Anthony W. Bannon

Behavioral characterization of neuropeptide Y knockout mice.

An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

Diversity of neuronal nicotinic acetylcholine receptors: lessons from behavior and implications for CNS therapeutics.

Although the molecular biology of neuronal nicotinic acetylcholine receptors (nAChRs) provides evidence for multiple receptor subtypes, few selective pharmacological tools exist to identify these subtypes in vivo. However, the diversity of behavioral effects of available nAChR agonists and antagonists reviewed in this paper suggests that neuronal nAChR subtypes may play distinct roles in a variety of behavioral outcomes. Further characterization of the behavioral effects of the activation of discrete nAChR subtypes may eventually provide information useful in designing selective nAChR ligands targeting a variety of CNS disorders.

The Vanilloid Receptor TRPV1 Is Tonically Activated In Vivo and Involved in Body Temperature Regulation

The vanilloid receptor TRPV1 (transient receptor potential vanilloid 1) is a cation channel that serves as a polymodal detector of pain-producing stimuli such as capsaicin, protons (pH <5.7), and heat. TRPV1 antagonists block pain behaviors in rodent models of inflammatory, neuropathic, and cancer pain, suggesting their utility as analgesics. Here, we report that TRPV1 antagonists representing various chemotypes cause an increase in body temperature (hyperthermia), identifying a potential issue for their clinical development. Peripheral restriction of antagonists did not eliminate hyperthermia, suggesting that the site of action is predominantly outside of the blood–brain barrier. Antagonists that are ineffective against proton activation also caused hyperthermia, indicating that blocking capsaicin and heat activation of TRPV1 is sufficient to produce hyperthermia. All TRPV1 antagonists evaluated here caused hyperthermia, suggesting that TRPV1 is tonically activated in vivo and that TRPV1 antagonism and hyperthermia are not separable. TRPV1 antagonists caused hyperthermia in multiple species (rats, dogs, and monkeys), demonstrating that TRPV1 function in thermoregulation is conserved from rodents to primates. Together, these results indicate that tonic TRPV1 activation regulates body temperature.

Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors.

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (Tb) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase Tb of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither Tb nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the Tb response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on Tb.

Repeated administration of vanilloid receptor TRPV1 antagonists attenuates hyperthermia elicited by TRPV1 blockade

Capsaicin, the active ingredient in some pain-relieving creams, is an agonist of a nonselective cation channel known as the transient receptor potential vanilloid type 1 (TRPV1). The pain-relieving mechanism of capsaicin includes desensitization of the channel, suggesting that TRPV1 antagonism may be a viable pain therapy approach. In agreement with the above notion, several TRPV1 antagonists have been reported to act as antihyperalgesics. Here, we report the in vitro and in vivo characterization of a novel and selective TRPV1 antagonist, N-(4-[6-(4-trifluoromethyl-phenyl)-pyrimidin-4-yloxy]-benzothiazol-2-yl)-acetamide I (AMG 517), and compare its pharmacology with that of a closely related analog, tert-butyl-2-(6-([2-(acetylamino)-1,3-benzothiazol-4-yl]oxy)pyrimidin-4-yl)-5-(trifluoromethyl)phenylcarbamate (AMG8163). Both AMG 517 and AMG8163 potently and completely antagonized capsaicin, proton, and heat activation of TRPV1 in vitro and blocked capsaicin-induced flinch in rats in vivo. To support initial clinical investigations, AMG 517 was evaluated in a comprehensive panel of toxicology studies that included in vivo assessments in rodents, dogs, and monkeys. The toxicology studies indicated that AMG 517 was generally well tolerated; however, transient increases in body temperature (hyperthermia) were observed in all species after AMG 517 dosing. To further investigate this effect, we tested and showed that the antipyretic, acetaminophen, suppressed the hyperthermia caused by TRPV1 blockade. We also showed that repeated administration of TRPV1 antagonists attenuated the hyperthermia response, whereas the efficacy in capsaicin-induced flinch model was maintained. In conclusion, these studies suggest that the transient hyperthermia elicited by TRPV1 blockade may be manageable in the development of TRPV1 antagonists as therapeutic agents. However, the impact of TRPV1 antagonist-induced hyperthermia on their clinical utility is still unknown.

Cocaine- and Amphetamine-Related Transcript Is Involved in the Orexigenic Effect of Endogenous Anandamide

Endocannabinoids acting at CB1 cannabinoid receptors (CB1) increase appetite. In view of the predominant presynaptic localization of CB1 in the brain, we tested the hypothesis that the orexigenic effect of endocannabinoids involves inhibition of the release of a tonically active anorexigenic mediator, such as the peptide product of the cocaine- and amphetamine-related transcript (CART). The CB1 antagonist rimonabant inhibited food intake in food-restricted wild-type mice, but not in their CART-deficient littermates. Mice deficient in fatty acid amide hydrolase (FAAH), the enzyme responsible for the in vivo metabolism of the endocannabinoid anandamide, have reduced levels of CART-immunoreactive nerve fibers and terminals in several brain regions implicated in appetite control, including the arcuate, dorsomedial and periventricular nuclei of the hypothalamus, the amygdala, the bed nucleus of the stria terminalis and the nucleus accumbens, and treatment of FAAH–/– mice with rimonabant, 3 mg/kg/day for 7 days, increased CART levels toward those seen in FAAH+/+ wild-type controls. In contrast, no difference in the density of CART-immunoreactive fibers was observed in the median eminence and the paraventricular nucleus of FAAH+/+ and FAAH–/– mice. Acute treatment of wild-type mice with the cannabinoid agonist HU-210 resulted in elevated CART levels in the dorsomedial nucleus and the shell portion of the nucleus accumbens. These observations are compatible with CART being a downstream mediator of the CB1-mediated orexigenic effect of endogenous anandamide.

Regulation of Protein Catabolism by Muscle-Specific and Cytokine-Inducible Ubiquitin Ligase E3α-II during Cancer Cachexia

The progressive depletion of skeletal muscle is a hallmark of many types of advanced cancer and frequently is associated with debility, morbidity, and mortality. Muscle wasting is primarily mediated by the activation of the ubiquitin-proteasome system, which is responsible for degrading the bulk of intracellular proteins. E3 ubiquitin ligases control polyubiquitination, a rate-limiting step in the ubiquitin-proteasome system, but their direct involvement in muscle protein catabolism in cancer remains obscure. Here, we report the full-length cloning of E3α-II, a novel “N-end rule” ubiquitin ligase, and its functional involvement in cancer cachexia. E3α-II is highly enriched in skeletal muscle, and its expression is regulated by proinflammatory cytokines. In two different animal models of cancer cachexia, E3α-II was significantly induced at the onset and during the progression of muscle wasting. The E3α-II activation in skeletal muscle was accompanied by a sharp increase in protein ubiquitination, which could be blocked by arginine methylester, an E3α-selective inhibitor. Treatment of myotubes with tumor necrosis factor α or interleukin 6 elicited marked increases in E3α-II but not E3α-I expression and ubiquitin conjugation activity in parallel. E3α-II transfection markedly accelerated ubiquitin conjugation to endogenous cellular proteins in muscle cultures. These findings show that E3α-II plays an important role in muscle protein catabolism during cancer cachexia and suggest that E3α-II is a potential therapeutic target for muscle wasting.

Antibodies to Nerve Growth Factor Reverse Established Tactile Allodynia in Rodent Models of Neuropathic Pain without Tolerance

A considerable body of evidence implicates endogenous nerve growth factor (NGF) in conditions in which pain is a prominent feature, including neuropathic pain. However, previous studies of NGF antagonism in animal models of neuropathic pain have examined only the prevention of hyperalgesia and allodynia after injury, whereas the more relevant issue is whether treatment can provide relief of established pain, particularly without tolerance. In the current work, we studied the effects of potent, neutralizing anti-NGF antibodies on the reversal of tactile allodynia and thermal hyperalgesia in established models of neuropathic and inflammatory pain in rats and mice. In the complete Freunds adjuvant-induced hind-paw inflammation, spinal nerve ligation and streptozotocin-induced neuropathic pain models, a single intraperitoneal injection of a polyclonal anti-NGF antibody reversed established tactile allodynia from approximately day 3 to day 7 after treatment. Effects on thermal hyperalgesia were variable with a significant effect observed only in the spinal nerve ligation model. In the mouse chronic constriction injury (CCI) model, a mouse monoclonal anti-NGF antibody reversed tactile allodynia when administered 2 weeks after surgery. Repeated administration of this antibody to CCI mice for 3 weeks produced a sustained reversal (days 4 to 21) of tactile allodynia that returned 5 days after the end of dosing. In conclusion, NGF seems to play a critical role in models of established neuropathic and inflammatory pain in both rats and mice, with no development of tolerance to antagonism. Antagonists of NGF, such as fully human monoclonal anti-NGF antibodies, may have therapeutic utility in analogous human pain conditions.

Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin

Mouse lines with targeted disruption of the cocaine amphetamine‐related transcript (CART), melanocortin receptor 3 (MCR3), or melanocortin receptor 4 (MCR4) were used to assess the role of each component in mediating the anorectic and metabolic effects of leptin, and in regulating the partitioning of nutrient energy between fat and protein deposition. Leptin was administered over a 3 day period using either intraperitoneal or intracerebroventricular routes of injection. The absence of MCR4 blocked leptins ability to increase UCP1 mRNA in both brown and white adipose tissue, but not its ability to reduce food consumption. In contrast, deletion of MCR3 compromised leptins ability to reduce food consumption, but not its ability to reduce fat deposition or increase UCP1 expression in adipose tissue. Leptin‐dependent effects on food consumption and adipocyte gene expression were unaffected by the absence of CART. Repeated measures of body composition over time indicate that the absence of either MCR3 or MCR4, but not CART, increased lipid deposition and produced comparable degrees of adiposity in both lines. Moreover, modest increases in fat content of the diet (4 to 11%) accentuated fat deposition and produced a rapid and comparable 10–12% increase in % body fat in both genotypes. The results indicate that nutrient partitioning, as well as the anorectic and metabolic responses to leptin, are dependent onintegrated but separable inputs from the melanocortin 3 and 4 receptor subtypes. Zhang, Y., Kilroy, G. E., Henagan, T. M., Prpic‐Uhing, V. Richards, W. G., Bannon, A. W., Mynatt, R. L., Gettys, T. W. Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin. FASEB J. 19, 1482–1491 (2005)

Structure-activity studies related to ABT-594, a potent nonopioid analgesic agent: Effect of pyridine and azetidine ring substitutions on nicotinic acetylcholine receptor binding affinity and analgesic activity in mice

Analogs of A-98593 (1) and its enantiomer ABT-594 (2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays.