Maleimide-functionalised Pt(IV) complexes with highly 

selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour 

targeting molecules like human serum albumi

Baban

Barnes

Bernhard K. Keppler

Carr

Choy

Christian R. Kowol

Dhar

Diana Groza

Feazell

Frei

Galanski

Gerrit Hermann

Grek

Gunda Köllensperger

Heffeter

Josef Mayr

Jungwirth

Kratz

Markus Galanksi

Miele

Orsolya Dömötör

Petra Heffeter

Pichler

Reithofer

Verena Pichler

Walter Berger

Wexselblatt

Wheate

Éva A. Enyedy

Pichler, Verena

Mayr, Josef

Heffeter, Petra

Dömötör, Orsolya

Enyedy, Éva Anna

Repository of the Academy's Library

Journal Name Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ►ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00  |  1 Maleimide-functionalised platinum(IV) complexes as synthetic platform for targeted drug delivery Verena Pichler,‡a Josef Mayr,‡a Petra Heffeter,*bc Orsolya Dömötör,d Éva A. Enyedy,d Gerrit Hermann,e Diana Groza,b Gunda Köllensperger,e Markus Galanksi,a Walter Berger,bc Bernhard K. Keppler,a and Christian R. Kowol*ab 5 Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Maleimide-functionalised Pt(IV) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour 10 targeting molecules like human serum albumin. The use of metal-based drugs in cancer therapy is well established due to the worldwide success of the platinum(II) drugs cisplatin, carboplatin and oxaliplatin.1,2 Nevertheless, therapy with these compounds is characterised by severe adverse 15 effects as a result of the unselective damage to healthy tissues and acquired or intrinsic resistance.3 One approach to overcome the drawbacks of platinum(II) chemotherapeutics is the development of platinum(IV) complexes. This class of compounds is kinetically more inert and it is widely accepted that they act as 20 prodrugs. Thus, platinum(IV) complexes open up the possibility to develop platinum drugs with reduced toxicity and enable potential oral application. The most prominent platinum(IV) drug is satraplatin which is currently investigated in advanced phase III clinical trials. 2,4 The additional axial ligands of platinum(IV) 25 complexes allow the coupling of bioactive molecules like estrogen,5 ethacrynic acid,6 folic acid,7 carbon nanotubes8 or gold nanoparticles.9  a University of Vienna, Institute of Inorganic Chemistry, Waehringer 30 Strasse 42, A-1090, Vienna, Austria. Fax:+43-1-4277-52680; Tel: +43-1-4277-52609; E-mail: christian.kowol@univie.ac.at b Research Platform “Translational Cancer Therapy Research” University of Vienna, Waehringer Strasse 42, A-1090, Vienna, Austria c Institute of Cancer Research, Medical University of Vienna, 35 Borschkegasse 8a, A-1090, Vienna, Austria. Fax:+43-1-4277-65196; Tel: +43-1-40160-57557; E-mail: petra.heffeter@meduniwien.ac.at d Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér. H-6720 Szeged, Hungary eDivision of Analytical Chemistry, Department of Chemistry, University 40 of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria † Electronic Supplementary Information (ESI) available: [Materials and Methods, Synthetic procedures, Preinvestigations for HSA binding, X-ray diffraction data, RP-HPLC studies with cysteine, SEC-ICP-MS 45 data]. See DOI: 10.1039/b000000x/ ‡ This authors contributed equally to this work. Fig 1. The concept of maleimide-functionalised platinum(IV) prodrugs.  These moieties are then released during the so called “activation 50 by reduction” process accompanied by simultaneous generation of the corresponding reactive platinum(II) drugs.  However, there is increasing evidence that also platinum(IV) compounds are not specifically activated only in the tumour tissue but also in healthy tissues, especially in the presence of 55 hemeproteins.10-12 Thus, it is of interest to further enhance the tumour-targeting of platinum(IV) drugs.  A well-known targeting molecule is albumin, the most abundant protein in human blood serum which accumulates in the tumour tissues, due to the EPR (“enhanced permeability and retention”) 60 effect.13 In addition, human serum albumin (HSA) possesses a single free thiol group (cysteine-34), which enables the selective and defined preparation of drug-HSA conjugates using maleimide as the coupling moiety. The favourable properties of HSA-coupled anticancer drugs were already clinically proven for 65 doxorubicin (INNO206; aldoxorubicin), currently in phase II clinical studies,14 and a HSA-based nanoparticle encapsulating paclitaxel (Abraxane®) approved for the treatment of breast cancer and non-small cell lung cancer (NSCLC).15 Herein, we report on the synthesis of the first platinum(IV) 70 complexes containing a maleimide moiety in axial position, which enables a simple and fast coupling of thiol-containing  Reduc on       Cell uptake DNA binding targe ng moiety targe ng moiety  2  |  Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]  Scheme 1. Synthesis of novel platinum(IV) complexes (a: NN  = 1,2-ethylenediamine, Y = Cl; b: NN = 1R,2R-diaminocyclohexane, Y2 = oxalate) Fig 2. ORTEP plot of 3a with atom labelling scheme, the thermal 5 ellipsoids have been drawn at 50% probability level.  targeting molecules. This was confirmed by investigations with cysteine and HSA. The starting compounds 1a and 1b were synthesised by standard 10 literature procedures, followed by oxidation with H2O2 (2a and 2b, Scheme 1).16 The maleimide-containing linker was generated by conversion of maleimidopropanoic acid to the isocyanate, which was subsequently coupled to the platinum centre by reaction with the hydroxido groups of the corresponding 15 platinum(IV) precursor in DMF yielding 3a (67%) and 3b (44%). As a reference complex, which is unreactive towards thiols, the succinimide functionalised compound 4b was synthesised by an analogous procedure in good yields (53%). All complexes were characterised by one- and two-dimensional NMR spectroscopy, 20 ESI-mass spectrometry and elemental analysis (see SI). The results of an X-ray crystallographic study of § 3a are shown in Fig. 2. The complex crystallised in the monoclinic space group C2/c, with octahedral coordination geometry. In the equatorial plane the bidentate ethane-1,2-diamine and two chlorido ligands 25 are coordinated to the platinum(IV) core, whereas in the axial position the two 2-maleimideethylcarbamoyl moieties are bound. The bond lengths are in good accordance with comparable compounds in literature,16,17 with Pt–Cl at around 2.3 Å and both Pt–O and Pt–N close to 2.0 Å (for bond lengths and angles see 30 Tables S1, S2, SI). The stability of the novel complexes in aqueous solution was measured with RP-HPLC over a period of 24 h. For both maleimide derivatives 3a and 3b slow hydrolysis could be  Fig 3. Binding studies of 3b with albumin using RP-HPLC. 35  observed (<1%/h, Fig. S1, SI). In comparison, the recorded chromatograms of the succinimide derivative 4b showed no change within 24 h suggesting that the changes in the case of 3a and 3b originate from slow hydrolysis of the maleimide moiety. 40 Subsequently, the complexes were incubated with an excess of cysteine to analyse the reactivity of the thiol group with the maleimide moiety (Fig. S2, SI). The results showed an immediate and complete reaction of cysteine with complexes 3a and 3b (<2 min). In contrast, the succinimide derivative 4b showed no 45 reaction during the same time (1.5 h, Fig. S3, SI), which proves the selective binding of cysteine to the maleimide moiety without any reaction with the platinum centre. The binding rate of 3a and 3b to HSA was measured in aqueous solution in 4:1 albumin-to-Pt ratio. The analyses indicated a rapid albumin linkage with a 50 half-time of ~1 h for both maleimide complexes (Fig. 3). To further investigate the albumin binding in detail, 3a, 3b, and 4b where incubated with HSA for 4 h at various concentrations in phosphate buffer, followed by addition of an excess 2,2’-dithiodipyridine which reacts with the remaining free thiol groups 55 of cysteine-34 and results in the generation of 2-thiopyridone, which can be quantified by UV-vis spectroscopy (see SI). In the case of 4b, the thiol groups of HSA remain unreacted at all tested concentrations, whereas an exponential decrease of free SH was found for the maleimide-containing complexes 3a and 3b (Fig. 60 4). This data reveals that around 80% of the maleimide-containing platinum(IV) compound is bound to HSA after 4 h incubation at a concentration of >30 µM. The original concentration of free SH groups from HSA was ~25 µM (see SI) and a binding-saturation 65 Fig 4. Free SH-content of HSA at various platinum complex/HSA ratios after 4 h incubation, 3a (○), 3b (▲), 4b (□) [cHSA = 99.2 µM; pH = 7.40, 20 mM phosphate puffer + 0.10 M NaCl, 2% (m/m) DMSO].  This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00  |  3 was reached at ca. 25–30 µM of the Pt-complex, which suggests a 1:1 Pt-HSA adduct possibly due to steric hindrance of the second maleimide group.  To investigate the drug protein binding properties of 3b in fetal calf serum (FCS), SEC-ICP-MS measurements of sulphur and 5 platinum were performed (Fig 5). The majority (>70%) of platinum was detected in the frame of 8–9 min, which corresponds to the retention time of albumin (see SI). Only small amounts of 3b were found in the low molecular weight fraction, while 4b was exclusively detected in this fraction. 10 To test the impact of albumin binding on the anticancer activity of the new compounds in vivo the murine CT-26 colon cancer model was used. The use of this syngeneic murine tumour model was necessary due to the recently reported importance of the immune system for the anticancer activity of oxaliplatin.10 Both 15 platinum complexes are well tolerated with no significant loss of body weight (data not shown). As depicted in Fig. 6, both drugs had potent anticancer potential resulting in significantly reduced tumour growth in case of 4b and even disease stabilisation in case of the maleimide-containing 3b. Thus, 3b had a significantly 20 higher anticancer activity (p <0.01 at day 15) against CT-26 Fig 5. Size-exclusion chromatography (SEC-)ICP-MS determination of 3b (50 µM) in fetal calf serum after 2h of incubation. Fig 6. In vivo anticancer activity. CT-26 cells were injected subcutaneously in the right flank of BALB/c mice. Mice were treated on 25 day 5, 8, and 12 (indicated by ▼) i.v. with 18 mg/kg 3b and 4b. Tumour volumes were calculated as described in the Supporting Information. Each experimental group contained four animals. Data are means ± SEM Statistical analysis was performed by two-way ANOVA with Bonferroni post-test (*, p <0.05; **, p <0.01; ***, p <0.001 compared to control 30 mice).  tumours than 4b resulting in a distinctly lower mean tumour burden (of 0.38 g vs. 0.18 g for 4b and 3b, respectively at day 15). Together, this suggests that the albumin binding of 3b leads either to prolonged plasma half-life of the drug and/or a more 35 selective accumulation in the malignant tissue due to the EPR effect.  Based on our data it seems feasible that the combination of maleimide-mediated platinum(IV) binding to serum albumin together with the “activation by reduction” principle can be used 40 to enhance the tumour-targeting of platinum drugs.  This work was supported by the “Fonds der Stadt Wien für innovative interdisziplinäre Krebsforschung” (to P.H. and C.R.K.), the OTKA project 103905 (to É.A.E.) and É.A. Enyedy 45 gratefully acknowledges the financial support of J. Bolyai research fellowship. We are thankful to V. Arion for collecting the X-ray diffraction data and G. Zeitler for animal care. Notes and references § Crystallographic details: 3a: C16H22Cl2N6O8Pt, Mr = 692.39, 0.12 x 50 0.10 x 0.02 mm, monoclinic, C2/c, a = 26-9636(16) Å, b = 7.7770(4) Å, c = 11.3395(6) Å, V = 2276.6(2) Å3calcd = 2020 g cm-3, T = 150(2) K,  = 0.71073 Å,  = 6454 mm-1, R1 = 0.0181, wR2 = 0.0438, GOF = 1.007; description of data collection and refinement see supporting information; CCDC XXX contains the supplementary 55 crystallographif data for this paper (The Cambridge Crystallographic Data Centre, www.ccdc.cam.ac.uk/data_request/cif)  1. M. Galanski, M. A. Jakupec and B. K. Keppler, Curr. Med. Chem., 2005, 12, 2075-2094. 60 2. N. J. Wheate, S. Walker, G. E. Craig and R. Oun, Dalton Trans., 2010, 39, 8113-8127. 3. P. Heffeter, U. Jungwirth, M. Jakupec, C. Hartinger, M. Galanski, L. Elbling, M. Micksche, B. Keppler and W. Berger, Drug resistance updates, 2008, 11, 1-16. 65 4. H. Choy, C. Park and M. Yao, Clin. Cancer Res., 2008, 14, 1633-1638. 5. K. R. Barnes, A. Kutikov and S. J. Lippard, Chem. Biol., 2004, 11, 557-564. 6. W. H. Ang, I. Khalaila, C. S. Allardyce, L. Juillerat-Jeanneret and P. 70 J. Dyson, J. Am. Chem. Soc., 2005, 127, 1382-1383. 7. S. Dhar, Z. Liu, J. Thomale, H. Dai and S. J. Lippard, J. Am. Chem. Soc., 2008, 130, 11467-11476. 8. R. P. Feazell, N. Nakayama-Ratchford, H. Dai and S. J. Lippard, J. Am. Chem. Soc., 2007, 129, 8438-8439. 75 9. S. Dhar, W. L. Daniel, D. A. Giljohann, C. A. Mirkin and S. J. Lippard, J. Am. Chem. Soc., 2009, 131, 14652-14653. 10. U. Jungwirth, D. N. Xanthos, J. Gojo, A. K. Bytzek, W. Korner, P. Heffeter, S. A. Abramkin, M. A. Jakupec, C. G. Hartinger, U. Windberger, M. Galanski, B. K. Keppler and W. Berger, Mol. 80 Pharmacol., 2012, 81, 719-728. 11. J. L. Carr, M. D. Tingle and M. J. McKeage, Cancer Chemother. Pharmacol., 2002, 50, 9-15. 12. J. L. Carr, M. D. Tingle and M. J. McKeage, Cancer Chemother. Pharmacol., 2006, 57, 483-490. 85 13. D. F. Baban and L. W. Seymour, Advanced drug delivery reviews, 1998, 34, 109-119. 14. F. Kratz, Current Bioactive Compounds, 2011, 7, 33-38. 15. E. Miele, G. P. Spinelli, F. Tomao and S. Tomao, International journal of nanomedicine, 2009, 4, 99. 90 16. M. R. Reithofer, S. M. Valiahdi, M. A. Jakupec, V. B. Arion, A. Egger, M. Galanski and B. K. Keppler, J. Med. Chem., 2007, 50, 6692-6699. 17. V. Pichler, S. M. Valiahdi, M. A. Jakupec, V. B. Arion, M. Galanski and B. K. Keppler, Dalton Trans., 2011, 40, 8187-8192. 95   4 6 8 10 12 14 160100020003000PBS3b4b********* ******days after injectiontumour volume (mm3)0 3 6 9 12 15 18time / min platinum signal sulfur signal10000 Cps

Maleimide-functionalised platinum(IV) complexes as synthetic platform for targeted drug delivery

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Maleimide-functionalised platinum(iv) complexes as a synthetic platform for targeted drug delivery

Maleimide-functionalised platinum(IV) complexes as synthetic platform for targeted drug delivery

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