New data concerning endogenous morphine,description of its localization in the central nervous system and development of biotechnological and therapeutic tools

C est depuis les années 80 que de nombreuses études ont démontré la présence de morphine endogène (ME), de structure identique à la morphine du pavot, dans le système nerveux central (SNC) de mammifères. Cependant, déterminer ses rôles au sein du SNC est difficile puisqu il existe que très peu de données la concernant (localisation cellulaire, protocoles de quantification). Dans ce contexte, mon objectifs était fournir de nouveaux outils permettant l'étude et la compréhension des rôles de la ME au sein du SNC. La première partie de mes résultats décrit la localisation de la ME au sein du SNC de souris adulte. Après avoir caractérisé l'anticorps anti-morphine que j ai utilisé, j ai montré que la ME et ses dérivés sont principalement retrouvés dans les interneurones GABAergiques et les astrocytes du SNC. La seconde partie de mes résultats présente les études réalisées sur deux protéines liant les alcaloïdes endogènes/exogènes : (i) la PhosphatidylEthanolamine Binding Protein, capable de lier la M6G et la M3G avec une affinité identique à son ligand de référence, la PhosphatidylEthanolamine. (ii) La créatine kinase, qui lie à haute affinité la ME et ses dérivés. Du plus, j ai décrit une dissociation de ce complexe par le lithium. Enfin ma dernière partie décrit que l'utilisation du lithium lors de prélèvements sanguins permet d'augmenter jusqu à quatre fois le rendement de détection de la ME. En conclusion, ce travail de thèse présente de nouveaux outils et de nouvelles pistes de recherche permettant l'étude et la compréhension des rôles de la ME au sein du SNC (cartographie, protéine de liaison, amélioration de la quantification).Since 80s, endogenous morphine (eM), structurally identical to the morphine from poppy, has been found in mammalian central nervous system (CNS). Only few informations are available about eM (cellular localization, models and quantification protocols) and thus, studies of its roles and implications in the brain physiology is difficult. During my thesis, my goal was to developed new tools in order to study and understand the roles of eM in the CNS. First, I have described the localization of eM in the CNS of adult mouse. Using a well characterized antibody, I have demonstrated that eM and its derived compounds are mainly found in astrocytes and GABAergic interneurons throughout the mouse CNS. Secondly, I described two endogenous / exogenous alkaloids binding proteins. (i) The PhosphatidylEthanolamine Binding Protein that is able to bind M6G and M3G in a similar manner as its reference ligand PhosphatidylEthanolamine. (ii) The creatine kinase (CK) that is able to bind eM and its derived compounds with high affinity. Such CK-eM complex is dissociated by a lithium treatment. Finally, in the last part of my results, I have described that the presence of lithium in the collection tube allows a better measuring of eM and exogenous morphine. To conclude, during my thesis I set up new tools and new research lines (eM CNS mapping, binding proteins and better quantification yield) that will allow to study and understand the roles of eM in the CNS of mammals

Morphine binds creatine kinase B and inhibits its activity

Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I-2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a mu M affinity for morphine and binds to morphine in vitro. The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B1-75 and CK-B184-258 were identified as two specific morphine binding-peptides. In vitro, morphine (1-100 mu M) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo

Corrigendum: Morphine binds creatine kinase B and inhibits its activity (vol 12, 464, 2018)

In the published article, there was an error in affiliation "2." Instead of "Global Drug Discovery, Global Therapeutic Research Groups, Gynecological Therapies, Bayer Healthcare, Berlin, Germany", it should be "Laboratoire de Spectrométrie deMasse BioOrganique, IPHC-DSA, CNRS UMR7178 and Université de Strasbourg, Strasbourg, France." Additionally, the current address of one author has been added as a footnote and is denoted using. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

Comparison of serum and lithium-heparinate plasma for the accurate measurements of endogenous and exogenous morphine concentrations

International audienceNo abstract availabl

Lithium reverses mechanical allodynia through a mu opioid-dependent mechanism

Background: Lithium is widely used to treat bipolar disorders and displays mood stabilizing properties. In addition, lithium relieves painful cluster headaches and has a strong analgesic effect in neuropathic pain rat models

Stable isotope-labelled morphine to study in vivo central and peripheral morphine glucuronidation and brain transport in tolerant mice

International audienceBackground and purpose: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels.Experimental approach: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms.Key results: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study.Conclusions and implications: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine