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A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemistry” targeted

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  • Int. J. Med. Sci. 2010, 7
    http://www.medsci.org
    326
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    2010; 7(6):326-339
    © Ivyspring International Publisher. All rights reserved
    Research Paper
    A cyclic-RGD-BioShuttle functionalized with TMZ by DAR
    inv
    “Click Chemi-
    stry” targeted to α
    v
    β
    3
    integrin for therapy
    Klaus Braun
    1
    *
    , Manfred Wiessler
    1
    *, Rüdiger Pipkorn
    2
    , Volker Ehemann
    3
    , Tobias Bäuerle
    1
    , Heinz
    Fleischhacker
    1
    , Gabriele Müller
    4
    , Peter Lorenz
    1
    , Waldemar Waldeck
    4
    1. German Cancer Research Center, Dept. of Imaging and Radiooncology, INF 280, D-69120 Heidelberg, Germany
    2. German Cancer Research Center, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany
    3. University of Heidelberg, Institute of Pathology, INF 220, D-69120 Heidelberg, Germany
    4. German Cancer Research Center, Division of Biophysics of Macromolecules, INF 580, D-69120 Heidelberg, Germany
    * The authors contributed equally to this work
    Corresponding author: Klaus Braun, Ph.D., German Cancer Research Center (DKFZ), Dept. of Imaging and Radiooncol-
    ogy, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: +49 6221-42 2495; Fax: +49 6221-42 3326; e-mail:
    k.braun@dkfz.de
    Received: 2010.07.02; Accepted: 2010.09.07; Published: 2010.09.21
    Abstract
    Clinical experiences often document, that a successful tumor control requires high doses of
    drug applications. It is widely believed that unavoidable adverse reactions could be minimized
    by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well
    as the bone-marrow. One new approach in this direction is the use of Targeted Therapies
    realizing a selective drug targeting to gain effectual amounts at the target site, even with
    drastically reduced application doses. MCF-7 breast cancer cells expressing the α
    v
    β
    3
    [al-
    p h a ( v ) b e t a ( 3 ) ] i n t e g r i n r e c e p t o r a r e c o n s i d e r e d a s a p p r o p r i a t e c a n d i d a t e s f o r s u c h a t a r g e t e d
    therapy. The modularly composed BioShuttle carrier consisting of different units designed to
    facilitate the passage across the cell membranes and for subcellular addressing of diagnostic
    and/or therapeutic molecules could be considered as an eligible delivery platform. Here we
    used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the α
    v
    β
    3
    integrin
    receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12
    µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which
    confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell
    killing.
    Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate
    composed of the cyclic RGD, a α
    v
    β
    3
    in t eg r i n -ligand, ligated to the cytotoxic drug TMZ. The
    ligation was carried out by the Diels Alder Reaction with inverse electron demand (DAR
    inv
    ).
    Key words: Click-Chemistry, Cycloaddition, BioShuttle, Ligation chemistry, Linker Systems,
    Adaptor Systems, inverse Diels Alder Reaction, RGD, Tetrazines, targeted Therapy, Temozolomide
    Introduction
    Breast cancer is one of the most common malig-
    nancies affecting women in developed countries.[1]
    Approximately three out of four women with breast
    cancer develop metastases in bone which, in turn,
    diminish the quality of life.[2] An optimal treatment
    concept for patients needs different therapy modali-
    ties and methods with an optimum in efficiency and
    the greatest possible protection. Attention should be
  • Int. J. Med. Sci. 2010, 7
    http://www.medsci.org
    327
    laid on an individual and not just standardized plan
    of treatment for every single patient and all available
    therapy options should be used, such as immunothe-
    rapy, surgery or chemotherapy sensibly using cytos-
    tatic active agents with acceptable adverse reactions.
    It is remarkable how dated medical treatment me-
    thods are persistently continued [reported during the
    “International Brain Tumor Research Conference 2010
    (http://www.kgu.de/index.php?id=4290)].
    Toxic side effects are documented for TMZ as
    adverse reactions in the bone-marrow. Moreover, it is
    known from clinical experience, that even higher ap-
    plication doses are necessary for successful tumor
    control. This approach seems obsolete now, because
    Targeted Therapy has reached the focus of scientific
    interest in order to minimize such unavoidable drastic
    side effects. Strategies were discussed during the
    aforementioned meeting to protect the bone-marrow,
    e.g. with gene-therapeutic methods. Another inter-
    esting field is the regional chemotherapy in which
    cytostatic drugs are being locally applied to certain
    body regions. The topical application increases the
    amount of active substances in the tumor and im -
    proves efficiency, while lowering the side effect rate at
    the same time.
    However, many cell immanent obstacles inhibit
    chemical therapy, such as the multidrug resistance
    (MDR) mediated against cytotoxic agents like TMZ,
    and apoptosis resistance with disruption of the com-
    plex programmed cell death pathway network. The
    Janicke group documented apoptosis resistant MCF-7
    breast cancer cells treated with ionizing radiation,
    however especially breast micro-metastases are diffi-
    cult to determine and even more difficult to treat ef-
    fectively.
    Therefore only a selective targeting of the drug
    can deliver an effectual amount of TMZ to its target
    site, even with drastically reduced application doses.
    How to perform this is exemplarily shown here by
    targeting and controlling breast cancer cells.
    Our considerations to overcome these resis-
    tance-inducing factors led to the application of li-
    gands, which are target-specific for cell-typical sur-
    face receptors, as described as follows.
    On these cells the α
    v
    β
    3
    [alpha(v)beta(3)] and α
    v
    β
    5
    [alpha(v)beta(5)]integrins are heterodimeric cell sur-
    face receptors which mediate adhesion between cells
    and the extracellular matrix.[3] The α
    v
    β
    3
    receptor has
    previously been implicated in a key role of tumor
    progression, metastasis and osteoclast bone resorp -
    tion. [4] Integrins, the corresponding ligands, are
    evolutionarily old and have critical roles during de-
    velopmental and pathological processes. The antibo -
    dies to α
    v
    β
    3
    integrin and its antagonists like
    arg-gly-a s p ( R G D ) -containing peptides, including
    osteopontin, bone sialoprotein, vitronectin and fibri-
    nogen are considered as efficient inhibitors which can
    control the tumor progression.[5]
    This α
    v
    β
    3
    integrin receptor is documented as an
    outstanding target in the field of tumor imaging [6-8]
    and is equally important as a chemotherapeutic target
    in the field of targeted therapy.[9]
    Endocytosis-mediated intracellular trafficking of
    ligands via the α
    v
    β
    3
    receptor of MCF-7 cells and the
    α
    v
    β
    5
    integrin receptor into the perinuclear region of
    HeLa cells is documented, which lack the functional
    α
    v
    β
    3
    receptor.[10] Interestingely HeLa cells, which
    express the α
    v
    β
    3
    integrin receptor at low level, possess
    lower invasive potential than MCF-7 cells. In our ex -
    periments we used MCF-7 human breast cancer cells
    and HeLa cervix cancer cells to investigate the new
    cRGD-BioShuttle as a delivery platform for targeting
    with TMZ in order to realize high local TMZ concen-
    trations at the MCF-7 and HeLa cells surfaces and,
    after uptake into the cells sufficient for cell killing.
    This paper intends to summarize the major ef-
    forts reached thus far and focuses on the design,
    synthesis and application of the
    cRGD-BioShuttle-TMZ conjugate. The whole mole-
    cule was synthesized via Diels Alder Reaction with
    inverse electron demand. It is composed of the cyclic
    RGD-containing the α
    v
    β
    3
    and α
    v
    β
    5
    integrin antagonist
    cRGD.
    Cell culture
    The estrogen sensitive MCF-7 adenocarcinoma
    breast cancer and HeLa cervix cancer cells (dkfz, tu-
    morbank) were maintained at 37°C in a 5% CO
    2
    at -
    mosphere in RPMI cell medium (Gibco, Germany)
    supplemented with 5% fetal calf serum (Biochrome,
    Germany). The cells were split twice a week.
    Chemical Procedures
    Synthesis of the RGD-BioShuttle
    Derivatization of temozolomide
    N-(2-Aminopropyl)-4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)be
    nzamide 4
    4-(6-(Pyrimidine-2-yl)-1,4-dihydro-1,2,4,5-tetrazi
    ne-3-yl)benzoic acid (3) was prepared from
    2-cyanopyrimidine 1 a nd 4 -cyano-b enz oic a cid 2 by
    reaction with hydrazine and then oxidized with so-
    dium nitrite to the tetrazine derivative 4 according to
    the following procedure [11]. The tetrazine derivative
    was converted with thionyl chloride under standard
    conditions to the chloride 5. To this suspension of the
    acid chloride (2 mmol) in 20 ml CH
    2
    C l
    2
    a solution of
  • Int. J. Med. Sci. 2010, 7
    http://www.medsci.org
    328
    N-Boc-1,3-diaminopropane (2 mmol) and TEA (2
    mmol) in 10 ml CH
    2
    C l
    2
    was slowly added at 0-5°C.
    The resulting solution was deeply coloured and
    maintained for 4 h at room temperature. Then the
    organic phase was washed with water, followed by
    1N HCl and again water. The organic layer was dried
    over Na
    2
    SO
    4
    and evaporated. The resulting residue
    was chromatographed on silica gel by elution with
    chloroform/ethanol (9:1) and further purified by re-
    crystallization from acetone. Yield: 50 to 70 % de-
    pending on the quality of the carboxylic acid. ESI MS:
    m/z 437.2 [M]
    +
    . The Boc-protected derivative was
    treated with TFA (5 ml) for 30 min at room tempera-
    ture and isolated by evaporation to a solid residue (6)
    (ESI: m/z 337.2 [M]
    +
    (as shown in Figure A).
    Figure A shows the mass of the
    N-(2-Aminopropyl)-4-(6-(pyrim idine-2-yl)-1,2,4,5-tetrazine
    -3-yl )b enz am ide (6 in scheme 1/Figure S1), as discussed by
    Wiessler [12].
    3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb
    oxylic acid chloride 7
    3-Methyl-4-o xo -3,4-dihydroimidazo[5,1-d][1,2,3,
    5]tetrazine-8-carboxylic acid was converted to the
    corresponding chloride 7 as documented by Ar-
    rowsmith [13]. The acid (2 mmol) was refluxed with
    thionyl chloride (10 ml) until the acid was completely
    dissolved. The excess of thionyl chloride was evapo-
    rated under vacuum and the resulting solid was
    stored over NaOH.
    3-Methyl-4-oxo-N-(3-(4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-y l )
    benzamido)propyl)-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-
    8-carboxamide (TMZ-tetrazine diene) 9
    Compound 8 (0.5 mmol) and the chloride 7 (0.5
    mmol) were dissolved in 5 ml chloroform and 5 ml
    TEA at 0-5 °C. After 4 h at room temperature, the so -
    lution was washed with water, 1 N HCl and again
    with water. The organic layer was dried over Na
    2
    SO
    4
    and evaporated. The residue was purified by chro -
    matography (silica gel) with chloroform/ethanol
    (9.5/0.5). Yield: 68%: ESI: m/z 536.3 [M+Na]
    +
    . (Figure
    B)
    Figure B shows the mass of
    3-methyl-4-oxo-N-(3-(4-(6-(pyrimidine-2-y l) -1,2,4,5-tetrazi
    ne-3-yl)benzamido)propyl)-3,4-di hy droim idazo [5 ,1 -d][1,2,3
    ,5]tetrazine-8-carboxamide {TMZ-tetrazine diene (9 in
    scheme 1/F ig ur e S1 )} [12].
    Derivatizations of the cRGD
    Synthesis of the Reppe anhydride 12
    The t
    etrac
    yclo-[5.4.2
    1,7
    .0
    2,6
    .0
    8,11
    ]3,5-d io xo -4-aza-
    9,12-t
    ridecadiene (Reppe-anhydride) 12 was prepared
    f r o m 4 2 m g o f ( 1 Z , 3 Z , 5 Z , 7 Z ) -cycloocta-1,3,5,7-tetraene
    10 and 44 mg maleic anhydride 11 in chloroform as
    documented by Reppe [14].

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A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemistry” targeted

Int. J. Med. Sci. 2010, 7 http://www.medsci.org 326 IInntteerrnnaattiioonnaall JJoouurrnnaall ooff MMeeddiiccaall SScciieenncceess 2010; 7(6):326-339 © Ivyspring International Publisher. All rights reserved Research Paper A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemi-stry” targeted to αvβ3 integrin for therapy Klaus Braun1* , Manfred Wiessler1*, Rüdiger Pipkorn2, Volker Ehemann3, Tobias Bäuerle1, Heinz Fleischhacker1, Gabriele Müller4, Peter Lorenz1, Waldemar Waldeck4 1. German Cancer Research Center, Dept. of Imaging and Radiooncology, INF 280, D-69120 Heidelberg, Germany 2. German Cancer Research Center, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany 3. University of Heidelberg, Institute of Pathology, INF 220, D-69120 Heidelberg, Germany 4. German Cancer Research Center, Division of Biophysics of Macromolecules, INF 580, D-69120 Heidelberg, Germany * The authors contributed equally to this work  Corresponding author: Klaus Braun, Ph.D., German Cancer Research Center (DKFZ), Dept. of Imaging and Radiooncol-ogy, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: +49 6221-42 2495; Fax: +49 6221-42 3326; e-mail: k.braun@dkfz.de Received: 2010.07.02; Accepted: 2010.09.07; Published: 2010.09.21 Abstract Clinical experiences often document, that a successful tumor control requires high doses of drug applications. It is widely believed that unavoidable adverse reactions could be minimized by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies” realizing a selective drug targeting to gain effectual amounts at the target site, even with drastically reduced application doses. MCF-7 breast cancer cells expressing the αvβ3 [al-p h a ( v ) b e t a ( 3 ) ] i n t e g r i n r e c e p t o r a r e c o n s i d e r e d a s a p p r o p r i a t e c a n d i d a t e s f o r s u c h a t a r g e t e d therapy. The modularly composed BioShuttle carrier consisting of different units designed to facilitate the passage across the cell membranes and for subcellular addressing of diagnostic and/or therapeutic molecules could be considered as an eligible delivery platform. Here we used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the αvβ3 integrin receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12 µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell killing. Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate composed of the cyclic RGD, a αvβ3 in t eg r i n -ligand, ligated to the cytotoxic drug TMZ. The ligation was carried out by the Diels Alder Reaction with inverse electron demand (DARinv). Key words: Click-Chemistry, Cycloaddition, BioShuttle, Ligation chemistry, Linker Systems, Adaptor Systems, inverse Diels Alder Reaction, RGD, Tetrazines, targeted Therapy, Temozolomide Introduction Breast cancer is one of the most common malig-nancies affecting women in developed countries.[1] Approximately three out of four women with breast cancer develop metastases in bone which, in turn, diminish the quality of life.[2] An optimal treatment concept for patients needs different therapy modali-ties and methods with an optimum in efficiency and the greatest possible protection. Attention should be

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