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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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      • 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.
      ,
      • Balabanian K.
      • Brotin E.
      • Biajoux V.
      • Bouchet-Delbos L.
      • Lainey E.
      • Fenneteau O.
      • Bonnet D.
      • Fiette L.
      • Emilie D.
      • Bachelerie F.
      Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice.
      ,
      • Bouchery T.
      • Dénécé G.
      • Attout T.
      • Ehrhardt K.
      • Lhermitte-Vallarino N.
      • Hachet-Haas M.
      • Galzi J.L.
      • Brotin E.
      • Bachelerie F.
      • Gavotte L.
      • Moulia C.
      • Bain O.
      • Martin C.
      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.

      EXPERIMENTAL PROCEDURES

      Chemistry

      Reagents were obtained from commercial sources and used without any further purification. Thin-layer chromatography was performed on silica gel 60F254 plates. Flash chromatography was performed on silica gel (puriFlash® 30 μm, Interchim) or C18 (puriFlash® 30 μm, Interchim) prepacked columns on a SpotII Ultima from Armen. NMR spectra were recorded on a Bruker AV400 spectrometer. Chemical shifts (δ) are reported in ppm, and coupling constants (J) are expressed in Hz. Analytical HPLC analyses were performed on an Eclipse XBD-C18 column (5 μm, 46 × 150 mm; Agilent) using the following conditions: flow rate, 1 ml/min; Solvent A, 0.1% aqueous TFA; Solvent B, 0.1% TFA in CH3CN; gradient, 5–100% B developed over 15 min; detection at 220/254/365 nm. Retention times (tR) from analytical reverse phase HPLC are reported in min. LC/MS spectra were obtained on an Agilent HPLC single quadrupole spectrometer (1200RRLC/1956b-SL) equipped with a THERMO Hypersyl column (1.9 μm, 1 × 30 mm) using an Agilent Multimode ion source. High resolution mass spectrometry spectra were obtained on an Accurate-Mass Q-ToF spectrometer from Agilent using electrospray ionization. For (E)-2-(4-chlorobenzoyl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile (CN-chalcone 4)
      The abbreviations used are: CN-chalcone 4
      carbonitrile-chalcone 4
      BALF
      bronchoalveolar lavage fluid
      BRET
      bioluminescence resonance energy transfer
      HP-βCD
      hydroxypropyl-β-cyclodextrin
      OVA
      ovalbumin
      TR
      Texas Red
      pCBA
      para-chlorobenzoylacetonitrile.
      , p-chloro-benzoyl-acetonitrile (1 g, 5.606 mmol) and 3-methoxy-4-(methoxymethyloxy) benzaldehyde (1.1 g, 5.606 mmol) were dissolved in dry CH2Cl2 (15 ml). Dry NEt3 (78 μl, 0.56 mmol) and pulverized activated 4-Å molecular sieves (1 g) were added. The mixture was stirred at room temperature, monitoring the progress of the reaction by TLC. After 20 h, the molecular sieve was filtered off, and the organic layer was concentrated in vacuo to dryness. The residual crude orange solid was recrystallized from aqueous EtOH to afford (E)-2-(4-chlorobenzoyl)-3-(3-methoxy-4-(methoxymethoxy)phenyl) acrylonitrile as a yellow solid (1.01 g, 50% yield). Rf = 0.38 (heptane-ethyl acetate: 7–3); mp = 140–1 °C; 1H NMR (CDCl3): d 3.51 (s, 3H), 3.96 (s, 3H), 5.32 (s, 2H), 7.23 (d, J = 8.6 Hz, 1H,), 7.43 (dd, J = 8.6, 2.1 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 2.1 Hz, 1H), 8.00 (s, 1H); 13C NMR (CDCl3): d 55.9, 56.3, 95.2, 106.5, 112.8, 116.2, 117.9, 125.7, 128.3, 129.1, 130.8, 134.8, 139.8, 150.3, 151.8, 156.1, 188.1.
      (E)-2-(4-Chlorobenzoyl)-3-(3-methoxy-4-(methoxymethoxy)phenyl)acrylonitrile was dissolved in THF (10 ml) in the presence of a 1 n HCl aqueous solution (5 eq, 14 ml). The resulting mixture was stirred at room temperature, monitoring the progress of the conversion by HPLC. After 12 h, the solvent was removed under reduced pressure, and the residue was dissolved in CH2Cl2 and washed with water until neutralization. The solvent was removed, and deprotected CN-chalcone 4 was recovered as a yellow solid in quantitative yield (886 mg). Rf = 0.27 (heptane-ethyl acetate: 8–2); mp = 162–3 °C (recrystallized from aqueous EtOH); 1H NMR (CDCl3): δ 3.98 (s, 3H), 6.26 (br s, 1H), 7.01 (d, J = 8.3 Hz, 1H,), 7.41 (dd, J = 8.3, 2.1 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.82 (d, J = 8.6 Hz, 2H), 7.91 (d, J = 2.1 Hz, 1H), 8.00 (s, 1H); 13C NMR (CDCl3): d 56.4, 105.9, 111.5, 115.3, 118.1, 124.7, 129.1, 129.5, 130.7, 134.6, 139.6, 147.0, 151.4, 156.0, 188.0. reverse phase HPLC purity >97%; high resolution mass spectrometry calcd. for C17H13ClNO3 314.0506; found: 314.0518 (M+ H)+. Chalcone 1 and chalcone 4 were provided by the French national chemical library.

      In Vitro and in Vivo Experiments

      Chalcone stock solutions were prepared in sterile DMSO and then stored at −20 °C until use. The human chemokines CXCL12 and CXCL12-TR were purchased from Alamc. The cAMP biosensor CAMYEL was kindly provided by Dr. Lily Jiang (Dallas, TX).

      Determination of Solubility and Stability

      Solubility and stability studies were performed on a Gilson HPLC system with a photodiode array detector, an autosampler, and a Valco injector. Data acquisition and processing were performed with Trilution LC version 2.0 software. Measurements were carried out at 21 ± 1 °C. A 2.6-μm Kinetex column (50 × 4.6 mm) was used for stability studies in biological media, and a 5-μm Luna C18(2) column (50 × 4.6 mm) was used for solubility and chemical stability analysis. Both columns were purchased from Phenomenex. The injection volume was 20 μl, the mobile phase flow rate was 2 ml/min, and the following program was applied for the elution: 0–0.2 min, 0% B; 0.2–2.7 min, 0–100% B; 2.7–3.2 min, 100% B; 3.2–3.4 min, 100–0% B; 3.4–6.1 min, 0% B. Solvent B was HPLC grade acetonitrile (Sigma-Aldrich CHROMASOLV). The aqueous solvent contained 0.1% trifluoroacetic acid, and the detection wavelength was 365 nm.

      Solubility

      Thermodynamic solubility was measured by dissolving the compounds up to saturation in a pH 7.4 phosphate-buffered saline (PBS) with the following composition: 137.5 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, and 1.4 mm KH2PO4 supplemented or not with 10% hydroxypropyl-β-cyclodextrin (HP-βCD). Samples were shaken for 24 h at 21 ± 1 °C. Saturation was confirmed by the presence of undissolved powder. After ultracentrifugation, the concentration in the supernatant was measured by an HPLC procedure using a calibration curve established for each compound by diluting a 10 mm DMSO stock solution to adapted concentrations. Due to rapid degradation of CN-chalcone 4, solubility was determined after 2 h of shaking. The indicated value given for CN-chalcone 4 is thus an estimate of the solubility.

      Chemical Stability

      Stability of compounds was assessed in PBS, pH 7.4, with or without 10% HP-βCD at 20 °C up to 24 h. For each compound, the 10 mm DMSO stock solution was diluted to a final incubation concentration of 10 μm with 0.1% DMSO. 20 μl of sample were removed at t0, 1, 2, 4, 6, 8, 10, and 24 h and directly injected onto the HPLC. The percentage of remaining test compound relative to t0 was measured by monitoring the peak area on the chromatogram.

      Stability in Mouse Serum

      Stability of chalcones was determined in mouse serum with or without 10% HP-βCD at 37 °C up to 16 h. For each compound, the 10 mm DMSO stock solution was diluted in serum to a final concentration of 20 μm with 1% DMSO. For the measurements with HP-β-cyclodextrin, a solution of PBS (pH 7.4) containing 10% (v/w) HP-βCD was saturated with compound powder. The saturated solution was then diluted in murine serum to a final compound concentration of 20 μm. The mixture was divided into five aliquots. The incubation of each aliquot was stopped at t0 and 30 min, 1, 2, and 16 h for chalcone 4 and at t0 and 15, 30, 45, and 60 min for CN-chalcone 4 by adding one volume of ice cold acetonitrile. Samples were stirred for 3 min, sonicated for 3 min, and then centrifuged at 4 °C before HPLC injection. The percentage of remaining test compound relative to t0 was measured by monitoring the peak area on the chromatogram.

      Stability in Lung Homogenate

      Stability of chalcones was determined in mouse lung homogenate with or without HP-βCD 10% at 37 °C. Lung homogenate was prepared separately with a Fastprep® (Q-BIOgene, Illkirch, France) in PBS (one lung homogenized in 1 ml of buffer). For each compound, the incubation solutions, the sampling, and the extraction conditions were prepared as described under “Stability in Mouse Serum.”

      Cell Culture

      Human embryonic kidney 293 cells expressing the fusion receptor EGFP-hCXCR4 (stable cell lines (
      • 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.
      )) were cultured to ∼80% confluence in 75-cm2 flasks in minimum Eagle's medium with Earle's salt supplemented with 10% fetal calf serum, 2 mm glutamine, and 1% antibiotics (penicillin/streptomycin) and replated twice a week. HepG2 cells were grown to ∼80% confluence in 75-cm2 flasks in MEM with Earle's salt supplemented with 10% fetal calf serum, 2 mm glutamine, 1% antibiotics (penicillin/streptomycin), 1 mm sodium pyruvate (Invitrogen), 0.1 mm non-essential amino acids (Invitrogen) and replated twice a week.

      Binding Experiments

      FRET-based binding experiments were carried out as described (
      • 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.
      ,
      • Maillet E.L.
      • Pellegrini N.
      • Valant C.
      • Bucher B.
      • Hibert M.
      • Bourguignon J.J.
      • Galzi J.L.
      A novel, conformation-specific allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with functionally selective properties.
      ). Human embryonic kidney 293 cells expressing the fusion receptor EGFP-hCXCR4 were harvested in PBS (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2PO4·7H2O, 1.4 mm KH2PO4, pH 7.4) supplemented with 5 mm EDTA, pH 7.4, centrifuged, and resuspended in HEPES-bovine serum albumin (BSA) buffer (10 mm HEPES, 137.5 mm NaCl, 1.25 mm MgCl2, 1.25 mm CaCl2, 6 mm KCl, 10 mm glucose, 0.4 mm NaH2PO4, 0.1% bovine serum albumin (w/v), pH 7.4). Cells were used at a concentration of 106 cells/ml. Apparent affinities of the different ligands were determined by real-time fluorescence monitoring ligand-receptor interactions. Time-based recording of binding was initiated by adding 100 nm CXCL12-TR (Texas Red-labeled CXCL12) to the 0.5-ml cell suspension. Fluorescence emitted at 510 nm (excitation at 470 nm) was recorded at 21 °C using a Fluorolog 2 spectrofluorimeter (SPEX) and sampled every 0.3 s. Binding of CXCL12-TR to EGFP-labeled CXCR4 was detected as a reversible decline of emission at 510 nm, due to energy transfer from excited EGFP to TR. For competition experiments, the fluorescent chemokine was preincubated for 1 h at room temperature with or without various concentrations of each chalcone. Then the premix was added, and fluorescence was recorded until equilibrium was reached (300 s). Data were analyzed using Kaleidagraph 3.08 software (Synergy Software, Reading, PA).

      cAMP Determination

      CXCR4 receptor coupling to adenylyl cyclase was assessed by measuring the dose-dependent inhibitory effects of chemokine with or without chalcone on forskolin-stimulated cAMP accumulation. To facilitate studies of cAMP regulation, we used a developed bioluminescence resonance energy transfer (BRET) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in cells (
      • Jiang L.I.
      • Collins J.
      • Davis R.
      • Lin K.M.
      • DeCamp D.
      • Roach T.
      • Hsueh R.
      • Rebres R.A.
      • Ross E.M.
      • Taussig R.
      • Fraser I.
      • Sternweis P.C.
      Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.
      ). HEK EGFP-hCXCR4 cells were cultured to ∼80% confluence in 75-cm2 flasks in MEM with Earle's salt supplemented with 10% fetal calf serum, 2 mm glutamine, and 1% antibiotics (penicillin/streptomycin). Cells were plated on 10-cm plates and transfected with 10 μg of CAMYEL plasmid using the calcium phosphate precipitation method. Two days later, cells were plated in 96-well solid white tissue culture plates (Greiner) at a density of 60,000 cells/well the day before cAMP assay. Cells were serum-starved in Hanks' balanced salt solution, pH 7.4 (Sigma-Aldrich) for 30 min at 37 °C before treatments (80 μl/well). The BRET assay was carried out with a Victor Light plate reader (PerkinElmer Life Sciences). Emission signals from Renilla luciferase and YFP were measured sequentially using a BRET1 filter set (475-30/535-30). Stimulations were initiated by injection of 20 μl of 5× concentrated ligands prepared in Hanks' balanced salt solution supplemented with 500 mm isobutylmethylxanthine. After 5 min, 10 μl of a Hanks' balanced salt solution of coelenterazine-H (2 μm) were added, and plates were incubated for 10 min at 37 °C before reading.

      Cytotoxicity

      The capacity of the cells to carry out reduction reactions after drug treatment was estimated with the fluorescent Alamar Blue reagent (AbD Serotec, Oxford, UK). Proliferating cells cause the change from the oxidized blue and non-fluorescent Alamar Blue form (resazurin) to a reduced, pink, highly fluorescent form (resorufin) that can be detected using fluorescence monitoring (excitation 560 nm; emission 590 nm). HepG2 or HEK 293 cells were seeded at a density of 30,000 cells/well on a 96-well plate and allowed to settle overnight before treatment. The dye was added to the cells together with the test substances (5-fold concentrated stock solution prepared in culture medium or in PBS with 10% HP-βCD), and the fluorescence was measured after 24-h incubation, using a Flex Station (Molecular Devices).

      Murine Model of Hypereosinophilia

      The in vivo activity of the compounds was assessed in an 8-day model of hypereosinophilia created by immunization and challenge to ovalbumin in mice. Briefly, 9-week-old male BALB/c mice were sensitized by intraperitoneal injection of a mixture of 50 μg of ovalbumin (OVA, grade V; Sigma-Aldrich) adsorbed on aluminum hydroxide (2 mg; Sigma-Aldrich) in 0.1 ml of saline on days 0, 1, and 2. Mice were challenged intranasally with 10 μg of OVA in 25 μl of saline (12.5 μl/nostril) on days 5, 6, and 7. Control mice received intraperitoneal and intranasal administrations of saline alone. Intranasal administrations were performed under anesthesia with 50 mg/kg ketamine and 3.33 mg/kg xylazine given intraperitoneally. Food and water were supplied ad libitum. Animal experimentation was conducted with the approval of the government body that regulates animal research in France. The chalcone compounds were administered either by the intraperitoneal or intranasal routes. In a first set of experiments, mice received either chalcone 4 or carbonitrile-chalcone 4 solubilized in PBS with 10% HP-βCD (C0926, Sigma) by intranasal injection 2 h before each nasal OVA or saline challenge. In a second set of experiments, mice received CN-chalcone 4, vanillin, or parachlorobenzoylacetonitrile intranasally, in PBS with HP-βCD 10%, 2 h before each OVA or saline challenge. In the last set of experiments, mice received chalcone 4 or CN-chalcone 4 in suspension in carboxymethylcellulose 1% or solubilized in PBS with HP-βCD 10% and administered by intraperitoneal injection 2 h before each OVA or saline challenge. Collection of bronchoalveolar lavage fluid (BALF) was performed 24 h after the last OVA challenge. Mice were deeply anesthetized by intraperitoneal injection of 150 mg/kg ketamine and 10 mg/kg xylazine. A plastic canula was inserted into the trachea, and airways were lavaged by 10 instillations of 0.5 ml of ice-cold saline supplemented with 2.6 mm EDTA (saline-EDTA). Total and differential cell counts in the BALF were performed after centrifugation (300 × g for 5 min at 4 °C) to pellet cells. Erythrocytes were lysed by hypotonic shock by the addition of 1.5 ml of distilled H2O, followed by the addition of 0.5 ml of 0.6 m KCl. Cells were centrifuged and resuspended in 500 μl of ice-cold saline-EDTA, and total cell counts were determined using a hemocytometer (Neubauer's chamber). Differential cell counts were assessed on cytologic preparation obtained by cytocentrifugation (Cytospin 3, Shandon Ltd.) of 200 μl of diluted BALF (250,000 cells/ml in ice-cold saline-EDTA). Slides were stained with Hemacolor (Merck), and counts were performed on at least 400 cells for each preparation. Differential counts were expressed as absolute numbers or as a percentage of the total number of cells.

      Cytochrome c Oxidase Activity

      Lung and heart were homogenized separately with an UltraTurax® (IMLAB, Lille, France) in PBS, pH 7.4 (one organ in 1 ml of buffer). Lung homogenate was diluted at 1:2 and heart homogenate at 1:5 in PBS. 100 μl of each diluted homogenate were placed in a microplate, and 100 μl of assay reagent (10 mm tetramethyl-p-phenylenediamine, 2 mm sodium ascorbate, 20 mm KH2PO4) were added. The microplate was placed immediately in a microplate spectrophotometer reader, and optical density kinetic (λ = 610 nm) was measured every 20 s for 30 min. Results are expressed as Vmax/100 μg of protein (OD/min/100 μg of protein).

      RESULTS

      Carbonitrile-chalcone 4 Neutralizes CXCL12 in Vitro and in Vivo

      CN-chalcone 4 was prepared by condensing para-chlorobenzoyl-acetonitrile (pCBA) with protected vanillin (VanP) as described in Scheme 1. CN-chalcone 4 is the homolog of chalcone 4, formerly described as a ligand of CXCL12, that inhibits chemokine binding to both CXCR4 and CXCR7 receptors (
      • Chow K.Y.
      • Brotin É.
      • Ben Khalifa Y.
      • Carthagena L.
      • Teissier S.
      • Danckaert A.
      • Galzi J.L.
      • Arenzana-Seisdedos F.
      • Thierry F.
      • Bachelerie F.
      A pivotal role for CXCL12 signaling in HPV-mediated transformation of keratinocytes. Clues to understanding HPV-pathogenesis in WHIM syndrome.
      ,
      • Galzi J.L.
      • Hachet-Haas M.
      • Bonnet D.
      • Daubeuf F.
      • Lecat S.
      • Hibert M.
      • Haiech J.
      • Frossard N.
      Neutralizing endogenous chemokines with small molecules. Principles and potential therapeutic applications.
      ,
      • 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.
      ). CN-chalcone 4 prevents binding of Texas Red-labeled CXCL12 (CXCL12-TR, 100 nm) to EGFP-tagged CXCR4, determined by fluorescence resonance energy transfer (
      • 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.
      ) (Fig. 1A) with similar affinity as chalcone 4 (Ki = 45 ± 57 nm for chalcone 4 versus Ki = 53 ± 31 nm for CN-chalcone 4). This inhibition of CXCL12 binding to CXCR4 consequently blocks CXCL12-evoked CXCR4 cellular signaling (inhibition of cAMP production, shown in Fig. 4B) and trafficking (data not shown).
      Figure thumbnail gr1
      FIGURE 1Carbonitrile-chalcone reduces inflammation in a mouse model of allergic eosinophilic airway inflammation. A, in vitro inhibition of CXCL12 binding to CXCR4 receptor by chalcone 4 (Chalc 4) and CN-chalcone 4 (CN-Chalc 4). Inhibition of CXCL12 binding to CXCR4 as a function of increasing concentration of chalcone 4 (gray squares) and CN-chalcone 4 (black circles) is monitored using FRET intensity variation. The fluorescence of cells expressing EGFP-labeled CXCR4 is followed at 510 nm as a function of time. Upon the addition of Texas Red-labeled CXCL12 (CXCL12-TR, 100 nm), fluorescence intensity at 510 nm declines as a result of interaction between CXCL12-TR and EGFP-CXCR4, which causes FRET. The ordinate axis reports the intensity of FRET as a percentage of the control value (100 nm CXCL12-TR alone). Ki values were derived from the IC50 values determined from competition curves using the Cheng and Prusoff relationship (
      • Cheng Y.
      • Prusoff W.H.
      Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.
      ). Ki values are 53 ± 31 nm (chalcone 4) and 45 ± 57 nm (CN-chalcone 4). Each data point represents the mean ± S.D. (error bars) of three experiments. B, in vivo dose-response effect of a topical treatment with chalcone 4 and CN-chalcone 4 in an 8-day mouse model of hypereosinophilia. BALB/c mice were sensitized and challenged with OVA or saline. Chalcone 4 (gray line) or CN-chalcone 4 (black line) solubilized in 10% HP-βCD were administered intranasally 2 h before each challenge. The percentage of inhibition of eosinophil is shown. Data points (squares) are means of n = 6 determinations. C and D, effect of topical (intranasal; i.n.) treatment with chalcone 4, CN-chalcone 4 (C), and chalcone 1 (Chalc 1) (D) in the 8-day mouse model of hypereosinophilia. BALB/c mice were sensitized and challenged with OVA or saline. Drugs (300 nmol/kg) were administered intranasally 2 h before each challenge in HP-βCD 10% (vehicle). Absolute numbers of macrophages, eosinophils, neutrophils, and lymphocytes in BALF are shown. Bars represent means, and error bars show S.E. values (n = 6/group). *, p ≤ 0.05 in comparison with the saline-treated OVA group.
      Figure thumbnail gr4
      FIGURE 4CN-chalcone 4 degradation products are inactive. A, CN-chalcone 4 (CN-Chalc 4) degradation products (up to 10 μm) do not inhibit CXCL12-TR binding to EGFP-CXCR4 detected by FRET as in A. Each data point represents the mean ± S.D. of three independent experiments performed in triplicates. B, CN-chalcone 4, but not its degradation products, dose-dependently inhibits CXCL12 action on forskolin-evoked cAMP responses in HEK EGFP-CXCR4 cells. The first two bars report the maximal production of cAMP (%) triggered by 1 μm forskolin and its inhibition by 3 nm CXCL12. The following bars show that chalcone 4 (Chalc 4) and CN-chalcone 4 (1, 3, and 10 μm), but not vanillin or pCBA (10 μm), inhibit the CXCL12 effect on cAMP production. Each bar represents the mean ± S.D. (error bars) of three independent experiments performed in triplicates. C, topical treatment with chalcone 4, CN-chalcone 4, vanillin, and pCBA in the 8-day mouse model of hypereosinophilia. BALB/c mice were immunized and challenged with OVA or saline. Treatments (300 nmol/kg) or HP-βCD 10% (vehicle), were administered intranasally 2 h before each challenge. Absolute numbers of macrophages, eosinophils, neutrophils, and lymphocytes in BAL are shown. Bars show means, and error bars show S.E. values (n = 6/group). *, p ≤ 0.05 in comparison with the saline-treated OVA group.
      The in vivo activity of CN-chalcone 4 was assayed in a recently developed 8-day mouse model of airway hypereosinophilia (
      • 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.
      ). In this model, mice are sensitized by intraperitoneal injection of OVA (50 μg) adsorbed on 2 mg of aluminum hydroxide in 0.1 ml of saline on days 0, 1, and 2. Mice are then challenged intranasally with 10 μg of OVA in 25 μl of saline (12.5 μl/nostril) on days 5, 6, and 7. Drugs to be tested can be administered either systemically by the intraperitoneal route or locally by the intranasal route. The intranasal route mimics the inhalation exposure used in humans, which is the preferred and well accepted administration for inflamed airway treatment, in particular asthma. Thus, unless otherwise stated, drugs were administered intranasally in this study. Due to limited solubility of chalcone 4 (9 ± 1 μm) and CN-chalcone 4 (16 ± 2 μm) in saline buffer, drugs were dissolved in physiological solutions complemented with HP-βCD (10%, w/w). Under such conditions, chalcone 4 and CN-chalcone 4 could be dissolved at maximal concentrations reaching 690 ± 44 and 493 ± 36 μm, respectively.
      Administering these solutions at 25 μl/mouse intranasally allows a maximal dose approximating 300–500 nmol/kg. Eosinophil counts in BALF are dose-dependently inhibited by both chalcone 4 and CN-chalcone 4 up to 50% at doses of 300–500 nmol/kg, which can be reached as limits of drug solubility (Fig. 1B).
      BALF infiltrate with macrophages, eosinophils, neutrophils, and lymphocytes was determined under treatment with intranasal chalcone 4 and CN-chalcone 4 at 300 nmol/kg. Fig. 1C shows that neither vehicle (10% HP-βCD) nor any of the chalcones by themselves elicit any cell recruitment in the airways. After ovalbumin challenge, a significant increase in the number of eosinophils and macrophages occurs (Fig. 1C, top). Polymorphonuclear neutrophils and lymphocytes were also significantly present in BALFs after OVA challenge, albeit at ∼10–20-fold lower levels than eosinophils (Fig. 1C, bottom). Significant reduction of eosinophil recruitment is noted with chalcone 4 administered intranasally (300 nmol/kg). In addition, we note that the new molecule CN-chalcone 4 is as potent as chalcone 4.
      In order to further document the specificity of chalcone 4 and CN-chalcone 4 action, we tested the activity of the unsubstituted chalcone backbone (chalc 1 in Scheme 1) already reported as inactive upon CXCL12 binding to CXCR4 (
      • 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.
      ). Fig. 1D shows that chalcone 1 does not promote any inflammatory response per se; nor does it significantly attenuate eosinophil or any other inflammatory cell recruitment in the airways.
      These results therefore indicate that the inactive chalcone chemotype can be substituted by functional groups to inhibit CXCL12 binding to CXCR4 to become active as an attenuator of allergen-induced inflammatory responses. CN-chalcone 4 is as active as chalcone 4 to neutralize CXCL12 in vitro and to inhibit eosinophilic airway inflammation in vivo. This shows that the introduction of the carbonitrile group in chalcone 4 does not affect the activity and potency of the compound.

      CN-chalcone 4 Is Active Locally but Not Systemically

      We then compared the in vivo activity of CN-chalcone 4 administered systemically by the intraperitoneal route with that of chalcone 4 (Fig. 2). Chalcone 4 and CN-chalcone 4 were solubilized in HP-βCD (10%), or in carboxymethylcellulose, allowing administrations either of 2 × 20 μmol/kg (Fig. 2A) or of a 350 μmol/kg (Fig. 2B) dose of compound, respectively. Chalcone 4 and CN-chalcone 4 could thus be administered intraperitoneally at doses 40- or 700-fold higher than those used for intranasal administration. These doses correspond to the maximal possible dose because the solubility of chalcones is limited by the concentration of vehicle (carboxymethylcellulose or HP-βCD) and because higher doses of vehicle lead to toxic effects on animals. Fig. 2 displays results of inflammatory cell recruitment in BALF of OVA-sensitized mice receiving the compounds or vehicles.
      Figure thumbnail gr2
      FIGURE 2CN-chalcone 4 is an antedrug. Systemic effect of chalcone 4 and CN-chalcone 4 in two vehicles. Systemic (intraperitoneal (i.p.)) treatment with chalcone 4 (Chalc 4) and CN-chalcone 4 (CN-Chalc 4) in the 8-day mouse model of hypereosinophilia is shown. BALB/c mice were sensitized and challenged with OVA or saline. Drugs (two administrations of 20 μmol/kg/day for 3 days in 10% HP-βCD (vehicle) (A) or 350 μmol/kg once a day for 3 days in 1% carboxymethylcellulose (CMC) as a vehicle (B)) were administered intraperitoneally 2 h before each OVA challenge. Absolute numbers of macrophages, eosinophils (top), neutrophils, and lymphocytes (middle) in BALF are shown. Bars show means, and error bars show S.E. values (n = 6/group). *, p ≤ 0.05 in comparison with the saline-treated OVA group. Dose intensity relationship of eosinophil recruitment in the intraperitoneal route is shown for chalcone 4 and CN-chalcone 4 up to the maximal dose (bottom).
      Fig. 2A shows the effect of twice daily treatment with 20 μmol/kg chalcone 4 or CN-chalcone 4 in 10% HP-βCD administered intraperitoneally. Neither HP-βCD alone nor any of the two drugs had any effect on inflammatory cell recruitment (macrophages, eosinophils, neutrophils, or lymphocytes) in the naive airways. When administered 1 h before the challenging doses of OVA, chalcone 4 shows anti-inflammatory properties by significantly reducing eosinophils, neutrophils, and T cell counts in BALF (Fig. 2A). By contrast, CN-chalcone 4 did not affect any inflammatory cell counts. Repeating the experiment with carboxymethylcellulose as the excipient allowed administration at doses as high as 350 μmol/kg (Fig. 2B). Again, only chalcone 4 exhibits dose-dependent anti-inflammatory activity in the airways with significant inhibition of macrophage recruitment in addition to inhibition of eosinophil, neutrophil, and lymphocyte influx. These experiments reveal that, in contrast to chalcone 4, CN-chalcone 4 is inactive at inhibiting airway inflammation in vivo when administered at a distance from the airways. Therefore, introducing a carbonitrile group in chalcone 4 affects its distribution or metabolism.

      CN-chalcone 4 Is Rapidly Degraded in Biological Media

      One major effect of the introduction of the carbonitrile group on chalcone 4 is detected on the stability of CN-chalcone 4 in biological media. Compound stability was assessed using HPLC detection (Fig. 3) after incubation in various media. In contrast to chalcone 4, which is stable for hours in buffer and tissue homogenates (Table 1), CN-chalcone 4 is rapidly degraded in phosphate-buffered saline (t½ = 6 h) and even more rapidly in murine serum (t½ = 20 min) or lung homogenate (t½ = 25 min). 10% HP-βCD not only solubilizes the molecule but also significantly improves its stability. CN-chalcone 4 half-life reaches 40 min in lung homogenate in the presence of HP-βCD. Whatever the experimental condition, the half-life of CN-chalcone 4 is significantly shorter than that of chalcone 4, possibly accounting for the lack of effect when using the intraperitoneal route rather than the intranasal administration.
      Figure thumbnail gr3
      FIGURE 3CN-chalcone 4 is rapidly hydrolyzed in murine serum. Chromatograms were obtained with a Luna C18(2) 5-μm, 4.6 × 50-mm column. Gradient elution was as follows: 0–0.2 min, 0% B; 0.2–2.7 min, 0–100% B; 2.7–3.2 min, 100% B; 3.2–3.4 min, 100–0% B; 3.4–6.1 min, 0% B (A, water, 0.1% trifluoroacetic acid; B, acetonitrile). A, chromatogram (at λ = 365 nm) of 20 μl of the reference compound CN-chalcone 4, dissolved at 10 μm in water/acetonitrile (1:1, v/v) (tR = 3.31 min). B, chromatogram (at λ = 280 nm) of 20 μl of the reference compounds vanillin (Van) and pCBA, dissolved at 100 μm in water/acetonitrile (1:1, v/v) (tR(Van) = 2.47 min and tR(pCBA) = 3.02 min). C, chromatogram (at λ = 280 nm and 365 nm) of 20 μl of CN-chalcone 4 dissolved at 20 μm and incubated for 1 h in murine serum. The solution was diluted with one volume of acetonitrile before injection in the HPLC. Peaks detected at tR = 2.47 min, tR = 3.02 min, and tR = 3.31 min correspond to vanillin, pCBA, and CN-chalcone 4 (CN-Chalc4), respectively.
      TABLE 1Stability of chalcone 4 versus CN-chalcone 4 in different media
      Stability half-life (t1/2)
      Chalcone 4CN-chalcone 4
      h
      PBS>10 (98% at 10 h)
      Data are taken from Ref. 24.
      6
      PBS, 10% β-cyclodextrine>10 (95% at 10 h)
      Data are taken from Ref. 24.
      16.3
      Murine serum>16 (96% at 16 h)0.3
      Murine serum, 10% β-cyclodextrine>16 (91% at 16 h)1.3
      Lung homogenate>6 (60%)
      Data are taken from Ref. 24.
      0.4
      Lung homogenate, 10% β-cyclodextrine>16 (66% at 16 h)0.6
      a Data are taken from Ref.
      • 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.
      .
      Because the carbonitrile group is an electron-attracting group, its presence on CN-chalcone 4 facilitates nucleophilic attack by water molecules, a reaction that ultimately leads to the hydrolysis of the molecule and production of pCBA and vanillin (Van) as shown in Scheme 2. This is indeed observed on reverse-phase HPLC chromatograms (Fig. 3B), showing that hydrolysis of CN-chalcone 4 is accompanied by the concomitant appearance of its two constituents, which represent the major degradation products in biological media (Fig. 3C).
      Figure thumbnail grs2
      SCHEME 2Hydrolysis reaction of CN-chalcone 4 toward pCBA and vanillin (Van).
      Because CN-chalcone 4 degradation occurs rapidly, we investigated the biological activity of its degradation products with regard to CXCL12 neutralization, cell signaling, and in vivo inhibition of eosinophil recruitment in the airways. Fig. 4A shows that CN-chalcone 4 dose-dependently prevents CXCL12 binding to CXCR4 with maximal inhibition beyond 1 μm. Neither vanillin nor pCBA exhibits any binding-neutralizing activity at concentrations up to 10 μm, indicating that binding inhibition is indeed due to CN-chalcone 4 itself.
      Inhibition of CXCL12 effect on cAMP formation in transfected HEK293 cells overexpressing the human CXCR4 receptor has also been characterized. As shown in Fig. 4B, forskolin (1 μm) evokes an increase in intracellular cAMP that is potently blocked by CXCL12 (3 nm). This blocking effect of CXCL12 is dose-dependently counteracted by either chalcone 4 or CN-chalcone 4 (1–10 μm) but not by the CN-chalcone 4 degradation products vanillin (10 μm) and pCBA (10 μm). The derived IC50 values are equal to 4.1 ± 0.3 and 6.9 ± 0.4 μm for chalcone 4 and CN-chalcone 4, respectively.
      In vivo effects of the degradation products have also been tested on OVA-sensitized and -challenged mice. Intranasal administration of vanillin and pCBA at the same dose as CN-chalcone 4 in Fig. 1 (300 nmol/kg) has no effect on eosinophil recruitment in the airways (Fig. 4C), showing their lack of activity in vivo that matches their lack of in vitro activity on CXCL12.
      Thus, CN-chalcone 4, which is as active as chalcone 4, is subject to spontaneous hydrolysis in buffered aqueous solutions and biological fluids. Its hydrolysis products, vanillin and pCBA, show no binding activity toward CXCL12 or CXCR4 and no biological activity either on cells or in the inflamed airways in our mouse model. All observed effects are therefore due to CN-chalcone 4 itself before degradation occurs. The short half-life of CN-chalcone 4 suggests that it may not diffuse over a long distance in vivo. This would result in the neutralization of CXCL12 only in tissues directly exposed to CN-chalcone 4, possibly those in the immediate vicinity of the administration site. This is the most plausible explanation of the in vivo activity observed after intranasal administration of CN-chalcone 4 and of the lack of activity of CN-chalcone 4 administered systemically by intraperitoneal injection.

      Chalcone 4, CN-chalcone 4, and Its Hydrolysis Products Have Low Toxicity

      We checked for cytotoxicity of chalcone 4, CN-chalcone 4, and its hydrolysis products vanillin and pCBA by measuring mitochondrial reduction of the Alamar Blue dye (Fig. 5A). HepG2 cells were incubated for 24 h with chalcone 4, CN-chalcone 4, vanillin, or pCBA at a 10 μm concentration. The positive cytotoxicity control molecule was simvastatin (100 μm) (
      • Waldhauser K.M.
      • Brecht K.
      • Hebeisen S.
      • Ha H.R.
      • Konrad D.
      • Bur D.
      • Krähenbühl S.
      Interaction with the hERG channel and cytotoxicity of amiodarone and amiodarone analogues.
      ).
      Figure thumbnail gr5
      FIGURE 5Cytotoxicity of chalcone derivatives on HepG2 cells using a resazurin reduction assay. Cell viability is expressed as the percentage of untreated control cells (A) and as 10% HP-βCD-treated cells (B). Cells were exposed to 10 μm compounds for 24 h. Each exposure was preceded by equilibrium setting for 16 h in growth medium at 37 °C, 5% CO2. The dye was added to the cells together with the test substances at a final concentration of 10%. Simvastatin (100 μm) was used as a positive control. All samples contained 0.1% DMSO. Data are expressed as means ± S.D. (error bars) (n = 3). Chalc 4, chalcone 4; CN-Chalc 4, CN-chalcone 4.
      In order to increase the low chalcone 4 solubility (9 ± 1 μm in physiological medium), we also used HP-βCD in this study. Although HP-βCD displays some toxicity on its own at 10% in culture medium, as was described previously (
      • Davis M.E.
      • Brewster M.E.
      Cyclodextrin-based pharmaceutics. Past, present and future.
      ,
      • Gould S.
      • Scott R.C.
      2-Hydroxypropyl-β-cyclodextrin (HP-β-CD). A toxicology review.
      ), there was no further cytotoxicity of any of the compounds (Fig. 5B) as compared with cells treated with HP-βCD alone.
      General toxicity was evaluated in vivo in mice that received CN-chalcone 4 intraperitoneally at a dose of 350 μmol/kg (100 mg/kg) per day during three consecutive days. As reported in Table 2, there was no body or spleen weight loss nor any modification of cytochrome c oxidase activity in lung and heart, indicating no toxicity of CN-chalcone 4 administered at the highest active dose.
      TABLE 2General toxicity
      WeightCytochrome c oxidase (Vmax)
      BodySpleenLung (×102)Heart (×103)
      %mg
      Carboxymethylcellulose99.9 ± 4124 ± 715.1 ± 1.625.6 ± 2.6
      CN-chalcone 499.6 ± 6125 ± 1113.8 ± 2.828.5 ± 3.2
      OVA99.8 ± 6128 ± 612.8 ± 2.023.1 ± 3.1
      OVA+ CN-chalcone 499.6 ± 6123 ± 412.7 ± 1.629.9 ± 3.5

      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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