Jean-Claude Louis

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.

Antihyperalgesic Effects of (R,E)-N-(2-Hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)phenyl)-acrylamide (AMG8562), a Novel Transient Receptor Potential Vanilloid Type 1 Modulator That Does Not Cause Hyperthermia in Rats

Antagonists of the vanilloid receptor TRPV1 (transient receptor potential vanilloid type 1) have been reported to produce antihyperalgesic effects in animal models of pain. These antagonists, however, also caused concomitant hyperthermia in rodents, dogs, monkeys, and humans. Antagonist-induced hyperthermia was not observed in TRPV1 knockout mice, suggesting that the hyperthermic effect is exclusively mediated through TRPV1. Since antagonist-induced hyperthermia is considered a hurdle for developing TRPV1 antagonists as therapeutics, we investigated the possibility of eliminating hyperthermia while maintaining antihyperalgesia. Here, we report four potent and selective TRPV1 modulators with unique in vitro pharmacology profiles (profiles A through D) and their respective effects on body temperature. We found that profile C modulator, (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)phenyl)acrylamide (AMG8562), blocks capsaicin activation of TRPV1, does not affect heat activation of TRPV1, potentiates pH 5 activation of TRPV1 in vitro, and does not cause hyperthermia in vivo in rats. We further profiled AMG8562 in an on-target (agonist) challenge model, rodent pain models, and tested for its side effects. We show that AMG8562 significantly blocks capsaicin-induced flinching behavior, produces statistically significant efficacy in complete Freunds adjuvant- and skin incision-induced thermal hyperalgesia, and acetic acid-induced writhing models, with no profound effects on locomotor activity. Based on the data shown here, we conclude that it is feasible to modulate TRPV1 in a manner that does not cause hyperthermia while maintaining efficacy in rodent pain models.

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.

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.

Influence of adrenocorticotropic hormone on the growth of isolated neurons in culture.

The present study reports the influence of adrenocorticotropic hormone (ACTH) on the development of cultured neurons from chick embryo cerebral hemispheres. Cultures were initiated in serum-supplemented medium and then transferred to serum-free hormonally-defined medium containing various concentrations of ACTH1--24. The effects of ACTH on the light microscopic features, metabolic activity and permeation properties were examined. The results demonstrate that ACTH exerts a trophic action on the neurons.

Presence of Somatostatin, Enkephalins, and Substance P‐Like Peptides in Cultured Neurons from Embryonic Chick Cerebral Hemispheres

Abstract: The presence of peptides in pure cultures of neurons from 8‐day‐old chick embryo cerebral hemispheres has been investigated by means of specific radioimmunoassays and chromatographic purification. Somatostatin, Met‐enkephalin, Leu‐enkephalin, and substance P immunoreactive substances have been detected in 8‐day‐old cultures grown in serum‐free culture medium. The peptides were present in the cellular extracts, as well as in the culture medium extracts. β‐Endorphin, thyroliberin, luteinizing hormone‐releasing hormone, and ACTH could not be detected. The largest amount was accounted by somatostatin (48 ± 2 ng/mg protein). Some 60% of the somatostatin‐immunoreactive material was found in the culture medium. Met‐enkephalin, Leuenkephalin, and substance P were present at lower concentrations: 1.61 ± 0.27, 0.24 ± 0.02, and 0.14 ± 0.005 ng/mg protein, respectively. The identities of somatostatin‐ and enkephalin‐immunoreactive materials were confirmed by high pressure liquid chromatography. The findings suggest that cultured neurons that express dopaminergic and GABAergic properties contain peptides similar, if not identical, to somatostatin, Met‐enkephalin, Leu‐enkephalin, and substance P.

Synthesis of choline phospholipids in neuronal and glial cell cultures by the methylation pathway.

The synthesis of choline in the nervous tissue has been a subject of debate (reviews [1,2]). Since the work of [3,4] it was believed that the stepwise methylation of ethanolamine and/or phosphatidylethanolamine in nervous tissue was non-existant or irrelevant until the suggestion that choline might be produced de novo in the rat brain [5-7] through methylation of phosphatidylethanolamine. Methyltransferase activity has been shown in rat brain synaptosomes [8,9], suggesting that nervous tissue may have the necessary machinery for the de novo synthesis of phosphatidylcholine. The possibility of obtaining cell cultures containing exclusively neurons or glial cells gave us the opportunity to check, in these isolated systems, whether neurons and/or glia can methylate phosphatidylethanolamine to phosphatidylcholine. The results obtained suggest that both cell types have this capacity and that the synthesis of choline phospholipids through the methylation pathway is much higher in glial cells than in neurons.

Effect of ethanol on superoxide dismutase activity in cultured neural cells

Superoxide dismutase (EC 1.15.1.1) activity was investigated in several types of neural cells cultivated in the presence of 100 mM ethanol. Superoxide dismutase was inhibited by acute treatment with ethanol. Chronic treatment with ethanol specifically inhibited superoxide dismutase in glial cells. In all instances withdrawal of ethanol produced a quick return to control values. Inhibition of superoxide dismutase by ethanol may increase toxic oxygen radicals in nervous tissue.

Separation from protein kinase C: a calcium-independent TPA-activated phosphorylating system

A calcium‐independent but 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐ or diacylglycerol‐activated phospholipid‐dependent phosphorylating activity has been separated from protein kinase C. This has been made possible by employing calcium‐dependent hydrophobic interaction chromatography. The material bound to phenyl‐Sepharose in the presence of calcium at low ionic strength was eluted with EGTA and was protein kinase C. While the unbound material passing through the phenyl‐Sepharose column showed no appreciable protein kinase C activity, instead it had a high phosphorylating activity manifested in the absence of calcium and in the presence of TPA plus phospholipid. The identification of this phosphorylating activity, distinct from protein kinase C, leads to important clues to cellular responses monitored by TPA in the absence of calcium.

Possible role of sialocompounds in the uptake of choline into synaptosomes and nerve cell cultures

Incubation of primary nerve cell cultures and of crude synaptosomal preparations with neuraminidase released sialic acid from both gangliosides and sialoglycoproteins. After this treatment, the pattern of ganglioside distribution was severely modified with a decrease of polysialogangliosides (GD1b, GT1b, GT1L, GQ1) and a dramatic increase in monosialoganglioside GM1. The choline influx into neuraminidase treated cells and organelles was reduced by 30–50% but the efflux was unmodified. In particular the high affinity mechanism of choline uptake disappeared and the low affinity mechanism was modified in both cases. The disappearance of the high affinity uptake mechanism was not followed by a decreased acetylcholine synthesis as it should be if the current theories on choline uptake and acetylcholine synthesis are correct. Our present data thus confirm our previous hypothesis that choline metabolism regulates choline uptake rather than the other way round as is suggested by the theories most widely accepted at present. Choline uptake was unaffected by pretreatment of cells and organelles with tetanus toxin suggesting that the effect of neuraminidase on the choline uptake were either mediated through glycoproteins or through gangliosides other than those which bind to tetanus toxin (GD1b and GT1b). Several speculative models for explaining the effect of neuraminidase on choline uptake are proposed.