Michael M. Morgan - US grants

Opioids are the most effective treatment for pain. Unfortunately, their potency decreases with repeated administration because of the development of tolerance. The broad long-term objective of this research is to improve the treatment of pain by learning how to block the development of tolerance to opioids. Although morphine tolerance has been studied extensively, the neural mechanisms underlying tolerance remain poorly understood. One problem is that opioids act at peripheral, spinal, and supraspinal sites and tolerance may be mediated by different mechanisms at each site. Another problem is that there are several different types of tolerance (behavioral, associative, and non-associative) and different mechanisms may underlie each type. The proposed studies overcome these problems by focusing on tolerance mediated by a brain structure known as the ventrolateral periaqueductal gray (vPAG). The vPAG is of particular interest because it is part of the primary descending pain modulatory system in the brain and the neural circuitry through which vPAG opioids produce antinociception has been well characterized. Moreover, previous research has shown that tolerance develops to administration of morphine directly into the vPAG. Thus, the vPAG provides a unique opportunity to identify both the specific classes of neurons and the mechanisms underlying different types of tolerance to opioids. The first aim of the proposed studies is to determine whether behavioral, associative, and non-associative factors contribute to vPAG mediated tolerance. This will be accomplished by microinjecting morphine into the vPAG in environmental situations that facilitate each type of tolerance. The second aim is to determine whether tolerance is caused by a change in opioid-sensitive GABAergic neurons or output neurons. Opioids in the PAG produce antinociception by inhibiting GABAergic neurons and disinhibiting output neurons. If the mechanism for tolerance resides within opioid-sensitive GABAergic neurons, then tolerance should not develop to microinjection of drugs that act directly on vPAG output neurons. This hypothesis will be tested by examining tolerance to repeated microinjection into the vPAG of the neuroexcitant kainate, the GABA antagonist bicuculline, and the cannabinoid CP-55,940. The proposed studies will provide a foundation for future research examining specific neural mechanisms for different types of vPAG mediated tolerance. In the long term, such knowledge should improve the treatment of pain by allowing therapies that disrupt tolerance to be developed.

Opioids such as morphine are the most powerful treatment for pain. Unfortunately, the analgesic effects of morphine decrease with repeated administration because of tolerance. Many mechanisms have been proposed to underlie the development of tolerance. Recent studies suggest that opioid binding at the mu-opioid receptor may be a key step in this process. Administration of high efficacy mu-opioid receptor agonists produce maximal receptor signaling, rapid desensitization of the mu-opioid receptor, and receptor internalization, but relatively little tolerance. In contrast, morphine produces minimal desensitization and receptor internalization, but tolerance is rapid and pronounced. Although these findings suggest that agonist efficacy and mu-opioid receptor internalization are important factors in tolerance to opioids, most of these data are derived from in vitro studies using brain slices or cultured cells lines. The objective of the proposed studies is to determine whether the knowledge gathered using these reduced preparations apply to tolerance mediated by the periaqueductal gray (PAG) in intact rats. In particular, the proposed studies will test the hypothesis that changes in mu-opioid receptor signaling in the PAG causes tolerance to the anti nociceptive effects of opioids. This hypotheSis will be tested by determining whether mu-opioid receptor internalization contributes to tolerance to morphine microinjections into the PAG. The strength of these studies lies in correlating physiological and anatomical changes in PAG neurons and behavioral measures of tolerance to the anti nociceptive effects of morphine. An understanding of the mechanisms underlying tolerance in intact rats will allow the development of better treatments for chronic pain patients who are tolerant to the analgesic effects of opioids.

DESCRIPTION (provided by applicant): Opioids such as morphine are the most effective treatment for pain. Unfortunately, this opioid analgesia is limited by the development of tolerance with repeated administration. Cannabinoids also have been shown to have analgesic effects, but this antinociception is much more mild than that produced by opioids. The combined administration of opioids and cannabinoids has the potential to enhance the analgesic effects of both drug classes and reduce the development of tolerance to opioids. Our preliminary data and published reports by others show that alternating opioid and cannabinoid treatment can enhance the analgesic effects of both opioids and cannabinoids. Our data show that repeated injections of the cannabinoid HU-210 into a brain structure called the periaqueductal gray (PAG) enhances the analgesic effect of a morphine injection into the PAG on a subsequent day. This finding suggests that the ability of cannabinoids to enhance morphine antinociception is mediated by the PAG. The first objective of this grant proposal (Aim 1) is to test the hypothesis that the antinociceptive effect of microinjecting a cannabinoid into the PAG will be enhanced in morphine tolerant rats. In addition, these PAG mediated effects will be compared to cannabinoid/opioid interactions in the rostral ventromedial medulla (RVM)-the primary output target for neurons in the PAG. Rats will be made tolerant to either morphine or HU-210 and then tested for enhanced analgesia to injection of either HU-210 or morphine into the PAG or RVM. The second objective (Aim 2) is to determine how this interaction between cannabinoids and opioids occurs. In particular, anatomical and electrophysiological techniques will be used to determine whether cannabinoids and opioids interact on the same or different neurons in the PAG. These studies will provide insights into the mechanisms underlying cannabinoid/opioid interactions. An understanding of these mechanisms will allow the development of pain treatments that both reduce tolerance to opioids and enhance the analgesic effects of opioids and cannabinoids. Thus, these simple, but innovative experiments could have a significant impact on improving the treatment for pain. PUBLIC HEALTH RELEVANCE: Persistent pain is a serious medical problem. Although the effectiveness of opioids to treat severe pain diminishes with repeated administration, prior treatment with a cannabinoid appears to enhance the analgesic effects of morphine. The proposed studies will examine possible mechanisms for this potentiation between opioids and cannabinoids so better pain treatments can be developed.

DESCRIPTION (provided by applicant): Psychostimulants, such as methylphenidate (MPH) and methamphetamine, are widely used and abused. The short-term adverse health effects of methamphetamine abuse are well known. Although much less is known about the long-term effects, changes in pain modulation appear to be a potentially important consequence. Our preliminary data show that chronic administration of MPH to young rats enhances morphine antinociception and facilitates the development of tolerance to morphine as adults. Behavioral and anatomical data suggest that dopamine, the neurotransmitter underlying the effects of psychostimulants, and opioids interact in the periaqueductal gray (PAG). The studies in this proposal focus on the PAG as a novel shared neurobiological substrate mediating changes in pain modulation associated with psychostimulant use. The ventrolateral PAG is critical for opioid analgesia and also contains intrinsic dopaminergic neurons that are a likely target of psychostimulant drugs. Thus, the PAG represents a site of convergence between dopamine and pain modulation. Changes in morphine antinociception and tolerance could be caused by changes in opioid potency or changes in basal pain states. This proposal uses a collaborative team science approach to examine PAG function at electrophysiological, anatomical, and behavioral levels. Experiments in Aim #1 will focus on changes in opioid tolerance, as well as opioid receptor potency and desensitization following psychostimulant administration. Experiments in Aim #2 will test the hypothesis that psychostimulants enhance chronic pain via changes in neural processing in the PAG. Finally, Aim #3 will test the hypothesis that combining chronic psychostimulant administration and chronic pain will produce unique interactions between dopamine and opioid systems in the PAG. These studies will clarify the cellular substrates for opioid/dopamine interactions in the PAG and how these cells fit into the central networks that underlie the long- term consequences of drug abuse on pain. We will test the overall hypothesis that the PAG is a critical substrate for the changes in nociception and analgesia produced by chronic psychostimulant use. The proposed studies will provide a better understanding of the cellular mechanisms that contribute to chronic pain in patients with a history of drug abuse so effective treatments can be developed. )

Abstract Chronic pain is a complex problem affecting over a billion people worldwide. An important goal for basic scientists is to develop and test treatments in animals that can be translated for use in chronic pain patients. Chronic pain patients suffer both because of the pain and the disruption of daily life routines (e.g., exercise, socializing). The development of treatments that restore normal activity will require the use of pain tests that mimic these effects. Most preclinical models of pain assess an animal's response to a noxious stimulus. Such pain-stimulated tests are useful in revealing whether a stimulus is noxious, but do not mimic the disruptive effects of pain on activity. Our objective is to use suppression of home cage wheel running in rats as a model of pain-suppressed activity in humans. Wheel running is a natural rodent behavior that requires no training other than placing a running wheel in the rat's home cage. Although a number of pain-suppressed tests have been developed including suppression of wheel running, home cage wheel-running could be a particularly useful model because wheel-running is a natural rodent behavior, assessment takes place in the rat's home cage (a low-stress environment), and measurements can be taken 24 hours a day. The proposed studies will reveal whether wheel-running is an accurate measure of clinical pain by testing rats with inflammatory and migraine pain. Five specific hypotheses will be tested: 1) Inflammatory and migraine pain will suppress wheel-running; 2) The affective dimension of inflammatory pain measured with wheel running will recover prior to noxious stimuli evoked responses; 3) Administration of pain treatments will restore pain-suppressed wheel running; 4) Wheel-running will distinguish between the antinociceptive (increased wheel running) and side effects (decreased wheel running) of drug treatments; and 5) Female rats will be more sensitive than male rats to pain-suppressed wheel running. These hypotheses will be tested by monitoring wheel- running in the home cage of male and female rats before and after administration of Complete Freund's Adjuvant (CFA) in the right hindpaw to induce inflammatory pain or administration of a TRPA1 agonist onto the dura mater to induce migraine pain. Wheel running will be assessed 23 hours a day for at least 7 days. Rats will be removed once a day to assess pain-evoked behavior (von Frey and Hargreaves tests) and to administer drugs (CFA, ketoprofen, morphine, amphetamine, sumatriptan). These studies will reveal whether pain-suppressed wheel running is a reliable measure of nociception and a valid model of pain-suppressed behavior in humans. Given the need to develop novel analgesics and improve upon existing preclinical pain assays, the proposed studies could provide a significant advance in pain research.

ABSTRACT Tens of thousands of people die each year from opioid overdose. Many of these people began taking opioids for pain. A critical treatment goal is to reduce the development of opioid dependence either by enhancing opioid analgesia so lower doses can be used or by blocking withdrawal symptoms. Opioid substitution therapy, in which long-lasting opioids such as methadone and buprenorphine are substituted for potent short acting opioids, requires continuous administration to mask opioid withdrawal without reducing opioid dependence. A potentially new approach is suggested by the finding that chronic opioid administration increases the formation of the mu-delta opioid receptor heterodimer (MDOR), and disrupting signaling from these heterodimers appears to enhance opioid antinociception and reduce dependence. These findings suggest that an MDOR antagonist may be especially effective in reducing dependence by limiting opioid tolerance and preventing opioid withdrawal. We created a novel series of potential selective peptide MDOR antagonists to test this hypothesis. These novel antagonists connect low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic-OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. Our preliminary in vitro data using radioligand binding and 35S-GTP?S shows selectively targeting of the MDOR, with a selectivity ratio of ~89 fold for our best compound, D24M. Microinjection of D24M into the mouse brain selectively increased opioid antinociception in models of acute and chronic pain while strongly decreasing morphine withdrawal. These preliminary findings suggest that D24M could reduce opioid dependence by enhancing opioid antinociception, reducing opioid tolerance, or directly inhibiting opioid withdrawal. Although this completely new class of ligand is promising, the efficacy and translatability of MDOR antagonists depends on the ability to reduce dependence in the absence of disruptive side effects. The UG3 phase of this application will vigorously assess the ability of D24M to reduce dependence in male and female mice and rats. Indications supported by mouse models will be tested using the highly novel home cage wheel-running test in rats to determine the effect of D24M on normal daily function. Male and female rats with and without chronic pain will be included to mimic the clinical situation of pain patients who transition to dependence. If these studies are successful in showing that the D24M can reduce dependence (the UG3 milestone), then new derivatives of D24M to improve MDOR potency, selectivity, metabolic stability, and blood-brain barrier (BBB) penetration via glycosylation and nanoparticle formulation will be developed (UH3 phase). The ultimate goal is to develop an optimized drug ready for Investigational New Drug (IND)-enabling studies and clinical testing. These proposed studies are high-risk, high-reward, with a brand new class of therapeutic drugs to be tested in highly innovative rodent behavioral models. Our plan to assess D24M, the novel MDOR antagonist, and develop new drugs to reduce opioid dependence is well suited to the UG3/UH3 mechanism.