Platinum–bisphosphonate complexes have proven to be inactive chemotherapeuticstargeted for malignant mesothelioma because of inappropriate hydrolysis
Nicola Margiotta a, Rosa Ostuni a, Sara Piccinonna a, Giovanni Natile a,⁎, Ilaria Zanellato b,Carla Doriana Boidi b, Ilaria Bonarrigo b, Domenico Osella b,⁎a Dipartimento Farmaco-Chimico, Università di Bari “A. Moro”, Via E. Orabona 4, 70125 Bari, Italyb Dipartimento di Scienze dell'Ambiente e della Vita, Università del Piemonte Orientale “A. Avogadro”, Viale T. Michel 11, 15125, Alessandria, Italy
a b s t r a c ta r t i c l e i n f o
Article history:Received 22 June 2010Received in revised form 23 December 2010Accepted 23 December 2010Available online xxxx
Keywords:BisphosphonatesMalignant mesotheliomaPlatinum complexesDrug targeting and deliveryBifunctional drug candidates
Bisphosphonates (BPs), the synthetic analogues of pyrophosphate, arewidely used in the treatment ofmetabolicbone diseases. BPs exhibit a preferential accumulation in malignant pleural mesothelioma (MPM) and,furthermore, nitrogen-containing BPs (n-BPs) show significant inhibition of MPM cell proliferation. Wesynthesised dinuclear platinum(II) complexes containing a n-BP moiety as bridging ligand and am(m)ines asterminal ligands (Pt–n-BP)s, with the aim of obtaining bifunctional mesothelioma-targeted drugs.We comparedthe antiproliferative effect of the single drugs (i.e. Pt-model and n-BPs) with that of the preformed Pt–n-BPcomplexes bymeans of the combination index (CI) in order to assess the synergistic/additive/antagonistic effectof the two constituents in the resulting conjugates. The combination of the two individual drugs was almostadditive, while the preformed Pt–n-BP produced an antagonistic effect. Furthermore, (Pt–n-BP)s neitherinhibited the mevalonate pathway (as n-BPs normally do) nor increased the Pt uptake. The minimal biologicalresults of these conjugates could be traced back to a slow and inappropriate hydrolysis, that does not split theadduct into active components.
Bisphosphonates (BPs) are the synthetic analogues of naturallyoccurring pyrophosphate. Depending on their molecular structures,these drugs can be divided into pyrophosphate-resembling (p-BPs,such as clodronate and medronate) and nitrogen-containing bisphos-phonates (n-BPs, such as risedronate and pamidronate). At a cellularlevel, n-BPs inhibit the mevalonate pathway, whereas the p-BPs canbe metabolically incorporated into non-hydrolysable analogues ofATP. Themain biological effect of all BPs is the inhibition of osteoclast-mediated bone resorption. These drugs are, therefore, widely used inthe treatment of metabolic bone diseases, such as osteoporosis. BPsalso inhibit the osteolytic complications of bone metastases of solidtumors and multiple myeloma [1].
The uptake of the bone scan agent 99mTc-medronate (99mTc-MDP,medronate stands formethylene diphosphonate) inmalignant pleuralmesothelioma (MPM) effusions [2–4] was found serendipitously andthis tropism is now well-established. Although the exact mechanismis still unclear, there are unambiguous indications of preferentialaccumulation of BPs in MPM with respect to other tumor cell linesimplanted in mice [5]. Furthermore, n-BPs effectively inhibit the
proliferation of mesothelioma cells in vitro and in vivo, and significantlyextend the survival time of mice bearing experimental models ofmesothelioma [6]. Malignant mesothelioma (MM) is an aggressiveasbestos-related tumor that affects the mesothelial surfaces of thepleura and theperitoneumcavities and, less commonly, thepericardiumand the tunica vaginalis [7]. MM was once rare, but its incidence isincreasing in several countries as a result of widespread exposure toasbestos. Microscopic features of MM may range from pure epithelioid(most common) to pure mesenchymal–sarcomatous, with any combi-nation of these two phenotypes [8]. Diagnosis and staging of MM areoften inaccurate, so that therapy usually addresses late-stage mesothe-liomas. Platinum-based protocols were identified as the most active inpolychemotherapeutic regimens for MM patients [9].
Since BPs target mesothelioma and, moreover, n-BPs inhibit itsgrowth, we speculated that platinum complexes containing n-BPscould target MM and exert a cytostatic/cytotoxic effect summingthe activities of the two constituting moieties (bifunctional drugs).Some (Pt–n-BP)s have already been investigated as specific bone-targeted antineoplastic drug candidates [10–14]. We report here thesynthesis of new di-platinum complexes containing am(m)ineligands (A) and a n-BP moiety as a bridging ligand (Fig. 1). Thesecompounds have been tested towards two primary MPM cell linesobtained from untreated patients, having epithelioid (BR95) orsarcomatous (MM98) phenotype.
The biological investigations have been extended to free n-BPs andPt–malonate model complex.
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Unless otherwise specified, all reagents were from Sigma-Aldrich(St. Louis, MO, USA).
2.2. Instrumental measurements
Electrospray ionisation mass spectrometry (ESI-MS) was per-formed with an electrospray interface and an ion trap massspectrometer (1100 Series LC/MSD Trap system Agilent, Palo Alto,CA) using H2O/MeOH as solvent.
Elemental analyses were carried out with an EA3000 CHNElemental Analyzer (EuroVector, Milano, Italy). Carboplatin (Sigma-Aldrich) was used as calibration standard (C %=19.412, H %=3.258,N %=7.456). Platinum content was determined bymeans of a SpectroGenesis ICP-OES spectrometer (Spectro Analytical Instruments, Kleve,Germany) equipped with a cross-flow nebulizer. Samples of com-plexes were directly mineralized in HNO3.
Thermogravimetric analysis (TGA)was performed using aMettler-Toledo thermobalance at a scanning rate of 5 °C/min from roomtemperature up to 600 °C under nitrogen flow.
NMR spectra were recorded on a Bruker Avance DPX instrumentoperating at 300 MHz (1H). Standard pulse sequenceswere used for 1H,31P{1H} (121.5 MHz), 195Pt{1H} (64.5 MHz) 1D and [1H,195Pt]-HMQC2Dspectra. Chemical shifts are given in ppm. 1H chemical shifts werereferenced to internal sodium 3-(trimethylsilyl)propionate (TSP), 31Pchemical shiftswere referenced to external H3PO4 (85%w/w), and 195Ptchemical shifts were referenced to external K2[PtCl4] in D2O fixed at−1628 ppm. A Crison Micro-pH meter Model 2002, equipped withCrison micro-combination electrodes (5 and 3 mm diameter) andcalibrated with Crison standard buffer solution at pH 2.00, 4.01, 7.02,and 9.26, was used for pH measurements. The pH readings for D2Osolutions are indicated as pH* values and are uncorrected for the effectof deuterium on glass electrodes [15].
2.3. Synthesis of the bisphosphonates
2-Ammonium-1-hydroxyethane-1,1-diyl-bisphosphonic acid(1, AHBP-H4) [10], 3-ammonium-1-hydroxypropane-1,1-diyl-bisphosphonic acid (2, pamidronate, PAM-H4) [16], and tetrasodium1-(pyridin-4-yl)-1-hydroxymethane-1,1-diyl-bisphosphonate (3, Na4[HPMBP]) [11] were prepared following already reported procedures.The elemental analyses and the spectroscopic characteristics of thesynthesised geminal bisphosphonates (or their corresponding acids)were consistent with the data reported in the literature.
2.4. Synthesis of mononuclear platinum complexes with malonate
[PtCl2(cis-1,4-DACH)] [17] (cis-1,4-DACH=cis-1,4-diaminocyclohex-ane), [Pt(OSO3)(H2O) (cis-1,4-DACH)] [18], and cis-[Pt(OSO3)(H2O)(NH3)2] [14] were prepared according to already reported procedures.
[Pt(MAL)(NH3)2] (4a) was prepared as previously reported [19].[Pt(MAL)(cis-1,4-DACH)] (4b) was prepared according to the
method of Hoeschele [20] with some modifications. Briefly, CH2
(COOH)2 (malonic acid,MAL-H2, 0.026 g, 0.25 mmol)was dissolved inwater (5 mL) and treated with Ag2O (0.058 g, 0.25 mmol). Theresulting suspension was stirred for 20 min at 50 °C in the dark andthen treated with [PtCl2(cis-1,4-DACH)] (0.096 g, 0.25 mmol). Afterstirring for 45 min at 50 °C in the dark, heating was interrupted andthe white suspension (due to precipitation of AgCl) was kept understirring at room temperature for 24 h (in the dark). AgCl was thenremoved by filtration through celite and the filtrate was evaporated todryness under reduced pressure at 50 °C. The white residue was thedesired [Pt(MAL)(cis-1,4-DACH)] compound (0.085 g, 83% yield).Anal. Calculated for [Pt(MAL)(cis-1,4-DACH)] (C9H16N2O4Pt): C, 26.28;H, 3.92;N, 6.81%. Found: C, 25.89;H, 3.75; N, 6.43%. ESI-MS: calculated for[C9H16N2O4NaPt]+: 434.3. Found: m/z (% relative to the base peak)=434.0 (100) [M+Na]+.
2.5. Synthesis of dinuclear platinum complexes with bridgingbisphosphonates
(2a), [{Pt(cis-1,4-DACH)}2(AHBP-H)]HSO4 (1b), and [{Pt(cis-1,4-DACH)}2(PAM-H)]HSO4 (2b) were prepared according to previously reportedprocedures [14].
[{cis-Pt(NH3)2}2(HPMBP-H)]HSO4 (3a): Na4[HPMBP-H]·7H2O(39 mg, 0.081 mmol) was dissolved in H2O (40 mL) and the resultingsolution was brought to pH 1.5 by addition of H2SO4 (1 M). Cis-[Pt(OSO3)(H2O)(NH3)2] (0.056 g, 0.162 mmol, in 10 mL of water) wasthen added to the acid solution and the resulting suspension was keptunder stirring in the dark at 40 °C for 10 h and then for 4 days at roomtemperature. The obtained solution was then treated with acetone(200 mL) which causes the precipitation of a yellow-greenish solid.This latter was left standing for 4 h at 4 °C and then isolated byfiltration of the mother solution, washed with acetone, and dried. Thecomplex is highly hygroscopic, as the corresponding free BP.Extensive hydration is a recurrent characteristic of compoundscontaining protonated basic (pyridinium ion) and/or acidic (HSO4
−)functionalities. However, 3a could be completely dried (as verified byTGA) by gentle warming at 50 °C (well below the decompositiontemperature) overnight under high vacuum. At the end of thisprocedure, the yield of anhydrous 3a was 72%. Anal. Calculated for[{cis-Pt(NH3)2}2(HPMBP)]HSO4, (C6H18N5O11P2Pt2S1 ): C, 8.78; H,2.21; N, 8.54, Pt, 47.56%. Found: C, 8.62; H, 2.39; N, 8.33%, Pt, 46.97%.ESI-MS: calculated for [M-SO4, i.e. C6H18N5O7P2Pt2]+=724.0. Found:m/z (% relative to the base peak)=723.9 (100).
[{Pt(cis-1,4-DACH)}2(HPMBP-H)]HSO4 (3b). Na4[HPMBP]·7H2O(0.039 g, 0.081 mmol) was dissolved in H2O (40 mL) and the resultingsolution was brought to pH 1.5 by addition of H2SO4 (1 M). The acidsolution was then treated with a suspension of [Pt(OSO3)(H2O)
Fig. 1. Am(m)ine (a, b) and bisphosphonate ligands (1, 2, and 3) and correspondingplatinum complexes (1a–3b). The malonato complexes 4a–b, selected as referencecompounds are also included.
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(cis-1,4-DACH)] (0.068 g, 0.16 mmol, in 10 mL of H2O) and theresulting suspension was left under stirring in the dark at 40 °C for48 h and then for 3 days at room temperature. The obtained solutionwas filtered and concentrated to a final volume of 20 mL and thentreated with acetone (200 mL) to induce the precipitation of a whitesolid which was left standing at 4 °C for 3 h. The white solid wasisolated by filtration of the mother solution, washed with acetone,and dried by gentle warming (+50 °C) overnight under high vacuum.At the end of this procedure, the yield of anhydrous 3b was41% yield. Anal. Calculated for [{Pt(cis-1,4-DACH)}2(HPMBP-H)]HSO4
(C18H34N5O11P2Pt2S1): C, 22.05; H, 3.49; N 7.14, Pt, 39.79%. Found:C, 22.24; H, 3.55; N, 7.27, Pt, 40.02%. ESI-MS: calculated for [M-SO4,i.e. C18H34N5O7P2Pt2]+=884.1. Found: m/z (% relative to the basepeak)=883.9 (100).
2.6. NMR experiments at different pH and calculation of pKa values
0.01 mmol of compounds 3a and 3bwere dissolved in 0.7 mL of D2Oand transferred into anNMR tube. The pH of the samplewas adjusted tothe required value by addition of DClO4 (0.9 M) or NaOD (2.0 or 0.5 M)solutions and the pH value was measured by using a 3 mm diameterelectrode for NMR tube. No control of the ionic strengthwas performed.Portions of the pH titration curve were fitted to the Henderson–Hasselbalch equation using the program KaleidaGraph (version 3.5,Synergy Software, Reading, PA, USA).
2.7. Stability of compound 1b–3b in physiological conditions
The stability of compounds 1b–3b in buffered solution at 37 °Cwasassessed by 31P-NMR spectroscopy. About 3 mg of each of thesecompounds were dissolved in 0.75 mL of D2O containing 100 mMKD2PO4 (pH=7.4) and 120 mM NaCl. The resulting three solutionswere transferred into NMR tubes and maintained at 37 °C. 31P-NMRspectra were recorded from time to time over a period of one week.The concentration of individual species in solution was deduced fromintegration values.
2.8. Cell testing
Compounds under investigation were dissolved in 0.9% (w/v) NaClaqueous solution and brought to pH 3 with HCl (final stockconcentrations ranging from 1 to 5 mM). [{cis-Pt(NH3)2}2(AHBP-H)]HSO4·2H2O (1a) and [{cis-Pt(NH3)2}2(PAM-H)]HSO4·H2O (2a)resulted to be barely soluble in the above described solution, thereforethey were directly dissolved in serum-free medium and thencomplemented with 10% fetal bovine serum (FBS, Euroclone, Pero,Italy) to a final stock concentration of about 0.4 mM.
Two primary cell lines, having epithelioid and sarcomatoidphenotype, derived from pleural effusion of previously untreatedpatients suffering from MPM, called BR95 [21,22] and MM98 [23],respectively, were used. Cells were grown in DMEM (Gibco, Invitro-gen Life Science, S. Giuliano Milanese) supplemented with L-glutamine 2 mM, penicillin 100 IU/ml, streptomycin (100 mg/L) and10% FBS at 37 °C in a 5% CO2 humified chamber.
All compounds were further diluted in complete medium andcontrols were used as reference.
Three treatment protocols were chosen: 24 h continuous treat-ment (CT), 24 h CT followed by PBS washing and 48 h of recovery (R)in fresh medium, and 72 h CT. Cell viability measurement followedthree PBS washings. Rough screening was made by the methyleneblue staining [24]. Briefly, cells were fixed and stained by 1 hincubation with 2 g/L methylene blue in 50% methanol-50% water,allowed to dry, washed, and eluted with 200 μL/well of 50% ethanol-50% 0.1 M HCl. Data were confirmed by the MTS assay (CellTiterAqueous Solution, Promega Corp., Madison, WI, USA), that wasassessed according to the manufacturer's instructions. In each
experiment, the MPM cells were challenged with the drug candidatesat different concentrations and the final data were calculated from atleast three replicates of the same experiment carried out in triplicate.
Absorbances were recorded at 620/405 nm formethylene blue and490/620 nm for MTS by a spectrophotometric plate reader (Sirio S,SEAC, Florence, Italy); the absorbances of 8 wells without cells wereused as blank.
Combination experiments were carried on using fixed doseratio protocols [25]. Briefly, for simultaneous administration, AHBP-H4 or PAM-H4 were co-diluted with [Pt(MAL)(cis-1,4-DACH)] (atmolar ratio 1:2 as in the corresponding Pt–n-BPs complexes 1b and2b), and the resulting stock solution was serially diluted (to a range ofthree orders of magnitude of concentrations) and tested for cellviability.
The clonal assay was performed as described by Franken et al. [26]:briefly, 200/well BR95 or 100/well MM98 were seeded in 6-wellplates on day−1, challenged by drug candidates from day 0 to day 2,and then allowed to recover in fresh medium, changed every 3 days,until the 10th day, when colonies were stained with methylene blue.Experiments were performed twice in duplicate. Colonies werecounted manually and reported as percentage of the control.
For the bypass of the mevalonate pathway inhibition, cells weretreated with concentration of compounds corresponding to IC25, IC50,and IC75, as determined from cell viability assays, and compared withthe same treatments performed in the presence of 25 μM geranyl-geraniol (GG-OH) in the culture medium. After 72 h, cell viability wasmeasured either by methylene blue or MTS.
2.9. Platinum uptake
About 1.5×106 cells were treated for 4 h with 5 μM 2b, 10 μM 4b,or with a combination of 5 μM PAM and 10 μM 4b. Cells were washedin PBS and harvested by trypsinisation and centrifugation. Afterremoval of the supernatant, cell pellets were stored at −80 °C. Thepellet was lysed in 300 μL of milliQ water and two 10 μl aliquots ofsuch cell suspension were used to determine protein concentration(BCA assay-microplate protocol, Thermo Scientifics), while 250 μLwere transferred into a glass tube containing 65% HNO3 andmineralized until complete drying at 120 °C. Platinum determinationwas performed with an Element 2 ICP-MS (Thermo Scientifics).Instrumental settings were optimised in order to yield maximumsensitivity for platinum. For quantitative determination, the mostabundant isotopes of platinum and indium (used as internal standard)were measured at m/z 195 and 115, respectively. Dry platinum-containing materials were dissolved in 5 ml of 2% HNO3 and addedwith 50 μg/ml of indium. After a few minutes of treatment in anultrasonic bath, a clear solution was obtained which was analysed fortotal platinum. Concentration values were corrected with respect toindium signal. Pt uptake was calculated as ng of Pt normalized to mgof proteins (determined by the BCA method) for each sample [27].
2.10. IC50 and CI calculations
Absorbance data were normalized to 100% cell viability for non-treated cells, half inhibiting concentration (IC50), defined as theconcentration of the drug reducing cell viability by 50%, was obtainedfrom the dose–response sigmoid using Origin Pro (version 8, MicrocalSoftware, Inc., Northampton, MA, USA). Combination index (CI) wascalculated for non-mutually exclusive drugs, according to theequation: CI=D1/Dx1+D2/Dx2+(D1D2)/(Dx1Dx2), where Dx1and Dx2 are the concentrations required for a given effect (expressedas Fraction Affected, FA) respectively for drug 1 and 2 alone, while D1and D2 are the concentrations required for the same FA effect for drug1 and 2 in a combination experiment [28]. 1b and 2bwere consideredas an intramolecular fixed-dose-ratio combination of their buildingblocks: 4b and either 1 or 2. The dose–response curve of each
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compound was compared to those of the single agents for com-puting CI. CI below 1 means synergism, about 1 additivity, over 1antagonism.
3. Results and discussion
3.1. Characterization of the new Pt complexes
[Pt(MAL)(cis-1,4-DACH)] (4b) was prepared by reacting silvermalonate with the precursor complex [PtCl2(cis-1,4-DACH)] (Schemeas Fig. S1).
Compound 4b was characterized by elemental analysis, ESI-MS,and 1H and 2D [1H-195Pt]-HMQC NMR. The 1H-NMR spectrum of 4b inD2O (Fig. S2a) shows broad singlets with platinum satellites at5.09 ppm (2JH-Pt=86 Hz) and 3.14 ppm (3JH-Pt=103 Hz) assigned tothe aminic and methinic protons of cis-1,4-DACH, respectively. Thesignal falling at 1.76 ppm and assigned to the eight methylenicprotons completes the assignments of the coordinated diamine.Finally, the signal at 3.66 ppm is assigned to themethylenic protons ofthe malonate ligand. Interestingly, the latter signal disappeared fromthe spectrum acquired 48 h after the preparation of the NMR sampleindicating that these protons are rather acidic and undergo progres-sive exchange with the deuterium of the D2O solvent. The 2D[1H–195Pt]-HMQC spectrum of 4b (Fig. S2b) shows a cross peak fallingat 3.14/−1872 ppm (1H/195Pt), which indicates a correlation betweenthe methinic protons of the coordinated cis-1,4-DACH ligand and thePt atom. Under the experimental conditions used to record the[1H–195Pt]-HMQC spectrum, it has not been possible to observe thecorrelation peak between the aminic protons and the metal centerbecause of the rather rapid chemical exchange of these protons withthe deuterium of the solvent. The 195Pt-NMR chemical shift is in therange typical for a Pt atom in an O2N2 coordination environment(−1872 ppm) [19,29,30].
The synthesis of the dinuclear complexes [{cis-Pt(NH3)2}2(HPMBP-H)]HSO4 (3a) and [{Pt(cis-1,4-DACH)}2(HPMBP-H)]HSO4 (3b),both having the bridging 1-(pyridinium-4-yl)-1-hydroxy-methane-1,1-diyl-bisphosphonate (HPMBP-H), was very similar.
The synthesis of 3a was performed starting from the sodium saltNa4[HPMBP] and two equivalents of cis-[Pt(OSO3)(H2O)(NH3)2]. The
elemental analysis and the ESI-MS data obtained from complex3a were in accordance with the presence of one n-BP and twocis-[Pt(NH3)2] moieties.
Table 1IC50 values calculated by means of n independent experiments. Viability was assessed by the methylene blue staining method or by the MTS assay.
1a 72 h 27.9 4.8 3 N100 – 32a 72 h 17.4 1.8 3 49 18 33a 72 h N50 – 3 21.1 3.9 31b 72 h 30.4 8.5 6 8.2 2.2 82b 72 h 18 3.7 3 2.9 0.6 33b 72 h 14.4 0.2 3 3 0.8 3[PtCl2(cis-1,4-DACH)] 72 h 3.0 1.0 3 1.1 0.5 3Cisplatin 72 h 3.5 0.6 3 5.9 1.8 3Carboplatin 72 h 32.2 6.8 5 57.5 11.6 7
Fig. 2. Clonogenic assay. Cells were treated with the reported FA concentrationscorresponding to IC10, IC25, IC50, and IC75 for 72 h CT, then allowed to recover in freshmedium for a further week. a) BR95; b) MM98.
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The 1H-NMR spectrum of 3a (Fig. S3) shows two doublets falling at8.74 (3JH-H=5.3 Hz) and 8.41 ppm assigned to the pyridinic protons2/6 and 3/5, respectively. The broad signal at 4.54 ppm is assigned tothe amminic protons coordinated to Pt. The acidic conditions(pH*=2) slow down the exchange of the aminic protons with thedeuterium of the solvent, allowing their detection in D2O. The31P-NMR spectrum (Fig. S3) shows a signal with unresolved platinumsatellites at 33.69 ppm assigned to the two phosphorus nuclei whichare magnetically equivalent and shifted at lower field (Δδ=20.86)with respect to the free HPMBP ligand at the same pH* [11]. The195Pt-NMR of 3a (Fig. S3) shows two signals (−1427 and−1447 ppm)which are at lower field with respect to the signal observed for [Pt(H2O)2(NH3)2]2+ (−1585 ppm) [19,29,31].
The structure in which one bisphosphonate unit bridges twoplatinum atoms in a W conformation is fully supported by thepresence of only one signal in the 31P{1H} spectrum and of two signalsin the 195Pt spectrum [10,11,13,14]. The plane of symmetry passingthrough the two platinum and the phosphonate carbon atom rendersthe two phosphorus atoms chemically equivalent. In contrast, theabsence of a plane of symmetry passing through the P–C–P atoms(due to the presence of two different substituents on the centralcarbon atom i.e. –OH and pyridinium) renders the two platinumatoms unequivalent. However, the two platinum atoms have quiteclose chemical shifts and it is not easy to decide which chemical shiftbelongs to the platinum on the same side of the pyridine residue andwhich chemical shift belongs to the platinum atom on the same side ofthe hydroxyl group. Considering that the pyridine ring should shift atlower field the closest Pt atom, the signal falling at −1427 ppm istentatively assigned to the Pt atom which is on the same side of thepyridine substituent.
A pH titration (31P-NMR experiments at different pH values) wasperformed on compound 3a in order to determine the pKa of thepyridinium functionality (Fig. S4). Only one inflexion point (occurringat pH=6.50 as calculated by fitting the points between pH 1 and 12 tothe Henderson–Hasselbalch equation) was observed in the titrationcurve. This pH value should correspond to the pKa of the pyridiniumgroup for compound 3a. At pH values higher than 12 compound 3astarted to decompose.
Compound 3b was prepared, similarly to 3a, starting from thesodium salt of the bisphosphonate, Na4[HPMBP], and two equivalentsof [Pt(OSO3)(OH2)(cis-1,4-DACH)]. The elemental analysis andthe ESI-MS data were in accord with one bisphosphonate per two[Pt(cis-1,4-DACH)] moieties. The 1H-NMR spectrum (Fig. S5) showstwo doublets placed at 8.66 (3JH–H=6.3 Hz) and 8.50 ppm(3JH–H=6.1 Hz) assigned to the pyridinic protons 2/6 and 3/5,respectively. The very small shift (in the average b0.05 ppm) foundfor the aromatic protons after coordination to platinum indicates thatthe pyridinic nitrogen is not involved in coordination to themetal. Themultiplets falling at 5.47 and 5.09 ppm are assigned to the aminicprotons of coordinated cis-1,4-DACH (geminal protons of each aminicgroup are made unequivalent by the lack of symmetry with respect tothe coordination plane). The acidity conditions (pH*=2) slow downthe exchange of the aminic protons with the deuterium of the solvent,allowing their detection in D2O.
The signals at 3.16 (broad singlet with broad Pt-satellites) and1.93 ppm were assigned to the methinic and methylenic protons ofcoordinated cis-1,4-DACH, respectively.
The 31P{1H}-NMR spectrum (Fig. S5) shows a signal withunresolved platinum satellites at 33.27 ppm (platinum satellites areusually very broad due to chemical shift anisotropy, CSA, relaxation of
Fig. 3. CI-FA plots: a) BR95 treated with a combination of AHBP (1) and [Pt(MAL)(cis-1,4-DACH)] (4b) or with 1b; b) MM98 treated with a combination of 1 and 4b or with 1b;c) BR95 treated with a combination of PAM (2) and 4b or with 2b; d) MM98 treated with a combination of 2 and 4b or with 2b.
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195Pt, especially at high magnetic fields, [30]) assigned to the twophosphorus nuclei which are magnetically equivalent and shifted atlower field (Δδ=20.17) with respect to the free HPMBP ligand at thesame pH*[11].
For a full characterization of complex3b, the 195Pt-NMR spectrumwasalso recorded (Fig. S5). Two broad signals at −1593 and −1612 ppmwere observed. These signals are broadened by the quadrupolar effects of14N (99.6% abundance) from the amine ligands and by CSA relaxation of195Pt [30]. The 195Pt signals are shifted at lower field with respect to thestarting [Pt(cis-1,4-DACH)(H2O)2]2+ substrate. The same variation hasalready been observed in platinum complexes with a diamine and adicarboxylate ligand with respect to the species with a diamine and twoaqua ligands [19,29,31]. As for compound 3a, the signal falling at−1593 ppm could be tentatively assigned to the Pt atom which is onthe same side of the pyridine substituent. The two amminic groups andthe bifunctional bisphosphonate saturate completely the coordination ofeach platinum center excluding the hydroxylic group from coordinationto platinum. This was not the case for complexes with other metal ionswhere the bisphosphonate could act as a tricoordinated ligand contrib-uting two oxygen atoms from the phosphonate groups and one from thedeprotonated hydroxylic group [32].
A pH titration (31P-NMR experiments at different pH values) wasalso performed on complex 3b in order to determine the pKa of acidicprotons. Differently from the free bisphosphonate ligand, where thetitration curve showed five inflexion points corresponding to thevarious deprotonation steps [11], only one inflexion point (occurringat pH=6.29 as calculated by fitting the points between pH 1 and 11 tothe Henderson–Hasselbalch equation) was observed in the titration ofcompound 3b (Fig. S6). The value is in accord with the pKa of thepyridinium functionality. This result further confirms the involvementof all phosphonate hydroxyl oxygens in coordination to platinum. Theplatinum-coordinated oxygen atoms cannot undergo either proton-ation or deprotonation steps as the pH is changed and therefore thereare no breaks in the curve of δP against pH apart from thatcorresponding to the deprotonation of the pyridinium residue.Moreover, compound 3b showed to be stable until pH 11.
3.2. The Pt–n-BP complexes inhibit mesothelioma cellular growth andproliferation
The cytotoxic effect for 24 h CT (continuous treatment) of the n-BPs(1–3) and model Pt-MAL (4a–b) on the two cell lines was almostnegligible (IC50N100 μM). Interestingly, values obtained after 24 hCT+48 h R (recovery) were similar (or even lower in the case of 2and complexes 2a and 2b, data not shown) to those recorded after 72 hCT (Table 1), indicating that the accumulation of the drug occurs withinthefirst 24 hwhereas the biological effectmanifests after 2–3 cell cycles(the time required for doubling the population of MM98 and BR95 celllines is about 24 h) as typically for antiproliferative Pt complexes[33,34]. For sake of clarity, only the IC50 values at 72 h CTwere reportedin Table 1 for complexes 1a–3b.
The sarcomatoid cell line MM98 resulted somewhat moresensitive to the compounds under investigation than the epithelialBR95 one, the reverse is true for the clinically employed drugscisplatin and carboplatin. The following trend in cytotoxicity wasobserved: i) n-BPs 1 and 2 were active while 3 was inactive. ii) Incontrast with the inactivity of 3, the Pt-n-BP derivatives 3a and 3bwere moderately active. iii) As a general rule, the substitution ofmalonate for chlorides greatly reduces the activity, as can be expectedfor the presence of a much more tightly bound leaving group.However, although cis-[Pt(MAL)(NH3)2] (4a) was 30–50 fold lesspotent than cisplatin, this does not apply to the case of cis-1,4-DACHderivatives for which the malonate complex 4b was equally activethan the dichlorido species [PtCl2(cis-1,4-DACH)]. Therefore, if thetwo ammines are replaced by a cis-1,4-DACH ligand, the substitutionof chelated malonate for monodentate chlorides has only a marginal
effect. We do not have a good explanation for such an interestingbehaviour, however, this should be taken into account whenexploring different leaving groups for particular purposes, such asthe targeting of platinum drugs.
The cis-1,4-DACH derivatives resulted to be far more active thanthe ammine derivatives also for the Pt–n-BP complexes (1b–3b versus1a–3a) against the MM98 cell line. In this context, it is also importantto recall that platinum complexes with cis-1,4-DACH carrier ligandare, in general, active also against cisplatin-resistant cell lines[18,20,35,36].
Since the viability data indicated that the cis-1,4-DACH offers thebestperformanceas carrier group, hereafterwepursuedamoredetailedinvestigation on the model compound [Pt(MAL)(cis-1,4-DACH)] (4b)and the corresponding n-BP derivates 1b–3b.
We performed a clonogenic assay in order to test whether 72 h CTat IC10, IC25, IC50, and IC75 concentrations could block long-termcellular proliferation evaluated by counting the number of coloniesafter one week recovery (Fig. 2). MM98 cells were again moresensitive to the treatment than BR95 cells, although controls showedsimilar plating efficiency (about 40%) [26]. Complexes 1b–3b wereable to reduce the number of colonies in a much more efficient waythan the corresponding free n-BP, indicating that the complexesmaintain the cytotoxic behaviour of the alkylating Pt family and aresomewhat more efficient than the model malonato complex [Pt(MAL)(cis-1,4-DACH)] (4b).
3.3. Evaluation of the building blocks activities of Pt(cis-1,4-DACH)–n-BPcomplexes
We evaluated the combination index (CI) which allows to comparethe single-drug dose–response curves to that of their combination.Since the Pt–n-BP complexes contain two Pt-moieties and one n-BP
Fig. 4. Bypass of the mevalonate pathway inhibition. Δ viability between cells treated inthe presence or absence of 25 μM GGOH for 72 h. Data were obtained from 3independent experiments using either MTS or methylene blue. a) BR95; b) MM98.
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skeleton, the ratio between the model compound [Pt(MAL)(cis-1,4-DACH)] and the n-BP was set to 2:1. Moreover, since free 3 wastotally inactive in the range of concentrations used in our experiments
(Table 1), we could not extrapolate the dose for the correspondingCI calculation, therefore this type of investigation could not beperformed for 3b.
1 was additive to [Pt(MAL)(cis-1,4-DACH)] on BR95 (Fig. 3a)and antagonistic on MM98 (Fig. 3b); 2 was synergistic to [Pt(MAL)(cis-1,4-DACH)] on BR95 (Fig. 3c) and additive/synergistic on MM98(3d). According to their CI values (open symbols in Fig. 3) 1b and 2bexhibited antagonistic behaviour with respect to their building blocks(4b and free n-BP). These results indicate that the assembling of the twobuilding blocks produces a conjugate not pharmacologically profitable.
3.4. Do Pt–n-BP complexes still act as bisphosphonates?
We evaluated the ability of the phosphonate complexes to inhibitthe mevalonate pathway. n-BPs are known to exert their cytostaticeffect through the depletion of intracellular prenyl-groups [37], whichare needed for the post-translational modification and activation ofsmall GTP-binding proteins, such as Ras, Rho, Rac, and Rap [38]. One ofthese terpenes, namely geranylgeraniol (GGOH), can be used tobypass the n-BPs inhibition of the mevalonate pathway. Thus, wetreated cells for 72 h in the presence or absence of GGOH (25 μM)with compounds under investigation at concentrations correspondingto their IC25, IC50, and IC75. The observed differences in viability (Δ)
Fig. 5. Platinum uptake (ng Pt/mg proteins). BR95 (white bars) and MM98 (grey bars)were treated for 4 h with 4b (10 μM), a combination of 5 μM 2 and 10 μM 4b, and 2b(5 μM). Results are means±SD of three independent experiments. The uptake of 4bwas compared to that of 2+4b and of 2b within the same cell line by means of thet-test (*, pb0.05).
Fig. 6. Top: Stability of compound 1b in physiological-like conditions (D2O, KD2PO4 buffer 100 mM, pH=7.4, 120 mM NaCl, 37 °C) obtained by 31P-NMR spectroscopy. Bottom: relativepercentage of the Pt–n-BP complexes in solution (obtained by integration from 31P-NMR spectra) plotted as a function of time. Curves represent smoothed fits of the data.
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are shown in Fig. 4. 1 and 2 gave positive Δ, indicating that, asexpected, they inhibit the mevalonate pathway. In contrast 4b andcompounds 1b–3b gave no difference on both cell lines. These resultssuggest that, unlike the respective n-BP precursors, compounds1b–3b do not longer act as inhibitors of the mevalonate pathway.
3.5. Intracellular uptake of Pt–n-BP complexes
Finally, we measured the uptake of platinum complexes on BR95and MM98 and the possible role of the free bisphosphonate. The Ptuptake was evaluated after 4 h incubation with 2b, the modelcomplex 4b, and the 2:1 combination of 2 and 4b, (5 μM 2, 10 μM4b). As depicted in Fig. 5, platinum uptake was generally greater for4b (a neutral compound capable to enter into the cell by passivediffusion) than for 2b (a mono-cationic compound due to protonationof the aminic group of the bisphosphonate) and greater for MM98than for BR95. This observation is in tune with the higherchemosensitivity of the former with respect to the latter cell line. 2was without effect on the uptake of 4b.
3.6. Hydrolysis of compounds 1b–3b
In order to test whether the minimal cytotoxic activity of the Pt–n-BPcompounds was due to slowness of their hydrolysis which inhibits thesplitting into active components, we monitored the stability of com-pounds 1b–3b in physiological-like conditions (D2O, KD2PO4 buffer
100 mM, pH=7.4, 120 mM NaCl, 37 °C) by 31P- and 195Pt-NMRspectroscopy.
Compound 1b resulted to be very stable and no release of freebisphosphonate (1) was observed in solution (the free ligand afforded asinglet at ~17.1 ppm in the 31P-NMR spectrum [10]) even after oneweek.However, after 4 h, the 31P-NMRspectrumshowed the appearance of twonewsets of signals, each one composed of a pair of doublets (one falling at28.15 and 22.98 ppm and the other at 26.43 and 21.51 ppm) whichaccount for nearly 90% of initial 1b (Fig. 6). Moreover, the 195Pt-NMRshowed, in addition to a broad signal falling at−1665 ppmand similar tothat of the starting complex (N2O2 coordination environment), two newverybroadpeaks falling at−2195and−2213 ppmwhichare compatiblewith a Pt(II) in a N2OCl or N3O coordination environment. Although thefull characterization of the two new species is beyond the scope of thispaper, on the basis of the NMR data we can confidently propose that thetwonewspecies, bothhavingunsymmetrical bisphosphonate, are formedfrom the precursor complex by displacement of one oxygen atom of thebridging bisphosphonate ligand by either a chloride (120 mM concen-tration) or by thebisphosphonate-boundaminic group (Fig. 6). Therefore,after 72 h in physiological-like conditions, hydrolysis of 1b does notrelease either free BP (1) or active [PtCl2(cis-1,4-DACH)].
In physiological-like medium also compound 2b undergoes atransformation. The 31P-NMR spectrum shows a decrease of theintensity of the signal of the starting compound (38.7 ppm atpH=7.4) with the simultaneous formation of a singlet at 25.4 ppmthat, after 72 h, accounts for nearly 25% of the total phosphorous
Fig. 7. Top: Stability of compound 2b in physiological-like conditions (D2O, KD2PO4 buffer 100 mM, pH=7.4, 120 mM NaCl, 37 °C) obtained by 31P-NMR spectroscopy. Bottom: relativepercentage of the Pt–n-BP complexes in solution (obtained by integration from 31P-NMR spectra) plotted as a function of time. Curves represent smoothed fits of the data.
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(Fig. 7). No signal assignable to free PAMwas detected (the free ligand2 gives a singlet at 19.0 ppm). The singlet at 25.4 ppm is indicativeof a symmetric bisphosphonate that can be assigned to a monomericPt–n-BP derivative obtained by release of a Pt(cis-1,4-DACH) moiety.This hypothesis is supported by ESI-MS experiments performed onthe NMR sample, showing the presence of a peak at m/z=420.9corresponding to the partially deuterated species C6H13Cl2DKN2Pt,[{PtCl2(cis-1,4-DACH)}+K]+.
The different behaviour of 2b, with respect to 1b, can be explainedwith the already reported [14] smaller tendency of the aminic groupof PAM-H to coordinate to platinum by displacing one oxygen atom ofthe bridging bisphosphonate. Moreover, the release in solution of thecytotoxic compound [PtCl2(cis-1,4-DACH)] could explain the highercytotoxicity of compound 2b as compared to 1b. Also the uptake dataare in tune with this view: the amount of intracellular Pt found afterthe challenge of cells with 2b is rough half than that found with thecorresponding model 4b (Fig. 5).
The behaviour of 3b in a physiological-like medium (Fig. 8)appears to have similarities with both 1b and 2b. Similarly to 1b,formation of a new species with unsymmetrical bisphosphonate isobserved (a pair of doublets falling at 33.17 and 21.44 ppm in the31P-NMR spectrum, accounting for nearly 70% of the total phospho-rous after 72 h). Such a species could be the product of displacementof one coordinated oxygen of the bridging bisphosphonate by chloride(for geometrical reasons the bisphosphonate-bound pyridine cannotcoordinate to a platinum atom which is still bound to the phosphonicgroups).
Similarly to the case of compound 2b, the ESI-MS spectrum,recorded on the NMR sample after 72 h, showed the presence of free[PtCl2(cis-1,4-DACH)] (m/z=420.9, corresponding to the partiallydeuterated species C6H13Cl2DKN2Pt, [{PtCl2(cis-1,4-DACH)}+K]+).
In summary, investigations on the hydrolysis process confirm that thedinuclear Pt–n-BP complexes do not split in the right way to generate thetwo active components (i.e. free n-BPs and [PtCl2(cis-1,4-DACH)]).
4. Conclusions
The aim of the present investigation was to combine two activemoieties, namely the cytostatic/cytotoxic n-BP and the cis-PtA core,for targeting mesothelioma (bifunctional drug strategy). BPs areknown to accumulate in the MPM tissue and n-BPs to induce celldeath. Moreover, Pt-based polychemotherapy is the first-line optionfor the treatment of this kind of tumor. The Pt–n-BP complexesshowed a modest activity which depends upon the type of n-BPbridging the two platinum subunits and upon the type of amineligands completing the coordination sphere of Pt(II). The Pt–n-BPcomplexes failed to inhibit the mevalonate pathway by targeting thefarnesyl pyrophosphate synthase [37]. This indicates that the twobuilding blocks (Pt-core and n-BP) do not act independently once theconjugate has reached the target, due to the slow and inadequatehydrolysis process.
We, therefore, plan to synthesize modified (Pt–n-BP)s, where thetwo building blocks are linked by an hydrolysable spacer with the aimof obtaining bifunctional drug candidates deprived of any antagonisticintramolecular interaction.
Fig. 8. Top: Stability of compound 3b in physiological-like conditions (D2O, KD2PO4 buffer 100 mM, pH=7.4, 120 mM NaCl, 37 °C) obtained by 31P-NMR spectroscopy. Bottom:relative percentage of the Pt–n-BP complexes in solution (obtained by integration from 31P-NMR spectra) plotted as a function of time. Curves represent smoothed fits of the data.
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BR95 MPM cells having epithelioid phenotypeCI Combination Indexcis-1,4-DACH cis-1,4-diaminocyclohexaneCSA Chemical shift anisotropyCT Continuous treatmentDMEM Dulbecco's Modified Eagle MediumESI ElectroSpray IonisationFA Fraction AffectedFBS Fetal Bovine SerumGG-OH Geranyl-geraniolHMQC Heteronuclear Multiple Quantum CoherenceHPMBP 1-(pyridin-4-yl)-1-hydroxymethane-1,1-diyl-bisphosphonateIC25, IC50, and IC75 Drug concentration causing 25, 50, and 75%
inhibition of cell growICP-OES Inductively Coupled Plasma Optical Emission SpectrometryICP-MS Inductively Coupled Plasma Mass SpectrometryMAL MalonateMM Malignant MesotheliomaMM98 MPM cells having sarcomatoid phenotypeMPM Malignant Pleural MesotheliomaMTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
ligand and am(m)ine terminal ligandsR RecoveryTGA Thermogravimetric AnalysisTSP 3-(trimethylsilyl)propionate
Acknowledgments
Weare indebted to Prof. P.-G. Betta (MesotheliomaBiobankRegionalReferenceCenter, AlessandriaNationalHospital) for providingMPMcelllines. We acknowledge the Italian Ministero dell'Università e dellaRicerca (MiUR), the European Union (COST D39/Metallo-Drug Designand Action), the Ambiente-Territorio-Formazione Association (ATF,Alessandria), and the Regione Piemonte (CIPE-project-code A370). I.B.thanks the Inter-University Consortium for Research on the Chemistryof Metal Ions in Biological Systems (C.I.R.C.M.S.B., Bari) for a researchgrant.
Appendix A. Supplementary data
Supplementary data to this article can be found online atdoi:10.1016/j.jinorgbio.2010.12.011.
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