NMR studies of interactions of new CB2 cannabinoid receptor ligands with cyclodextrins hosts. Correlation with micellar electrokinetic chromatography and reversed phase high performance liquid chromatography

  • Nathalie Azaroual
  • Jamal El Bakali
  • Delphine Le Broc
  • Carole Deghaye
  • Amaury Farce
  • Philippe Chavatte
  • Régis Millet
  • Claude Vaccher
  • Emmanuelle Lipka-BelloliEmail author
Original Article


Three selective CB2 cannabinoid receptor ligands have recently been discovered to be promising anti-inflammatory agents but their low water solubility hinder their per os administration. The popularity of the cyclodextrins, from a pharmaceutical standpoint lies on their ability to interact with poorly water-soluble drugs and improve their solubility. Herein, three experimental approaches for calculating the stability constant of complexes between the selective CB2 ligands and either the β-CD or the HP-β-CD, were tested: nuclear magnetic resonance, micellar electrokinetic chromatography and high performance liquid chromatography in reversed phase. In NMR studies the calculated K values were relatively high and were between 1486 and 3571 M−1 with β-CD. With HP-β-CD they were between 1203 and 2650 M−1. Concerning the two others techniques the K values were found lower. In MECK studies with β-CD they were between 308 and 792 M−1 and with HP-β-CD between 124 and 764 M−1. Finally in RP-HPLC studies with β-CD, they were between 539 and 1144 M−1 and with HP-β-CD between 196 and 396 M−1. These calculated constants suggest that a complexation phenomenon occurs. A model for inclusion of one of the CB2 ligands in the β-CD was then proposed from molecular modeling studies.


IBD 4-oxo-1 4-Dihydropyridine Solubility Formation constant Molecular modeling studies 



This work was financially supported by a grant from the Nord-Pas-de-Calais Regional Council and University of Lille 2. The 500 MHz NMR facilities were funded by the Région Nord-Pas de Calais (France), the Ministère de la Jeunesse, de l’Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER).


  1. 1.
    Massa, F., Marsicano, G., Hermann, H., Cannich, A., Monory, K., Cravatt, B.F., Ferri, G.L., Sibaev, A., Storr, M., Lutz, B.: The endogenous cannabinoid system protects against colonic inflammation. J. Clin. Invest. 113, 1202–1209 (2004)Google Scholar
  2. 2.
    Kimball, E.S., Schneider, C.R., Wallace, N.H., Hornby, P.J.: Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis induced by oil of mustard and by dextran sulfate sodium. Am. J. Physiol. Gastrointest. Liver Physiol. 291, 364–371 (2006)CrossRefGoogle Scholar
  3. 3.
    Singh, U.P., Singh, N.P., Singh, B., Price, R.L., Nagarkatti, M., Nagarkatti, P.S.: Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10(-/-) mice by attenuating the activation of T cells and promoting their apoptosis. Toxicol. Appl. Pharmacol. 258, 256–267 (2012)CrossRefGoogle Scholar
  4. 4.
    Storr, M.A., Keenan, C.M., Emmerdinger, D., Zhang, H., Yüce, B., Sibaev, A., Massa, F., Buckley, N.E., Lutz, B., Göke, B., Brand, S., Patel, K.D., Sharkey, K.A.: Targeting endocannabinoid degradation protects against experimental colitis in mice: involve-ment of CB1 and CB2 receptors. J. Mol. Med. (Berl) 86, 925–936 (2008)CrossRefGoogle Scholar
  5. 5.
    Andrzejak, V., Muccioli, G.G., Body-Malapel, M., El Bakali, J., Djouina, M., Renault, N., Chavatte, P., Desreumaux, P., Lambert, D.M., Millet, R.: New FAAH inhibitors based on 3-carboxamido-5-aryl-isoxazole scaffold that protect against experimental colitis. Bioorg. Med. Chem. 19, 3777–3786 (2011)CrossRefGoogle Scholar
  6. 6.
    Alhouayek, M., Lambert, D.M., Delzenne, N.M., Cani, P.D., Muccioli, G.G.: Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation. FASEB J. 25, 2711–2721 (2011)CrossRefGoogle Scholar
  7. 7.
    El Bakali, J., Muccioli, G.G., Renault, N., Pradal, D., Body-Malapel, M., Djouina, M., Hamtiaux, L., Andrzejak, V., Desreumaux, P., Chavatte, P., Lambert, D.M., Millet, R.: 4-Oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands: structural insights into the design of a novel inverse agonist series. J. Med. Chem. 53, 7918–7931 (2010)CrossRefGoogle Scholar
  8. 8.
    El Bakali, J., Gilleron, P., Body-Malapel, M., Mansouri, R., Muccioli, G.G., Djouina, M., Barczyk, A., Klupsch, F., Andrzejak, V., Lipka, E., Furman, C., Lambert, D.M., Chavatte, P., Desreumaux, P., Millet, R.: 4-Oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands part 2: discovery of new agonists endowed with protective effect against experimental colitis. J. Med. Chem. 55, 8948–8952 (2012)CrossRefGoogle Scholar
  9. 9.
    Dahan, A., Hoffman, A.: Rationalizing the selection of oral lipid based drug delivery systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs. J. Control. Rel. 129, 1–10 (2008)CrossRefGoogle Scholar
  10. 10.
    Rupp, C., Steckel, H., Müller, B.W.: Solubilization of poorly water-soluble drugs by mixed micelles based on hydrogenated phosphatidylcholine. Int. J. Pharm. 395, 272–280 (2010)CrossRefGoogle Scholar
  11. 11.
    Merisko-Liversidge, E., Liversidge, G.G., Cooper, E.R.: Nanosizing: a formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci. 18, 113–120 (2003)CrossRefGoogle Scholar
  12. 12.
    Brewster, M.E.: Cyclodextrins as pharmaceutical solubilizers. Adv. Drug Deliv. Rev. 59, 645–666 (2007)CrossRefGoogle Scholar
  13. 13.
    Loftsson, T., Hreinsdóttir, D., Másson, M.: Evaluation of cyclodextrin solubilization of drugs. Int. J. Pharm. 302, 18–28 (2005)CrossRefGoogle Scholar
  14. 14.
    Stella, V., Rao, V., Zannou, E., Zia, V.: Mechanisms of drug release from cyclodextrin complexes. Adv. Drug Deliv. Rev. 36, 3–16 (1999)CrossRefGoogle Scholar
  15. 15.
    Job, P.: Recherches sur la formation de complexes minéraux en solution et leur stabilité. Ann. Chim. 9, 113–203 (1928)Google Scholar
  16. 16.
    Sharff, A.J., Rodseth, L.E., Quiocho, F.A.: Refined 1.8-.ANG structure reveals the mode of binding of β-cyclodextrin to the maltodextrin binding protein. Biochemistry 32, 10553–10559 (1993)CrossRefGoogle Scholar
  17. 17.
    Clark, M., Cramer III, R.D., Van Opdenbosch, N.: Validation of the general purpose tripos 5.2 force field. J. Comput. Chem. 10, 982–1012 (1989)CrossRefGoogle Scholar
  18. 18.
    Jones, G., Willett, P., Glen, R.C., Leach, A.R., Taylor, R.: Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748 (1997)CrossRefGoogle Scholar
  19. 19.
    Schneider, H.: Supermolecular chemistry. Chap 36. NMR spectroscopy and molecular-mechanics calculations in supermolecular chemistry. Rec. Trav. Chem. 112, 412–419 (1993)CrossRefGoogle Scholar
  20. 20.
    Ganza-Gonzalez, A., Vila-Jato, J., Anguiano-Igea, S., Otero-Espinar, F., Blanco-Mendez, J.: A proton nuclear magnetic resonance study of the inclusion of naproxen with β-cyclodextrin. Int. J. Pharm. 106, 179–185 (1994)CrossRefGoogle Scholar
  21. 21.
    Piel, G., Labres, L., Evrard, B., Van Hees, T., de Hassonville, S., Delattre, L.: A nuclear magnetic resonance study of the miconazole-β-cyclodextrin inclusion complex in an acidic medium: determination of the structure and stability constant. STP Pharm. Sci. 11, 235–238 (2001)Google Scholar
  22. 22.
    Benesi, H.A., Hildebrand, J.H.: A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J. Am. Chem. Soc. 71, 2703–2707 (1949)CrossRefGoogle Scholar
  23. 23.
    Masson, M., Sigurjonsdottir, J., Jonsdottir, S., Loftsson, T.: Estimation of 19F-NMR as a tool for investigation of drug-cyclodextrin complexes. Drug Dev. Ind. Pharm. 29, 107–112 (2003)CrossRefGoogle Scholar
  24. 24.
    Polyakov, N., Leshina, T., Konovalova, T., Hand, E., Kispert, L.: Inclusion complexes of carotenoids with cyclodextrins: 1H-NMR, EPR and optical studies. Free Radic. Biol. Med. 36, 872–880 (2004)CrossRefGoogle Scholar
  25. 25.
    Terabe, S., Otsuka, K., Ando, T.: Electrokinetic chromatography with micellar solution and open-tubular capillary. Anal. Chem. 57, 834–841 (1985)CrossRefGoogle Scholar
  26. 26.
    Scriba, G.E.: Fundamental aspects of chiral electromigration techniques and application in pharmaceutical and biomedical analysis. J. Pharm. Biomed. Anal. 55, 688–701 (2011)CrossRefGoogle Scholar
  27. 27.
    Gazpio, C., Sanchez, M., Garcia-Zubiri, I.X., Velaz, I., Martinez Oharriz, C., Martin, C., Zornoza, G.: HPLC and solubility study of the interaction between pindolol and cyclodextrins. J. Pharm. Biomed. Anal. 37, 1–9 (2005)CrossRefGoogle Scholar
  28. 28.
    Lopez-Nicolas, J.M., Bunez-Delicado, E., Perez-Lopez, A.J., Carbonell Barrachina, A., Cuadra-Crespo, P.: Determination of stoichiometric coefficients and apparent formation constants for β-cyclodextrin complexes of trans-resveratrol using reversed-phase liquid chromatography. J. Chrom. A 1135, 158–165 (2006)CrossRefGoogle Scholar
  29. 29.
    Bielejewska, A., Duszczyk, K., Sybilska, D.: Influence of organic solvent on the behaviour of camphor and α-pinene enantiomers in reversed-phase liquid chromatography systems with α-cyclodextrin as chiral additive. J. Chrom. A 931, 81–93 (2001)CrossRefGoogle Scholar
  30. 30.
    Darrouzain, F., Matoga, M., Cavalli, E., Thomassin, M., Ismaili, L., Guillaume, Y.C.: Thermodynamic approach for studying both the retention and complexation mechanisms with hydroxypropyl-β-cyclodextrin of a phenoxy-propionic acid herbicide series. Talanta 64, 836–843 (2004)CrossRefGoogle Scholar
  31. 31.
    Claude, B., Morin, Ph, Lafosse, M., Andre, P.: Evaluation of apparent formation constants of pentacyclic triterpene acids complexes with derivatized β- and γ-cyclodextrins by reversed phase liquid chromatography. J. Chrom. A 1049, 37–42 (2004)Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Nathalie Azaroual
    • 1
  • Jamal El Bakali
    • 1
  • Delphine Le Broc
    • 2
  • Carole Deghaye
    • 1
  • Amaury Farce
    • 1
  • Philippe Chavatte
    • 1
  • Régis Millet
    • 1
  • Claude Vaccher
    • 1
  • Emmanuelle Lipka-Belloli
    • 1
    • 3
    Email author
  1. 1.UDSL, EA 4481Université Lille Nord de FranceLilleFrance
  2. 2.UDSL, EA 4483Université Lille Nord de FranceLilleFrance
  3. 3.Laboratoire de Chimie Analytique, Faculté des Sciences Pharmaceutiques et BiologiquesUniversité de Lille 2Lille CedexFrance

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