rat gene (scrambled siRNA: 5'- UACUUUUCGGUCCCGACGUCATT-3'/5'-UGACGUC

GGGACCGAAAAGUATT-3') was used as a control. All siRNAs were synthesized by Dharmacon, Inc (Chicago, IL). To deliver siRNA into cells, polyethyleneimine (PEI; Fermentas Life Sciences, Glen Burnie, MD), a cationic polymer, was used as a delivery vehicle to prevent degradation and enhance cell membrane penetration of siRNA. siRNAs and vehicle were injected i.th.

The rats received i.th. injections of morphine (10 μg/10 μί) twice daily for 7 days beginning day 5 after siRNA injection. After behavioral testing on day 8 post- morphine, the tissues were collected. mTOR expression was detected using Western blotting analysis. The data showed that mTOR siRNA (5 μg/10 μί) significantly decreased the level of mTOR protein (but not of p70S6K or β-actin) in the L₄_5 segments (but not in cervical segments) of spinal cord compared with that of naive rats or rats given the scrambled siRNA (5 μg/10 μί) or vehicle (FIG. 4). Behavioral observation revealed that mTOR siRNA, but not scrambled siRNA or vehicle, attenuated the decrease of morphine antinociceptive effect (FIG. 4). Neither mTOR siRNA nor vehicle alone markedly altered basal tail-flick latency (FIG. 4) or affected motor function (Table 2). These findings further support the role of spinal mTOR in the development of chronic morphine tolerance.

EXAMPLE 4

Activation of spinal mTOR and its downstream effectors in dorsal horn neurons after repeated i.th. morphine injection

To examine whether mTOR and its downstream effectors (p70S6K and 4E- BP1) were activated in dorsal horn after repeated i.th. morphine injection, Western blot was first used to detect the levels of their phosphorylation in dorsal horn. The chronic morphine tolerance model and i.th. catheter implantation were prepared as described above. The fifth lumbar (L₅) spinal cord, the second cervical (C₂) spinal cord, and bilateral L₄_5 DRGs were collected at different time points (FIG. 5). Six polyclonal rabbit antibodies (all from Cell Signaling Technology), including anti- mTOR (1 : 1,000), anti-phosphorylated (p)-mTOR (Serine 2448; 1 : 1,000), anti- p70S6K (1 : 1,000), anti-p-p70S6K (threonine 421/serine 424; 1 : 1,000), anti-4E-BPl (1 : 1,000), and anti-p-4E-BP 1 (serine 65; 1 : 1,000), were used. Basal levels of p- mTOR, p-p70S6K, and p-4E-BPl were very low in dorsal horn (FIG. 5A).

Repeated i.th. infections of morphine (Mor), but not saline, increased the level of p-mTOR in L₅ spinal cord, but not in C₂ spinal cord (FIG. 5 A) or DRG (FIG. 5B) after 8 days. Repeated i.th. injections of morphine also time-dependently increased p- mTOR, p-p70S6K, and p-4E-BPl in L₅ spinal cord (FIG. 5C), but not in the dorsal root ganglion (FIG. 5B). These increases started on day 1, reached a peak on day 5, and persisted for at least 8 days. Interestingly, chronic morphine exposure did not significantly affect the expression of total mTOR, p70S6K, or 4E-BP 1 proteins in dorsal horn (FIG. 5C).

EXAMPLE 5

Activation of spinal mTOR. p70S6K. and 4E-BP1 repeated i.th. morphine injection requires spinal mu receptor

Using an in vitro dorsal horn neuronal culture, it was first examined whether spinal mu receptor activation induced activation of mTOR and downstream effectors in dorsal horn neurons. The cultured neurons were challenged by morphine, DAMGO (a selective mu receptor agonist), and CTAP (a selective mu receptor antagonist). Morphine and DAMGO led to time-dependent and dose-dependent increases in expression of p-mTOR, p-p70S6K, and p-4E-BPl (FIG. 6). Neither morphine nor DAMGO affected expression of total mTOR, p70S6K, or 4E-BP 1 proteins (data not shown). Pre-treatment with CTAP (10 μΜ) abolished morphine- and DAMGO- induced increases in p-mTOR, p-p70S6K, and p-4E-BPl (FIG. 6). CTAP alone did not affect basal expression of p-mTOR, p-p70S6K, and p-4E-BPl (data not shown). The results indicate that stimulation of spinal mu receptor can activate mTOR and its two downstream effectors.

To further define whether morphine-induced activation of mTOR signal pathway is specifically mediated by mu receptor in dorsal horn neurons, the effect of blocking spinal delta and kappa receptor on morphine-induced activation of mTOR, p70S6K, and 4E-BP 1 in dorsal horn neurons was examined. Cultured dorsal horn neurons were challenged by morphine, BNI (a selective kappa receptor antagonist), or naltrindole (Nal, a delta receptor antagonist). Morphine (20 μΜ) produced a significant increase in expression of p-mTOR, p-p70S6K, and p-4E-BPl (FIG. 6E). Neither BNI nor naltrindole affected morphine-induced increases in their expression (FIG. 6E). These data suggest that morphine-stimulated activation of mTOR, p70S6K, and 4E-BP1 in dorsal horn neurons might not require activation of spinal delta and kappa receptors.

Finally, the role of spinal mu receptor in the activation of spinal mTOR and its downstream effectors following repeated i.th. morphine injection in vivo was examined. The rats received i.th. injections of saline (10 μί, twice daily) + vehicle (10 μΐ_^ saline, once daily); morphine (10 μg/10 μΐ_^, twice daily) + vehicle; saline + CTAP (0.1 ng/10 μΐ_^, dissolved in saline, once daily); or morphine + CTAP for 7 days. After the behavioral tests on day 8, L₄_₅ segments were collected for Western blot analysis.

CTAP prevented the development of morphine tolerance (FIG. 7). More importantly, CTAP blocked the morphine-induced increases in expression of p- mTOR, p-p70S6K, and p-4E-BPl in dorsal horn (FIG. 7). The in vitro and in vivo evidence suggests that spinal mu receptor participates in activation of mTOR and its downstream effectors in chronic morphine tolerance. EXAMPLE 6

Inhibition of spinal PI3K and Akt attenuates morphine- and DAMGO-induced increases in p-mTOR. p-p70S6K. and p-4E-BP l in dorsal horn neurons

The role of spinal PI3K and Akt in morphine-induced activation of mTOR and its downstream effectors in dorsal horn neurons in vitro was examined. The neurons were challenged by morphine, DAMGO, Ly294002 (a PI3K inhibitor), or Akt inhibitor IV (an Akt inhibitor).

Pretreatment with Ly294002 (10 μΜ in 20% DMSO) or Akt inhibitor IV (5 μΜ in 20% DMSO) reduced morphine- and DAMGO-induced increases in p-mTOR, p-p70S6K, and p-4E-BPl (FIG. 8). Neither Ly294002 nor Akt inhibitor TV alone altered basal expression of p-mTOR, p-p70S6K, or p-4E-BPl . These findings suggest that spinal PI3K and Akt are involved in morphine-induced activation of dorsal horn mTOR and its downstream effectors in dorsal horn neurons.

The roles of spinal PI3K and Akt in the development of chronic morphine tolerance in vivo also were examined. The rats received i.th. injections of morphine (10 μ^ΙΙΟ twice daily) plus vehicle (20% DMSO, 10 μΐ, once daily), Ly294002

(20 μg/10 μΐ_^ once daily), or Akt inhibitor IV (3 μg/10 μΐ_^ once daily) for 7 days. The tail-flick test was performed prior to the start of morphine injections and at 0.5 h after i.th injection of 10 μg morphine on days 1 and 8.

The results showed that Ly294002 and Akt inhibitor IV attenuated the decrease in morphine antinociceptive effect (FIG. 9). Given alone, neither changed the baseline of tail flick latency (FIG. 9) or affected motor functions (Table 3). After the behavioral test, L₅ segment was collected for Western blot analysis. Consistent with the results from the in vitro experiment (FIG. 8), i.th. administration of

Ly294002 or Akt inhibitor IV blocked the increases in expression of dorsal horn p- mTOR and p-p70S6K after repeated i.th. morphine injection (FIGS. 9B and 9C). Given alone, neither altered basal expression of p-mTOR or p-p70S6K in dorsal horn (FIG. 9). Taken together, these data suggest that spinal PI3K and Akt play an important role in activation of dorsal horn mTOR and downstream effectors in chronic morphine tolerance.

EXAMPLE 7

Intrathecal injection of rapamycin does not produce

significant systemic side effects

mTOR inhibitors are FDA-approved immunosuppressive agents used for clinical organ translation. Thus, the question of whether i.th. injection of rapamycin affected immuno-function was examined. Spleen was collected from rats on day 8 after they had been given i.th. injections of vehicle (10 μϊ_^ of 50% DMSO) or rapamycin (10 μg/10 μί) once daily for 7 days. The proliferation of splenocytes in response to ConA (1 μg/mL) or LPS (10 μg/mL) was determined by [³H] -thymidine. In addition, the number of spleen cells was counted. Rapamycin given i.th. did not produce a significant reduction in total number of spleen cells (Table 4) and did not affect ConA- or LPS-induced proliferation of splenocytes (FIG. 10). Spleen weight, body weight, and blood samples were also examined. As indicated in Table 4, rapamycin given i.th. did not lead to marked changes in body weight, spleen weight, total number of red blood cells, total number of white blood cells, total number of lymphocytes, total number of platelets, or amount of hemoglobin. These findings indicate that mTOR inhibitors given i.th. do not produce significant systemic side effects. Table 4. Effect of i.th. rapamycin on body weight, spleen weight, number of spleen cells, and number of blood cells

EXAMPLE 8

Discussion of spinal mTOR attenuating the development of morphine tolerance

It has been found that intrathecal administration of rapamycin, a specific and selective mTOR inhibitor, significantly attenuated the development and maintenance of morphine tolerance and associated pain hypersensitivity. Intrathecal rapamycin did not affect motor function and systemic side effects, such as immuno-suppression.

mTOR inhibitors are FDA-approved immunosuppressive drugs used in organ

transplantation. The findings disclosed herein indicate that inhibition of spinal mTOR with an mTOR inhibitor can be used as an adjuvant with morphine in the treatment of chronic pain.

Moreover, the in vitro and in vivo evidence indicates that activation of the mTOR signaling pathway caused by repeated morphine injection is mediated by the mu receptor/PI3K and Akt pathway. A novel intracellular pathway, mu receptor- PI3K-Akt-mTOR, has been found in dorsal horn neurons during the development and maintenance of chronic morphine tolerance.

EXAMPLE 9

Bone Cancer Pain Model

Materials and Methods

Animal preparation

Male Copenhagen rats weighing 200-225 g (Harlan, Frederick, MD) were housed on a standard 12-h light/dark cycle, with water and food pellets available ad libitum. To minimize intra-and inter- individual variability of behavioral outcome measures, animals were trained for 1 -2 days before behavioral testing was performed. Animal experiments were approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University School of Medicine and were consistent with the ethical guidelines of the National Institutes of Health and the International Association for the study of Pain. All efforts were made to minimize animal suffering and to reduce the number of animals used. The experimenters were blind to drug treatment condition in the behavioral testing.

Experimental drugs

The AT-3.1 prostate cancer cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). Rapamycin and ascomycin were purchased from LC laboratories (Woburn, MA) and dissolved in 50% DMSO. DL-2- amono-5-phosphonovaleric acid (DL-AP5) was purchased from Tocris Bioscience (Ellisville, MO) and dissolved in saline. All drug dosage used was based on data from previous studies (Jimenez-Diaz et al. 2008; Geranton et al. 2009; Asante et al. 2009; Xu et al. 2010; Xu et al. 201 1) and preliminary studies.

Cancer cell preparation

The prostate cancer cells were maintained in T-75 plastic flasks (Corning Glass), grown in RPMI 1640 medium (Sigma) with L-glutamine supplemented with 250 nM dexamethasone and 10% fetal bovine serum (Hyclone), and cultured in a water-saturated incubator in an atmosphere of 5% CO₂: 95% air. For passage, cells were detached by rinsing gently with calcium and magnesium- free Hank's balanced salt solution and a trypsin solution containing 0.05% trypsin and 0.02% EDTA. For injection, the detached cells were first collected by centrifuging 10 mL of medium for 3 min at 1,200 rpm. The resulting pellet was washed twice with 10 mL of calcium- and magnesium-free Hank's solution and re-centrifuged for 3 min at 1,200 rpm. The final pellet was re-suspended in 1 mL of Hank's solution. The cells were counted using a haemocytometer. Cells were diluted at final concentration of 4.5 x 10^s cells/^' 1 μί Hank's solution for injection.

Intrathecal catheter implantation

Following complete induction of anesthesia with 2% isoflurane, a 1-cm midline incision was made from the back, and the muscles were retracted to expose the L4 - L5 vertebrae. Sterile polyethylene tubing (PE-10 catheter) was inserted into the subarachnoid space and advanced 3.6 cm rostral ly at the level of spinal cord lumbar enlargement segments. The catheter was secured to the paraspinal muscle of the back and then tunneled subcutaneously to exit in the dorsal neck region, where it was secured to the superficial musculature and skin. The rats were allowed to recover for 5-7 days; rats that showed neurologic deficits or the presence of fresh blood in the cerebral spinal fluid postoperatively were excluded from the study. The position of the PE- 10 catheter was confirmed after behavioral testing. All drugs were administrated i.th. in total 10 μΐ_^ volume followed by 12 μ ί. of saline to flush the catheter.

PCC injection

Five-7 days after catheter implantation, rats were anesthetized with 2% isoflurane. A 1-cm long rostro-caudal incision was made in the skin over the upper medial half of the tibia on the right leg. The tibia was carefully exposed with minimal damage to the muscle. Using a 23-gauge needle, the bone was pierced 5 mm below the knee joint medial to the tibial tuberosity. A 15 μΐ_^ volume of PCC^'s (4.5 x 10^s cells ) or vehicle ( Hank's solution only) was injected into the bone cavity using a 50 μΙ_ Hamilton syringe. After injection, the syringe remained for 2 min and was then slowly pulled out. Bone hole was filled with bone wax ( Ethicon) and the skin was sutured using 4-0 silk threads.

Radiology and histochemical staining

To confirm if cancer was dev eloped in the tibia, rats were anesthetized and the legs radiographed 1 2 days after behavioral testing. The tibias were the collected, dcmincralizcd in EDTA (10%) for 2-3 weeks, and embedded in paraffin. The sections with the thickness of 5 mm were cut using a microtome and stained w ith Harris' hematoxylin and cos in (HE) to verify cancer cell infiltration and bone destruction.

Behavioral testing

To measure paw withdrawal latency to noxious heat stimuli, each animal was placed in a plastic chamber on a glass plate located above a light box. Radiant heat from a Model 336 Analgesic Meter (IITC, Inc. /Life Science Instruments, Woodland Hills, CA) was applied by aiming a beam of light through a hole in the light box through the glass plate to the middle of the plantar surface of each hind paw. When the animal lifted its foot, the light beam was turned off. The length of time between the start of the light beam and the foot lift was defined as the paw withdrawal latency. Each trial was repeated five times at 5 -min intervals for each paw. A cut-off time of 20 s was used to avoid paw tissue damage. The thermal test was performed 1 day prior to PCC inoculation (BASALINE^®) and on day 3, 5, 7, 9, and 12 after PCC inoculation.

To measure paw withdrawal threshed to mechanical stimuli, each animal was placed in a plastic chamber on an elevated mesh screen. Von Frey filaments in log increments of force (3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93, 5.18 g) were applied to the plantar surface of the left and right hind paws. The 4.31-g stimulus was applied first. If a positive response occurred, the next smaller von Frey hair was used; if a negative response was observed, the next higher von Frey hair was used. The test was ended when (1) a negative response was obtained with the 5.18-g hair, (2) four stimuli were applied after the first positive response, or (3) nine stimuli were applied to one hind paw. The pattern of positive and negative paw withdrawal responses to the von Frey filament stimulation was converted to a 50% threshold value using the formula provided by Dixon (Dixon 1980). The mechanical test was performed 1 day prior to PCC inoculation (BASALINE^®) and on day 3, 5, 7, 9, and 12 after PCC inoculation.

To observe locomotor function, the following three reflex tests were performed (Tao et al. 2000; Tao and Johns 2002; Zhang et al. 2003). (1) Placing reflex: The rat was held with the hind limbs slightly lower than the forelimbs, and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. The experimenter recorded whether the hind paws were placed on the table surface reflexively; (2) Grasping reflex: The rat was placed on a wire grid and the

experimenter recorded whether the hind paws grasped the wire on contact; (3) Righting reflex: The rat was placed on its back on a flat surface and the experimenter noted whether it immediately assumed the normal upright position. Scores for placing, grasping and righting reflexes were based on counts of each normal reflex exhibited in five trials.

Western blot analysis

The ipsilateral and controlateral L4-5 dorsal horns and L4-5 DRGs were collected after behavioral testing or different time points post-PCC injection. The tissues were homogenized in hornogenization buffer [10 mM Tris-HCI (pH 7.4), 5 mM NaF, I mM sodium orthovanadate, 320 mM sucrose, I mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 M leupeptin, and 2 μΜ pepstatin A]. After centrifugation at 1,000 x g for 20 min at 4°C, the supernatant was collected and the pellet (PI, nuclei and debris fraction) discarded. The samples were heated for 5 min at 95°C and then loaded onto 4% stacking/10% separating SDS-polyacrylamide gels. The proteins were eletrophoretically transferred onto nitrocellulose membrane. The blotting membranes were blocked with 3% non-fat dry milk for 1 h and incubated overnight at 4°C with rabbit anti-phospho-mTOR (1 :500; Cell Signaling Technology, Inc, Danvers, MA), rabbit anti-mTOR (1 :500; Cell Signaling Technology, Inc.), rabbit anti-phospho-p70S6K (1 :500; Cell Signaling Technology, Inc.), rabbit anti- p70S6K (1 :500; Cell Signaling Technology, Inc.), and mouse anti- -actin (1 :2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). β-actin was used as a loading control. The proteins were detected using anti-rabbit or anti-mouse secondary antibody and visualized with chemiluminescence reagents provided with the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ) and exposure to film. The intensity of blots was quantified with densitometry. The blot density from naive animals (0 d) was set as 100%.

Double-labeling immunofluorescence histochemistry

Double-labeling immunofluorescence histochemistry was carried out as described previously (Xu et al. 2010). Briefly, the rats were deeply anesthetized and perfused transcardially with 100 mL of 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After the perfusion, the lumbar enlargement segments were harvested, postfixed at 4°C for 4 h, and cryoprotected in 30% sucrose overnight. The transverse sections were cut on a cryostat at a thickness of 15 μιη. The sections were incubated overnight at 4°C with a mixture of rabbit polyclonal anti-mTOR (1 : 1000) and mouse monoclonal anti-NRI (1 :200, Chemicon, Temecula, CA) or a mixture of rabbit polyclonal anti-p70S6K (1 : 1000) and mouse monoclonal anti-NRI (1 :200,

Chemicon). The sections were then incubated with a mixture of goat anti-rabbit IgG conjugated with Cy3 (1 :300) and monkey anti-mouse IgG conjugated with Cy2 (1 :300, Jackson ImmunoResearch) for 1 h at 37°C. Control experiments included pre-absorption of the primary antiserum with excess of the corresponding antigen (Cell Signaling Technology), substitution of normal rabbit serum for the primary antiserum, and omission of the primary antiserum in parallel as described previously (Xu et al. 2010). Statistical Analysis

The results from the behavioral tests and Western blotting were statistically analyzed with a one-way or two-way analysis of variance (ANOVA). Data are presented as mean ± SEM. When ANOVA showed significant difference, pairwise comparisons between means were tested by the post hoc Tukey method. Significance was set at < 0.05. The statistical software package SigmaStat (Systat, Port Richard, CA) was used to perform all statistical analyses.

EXAMPLE 10

Bone cancer pain model produced by PCC injection in rats

Given that the changes of neuronal plasticity in spinal dorsal horn and dorsal root ganglion arc different between cancer pain and neuropathic pain or inflammatory pain ( Schwei et al. 1999; Clohisy and Mantyh 2003; Svendsen et al. 2005 ) and that m^'T^'OR inhibitors clinically have been used to treat cancer (Yee et al. 2006; Garcia and Danielpour 2008; Al-Batran et al. 201 1 ; Molina et al. 201 1 ), the presently disclosed subject matter addressed whether niTOR and its downstream effectors have a functional role in cancer-related pain. A rat model of bone cancer pain produced by prostate cancer cell (PCC) injuction of the tibia was used to first determine whether i.th. pre-and post-administration of rapamycin affected the development and maintenance of PCC-induced pain hypersensitivity. It was further examined whether PCC-induced peripheral noxious input changed the activity of mTOR and p70S6K in spinal cord and DRG.

Consistent with a previous study (Zhang et al. 2005), the animals injected with PCCs exhibited a general health comparable with the rats injected with Hanks' solution including well groomed coats, normal muscle strength, body temperature, and general sensory functions (e.g., olfactory, auditory and visual functions). In addition, body weight and locomotor behavior were indistinguishable between two treated groups during the 12-day observation period (data no shown). In most rats injected with PCCs by day 12, however, the size around the knee on the ipsilateral side was increased.

PCC injection into a tibia produced significant mechanical allodynia as evidenced by a significant decrease in paw withdrawal threshold and thermal hyperalgesia as evidenced by a significant decrease in paw withdrawal latency on the ipsilateral side compared with pre-inoculation baseline values (n = 6; FIGS. 1 1A and 1 IB). These pain hypersensitivies occurred between 5-7 days and remained pronounced for at least 12 days (FIGS. 1 1A and 1 1 B). No marked changes in paw withdrawal threshold and latency were observed on the contralateral side following PCC injection (FIGS. 11 A and 1 IB). As expected, Hanks solution injection did not alter basal paw withdrawal responses to mechanical and thermal stimuli on either ipsilateral or contralateral side (n = 6; FIGS. 1 1A and 1 IB).

After behavioral test, rats were radiographed. Significant cortical destruction of the proximal epiphysis was observed on days 12 post-PCC injection (FIG. 1 1C). No radiological change was found in rats with Hanks' solution injection (FIG. 11C). Furthermore, histological examination showed that PCCs were densely packed in the narrow cavity and induced the destruction of trabeculae on days 12 post-PCC injection (FIG. 1 1 D).

Effect of i.th. rapamycin on the development of PCC-induced bone cancer pain

To evaluate the role of spinal mTOR in the development of bone cancer pain, rapamycin was given i.th. once daily for 7 days following injection of Hanks' solution or PCCs into the tibia. To examine its specificity, ascomycin, which does not inhibit mTOR activity (Hou and Klann 2004; Jimenez-Diaz et al. 2008), was used as a control. The i.th. administration of rapamycin at 10 μg (n = 5), but not ascomycin at 10 μg (n = 5), abolished PCC-induced mechanical allodynia and thermal hyperalgesia (FIG. 12). The effects of rapamycin were dose-dependent (FIG. 13). On day 7 post- PCC inoculation, the 10 μg dose of rapamycin increased paw withdrawal threshold by 4.84-fold (p < 0.01), the 1 μg dose of rapamycin by 3.74-fold (n = 5, p < 0.05), and the 0.1 μg dose of rapamycin by 1.98-fold (n = 5, P > 0.05) compared to the corresponding group treated with PCC and vehicle (50% DMSO) (n = 5, FIG. 13A). Similarly, on day 7 post-PCC injection, the 10 μg dose of rapamycin increased paw withdrawal latency by 1.35-fold (n = 5, p < 0.01), the 1 μg dose of rapamycin by 1.22-fold (n = 5, p < 0.05), and the 0.1 μg dose of rapamycin by 1.04-fold (n = 5, p > 0.05) compared to the corresponding group treated with PCC and vehicle (n = 5, FIG. 13B). Neither the vehicle, nor rapamycin or ascomycin at the 10 μg dose

significantly altered basal paw withdrawal responses to mechanical and thermal stimuli applied to the contralateral hind paw (FIG. 12). Likewise, rapamycin at the 10 μg dose given alone did not affect basal paw withdrawal responses to mechanical and thermal stimuli in Hanks' solution-treated rats (n = 5, FIG. 12). Effect ofi.th. rapamycin on the maintenance of PCC-induced bone cancer pain

To further examine the role of spinal mTOR in the maintenance of bone cancer pain, rapamycin, ascomycin, or vehicle was given i.th. once daily for 5 days beginning on day 7 after rats were injected with Hanks' solution or PCCs. The i.th. rapamycin at 10 μg (n = 6), but not ascomycin at 10 μg (n = 6), significantly reduced PCC-induced mechanical allodynia and thermal hyperalgesia during the maintenance period (FIG. 14). The effects of rapamycin were dose-dependent (FIG. 15). On day 12 post-PCC inoculation, the 10 μg dose of rapamycin increased paw withdrawal threshold by 5.2-fold (p < 0.01), the 1 μg dose of rapamycin by 2.34-fold (n = 6, p < 0.05), and the 0.1 μg dose of rapamycin by 0.97-fold (n = 6, P > 0.05) compared to the corresponding group treated with PCC and vehicle (50% DMSO) (n = 6, FIG. 15A . Similarly, on day 12 post-PCC inoculation, the 10 μg dose of rapamycin increased paw withdrawal latency by 1.32-fold (n = 6, p < 0.0 1), the 1 μg dose of rapamycin by 1.13-fold (n = 6, p < 0.05), and the 0.1 μg dose of rapamycin by 1.01- fold (n = 6, p > 0.05) compared to the corresponding group treated with PCC and vehicle (n = 6, FIG. 15B). As expected, neither the vehicle, nor rapamycin or ascomycin at the 10 μg dose significantly altered basal paw withdrawal responses to mechanical and thermal stimuli applied to the contralateral hind paw during the maintenance period (FIG. 14). Rapamycin at the 10 μg dose given alone also did not affect basal paw withdrawal responses to mechanical and thermal stimuli in Hanks' solution-treated rats during the maintenance period (n = 6, FIG. 14).

Effect of i.th. rapamycin on locomotor function

To exclude the possibility that the effect of i.th. rapamycin on PCC-induced pain behaviors was caused by impaired locomotor functions (or reflexes), the locomotor functions of experimental animals was examined. As shown in Table 5, the rats treated with either rapamycin or ascomycin exhibited normal locomotor functions, including placing, grasping, and righting reflexes. Convulsions and hypermobility were not observed in any of the treated rats. In addition, any significant difference in general behaviors was not observed, including spontaneous activity, between the saline-treated and the drug-treated rats. Table 5. Mean (SEM) changes in locomoter function (N= 5/ group; 5 trials)

Functional Test

Treatment Placing Grasping Righting

Vehicle (50% DMSO) 5 (0) 5 (0) 5 (0)

Rapamycin (10 μg) 5 (0) 5 (0) 5 (0)

Ascomycin (10 μg) 5 (0) 5 (0) 5 (0)

Activation of mTOR and p70S6K in spinal dorsal horn and dorsal root ganglion following PCC injection

Pharmacological studies described hereinabove suggest that mTOR and its downstream effectors are activated at the spinal cord level following PCC injection. To further investigate this observation, Western blot analysis was carried out to examine the activity of mTOR and p70S6K by assessing their phosphorylation levels in L 4_₅ spinal dorsal horns and L ₄_₅ DRGs. It was found that PCC injection produced time-dependent changes in the levels of phosphorylated (p-) mTOR and p-p70S6K in the ipsilateral dorsal horn when compared with naive rats (0 d) (FIG. 16; n = 5/time point). Significant increases in p-mTOR and p-p70S6K began at day 3 and were maintained for at least 12 days after PCC injection (FIGS. 16A and 16B). In contrast, the amounts of p-mTOR and p-p70S6K in the ipsilateral DRGs were detected to dramatic increase only at 3 day post-PCC injection (FIGS. 17 A and 17B). As expected, the expression levels of p-mTOR and p-p70S6K in the contralateral dorsal horn and DRG were not markedly altered during the observed period (FIGS. 16C-16D and 17C-17D). Hanks' solution injection did not change the basal level of p-mTOR and p-p70S6K in either side of dorsal horn or DRG (data not shown). These results indicate that dorsal horn mTOR and its downstream effector p70S6K are activated during the development and maintenance of bone cancer pain.

Also, the question of whether bone cancer pain insult altered expression of total mTOR and p70S6K proteins in L ₄_5 dorsal horns and L ₄_5 DRGs was examined. Quantitative Western blot analysis indicated that PCC injection did not lead to significant changes in the levels of total mTOR and p70S6K in either side of dorsal horn or DRR within a 12-day period ( FIGS. 1 and 17), indicating that PCC-induced bone cancer pain input alters phosphorylation status of mTOR and p70S6K rather than expression of their total proteins in dorsal horn and DRG. Role of spinal NMDA receptors in PCC-induced activation of mTOR and its downstream effectors in spinal cord

Finally, how the mTOR and its downstream pathway were activated in dorsal horn after PCC injection was defined. Preclinical and clinical research has shown that blocking M DA receptors produces a significant analgesic effect on bone cancer pain ( McDonnell et al. 2001 ; Lucas and Lipman 2002; Wcinbroum et al. 2003; Saito et al. 2006 ), suggesting that NMDA receptors arc activated under bone cancer pain conditions. To test whether spinal M DA receptors arc involved in PCC-induced activation of dorsal horn mTOR and its downstream pathway, the rats were pre- infused i.th. with saline or DL-AP5, a selective N M DA receptor antagonist, at a constant flow rate of 2 μg/l μ L/h via a syringe pump. One hour after infusion. Hanks' solution or PCCs were injected into the tibia. Behavioral testing was carried out 1 day- be fore drug infusion and on day 3, 5, and 7 post- PCC or Hanks' solution injection. Consistent with the previous reports (Weinbroum et al. 2003; Saito et al. 2006), i.th. infusion of DL-AP5 abolished PCC-induced mechanical allodynia and thermal hyperalgesia on the ipsilateral side ( FIGS. 18A and 18C). DL-AP5 infused alone did not alter basal paw withdrawal responses to mechanical and thermal stimuli on cither side of hind paw (FIG. 18). After behavioral testing, L 4.5 dorsal horn on the ipsilateral side was collected. As expected, PCC injection significantly increased the levels of p-mTOR ( 203 ± 0.84%, n = 5, P < 0.01 ) and p-p70S6K (357 ± 1.81%, n = 5, P < 0.01) from the ipsilateral (but not contralateral ) dorsal horn 7 days post-PCC injection (FIGS. 18E and 1 8F). In contrast, those indices were unchanged in the rats treated with PCC and DL-AP5 at the same time point (n = 5, FIGS. 18A and 18F). Compared to the rats treated with Hanks' solution and saline (n = 5), DL-AP5 given alone did not significantly affect basal levels of p-mTOR and p-p70S6K in dorsal horn from Hanks' solution-treated rats (n = 5, FIGS. 18E and 18F).

Our double-labeling studies showed that mTOR and p70S6K co-localized with NR1, a subunit of NMDA receptors, in dorsal horn neurons (FIG. 19).

Approximately 62.5% of mTOR-positive neurons and 40% of p70S6K-positive neurons in dorsal horn were positive for NR1 (n = 3). Cellular distributions of p- mTOR and p-70S6K in dorsal horn from Copenhagen rats were unable to be obtained, although their localizations were detected in dorsal horn from Sprague-Dawley rats in our previous study (Xu et al. 2010). Taken together, these findings suggest that NMDA receptors mediate PCC- induced activation of mTOR and its downstream effectors in dorsal horn under bone cancer pain conditions. EXAMPLE 11

Discussion of bone cancer pain model produced by PCC injection in rats

The presence of bone metastases is the most common cause of cancer-related pain. Although opioids and non-steroidal anti-inflammatory drugs have been used in cancer pain treatment, they have limited effects and produce serious side effects. Uncovering the mechanisms that underlie pain hypersensitivity in cancer-related pain may lead to novel therapeutic strategies for prevention and/or treatment of this disorder. The inoculation of cancer cells into bone produces persistent pain hypersensitivities in rodent animal models that mimic clinical pain in the patients with bone metastases. Herein, it has been found that spinal cord mTOR may be required for the induction and maintenance of PCC-induced bone cancer pain. mTOR may be a new target for the treatment of cancer-related pain.

Several bone cancer pain models have been developed. Schwei M et al. first reported a mouse model by injecting osteolytic sarcoma cells, derived from a spontaneous connective tissue cancer, into the femur of syngeneic C3H mice (Schwei et al. 1999). Medhurst SJ et al. then developed a female rat model by injecting

MRMT-1 rat mammary gland carcinoma cells into the tibia (McDonnell et al. 2001). Because rats are more frequently used in studies than mice and sexual hormones have significant effects on pain (Craft et al. 2004), a male rat model has recently been established by injecting AT-3.1 PCCs into the tibia (Zhang et al. 2005). Consistent with the previous studies (Zhang et al. 2005; Zhang et al. 2008), it was observed that PCC injection produced significantly and persistent mechanical allodynia and thermal hyperalgesia on the ipsilateral side. The degree of mechanical allodynia and the time at which mechanical allodynia and thermal hyperalgesia occurred are a little different from those in previous reports (Zhang et al. 2005; Zhang et al. 2008). These discrepancies may be related to differences in concentrations of the injected PCCs and locations of von Frey filament application in hind paw (plantar side vs lateral edge). mTOR and its downstream effectors in dorsal horn and DRG are activated under PCC-induced cancer pain conditions. It was found herein that phosphorylation of mTOR and p70S6K in the ipsilateral L₄_₅ dorsal horns was significantly increased on days 3, 5, 7, and 12 post-PCC injection. These increases occur predominantly in dorsal horn neurons because dorsal horn neurons express mTOR and p70S6K (Xu et al. 2010) and blocking NMDA receptors (that are expressed in dorsal horn neurons) abolish PCC-induced increases in dorsal horn p-mTOR and p-p70S6K. The possibility, however, cannot be ruled out that these increases also might occur in dorsal horn glial cells, as mTOR, p70S6K, and their phosphorylated counterparts also are distributed in dorsal horn glia (Jimenez-Diaz et al. 2008; Xu et al. 201 1) and PCC injection increases activation of dorsal horn astrocytes and microglia (Zhang et al. 2005). It is worth noting that time-dependent increases in p-mTOR and p-p70S6K in dorsal horn are inconsistent with induction of PCC-induced mechanical allodynia and thermal hyperalgesia. This observation might be related to the fact that protein translation and neuronal plastic changes take some time to occur. PCC injection may first activate mTOR and its downstream effectors. This activation then participates in neuronal plastic changes in dorsal horn that are considered to underlie the mechanisms of cancer-related pain development. In the ipsilateral L₄_5 DRGs, PPC- induced increases in p-mTOR and p-p70S6K were observed only on day 3 post-PCC injection. Significance of this transient increase in the DRG is unknown.

NMDA receptors play a critical role in the activation of dorsal horn mTOR and p70S6K following PCC injection. The presently disclosaed subjet matter demonstrates that PCC-induced increases in p-mTOR and p-p70S6K in dorsal horn could be completely blocked by the NMDA receptor antagonist. Morphologic evidence further demonstrates co-localization of NMDA receptor subunit NRl with mTOR and p70S6K in dorsal horn neurons. Taken together, the presently disclosed in vivo findings suggest that NMDA receptors mediate PCC-induced activation of dorsal horn mTOR and p70S6. This conclusion is further supported by in vitro experiments in which brief glutamate /NMDA (an NMDA receptor agonist) stimulation induces activation of mTOR signaling pathway in brain neurons (Lenz and Avruch 2005). How NMDA receptor activation triggers activation of dorsal horn mTOR and its downstream signals is unclear. An increase in [Ca²⁺]_;- following NMDA receptor activation might activate the PI3K/Akt pathway (Zheng and Quirion 2009). Phosphorylated Akt can activate mTOR in central neurons (Jaworski and Sheng 2006) and peripheral inflammation initiates spinal activation of

PI3K/Akt/mTOR signaling pathway (Xu et al. 2011). It is very likely that PI3K/Akt participates in NMDA receptor-triggered dorsal horn activation of mTOR and its downstream effectors. In addition to NMDA receptors, an increase of [Ca²⁺]_;- also might be a result of an inflow from extracellular Ca²⁺ through the voltage-dependent calcium channels and Ca²⁺-permeable AMPA/KA receptors and be the mobilization from intracellular stores through the activation of group 1 metabotropic glutamate receptor activation. Particularly, group I metabotropic glutamate receptors are coupled to PI3K/Akt/mTOR pathway in brain neurons (Hou and Klann 2004; Page et al. 2006).

In conclusion, it was demonstrated that PCC-induced dorsal horn activation of mTOR pathway was mediated by NMDA receptors under cancer pain conditions. It also was shown that i.th. rapamycin dose-dependently attenuated PCC-induced mechanical allodynia and thermal hyperalgesia during both development and maintenance of cancer-related pain. Moreover, i.th. rapamycin at doses used did not affect acute basal nociceptive responses and locomotor functions. Recent work indicates that i.th. rapamycin also did not produce significant systemic side effects such as immuno-suppression (data not shown). Given that mTOR inhibitors are FDA-approved drugs used in organ transplantation and have been used in treating cancer in clinic (Yee et al. 2006; Garcia and Danielpour 2008; Dean et al. 2008; AI- Batran et al. 2011; Molina et al. 2011), mTOR may represent a promising novel strategy for treating cancer-related pain.

EXAMPLE 12

Systemic administration of rapamycin

To determine the effect of systemic administration of rapamycin, mice received a subcutaneous injection of morphine and an intraperitoneal injection of rapamycin. In one set of experiments, mice received a subcutaneous injection twice a day and an intraperitoneal injection of rapamycin once a day for 5 days and in another set of experiments, mice received a subcutaneous injection twice a day for 10 days and an intraperitoneal injection of rapamycin once a day starting on day 6.

For the effect of systemic administration of rapamycin on the development and maintenance of morphine tolerance, a tail-flick test was carried out before and after the morphine injections. Both the subcutaneous injection twice a day and an intraperitoneal injection of rapamycin once a day for 5 days (FIG. 20, Panel A) and subcutaneous injection twice a day for 10 days and an intraperitoneal injection of rapamycin once a day starting on day 6 (FIG. 20, Panel B) showed that the systemic administration of rapamycin attenuated the development and maintenance of morphine tolerance.

For the effect of systemic administration of rapamycin on morphine-induced cold allodynia during the development and maintenance periods of morphine tolerance, a cold plate test was carried out in which the withdrawal response to cold stimuli was measured. FIG. 21 shows that both the subcutaneous injection twice a day and an intraperitoneal injection of rapamycin once a day for 5 days (Panel A) and subcutaneous injection twice a day for 10 days and an intraperitoneal injection of rapamycin once a day starting on day 6 (Panel B) resulted in the systemic

administration of rapamycin blocking morphine-induced cold allodynia during the development and maintenance periods of morphine tolerance.

To determine the effect of systemic administration of rapamycin on morphine- induced mechanical allodynia, a mechanical test was performed in which the paw withdrawal frequency was measured. Both the subcutaneous injection twice a day and an intraperitoneal injection of rapamycin once a day for 5 days (FIG. 22, Panel A) and subcutaneous injection twice a day for 10 days and an intraperitoneal injection of rapamycin once a day starting on day 6 (FIG. 22, Panel B) showed that the systemic administration of rapamycin blocked morphine- induced mechanical allodynia during the development and maintenance periods of morphine tolerance.

These experiments show that systemic administration of a presently disclosed mTOR inhibitor, such as rapamycin, can attenuate the development and maintenance of opioid tolerance as well as decrease the side effects of opioid administration.

EXAMPLE 13

Co-localization of MOR mR A with p-mTOR and mTOR in dorsal horn neurons

To determine if the mu receptor (MOR) mRNA co-localized with mTOR in dorsal horn neurons, MOR mRNA was detected using in situ hybridization histochemistry and p-mTOR and mTOR were detected by using

immunohistochemistry in dorsal horn neurons (FIG. 23). It was determined that p- mTOR and mTOR co-localized with MOR mRNA, which is further evidence that spinal mu receptor participates in activation of mTOR and its downstream effectors in chronic morphine tolerance. REFERENCES

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Claims

THAT WHICH IS CLAIMED:

1. A method for preventing or treating opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain in a subject in need of treatment thereof, the method comprising administering a mammalian Target of Rapamycin (mTOR) inhibitor to a subject in an amount effective to prevent or treat opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain.

2. The method of claim 1, wherein administering the mTOR inhibitor results in a decrease in one or more of the: phosphorylation of mTOR, activity of mTOR, and protein kinase activity of mTOR.

3. The method of claim 1, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, derivatives of rapamycin, and isomers of rapamycin.

4. The method of claim 1, wherein the mTOR inhibitor is an siRNA of mTOR.

5. The method of claim 1, wherein mTOR is spinal mTOR.

6. The method of claim 1, wherein the opioid tolerance, opioid-induced hyperalgesia, and/or cancer pain is associated with the mu receptor//PI3K/Akt/mTOR signaling pathway.

7. The method of claim 6, wherein administering the mTOR inhibitor results in a decrease in one or more of the: activity of the mu receptor, activity of the P13K protein, activity of the Akt protein, phosphorylation of p70S6K, and phosphorylation of 4E-BP 1.

8. The method of claim 1, wherein the opioid is morphine.

9. The method of claim 1, wherein the mTOR inhibitor is administered in combination with an opioid to the subject.

10. The method of claim 1, wherein the mTOR inhibitor is administered prior to administering an opioid to the subject.

11. The method of claim 1 , wherein administration of the mTOR inhibitor is by intrathecal injection.

12. The method of claim 1 , wherein the mTOR inhibitor prevents or treats opioid tolerance in dorsal horn neurons.

13. The method of claim 1, wherein the subject is human.

14. The method of claim 1, wherein the subject has chronic pain.

15. The method of claim 14, wherein the chronic pain is due to one or more of: allodynia, hyperalgesia, and cancer.

16. The method of claim 15, wherein the allodynia is mechanical.

17. The method of claim 15, wherein the hyperalgesia is thermal.

18. A method for preventing or treating chronic pain associated with the mu receptor//PI3K/Akt/mTOR signaling pathway in a subject in need of treatment thereof, the method comprising administering an mTOR inhibitor to a subject in an amount effective to prevent or treat chronic pain associated with the mu

receptor//PI3K/Akt/mTOR signaling pathway.

19. The method of claim 18, wherein administering the mTOR inhibitor results in a decrease in one or more of the: phosphorylation of mTOR, activity of mTOR, and protein kinase activity of mTOR.

20. The method of claim 18, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, sirolimus, temsirolimus, everolimus, derivatives of rapamycin, and isomers of rapamycin.

21. The method of claim 18, wherein the mTOR inhibitor is an siRNA of mTOR.

22. The method of claim 18, wherein mTOR is spinal mTOR.

23. The method of claim 18, wherein administering the mTOR inhibitor results in a decrease in one or more of the: activity of the mu receptor, activity of the P13K protein, activity of the Akt protein, phosphorylation of p70S6K,

phosphorylation of 4E-BP 1 , and activity of an NMDA receptor.

24. The method of claim 18, wherein the mTOR inhibitor is administered along with an opioid or prior to administration of an opioid to the subject.

25. The method of claim 24, wherein the opioid is morphine.

26. The method of claim 18, wherein administration of the mTOR inhibitor is by intrathecal injection.

27. The method of claim 18, wherein the mTOR inhibitor prevents or treats chronic pain in dorsal horn neurons.

28. The method of claim 18, wherein the subject is human.

29. The method of claim 18, wherein the chronic pain is due to one or more of allodynia, hyperalgesia, and cancer.

30. The method of claim 29, wherein the allodynia is mechanical.

31. The method of claim 29, wherein the hyperalgesia is thermal.