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An Antedrug of the CXCL12 Neutraligand Blocks Experimental Allergic Asthma without Systemic Effect in Mice*

  • François Daubeuf
    Footnotes
    Affiliations
    Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France

    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France
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  • Muriel Hachet-Haas
    Footnotes
    Affiliations
    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France

    Laboratoire de Biotechnologie et Signalisation Cellulaire, Ecole de Biotechnologie de Strasbourg, UMR 7242 CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie, Bd. Sébastien Brant, 67412 Illkirch, France
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  • Patrick Gizzi
    Footnotes
    Affiliations
    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France

    Laboratoire de Biotechnologie et Signalisation Cellulaire, Ecole de Biotechnologie de Strasbourg, UMR 7242 CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie, Bd. Sébastien Brant, 67412 Illkirch, France
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  • Vincent Gasparik
    Footnotes
    Affiliations
    Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France

    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France
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  • Dominique Bonnet
    Affiliations
    Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France

    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France
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  • Valérie Utard
    Affiliations
    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France

    Laboratoire de Biotechnologie et Signalisation Cellulaire, Ecole de Biotechnologie de Strasbourg, UMR 7242 CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie, Bd. Sébastien Brant, 67412 Illkirch, France
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  • Marcel Hibert
    Affiliations
    Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France

    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France
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  • Nelly Frossard
    Affiliations
    Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France

    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France
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  • Jean-Luc Galzi
    Correspondence
    To whom correspondence should be addressed. Tel.: 33-3-6-68-02-84-21;
    Affiliations
    Laboratory of Excellence MEDALIS, Université de Strasbourg, 67400 Illkirch, France

    Laboratoire de Biotechnologie et Signalisation Cellulaire, Ecole de Biotechnologie de Strasbourg, UMR 7242 CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie, Bd. Sébastien Brant, 67412 Illkirch, France
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  • Author Footnotes
    * This work was supported by funds from CNRS, the Institut National pour la Santé et la Recherche Médicale, and the Agence Nationale pour la Recherche (ANR).
    1 Both authors contributed equally to this work.
    2 Supported by a fellowship from the Fonds de Recherche en Santé Respiratoire from Société de Pneumologie de langue française (SPLF) and the Region Alsace.
    3 Supported by a fellowship from ANR.
    4 Supported by a fellowship from CNRS.
Open AccessPublished:February 28, 2013DOI:https://doi.org/10.1074/jbc.M112.449348
      The chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair, in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue and should not diffuse in the systemic circulation, where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by the intranasal route, but not when administered systemically by the intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands although the target protein is widely distributed in the organism.

      Introduction

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      Small neutralizing molecules to inhibit actions of the chemokine CXCL12.
      ,
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      • Brotin E.
      • Biajoux V.
      • Bouchet-Delbos L.
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      Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice.
      ,
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      The chemokine CXCL12 is essential for the clearance of the filaria Litomosoides sigmodontis in resistant mice.
      ). Chalcone 4 blocks responses of CXCR4 to CXCL12 in vitro without affecting the basal level receptor activity and displays anti-inflammatory effects in a murine model of asthma in vivo.
      Figure thumbnail grs1
      SCHEME 1A, synthesis of CN-chalcone 4 (CN-Chalc4) following a Knoevenagel condensation of pCBA with 3-methoxy-4-(methoxymethyloxy)benzaldehyde (VanP). B, structure of reference compounds chalcone 4 (Chalc 4) and unsubstituted chalcone (Chalc 1).
      In order to favor desired local anti-inflammatory action of the neutraligand at the expense of undesired distant effects, we explored the possibility of generating a short lived neutraligand prone to efficient biodegradation before it distributes in the body and reaches unwanted tissues. To this end, we describe here the synthesis of a molecule using previously unexploited Knoevenagel/retro-Knoevenagel reactions to generate so called soft drugs or antedrugs (
      • Khan M.O.
      • Park K.K.
      • Lee H.J.
      Antedrugs. An approach to safer drugs.
      ). Characterization of the functional properties of the new short lived carbonitrile-chalcone, in vitro and in vivo, shows that it is active when administered locally and inactive after systemic administration.

      DISCUSSION

      Our results show the anti-inflammatory effect of a rapidly hydrolyzable CXCL12 neutraligand in an airway hypereosinophilia model. Carbonitrile-chalcone 4 is an efficient blocker of CXCL12 binding to CXCR4 and of the associated inhibition of cAMP production. However, in biological fluids, CN-chalcone 4 is rapidly degraded into two inactive metabolites, vanillin and pCBA, the two compounds that served as synthetic building blocks for its production. When administered locally in the airways by the intranasal route, CN-chalcone 4 efficiently inhibits eosinophil, neutrophil, and T cell recruitment at a low dose. By contrast, it remains without any anti-inflammatory effect in the airways when administered systemically by the intraperitoneal route even at doses 100–1000-fold higher. This is opposed to the systemic effect of chalcone 4 and demonstrates that CN-chalcone 4 behaves as an antedrug or soft drug acting at the administration site that is degraded prior to wider distribution.
      Three groups, including ours (
      • Gasparik V.
      • Daubeuf F.
      • Hachet-Haas M.
      • Rohmer F.
      • Gizzi P.
      • Haiech J.
      • Galzi J.L.
      • Hibert M.
      • Bonnet D.
      • Frossard N.
      Prodrugs of a CXC chemokine-12 (CXCL12) neutraligand prevent inflammatory reactions in an asthma model in vivo.
      ,
      • Gonzalo J.A.
      • Lloyd C.M.
      • Peled A.
      • Delaney T.
      • Coyle A.J.
      • Gutierrez-Ramos J.C.
      Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 α in the inflammatory component of allergic airway disease.
      ,
      • Hachet-Haas M.
      • Balabanian K.
      • Rohmer F.
      • Pons F.
      • Franchet C.
      • Lecat S.
      • Chow K.Y.
      • Dagher R.
      • Gizzi P.
      • Didier B.
      • Lagane B.
      • Kellenberger E.
      • Bonnet D.
      • Baleux F.
      • Haiech J.
      • Parmentier M.
      • Frossard N.
      • Arenzana-Seisdedos F.
      • Hibert M.
      • Galzi J.L.
      Small neutralizing molecules to inhibit actions of the chemokine CXCL12.
      ,
      • Lukacs N.W.
      • Berlin A.
      • Schols D.
      • Skerlj R.T.
      • Bridger G.J.
      AMD3100, a CxCR4 antagonist, attenuates allergic lung inflammation and airway hyperreactivity.
      ), described that when CXCR4 signaling is inhibited, either with antibodies (
      • Gonzalo J.A.
      • Lloyd C.M.
      • Peled A.
      • Delaney T.
      • Coyle A.J.
      • Gutierrez-Ramos J.C.
      Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 α in the inflammatory component of allergic airway disease.
      ), with CXCR4 antagonists (
      • Lukacs N.W.
      • Berlin A.
      • Schols D.
      • Skerlj R.T.
      • Bridger G.J.
      AMD3100, a CxCR4 antagonist, attenuates allergic lung inflammation and airway hyperreactivity.
      ), or with CXCL12-neutralizing small molecules (
      • Hachet-Haas M.
      • Balabanian K.
      • Rohmer F.
      • Pons F.
      • Franchet C.
      • Lecat S.
      • Chow K.Y.
      • Dagher R.
      • Gizzi P.
      • Didier B.
      • Lagane B.
      • Kellenberger E.
      • Bonnet D.
      • Baleux F.
      • Haiech J.
      • Parmentier M.
      • Frossard N.
      • Arenzana-Seisdedos F.
      • Hibert M.
      • Galzi J.L.
      Small neutralizing molecules to inhibit actions of the chemokine CXCL12.
      ), invasion of lungs by eosinophils is reduced by ∼50%. This piece of evidence highlights a functional role of CXCR4 and of its ligand either in the allergic response onset or in its maintenance. The question as to whether airway inflammation stimulates CXCL12 production continues to be debated because immunohistochemical detection in lung tissue shows no change (
      • Gonzalo J.A.
      • Lloyd C.M.
      • Peled A.
      • Delaney T.
      • Coyle A.J.
      • Gutierrez-Ramos J.C.
      Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 α in the inflammatory component of allergic airway disease.
      ), whereas immunochemical determination in BALF (
      • Negrete-García M.C.
      • Velazquez J.R.
      • Popoca-Coyotl A.
      • Montes-Vizuet A.R.
      • Juárez-Carvajal E.
      • Teran L.M.
      Chemokine (C-X-C motif) ligand 12/stromal cell-derived factor-1 is associated with leukocyte recruitment in asthma.
      ) and gene expression in lung (
      • Fulkerson P.C.
      • Zimmermann N.
      • Hassman L.M.
      • Finkelman F.D.
      • Rothenberg M.E.
      Pulmonary chemokine expression is coordinately regulated by STAT1, STAT6, and IFN-γ.
      ) indicate that CXCL12 is up-regulated. The expression of CXCR4, on the other hand, is higher in BAL CD4+ T cells of human asthmatics as compared with their peripheral blood CD4+ lymphocytes (
      • Thomas S.Y.
      • Banerji A.
      • Medoff B.D.
      • Lilly C.M.
      • Luster A.D.
      Multiple chemokine receptors, including CCR6 and CXCR3, regulate antigen-induced T cell homing to the human asthmatic airway.
      ) and is up-regulated by the proinflammatory cytokine IL-4 in CD4+ T cells, including Th2 cells (
      • Gonzalo J.A.
      • Lloyd C.M.
      • Peled A.
      • Delaney T.
      • Coyle A.J.
      • Gutierrez-Ramos J.C.
      Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 α in the inflammatory component of allergic airway disease.
      ,
      • Eddleston J.
      • Christiansen S.C.
      • Zuraw B.L.
      Functional expression of the C-X-C chemokine receptor CXCR4 by human bronchial epithelial cells. Regulation by proinflammatory mediators.
      ,
      • Jourdan P.
      • Abbal C.
      • Noraz N.
      • Hori T.
      • Uchiyama T.
      • Vendrell J.P.
      • Bousquet J.
      • Taylor N.
      • Pène J.
      • Yssel H.
      IL-4 induces functional cell-surface expression of CXCR4 on human T cells.
      ,
      • Wang J.
      • Harada A.
      • Matsushita S.
      • Matsumi S.
      • Zhang Y.
      • Shioda T.
      • Nagai Y.
      • Matsushima K.
      IL-4 and a glucocorticoid up-regulate CXCR4 expression on human CD4+ T lymphocytes and enhance HIV-1 replication.
      ). This renders significant response to CXCL12 likely to occur in the airway, whatever the regulation of CXCL12 expression. In addition, CXCR4 is also expressed in eosinophils (
      • Nagase H.
      • Miyamasu M.
      • Yamaguchi M.
      • Fujisawa T.
      • Ohta K.
      • Yamamoto K.
      • Morita Y.
      • Hirai K.
      Expression of CXCR4 in eosinophils. Functional analyses and cytokine-mediated regulation.
      ,
      • Nagase H.
      • Miyamasu M.
      • Yamaguchi M.
      • Kawasaki H.
      • Ohta K.
      • Yamamoto K.
      • Morita Y.
      • Hirai K.
      Glucocorticoids preferentially upregulate functional CXCR4 expression in eosinophils.
      ). Eosinophils have a migratory response to CXCL12 comparable with that evoked by eotaxin.
      The mode of action of neutraligands opens the way to new therapeutic strategies especially for airway diseases, because (i) chalcone 4 and its analogs are active through the intranasal route, and (ii) they act on a new target, namely CXCL12, the ligand of CXCR4 and CXCR7 chemokine receptors. Thus, the mode of action of chalcone 4 (
      • Hachet-Haas M.
      • Balabanian K.
      • Rohmer F.
      • Pons F.
      • Franchet C.
      • Lecat S.
      • Chow K.Y.
      • Dagher R.
      • Gizzi P.
      • Didier B.
      • Lagane B.
      • Kellenberger E.
      • Bonnet D.
      • Baleux F.
      • Haiech J.
      • Parmentier M.
      • Frossard N.
      • Arenzana-Seisdedos F.
      • Hibert M.
      • Galzi J.L.
      Small neutralizing molecules to inhibit actions of the chemokine CXCL12.
      ) and its analogs chalcone 4-phosphate (
      • Gasparik V.
      • Daubeuf F.
      • Hachet-Haas M.
      • Rohmer F.
      • Gizzi P.
      • Haiech J.
      • Galzi J.L.
      • Hibert M.
      • Bonnet D.
      • Frossard N.
      Prodrugs of a CXC chemokine-12 (CXCL12) neutraligand prevent inflammatory reactions in an asthma model in vivo.
      ) and CN-chalcone 4 (this work) appears as complementary to that of classical receptor antagonists because the blockade of the chemokine is without any effect on the receptor. In particular, it is neither a partial agonist of CXCR4 nor an activator of CXCR7 (
      • Kalatskaya I.
      • Berchiche Y.A.
      • Gravel S.
      • Limberg B.J.
      • Rosenbaum J.S.
      • Heveker N.
      AMD3100 is a CXCR7 ligand with allosteric agonist properties.
      ,
      • Kim H.Y.
      • Hwang J.Y.
      • Kim S.W.
      • Lee H.J.
      • Yun H.J.
      • Kim S.
      • Jo D.Y.
      The CXCR4 antagonist AMD3100 has dual effects on survival and proliferation of myeloma cells in vitro.
      ,
      • Trent J.O.
      • Wang Z.X.
      • Murray J.L.
      • Shao W.
      • Tamamura H.
      • Fujii N.
      • Peiper S.C.
      Lipid bilayer simulations of CXCR4 with inverse agonists and weak partial agonists.
      ), as was described for AMD 3100 and in other instances with RANTES (regulated on activation normal T cell expressed and secreted) analogs acting on the CCR5 receptor (
      • Wells T.N.
      • Proudfoot A.E.
      Chemokine receptors and their antagonists in allergic lung disease.
      ). Therefore, the mechanism of action of chalcone 4 and its analogs deserves to be exploited in drug development programs.
      Another concern was raised regarding the large tissue distribution of CXCR4, which can be the cause of possible side effects of CXCR4-targeting drugs. The use of systemically administered AMD 3100 confirmed the risk of side effects resulting from general CXCR4 inhibition. This was illustrated on leukocyte maturation in the bone marrow (
      • Lukacs N.W.
      • Berlin A.
      • Schols D.
      • Skerlj R.T.
      • Bridger G.J.
      AMD3100, a CxCR4 antagonist, attenuates allergic lung inflammation and airway hyperreactivity.
      ) and on cardiac function (
      • Flomenberg N.
      • Devine S.M.
      • Dipersio J.F.
      • Liesveld J.L.
      • McCarty J.M.
      • Rowley S.D.
      • Vesole D.H.
      • Badel K.
      • Calandra G.
      The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone.
      ,
      • Flomenberg N.
      • DiPersio J.
      • Calandra G.
      Role of CXCR4 chemokine receptor blockade using AMD3100 for mobilization of autologous hematopoietic progenitor cells.
      ,
      • Rusconi S.
      • Lo Cicero M.
      • Viganò O.
      • Sirianni F.
      • Bulgheroni E.
      • Ferramosca S.
      • Bencini A.
      • Bianchi A.
      • Ruiz L.
      • Cabrera C.
      • Martinez-Picado J.
      • Supuran C.T.
      • Galli M.
      New macrocyclic amines showing activity as HIV entry inhibitors against wild type and multi-drug resistant viruses.
      ). We therefore generated a short lived readily hydrolyzable analog of the initial compound, chalcone 4, and show here that CN-chalcone 4 is as active as chalcone 4 on airway inflammation when administered by the intranasal route, whereas it is inactive when delivered systemically using the intraperitoneal route. It therefore typically behaves as an antedrug or soft drug.
      The general principles and reactions that are used for antedrug structures include various cleavable chemical functions, such as carboxylic esters and amides, oximes, thioester, spiroenones, or lactones (
      • Khan M.O.
      • Park K.K.
      • Lee H.J.
      Antedrugs. An approach to safer drugs.
      ,
      • Khan M.O.
      • Lee H.J.
      Synthesis and pharmacology of anti-inflammatory steroidal antedrugs.
      ). In designing carbonitrile-chalcone 4, we here make use of the Knoevenagel and retro-Knovenagel reactions (
      • Rajasekaran K.
      • Sarathi A.
      • Ramalakshmi S.
      Micellar catalysis in the retro-Knoevenagel reaction of ethyl-α-cyanocinnamates.
      ) yielding the desired compound due to a reversible aldolization reaction (
      • Guthrie J.P.
      • Cossar J.
      • Cullimore P.A.
      • Kamkar N.M.
      • Taylor K.F.
      The retroaldol reaction of chalcone.
      ) that has never been exploited in the antedrug field before. The biologically active compound, carbonitrile-chalcone 4, is readily hydrolyzed in aqueous media with a half-life of a few tens of min and yields vanillin and pCBA, which both serve as synthetic building blocks for the preparation of carbonitrile-chalcone 4. The probable hydrolysis mechanism involves the addition of one water molecule according to a Michael addition on the α-β unsaturated conjugated system. Hydration of the double bond is presumably facilitated by the presence of the electron-attracting nitrile group. The resulting enolic structure then evolves toward production of the initial reactants vanillin and pCBA according to a retroaldolization reaction (
      • Roberts D.L.
      • Rowland R.L.
      Macrocyclic diterpenes α- and β-4,8,13-duvatriene-1,3-diols from tobacco.
      ). We show here that neither the reactants nor carbonitrile-chalcone 4 display any toxic effect in vivo or in HepG2 cells in vitro.
      In conclusion, our results show a strong activity of a chalcone 4 derivative, carbonitrile-chalcone 4, displaying only local and no systemic effect due to a short lifetime in biological fluids, therefore playing the role of an antedrug, which is particularly interesting when the airways are considered. The various chalcone 4 derivatives that we have generated in this and previous works will serve as tools to understand CXCR4, CXCR7, and CXCL12 functions in the airway inflammation process. In particular, the sequence of events and their dependence on CXCL12 activity will be important elements in the characterization of CXCL12 as a drug target in airway inflammation. The mechanism of action of chalcone 4 and its analogs deserves to be exploited in drug development programs because blockade of the chemokine is without any effect on the receptor spontaneous activity as opposed to the most widely encountered pharmacological action of G protein-coupled receptor antagonists.

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