Lindsay B. Hough - US grants

The neurotransmitter histamine (HA) causes analgesia when administered directly into the various brain areas, including the periaqueductal grey (PAG), an important site of action of morphine (MOR). Recent studies from this lab show that: 1) HA H2 receptor antagonists attenuate MOR analgesia, and 2) MOR enhances brain HA turnover, consistent with the hypothesis that activation of brain H2 receptors is important in the expression of MOR analgesia. The present proposal will clarify the role of histaminergic neurons in analgesic responses, and will identify the sites and mechanisms of action of HA-related drugs on these responses. To test directly the hypothesis that activation of brain H2 receptors is important for the expression of MOR analgesia, intraventricular (ivt) does-response curves will be determined for 11 H2 antagonists (of varying chemical structure and H2 potency) as inhibitors of MOR analgesia. The H2 antagonist cimetidine potentiates, rather than inhibits Mor analgesia; it will also be determined if this effect is a result of this drug's documented ability to inhibit MOR metabolism. To identify the CNS site (s) where H2 antagonists act to inhibit MOR analgesia, the effects of combinations of intracerebral, intrathecal and systemic administration of H2 antagonists and MOR will be studied. To pharmacologically classify the analgesia elicited by ivt and intracerebral HA, the effects of HA agonists (alone) and antagonists (in the presence of HA) will be assessed on antinociceptive responses. To determine the anatomical and pharmacological relationship(s) between histaminergic and opiate analgesia, the effects of acute and chronic combinations of HA and MOR will be determined. To characterize the neurochemical effects of MOR on brain HA dynamics, the actions of systemic MOR will be assessed on the levels and turnover of HA and its metabolites in brain regions and spinal cord. To learn the relationship between MOR analgesia and MOR's actions on HA turnover, pharmacological and microinjection experiments with MOR and HA turnover will be performed. To test the hypothesis that agents capable of altering histaminergic activity should modify analgesic responses, the effects of drugs that modify HA synthesis, HA metabolism, and neuronal HA release will be determined on antinociceptive tests in the presence and absence of MOR. These studies will enhance our knowledge of antinociceptive mechanisms, and may lead to the development of novel, clinically useful agents for the relief of pain.

[unreadable] DESCRIPTION (provided by applicant): A new class of pain-relieving drugs, derived from histamine antagonists, has been discovered. The prototype (named improgan) shows the following characteristics after injection into the brain: A) highly effective attenuation of thermal and mechanical nociception in two rodent species, B) absence of impairment of motor function, C) independence from known opioid or histamine receptors, and D) lack of tolerance with daily dosing. The experiments below in rats and mice will reveal the mechanism of action of improgan, and evaluate the efficacy of this drug in clinically relevant pain models: (1) The improgan receptor has not yet been discovered. Radioligand binding studies with 3H-cimetidine will test the hypothesis that this ligand binds to the brain improgan receptor. Validation of this assay will lead to discovery of the improgan receptor, and to the development of new drugs acting on this receptor. (2) The effects of improgan on inflammatory and neuropathic nociceptive models will evaluate the efficacy of this drug in clinically relevant pain. (3) "Off-cells" in the rostral ventromedial medulla (RVM) are crucial for RVM-mediated analgesia, and improgan appears to activate these cells. Combinations of single unit recording, microinjections, behavioral testing and iontophoresis will be performed to reveal the neurophysiological basis for improgan antinociception. (4) Improgan antinociception is blocked by cannabinoid CBj antagonists, yet this drug lacks affinity for the CBi receptor. In vivo studies with cannabinoid drugs and anti-sense oligonucleotides will verify mechanistic roles for CBi receptors and endogenous cannabinoids (endocannabinoids) in improgan antinociception. These collaborative experiments between pharmacologists, neurophysiologists and chemists will discover the mechanism of action of this novel class of agents and lead to the development of new, non-opioid pharmacotherapies for pain. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A new class of pain-relieving drugs, derived from histamine antagonists, has recently been discovered. The prototype (named improgan) shows the following characteristics after direct injection into the brain: a) highly effective, morphine-like antinociception on thermal and mechanical tests, b) no impairment of motor coordination or locomotor activity, e) mechanism that is independent of known receptors for histamine and opioids, d) lack of tolerance with daily dosing, and e) unique structure-activity relationships among chemical congeners. Several impediments exist in the development of these agents: l) the mechanism of action is unknown, 2) high-potency congeners have not been discovered, 3) many have H2 or H3-blocking side effects (but do not produce analgesia through these receptors), and 4) the compounds do not penetrate the blood brain barrier after systemic dosing. The experiments below in rats and mice will discover new analgesic congeners of improgan with enhanced potency, reduced side effects and improved brain-penetrating properties: Aim l) Synthesize and test new improgan-like analgesics which lack H2 and H3 receptor side effects, and possess enhanced analgesic potency. Pilot results indicate that open chain; furan-containing congeners will show these properties. Aim 2) Synthesize and test brain-penetrating improgan-like analgesics. Pilot studies show the feasibility of discovering such compounds. Aim 3) Determine the in vitro actions of these new drugs on two new radioligand binding assays being developed to predict improgan analgesia. Aim 4) Study the activity of selected new compounds in vivo: a) classify their analgesic mechanism by treatments with transmitter agonists and antagonists, and b) assess their clinical potential in several different nociceptive tests and after various routes of administration. This project, which is a collaborative effort between medicinal chemists and pharmacologists, will produce new, brain-penetrating, non-opioid analgesics with the efficacy and potency of morphine. These experiments will help to discover the mechanism of action of this novel class of agents, and lead to the development of new pharmacotherapies for pain.

DESCRIPTION (provided by applicant): Morphine acts on mu opioid receptors in the brain and spinal cord to produce dramatic pain relief, but mu-associated side effects (including tolerance and substance abuse) limit its use. Despite substantial information on mu signaling, the mechanisms by which morphine activates brain stem analgesic circuits remain unknown. The goal of this proposal is to validate a new mechanism for morphine analgesia and to investigate new analgesic agents which may mimic morphine actions but lack side effects. Cytochrome P450s are a family of drug-metabolizing enzymes that also perform endogenous metabolic oxidations in the brain. Many P450s have arachidonic acid (AA) epoxygenase activity, i.e. they convert AA to epoxyeicosatrienoic acids (EETs). Based on three new findings and a literature review, an epoxygenase hypothesis is proposed for opioid analgesic action in the brain stem: Morphine ->[unreadable] receptor ->? AA ->(P450) ->? EET s ->? K+ ->? Analgesic Circuits Experiments to validate and exploit this hypothesis include: 1) Pilot studies show that newly-developed brain P450-deficient transgenic mice (BCPRN) have defective morphine antinociception. To validate this opioid analgesic phenotype, morphine experiments with BCPRN and control mice will investigate the importance of morphine dose, routes of administration, nociceptive tests and gender. 2) P450 epoxygenase inhibitors block morphine antinociception. Since morphine acts in the ventrolateral periaqueductal gray, the rostral ventromedial medulla, and the spinal dorsal horn, microinjections of P450 inhibitors and morphine into these regions of the rat CNS will identify the P450-relevant sites. Similar microinjections of morphine into BCPRN and control mice will confirm results from rats. 3) Morphine's mu-mediated side effects include modulation of respiration, locomotor activity, rotorod performance, and body temperature. Experiments with morphine and P450 inhibitors in rats and with morphine in BCPRN mice will discover which of these side effects utilize P450 mechanisms. 4) Additional experiments in rats with P450 inhibitors and with inhibitors of EET metabolism will further confirm and characterize the role of P450 expoxygenases in morphine analgesia. 5) Pilot results show that EETs, the products of AA epoxidation, have antinociceptive properties. Experiments to validate EET analgesia, to search for EET side effects, and to characterize EET mechanisms may reveal EETs to be a new class of experimental analgesic agents. The proposed studies will increase understanding of the mechanisms by which morphine relieves pain and produces unwanted side effects. Confirmation of the epoxygenase hypothesis may lead to new approaches for developing non-opioid, non-addicting pain relievers. PUBLIC HEALTH RELEVANCE: There is an urgent need to discover new kinds of pain-relievers that lack addictive properties. This proposal will uncover new biochemical mechanisms for pain relief, and test a new group of chemicals for pain-relieving properties. This research could lead to the development of new, non-addicting pain relievers.