Laboratory of Pharmacology of Pain

Head of the Lab
Catherine ROUGEOT


The main goal of the research group is focused on the study of the intercellular regulatory mechanisms of neuroendocrine networks and specifically of mu- and delta-opioidergic pathways, which control pain perception in mammals

The center of the sensory nerve signal transmission control is primarily located at the spinal cerebrospinal tract, schematized Fig1-a. Briefly, the pain signals emanating from the nerve endings of the skin tissues, muscles and joints and viscera (= detection system) activated in response to mechanical, thermal or chemical stimuli are conveyed by afferent nerve pathways to the level of the dorsal horn of the spinal cord. In the spinal tissue (= transmission system) also converge specialized interneurons that release enkephalins in response to noxious stimulus (= negative control system). By binding with high affinity to opioid membrane receptors (MOR and DOR) enkephalins will activate them and therefore inhibit the transmission of pain signals to the brain. The perception of pain is reduced. However, the ectoenkephalinases NEP (NeutralEndoPeptidase) and AP-N (AminoPeptidase-N), located in close proximity to the opioid membrane receptors, by destroying circulating enkephalins within nerve synaptic spaces, will quickly neutralize their action. This mechanism of pain perception control that takes place at peripheral, spinal and central nervous systems ensures under physiological conditions the dynamic equilibrium: pronocicetion / antinociception and the behavioral responses appropriate to the nature, duration and intensity of the pain stimulus (Fig. 1-a).

Fig. 1-a: Representative diagram of the inhibitory action of enkephalinergic pathways on the transmission
of nociceptive signals at the level of the dorsal horn of the spinal cord.

Combining a multi-disciplinary approach including genetic, biochemistry, molecular and behavioral pharmacology, we discovered the existence of physiological inhibitors of the enkephalin-inactivating Zn-ectopeptidases, NEP and AP-N in mammals. Rat Sialorphin (QHNPR-pentapeptide), was identified using integrative post-genomic and Human Opiorphin (QRFSR-pentapeptide) was then characterized using functional biochemistry. Integrative pharmaco-chemical studies allowed us to elucidate their structure and function relationship.

Thus, in vivo, Opiorphin or Sialorphin by increasing the bioavailability of endogenous enkephalins released in response to noxious stimulus potentiates their physiological action in terms of amplitude and duration of action and thus reinforces the negative feedback control exerted by enkephalin on the transmission pain signals (Fig. 1-b). The transmission of nociceptive messages to the brain is inhibited and thus the perception of pain is highly attenuated or blocked.

Fig. 1-b: Representative diagram of the mechanism of human Opiorphin
or rat Sialorphin action on enkephalinergic pathways.

Major results and objectives

Rat Sialorphin: a ligand and a physiological regulator of NEP-enkephalinase

Sialorphin is a hormonal peptide messenger of intercellular communication in the rat that was identified using a pharmaco-biochemical post-genomic approach. Its functional characterization was based on the molecular identification of membrane receptor sites that bind circulating Sialorphin, in vivo (Rougeot et al., 1994, Eur. J. Biochem., 219: 765; Rougeot et al., 1997, Am. J. Physiol., 273: R1309; Rougeot et al., 2000, Peptides, 21: 443) (Fig. 2).

Fig. 2: Visualization of the main systemic targets
for circulating Sialorphin in the adult male rat.

Red grains, organs or tissues; black grains, cells.

We discovered that Sialorphin is a physiological ligand of rat NEP and a competitive inhibitor of membrane bound NEP.

Using two behavioral models of peripheral injury-induced acute pain in the adult male rat, we demonstrated that Sialorphin elicits a powerful anti-nociceptive activity. The analgesic effects induced by Sialorphin require the specific activation of endogenous mu- and delta-opioïd receptors, which are involved in the transmission of enkephalinergic signals. Altogether, ex vivo and in vivo data suggest that by protecting endogenous enkephalins released in response to pain stimuli from inactivation by the ectoenkephalinases, Sialorphin potentiates their physiological actions, and in particular anti-nociception (Rougeot et al., 2003, PNAS, 100: 8549).

Human Opiorphin: a natural inhibitor of enkephalin-inactivating Zn-ectopeptidases NEP and AP-N, and a novel modulator of enkephalin-dependant opioid pathways.
A functional biochemical approach allowed us to discover the existence in human of a pentapeptide with similar properties to rat Sialorphin as a physiological inhibitor of the enkephalin-inactivating Zn-ectopeptidases. Indeed, using molecular pharmacology (recombinant pure hNEP-hAP-N) and cellular pharmacology approaches (human cell lines expressing hNEP and hAP-N), we demonstrated that Opiorphin is a dual inhibitor of both hNEP and hAP-N ectopeptidases. The first discovered to date in humans. In addition, we demonstrated that opiorphin protects enkephalins from degradation by these membrane ectoenkephalinases and highly increases their specific binding capacity and affinity for mu- and delta-opioid membrane receptors (Tòth et al. 2012, Regulatory peptides 178: 71)

The behavioral pharmacological responses induced, in vivo in the rat, by human Opiorphin were then searched, notably those involving the nociceptive neurotransmission pathways, which are under the negative feedback control of enkephalins. This research program benefited from the support of the Agence Nationale pour la Recherche (Emergence et maturation de projets de biotechnologie à fort potentiel de valorisation, 2006-2008).
Using a behavioral model of mechanical acute pain in rat, we demonstrated that Opiorphin displays pain-suppressive potency comparable to morphine (Wisner et al. 2006, Proc. Natl. Acad. Sci. USA. 103: 17974); Rougeot et al. 2007, Med Sci. 23: 33, Rougeot et al. 2008, Proc. Int. Peptide Symp.) (Fig. 3).

Fig.3: Comparison of the behavioral responses of Opiorphin or morphine in the Pin Pain test.
***P=0.0002 vs vehicle by Mann-Whitney U-test.

Furthermore, we demonstrated that Opiorphin elicited minimal adverse morphine-associated effects at systemically active doses that produced analgesia in several experimental models of pain in rats. The analgesic responses induced by Opiorphin, which require activation of endogenous mu-opioid pathways, is comparable to that induced by the morphine mu-opioid agonist, in terms of effective doses and delay of action. However, in contrast to morphine, Opiorphin does not develop significant abuse liability, anti-nociceptive drug tolerance or anti-peristaltism (Rougeot et al. 2010, J. Physiol. Pharmacol. 61(4): 483). Thus, the action of Opiorphin is restricted to opioidergic pathways specifically and dynamically activated by endogenous enkephalins whose amplitude and duration of secretion is dependent on the intensity and nature of the stimulus. Unlike morphine or other opioid agonist that will stimulate all the opioid receptors in the body (including those not involved in the pain control) and will saturate at high dose and/or repeated dose these receptors thus preventing the regulation of their expression and transport to the cell membrane.

In addition to pain control, endogenous opioid pathways are also implicated in the modulation of emotion-related behaviors. Thus, we explored the motivational responses induced by opiorphin using the forced swim test, the standard rat model of depression. We found that at 1-2 mg/kg i.v. doses, opiorphin elicited antidepressant-like effects by activating endogenous δ-opioidergic pathways. The antidepressive behavioral responses exerted by opiorphin are specific at systemically active doses. Treated-rats did not develop either hypo- or hyper-active responses in a locomotor test or amnesic behavioral response in the passive avoidance rat model. In addition, opiorphin did not induce either anxiolytic-, or anxiogenic-like responses in the conditioned defensive burying test (Rougeot et al. 2010, J. Physiol. Pharmacol. 61(3): 355).

Taking the data together, Opiorphin, is a systemically active key modulator of both pain perception and motivational adaptation responses to physical and psychological stimuli in mammals. Its key integrative role is mediated through enkephalin-related activation of mu and delta opioidergic pathways.
This discovery is of crucial importance from a physiological and physio-pathological point of view, given the extent of the functions mediated by the endogenous opioidergic pathways.

In a physiological scenario because of its mechanism of action, human Opiorphin would be involved in the process of adaptation mediated by enkephalins and in particular in the regulation of the homeostatic equilibrium: pronociception-antinociception and depression-motivation. To explore the profile of secretion and distribution of Opiorphin in healthy human biological media, a clinical research protocol, which received ethic committee agreement (Paris-Cochin), was established. The results of this propram, obtained in physiological situations provide the bases for further approaches to identify human pathological states up- or down-regulating the levels of circulating Opiorphin.
Finally, because of its in vivo properties, Opiorphin may have important therapeutic applications as a potential initiator of molecular pathways, which could be exploited to develop new drug-candidates. Thus, we design conformational restricted Opiorphin mimetics, which retain the activity profile of the native peptide and present increased metabolic stability (Rougeot C. 2009; patent N° WO/2009/124948). Opiorphin or optimized derivatives is a promising single candidate to treat disorders that include both pain and mood disorders, particularly depression.

Institut Pasteur and a Swiss based group of companies, Stragen Pharma, signed (January 2011) a collaborative research and worldwide licence option agreement for the first in class pain management novel drug.

Technical know-how
Topics : Biochemistry, Molecular and Integrative Pharmaco-Chemistry

* Protein and small peptide purifications (HPLC).
* Quantitative Immuno-assays (ELISA, RIA…).
* Pharmacokinetics and biodistribution analyses (in vitro, ex vivo and in vivo).
* FRET-based peptidase assays.



Last update of this site : September 2012