Field of the invention

The present invention relates to novel compounds containing a platinum isotope and a bone targeting agent capable of associating with bone tissue and the application of the compounds in the treatment of cancers such as bone cancer.

Background of the invention

Most cancers metastasize to bone since its physiological environment facilitates the formation and growth of tumors. Bone is often affected by malignant cancers, which then become the primary cause of mortality. Distant metastases are the leading cause of death for both breast and prostate cancer patients, with 65-75% and 90% of these patients developing bone metastases in advanced stages, respectively. Bone metastases are often associated with accelerated bone resorption leading to complications such as skeletal-related events (SREs), bone pain or hypercalcemia. Unfortunately, current treatments for bone metastases are limited. Bisphosphonates (BP) and denosumab are most commonly used for palliative treatment to prevent or limit SREs. Although such treatments inhibit osteoclast activity and prevent the progression of metastases, they do not kill cancer cells effectively and do not improve the quality of life substantially. Consequently, the development of effective therapies to treat bone metastases is still a major clinical challenge.

Cancer cells modulate the bone microenvironment to support tumor growth and accelerate tumor progression. The elimination of cancer cells from bone metastases is crucial for treatment efficacy, which requires precise and efficient delivery of antitumor therapeutics to bone metastases. Targeted drug delivery aims at selective and effective accumulation of pharmacologically active compounds at desired target site(s), thus minimizing undesired side effects and maximizing therapeutic efficacy. Among various types of radiosensitizers, platinum-based (Pt) drugs belong to a class of radiosensitizer that influence the nature or repair of DNA damage.

Platinum-based (Pt) drugs are a specific class of chemotherapeutic drugs which exhibit antitumor properties by interacting with DNA and suppressing its replication. Pt-based drugs are known to induce systemic toxicity, while the lack of tumor specificity restricts their application for long-term treatment. Consequently, there remains a need for improved compounds and their preparation in the treatment of various cancers such a bone cancers. Margiotta et al. formulated various anticancer Pt-bisphosphonate complexes to deliver Pt to bone (40-42), since BPs exhibit a strong affinity for hydroxyapatite bone mineral (43). Recent advances in imaging technologies allow for early detection of malignant tissues directly in bone marrow (54-56) or detection of indirect tumor activity related to bone remodeling (57,

58). Such early assessment of developing bone metastases would enable patient-specific bone-targeted treatment. However, treatment options for patients with bone metastases are scarce and palliative rather than curative (8, 9). Consequently, the design of novel theranostic compounds which detect and treat bone metastases in cancer patients would overcome this unmet need (31).

The present inventors have focused on the design of radioactive chemotherapeutics based on ^195m pt which exhibit diagnostic, chemo- and radiotherapeutic efficacy to facilitate effective treatment of bone metastases. The high metabolic activity of metastatic bone offers the opportunity for bone tumor-targeting of bone-seeking ^195m Pt-based therapeutics.

Nadar et al. (Nadar et al, Clin. Exp. Metastasis 2017, 34, 491-524, page 514) have disclosed and discussed the use of ^195m pt-BP as a theranostic agent, but only combined the chemotherapeutic effect of Pt with the gamma radiation of the ^195m pt isotope as a diagnostic in SPECT imaging.

Summary of the invention

The present inventors have found that the integration of radioactive Pt isotopes, preferably 1 ^95m pt _or ^i93m p _t , _more preferably ^195m pt in bone tissue-targeting agents further enhances the therapeutic/diagnostic properties of Pt-based drugs. More in particular, the use of Pt-isotope containing compounds combines the chemotherapeutic effects of Pt with the radiotherapeutic effects of the Pt isotope. The combination of the Pt isotope with a bone tissue-targeting agent (bone-seeking agent) delivers the Pt isotope at the desired location (bone of high metabolic activity), where the Pt can exert its function as a chemotherapeutic in (synergistic) combination with its radiotherapeutic function as the Pt isotope as the ^195m pt radionuclide emits Auger electrons. In addition to these therapeutic properties, these compounds have also diagnostic applications, as ^195m pt or ^193m pt emits gamma rays that can be detected outside the patient. This gamma emission allows SPECT imaging. SPECT imaging provides for the detection of the location and distribution of the compound in the patient. The compound of the invention thus provides information on cancer location and/or distribution of the compound in the body and/or compound targeting efficiency.

Brief description of the Figures

Fig. 1. Synthesis and Characterization of Pt-BP complex.

Fig. 2. FTIR spectra of Pt-BP complex.

Fig. 3 NMR characterization of Pt-BP complex.

Fig. 4. In vivo phenotypic effects of Pt-BP in zebrafish embryos. Fig. 5. Ototoxicity caused by Pt-BP treatment of zebrafish embryos.

Fig. 6. ^195m Pt radioactivity in mice.

Fig. 7. Biodistribution profile of Pt-BP and Pt(N0 ₃ Men) in vivo.

Fig. 8. Biodistribution profile of ^195m pt-BP and ^195m Pt(N03)2(en) in vivo.

Fig. 9. Quantification of ^195m pt-BP and ^195m Pt(N03)2(en) biodistribution in vivo.

Fig. 10. Spatial distribution of Pt in bone.

Fig. 11. Biodistribution profile of ^195m pt-BP and ^195m Pt-Cisplatin in vivo (bone tumor-bearing mice).

Fig. 12. Quantification of ^195m pt-BP and ^195m Pt-Cisplatin biodistribution in vivo (bone tumor bearing mice)

Fig. 13. Representative histological images of tumor regions in metastatic mice model.

Fig. 14. Representative histochemical and immunohistochemical images of tibial lesions in a prostate cancer cell-induced bone metastasis model 14 days after treatment.

Detailed description of the invention

The present invention hence pertains in a first aspect to a compound comprising a Pt isotope, preferably ^195m pt or ^193m pt, more preferably ^195m pt isotope, the compound further comprising a bone-seeking agent preferably capable of targeting bone of high metabolic activity.

Hence the invention provides a compound that is a complex or a conjugate of a Pt-isotope in combination with a bone-seeking (or bone-targeting) agent that is capable of associating with bone. The bone seeking agent is preferably a compound or complex that is bone seeking, i.e. when administered to a subject, there is a tendency of association with bone (or mineralized tissue) more than with other tissues of the subject.

Without being bound by theory, the inventors consider that the compound of the present invention preferably displays the following functionalities and advantages: the compound is targeted to bone (or mineralized tissue) of high metabolic activity; the compound dissociates in the cell environment; the compound upon dissociation is capable of releasing a Pt-isotope compound/molecule, which is similar or identical to platinum-based chemotherapeutic molecules. Platinum-based chemotherapeutic molecules have a tendency to bind to DNA or cell nucleus material. the activated Pt-isotope is close enough to cellular DNA to cause severe damage to the DNA by Auger electrons and/or gamma rays, and kill the cell. The present invention solves a major problem with Pt-based chemotherapeutics. Pt-based chemotherapeutics are generally delivered systemically, thereby causing serious side-effects in various organs unaffected by cancer, since the chemotherapeutic affects also healthy cells. Although the invention encompasses any Pt-isotope capable of emitting Auger electrons, there is a preference for ^195m Pt.or ^193m pt, more preferably ^195m pt. It is thought, without being limiting, by the present inventors that the use of ^195m pt would cause additional damage, not only to cancer cells but also to healthy cells and induce or enhance unwanted side effects. The in-cell dissociation/activation of the compound of the invention in combination with the ^195m Pt can bring the compound to the desired location (DNA in mineralised tissue such as bone) where the Pt can exert a chemotherapeutic effect and the Auger electrons and/or gamma rays of the ^195m pt isotope exert a radiotherapeutic/diagnostic effect.

The present invention thus demonstrates that significant advantages are being achieved with this radiation therapy wherein the radiation damage is preferentially brought to the location that needs to be affected.

It is theorized that the short range of Auger electrons causes that only cells with the Auger emitter at the right location in the cell will be harmed. In the compounds of the present invention this is achieved by targeting the bone and targeting the DNA after which the Auger emitter is close enough to the cell nucleus material and DNA to be effective, considering the very short range at which Auger emitters are damaging. If the compound does not enter the cell, it nevertheless may be effective, provided the emitter is located in proximity of the cell or nucleus of interest. At larger distances, the Auger emitter range of damage is typically too short to affect the cell. The compound of the present invention is radiotherapeutically active by killing cancer cells.

Examples of bone seeking agents include phosphorus-containing compounds such as those which contain C-O-P bonds (phosphates such as those described in U.S. Patent No. 4,582,700), P-O-P bonds (pyrophosphates or polyphosphates such as those described in U.S. Patent No. 4,016,249), C-C-P bonds (phosphonates such as those described in U.S. Patent Nos. 4,233,284 and 4,642,229), P-C-P bonds (diphosphonates or bisphosphonates such as those described in U.S. Patent No. 4,233,284), P-N-P bonds (imidodiphosphonates such as those described in U.S. Patent No. 3,974,268), or combinations or derivatives thereof. The targeting agents may be used in a variety of forms, e.g., mono-, di- and polyphosphonates. Of the phosphonates, particularly examples are methylene diphosphonate (MDP), 4,5-diamino-1-hydroxypentane-1 ,1-diphosphonate, 3-amino-1-hydroxypropane-1 ,1- diphosphonate (ADP) and 2-amino-1-hydroxyethane-1 ,1-diyl-bisphosphonate (AHBP).

A typical Example in the context of the present invention are pyrophosphates and phosphonates, preferably bisphosphonates. Bisphosphonates and pyrophosphates have been described as expressing similar behavior for the local delivery of Pt-containing compounds and these ligands influenced the loading and release of platinum. Nanoparticles retaining these bisphosphonates or pyrophosphates ligands at their surface release chemotherapeutically active residues of the Pt compounds. As such, these Pt-loaded nanocarriers not only deliver their payload at the tumor sites in a controlled manner, but also improve the cytotoxicity of the unmodified complexes that can thus be considered as platinum prodrugs. (M. lafisco, N. Margiotta, J Inorg Biochem 2012,

117, 237. M. lafisco, B. Palazzo, M. Marchetti, N. Margiotta, R. Ostuni, G. Natile, M.

Morpurgo, V. Gandin, C. Marzano, N. Roveri, Journal of Materials Chemistry 2009, 19. B. Palazzo, M. lafisco, M. Laforgia, N. Margiotta, G. Natile, C. L. Bianchi, D. Walsh, S. Mann, N. Roveri, Advanced Functional Materials 2007, 17, 2180; M. lafisco, B. Palazzo, G. Martra, N. Margiotta, S. Piccinonna, G. Natile, V. Gandin, C. Marzano, N. Roveri, Nanoscale 2012, 4, 206. K. Farbod, K. Sariibrahimoglu, A. Curci, A. Hayrapetyan, J. N. Hakvoort, J. J. van den Beucken, M. lafisco, N. Margiotta, S. C. Leeuwenburgh, Tissue Eng Part A 2016, 22, 788. M. Benedetti, F. De Castro, A. Romano, D. Migoni, B. Piccinni, T. Verri, M. Lelli, N. Roveri, F. P. Fanizzi, J Inorg Biochem 2016, 157, 73.

The Pt may be complexed, bound or conjugated via covalent, ionic and/or coordination bonds to the targeting agent. The compound may contain other ligands, ions etc. In certain embodiments of the invention, the compound can comprise a ligand ( or a chelate). There is a preference for a nitrogen containing ligand. The ligand may be a monodentate, bidentate tridentate or tetradentate. There is a preference for a nitrogen-containing bidentate ligand. Suitable and preferred are ligands selected from the group consisting of ethylenediamine (en), diazabicyclo [2.2.2] octane (DABCO), N,N,N' ,N'-tetramethyl ethylenediamine (TMEDA) , N,N,N' ,N '-tetraethyl ethylenediamine (TEEDA) ,1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), 2,2’-bipyridine (bipy), 5- or 6 membered aliphatic cyclic diamines such as 1,2-cyclohexanediamine, 1,4-cyclohexanediamine, 1,3- cyclopentanediamine, 1 ,4-cycloheptanediamine.

The ligands and/or targeting agents may be protonated or quaternized to improve solubility, for instance under physiological conditions.

In a specific embodiment, the compound can have the formula (I): wherein Pt comprises a Pt isotope, preferably ^195m Pt.or ^193m pt, more preferably ^195m Pt.; wherein R1 is C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkynyl, C5-C6 (het)aryl, preferably substituted with a (quaternized) amine group; wherein R2 is OH, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkynyl, C5-C6 (het)aryl, preferably an OH group; wherein L1 , L2, L3, L4 are monodentate ligands; wherein L1 together with L2 and/or L3 together with L4 are independently a bidentate ligand; or wherein L1-L4 are a quadridentate ligand, capable of forming a coordination complex with Pt.

The Pt in formula I comprises a Pt isotope, preferably ^195m Pt.or ^193m pt, more preferably ^195m pt isotope. Not all Pt atoms in a composition the compound of formula I will be ^195m pt. There will be a fraction in conjunction with other Pt isotopes, and the fraction may diminish due to decay of the ^195m Pt. this is an inherent feature of radioactive isotopes. The Pt in formula I may further contain Pt as a stable element or other Pt isotopes,. In the Pt of formula I, a fraction of the Pt will be ^195m Pt, depending on the ^195m pt specific activity of Pt used for the synthesis of the compound, and wherein the ^195m pt fraction may reduce over time due to nuclear decay of the

195mp†

In more preferred embodiments the ligand is ethylenediamine (en).

Good results have been obtained with a model targeting agent based on 2--amino-1- hydroxyethane-1 ,1-diyl-bisphosphonate (AHBP) which provides a good indication of the versatility and wide applicability of the compound of the invention.

The compound of the invention further finds use as a medicament, a theranostic, chemotherapeutic and/or a diagnostic agent, in particular in the field of bone cancer.

The bone cancer can be a primary bone cancer, a metastatic bone cancer. The use of the compound for bone cancer is foreseen in the treatment of bone cancers selected from the group consisting of osteosarcoma, chondrosarcoma, Ewing tumor, giant cell tumor of bone, chordoma or wherein the bone cancer is metastasized from primary tumors such as prostate, breast, kidney, lung and thyroid primary tumors.

The use of the compound is foreseen in a pharmaceutical comprising which may comprise suitable and common additives such as carriers, diluents, excipients etc.

The present inventors have designed, synthesized and tested novel radioactive Pt-BP ( ^195m Pt- BP) complexes to enable targeted delivery of ^195m pt to bone of high metabolic activity. First, novel bone-targeted ^195m pt-BP was synthesized using ^195m Pt(NC>3)2(en) (en = ethylenediamine) as Pt precursor. Second, Pt release from Pt-BP complexes was investigated at physiological (pH 7.4) and acidic conditions (pH 5.25). This latter condition mimics the acidic cancer environment. Third, the in vivo bone-seeking and Pt-DNA adduct forming capacity of Pt-BP complexes 24h post-administration using high-resolution inductively coupled plasma-mass spectrometry (ICP-MS) was confirmed. Fourth, the long-term stability and bone-seeking capacity of ^195m pt-BP was monitored over a 7 day period using preclinical micro-SPECT/CT imaging in mice. Subsequently, the spatiotemporal distribution of ^195m pt-BP within the mice tibia using advanced laser ablation ICP-MS imaging was shown. Finally, the in vivo bone tumor-targeting capacity and radiotherapeutic efficacy was monitored over a 7 day period using preclinical micro-SPECT/CT imaging and immunohistochemistry in bone tumor bearing mice.

Examples

General

Study design

The objective of the study was to evaluate Pt-BP as potential therapeutic (Pt) or theranostic agent ( ^195m pt) to selectively deliver Pt or ^195m pt to metabolic active region of bone. All experiments were performed with five animals per group to reach statistical significance. Mice were randomized into different groups before administration of pt/ ^195m pt compounds.

Blindings were applied to the housing of the mice. In addition, biotechnicians were also blinded (non-informed) before administration of Pt compounds. Image acquisition in vivo and ex vivo was performed in a non-blinded manner. Data analysis was also performed in a non- blinded manner.

In vivo studies

All in vivo work was conducted in accordance with ISO standards and the principles set forth by the Revised Dutch Act on Animal Experimentation. The in vivo experiments were approved by the institutional Animal Welfare Committee of the Radboud University Medical Center (Radboudumc), Nijmegen, the Netherlands. For both the experiment, 10 male C57BI/6N mice (Charles River), with an average weight of ~25 g and an age of approximately 6-8 weeks were housed in filter-topped cages (5 mice per cage) under non-sterile standard conditions provided with standard animal food and water ad libitum. The mice were allowed to adapt to laboratory conditions for 1 week before experimental use.

Statistical Analysis

All results except ratios are depicted as mean ± standard deviation. The statistical analyses were performed using GraphPad Prism (version 6.0) software. Two-way analysis of variance (ANOVA) with a Bonferroni (multiple comparisons) post-hoc test was used to determine the differences among the two groups. For ratios, paired t-test was used to determine the differences among the two groups. For the ototoxicity assay, one-way analysis of variance (ANOVA) was performed followed by the Dunnett’s method for multiple comparisons.

Zebrafish care and handling

The zebrafish (Danio rerio) strains were kept under standard conditions (28 °C in E3 buffer) until 48 hours post fertilization (hpf). All animal experiments were conducted at larval stages before the point of independent feeding and were in agreement with the animal protection law (Tierschutzgesetz).

Materials

Na2SC>4 and Ba(OH)2 were purchased from Sigma-Aldrich. 2-amino-1-hydroxyethane-1,1-diyl- bisphosphonic acid (AHBP-FU) was prepared following procedures reported previously (40). Milli-Q water was used to dissolve the compounds. All other reagents were purchased from Sigma-Aldrich and used without further purification.

Example 1 : Preparation and Characterization of

Pt(NC>3)2(en) (en = ethylenediamine) was synthesized according to a previously reported procedure. Briefly, i^PtCU (100 mg, 0.241 mmol) was dissolved in Milli-Q water (2.5 ml) and stirred at room temperature. Upon complete dissolution, the solution was filtered using a sintered glass filter to remove undissolved platinum. Subsequently, potassium iodide (Kl; 239.9 mg, 1.445 mmol) was added to the filtrate at room temperature, agitating for approximately 5 min. 16.08 pi (0.441 mmol) of ethylenediamine diluted in 400 mI of Milli-Q water was then added slowly and drop-wise under constant magnetic stirring and left at room temperature for approximately 2 h. The obtained yellow precipitate was filtered with a sintered glass filter. The solid phase was washed with cold Milli-Q water, absolute ethanol, and finally with diethyl ether, followed by drying under vacuum. The obtained Ptl2(en) (58.6 mg, 0.115 mmol) was suspended in 16.4 ml of Milli-Q water at 55 °C in the dark and treated with AgNOs (39.12 mg, 0.23 mmol) previously dissolved in 131 mI Milli-Q water protected from light. The mixture was stirred for approximately 4 h in the dark. The flask was then cooled down to room temperature prior to filtration through a plug of Celite ^® to remove Agl. The filtrate was dried using a rotary evaporator (at 40 °C) followed by vacuum drying. Electrospray Ionisation-Mass Spectrometry (ESI-MS) was carried out to measure the molecular weight of the obtained Pt(N0 ₃ ) ₂ (en) using an electrospray interface and ion trap mass spectrometer (1100 Series LC/MSD Trap system Agilent, Palo Alto, CA): Anal. Calc for Pt(N0 ₃ ) ₂ (en): ^Hsl hOePt, M _w = 351.17 g mol ¹ ). Spectroscopic characterization of the complex with Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR; Spectrum Two ^™ , Perkin Elmer) was consistent with literature data (data not shown).

Example 2: Preparation of ^195m Pt(NO _¾ ) _? (en)

The ^195m Pt was produced as previously reported (E. A. Aalbersberg, B. J. de Wit-van der Veen, O. Zwaagstra, K. Codee-van der Schilden, E. Vegt, W. V. Vogel, Preclinical imaging characteristics and quantification of Platinum- 195m SPECT. EurJ Nucl Med Mol Imaging 44, 1347-1354 (2017).) The specific activity of ^195m Pt(N03)2(en) was 48,5 MBq/mg Pt. The radionuclide purity of ^195m Pt(NC>3)2(en) as received from NRG (Petten, The Netherlands) is reported in Table S1.

Table S1. Characteristics of the radionuclide purity of ^195m Pt(NC>3)2(en)

Example 3: Pt-BP pH stability assessed by NMR

³¹ P NMR spectra were recorded on a Bruker Avance III 700 MHz instrument. Standard pulse sequences were used for ³¹ P{1H} (121.5 MHz) spectra. Chemical shifts ( ³¹ P) were referenced to external H ₃ PO ₄ (85% w/w; 0 ppm). A Crison Micro-pH meter Model 2002, equipped with Crison microcombination electrodes (5 and 3 mm diameter) and calibrated with Crison standard buffer solution at pH 4.01, 7.02, and 10.00, was used for pH measurements. The pH readings from the pH meter for D2O solutions are indicated as pD values and are uncorrected for the effect of deuterium on glass electrodes. The stability of Pt-BP in buffered solutions at 37 °C was assessed by ³¹ P NMR spectroscopy as previously reported(73). Pt-BP (~4 mg) was dissolved in 0.8 ml_ of D2O containing (/) 50 mM 4-(2hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES) buffer (pD = 7.4) and 120 mM NaCI or (ii) 50 mM 2- (N-morpholino)ethanesulfonic acid (MES) buffer (pD = 5.25) and 120 mM NaCI. The resulting two solutions were transferred into NMR tubes and maintained at 37 °C. ³¹ P NMR spectra were recorded over a period of 15 days. The relative concentrations of the individual species in solution were deduced from integration of the ³¹ P signals. Example 4: Preparation and characterization of Pt-BP complex

A solution containing 2-amino-1-hydroxyethane-1,1-diyl-bisphosphonic acid (AHBP-hU)

(3.33 mg, 0.015 mmol; in 2.2 ml Milli-Q water) was maintained at 40 °C and then treated with Ba(0H) ₂ 8H ₂ 0 (4.97 mg, 0.01575 mmol). The mixture was left under stirring at 40 °C and then treated with a solution of Pt(NC>3)2(en) (12.5 mg, 0.033 mmol) and Na2SC>4 (2.24 mg, 0.01575 mmol) in 1.7 ml Milli-Q water. The obtained suspension was subsequently added drop-wise to the main vial containing AHBP-hU and Ba(0H) ₂ 8H ₂ 0 agitating at 40 °C. A white suspension was formed which was left overnight under constant stirring at 40 °C. Subsequently, the suspension was cooled down for approximately 1 h in an ice bath to facilitate the precipitation of BaS0 ₄ prior to filtration through a plug of Celite ^® . The volume of the filtrate, which contained the final product, was reduced using a rotary evaporator (at 40 °C) and the pH of the concentrated filtrate was brought to ~1 using H2SO4 (95-97%). Addition of methanol induced the precipitation of the desired product as a white precipitate that was filtered and washed with methanol and diethyl ether, and subsequently dried under vacuum. Elemental analyses were carried out using a Hewlett Packard 185 C and N analyzer. ESI-MS was carried out to measure the molecular weight of the obtained Pt-BP using an electrospray interface and ion trap mass spectrometer. Anal. Calc for [<Rΐ(bh)> ₂ (m-AHBR- H ₂ )](HS0 ₄ )-3H ₂ 0 (C ₆ H29N ₅ Oi4P2Pt2S, M _w = 879.4 g-mo ): C, 8.19%; N, 7.96%. Found: C, 7.99%; N, 7.45%. Spectroscopic characterization of the Pt-BP was carried out using ¹ H and ¹³ P Nuclear Magnetic Resonance (NMR) spectroscopy and ATR-FTIR. Fig. 1. FTIR spectra of Pt-BP complex. Pt-BP complex infrared spectra prepared using Pt(N0 ₃ Men) as the precursor versus Pt-BP complex prepared using Pt(0S0 ₃ )(0H ₂ )(en) as the precursor

Example 5: Preparation of radioactive Pt-BP complex

The synthesis procedure was followed as described above for the cold platinum- bisphosphonate complex. The ^195m Pt(N0 ₃ ) ₂ (en) solution was received from NRG (Petten, the Netherlands). The pH of ^195m Pt(N0 ₃ ) ₂ (en) solution was neutralized to pH 7 using 1M NaOH and reconstituted in sterile saline solution.

Example 6: Synthesis and Characterization of Pt-BP complex

The previously reported synthesis of dinuclear bis<ethylenediamineplatinum(ll)>-2-amino-1- hydroxyethane-1 ,1-diyl-bisphosphonate complex (Pt-BP) ( N. Margiotta, R. Ostuni, D. Teoli,

M. Morpurgo, N. Realdon, B. Palazzo, G. Natile, Bisphosphonate complexation and calcium doping in silica xerogels as a combined strategy for local and controlled release of active platinum antitumor compounds. Dalton Trans, 3131-3139 (2007); B. Palazzo, M. lafisco, M. Laforgia, N. Margiotta, G. Natile, C. L. Bianchi, D. Walsh, S. Mann, N. Roveri, Biomimetic Hydroxyapatite-Drug Nanocrystals as Potential Bone Substitutes with Antitumor Drug Delivery Properties. Advanced Functional Materials 17, 2180-2188 (2007) K. S. Lovejoy, S. J. Lippard, Non-traditional platinum compounds for improved accumulation, oral bioavailability, and tumor targeting. Dalton Trans, 10651-10659 (2009); K. Farbod, K. Sariibrahimoglu, A. Curci, A. Hayrapetyan, J. N. Hakvoort, J. J. van den Beucken, M. lafisco, N. Margiotta, S. C. Leeuwenburgh, Controlled Release of Chemotherapeutic Platinum-Bisphosphonate Complexes from Injectable Calcium Phosphate Cements. Tissue Eng Part A 22, 788-800 (2016) by using Pt(NC>3)2(en) as Pt precursor instead of Pt(OSC>3)(OH2)(en). This Pt(NC>3)2(en) facilitates the synthesis of radioactive Pt-BP complexes using ^195m pt radioisotopes for future applications as a theranostic agent (Fig 6 ). Fig 6 described the Synthesis and Characterization of Pt-BP complex. (A) Chemical structures of platinum complexes. (B) ³¹ P-NMR (121.5 MHz) spectra of Pt-BP at different time points in near- physiological conditions (D2O, HEPES buffer 50 mM, pD = 7.4, 120 mM NaCI, 37 °C). (C) ³¹ P-NMR (121.5 MHz) spectra of Pt-BP at different time points at pD = 5.25 (D2O, MES buffer 50 mM, 120 mM NaCI, 37 °C). (D) Schematic representation of Pt release from Pt-BP as assessed by ³¹ P-NMR spectroscopy. Pt-BP undergoes hydrolysis to release a Pt(en) moiety and a symmetric monomeric Pt-n-BP derivative (M). The second Pt(en) moiety is released by the hydrolysis of M with the release of free BP. At intermediate time points, Pt-BP also forms new species with asymmetric bisphosphonate groups as shown by the black square and diamond symbols.

Characterization via elemental analysis, Electrospray Ionisation-Mass Spectrometry (ESI-MS) measurements, and spectroscopic features show consistency of the Pt-BP product with the one obtained using Pt(OSC>3)(OH2)(en) as a Pt precursor. The ¹ H-NMR spectrum of Pt-BP shows two broad singlets at 5.93 and 5.43 ppm assigned to the aminic protons of coordinated ethylenediamine. The acidic conditions decelerated the exchange of the aminic protons with the deuterium of the solvent, allowing detection of the aminic protons in aqueous solution.

The doublet centered at 3.34 ppm is assigned to the protons of the methylene group of the bisphosphonate. The ³¹ P-NMR spectrum of the Pt-BP in D2O shows a singlet with unresolved Pt satellites positioned at 37.60 ppm. This singlet can be assigned to the two phosphorus atoms of the phosphonic groups, which are magnetically equivalent and shifted at lower field (Dd = 21 ppm) with respect to the free 2-amino-1-hydroxyethane-1 ,1-diyl-bisphosphonic acid (AHBP) ligand at the same pH (40).

Example 7: Phenotypic assessment of drug-treated zebrafish embryos 48 hpf wildtype zebrafish embryos were separated into 6 groups (n=20 embryos/group), i.e. , control (untreated) and embryos treated with 5 mM, 10 pM, 50 pM, and 100 pM Pt-BP as well as 30 pM cisplatin. Both types of compounds were added externally to the medium and the embryos were incubated at 33 °C for the duration of the experiment. The embryos were assessed each day for phenotypic toxicity until 2 days post treatment (48 hpt) and imaged live in an Olympus MVX10 microscope.

Fig. 4. In vivo phenotypic effects of Pt-BP in zebrafish embryos. Representative images of embryos treated with different concentrations of Pt-BP. Until 2 days post treatment (dpt), Pt- BP-treated embryos did not show any phenotypic abnormality in comparison to untreated and cisplatin-treated embryos. Scale bar: 50 pm.

Example 8: Ototoxicity assay

The lateral line neuromast hair cells in embryos were stained with a vital dye to analyse the loss of hair cells after Pt-BP treatment. Cisplatin was used as a control, as it was known to affect hair cells in humans. The fluorescent dye 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI) [Molecular Probes, Eugene, OR] was used to stain hair cells within neuromasts as described previously (75). Larvae were incubated in embryo medium containing 0.005 % DASPEI for 15 min, anesthetized in Tricaine MS222 (10 Ig/ml) for 5 min, rinsed once in fresh embryo medium, and imaged using Vertebrate automated screening technology (VAST, Union Biometrica), combined with high-speed and super-resolution Zeiss cell observer spinning disk confocal system. The quantification of number of neuromast hair cells in the treated embryos was done manually with confocal microscopy (Zeiss LSM780). Results in Fig 5: Ototoxicity caused by Pt-BP treatment of zebrafish embryos. (A) Embryos were treated with Pt-BP (5, 10, and 50 pM) and cisplatin (positive control, 30 pM) and lateral line neuromasts were stained using DASPEI. (B) Quantification of the lateral line neuromast cells stained by DASPEI after 48 h Pt-BP and Cisplatin treatments. ***P < 0.001; ****p < 0.0001 by one-way ANOVA, followed by Dunnett’s method for multiple comparisons. Scale bar: 50 pm.

Example 9: Pt release from Pt-BP at physiological and acidic, tumor-mimicking pH

In vitro investigations on the stability of Pt-BP showed that the complex is very stable in aqueous solution (pH 7.0, room temperature) for several months (42), while structural changes became apparent at pH > 8.0 with the formation of new products having inequivalent phosphorous atoms (as revealed by ³¹ P NMR spectroscopy). However, no release of free bisphosphonate was observed, indicating that Pt-BP rearranges rather than decomposes in water at pH > 8.0.

To investigate the stability of Pt-BP in near-physiological conditions, we utilized a medium consisting of D ₂ 0 in HEPES buffer (50 mM, pH = 7.4) and NaCI (120 mM) at 37 °C, in which we analyzed Pt-BP stability by ³¹ P-NMR spectroscopy. Fig. 6B shows that at pD = 7.4 and 37 °C, Pt-BP underwent rapid hydrolysis leading to formation of monomeric Pt-n-BP derivative M with the release of a single Pt(en) moiety. This observation was confirmed by the decrease in intensity of the Pt-BP signal in the ³¹ P spectrum (36.40 ppm; indicated with Pt-BP in Fig. 6B) with simultaneous formation of a singlet at 23.6 ppm (M in Fig. 6B; assigned to a symmetric monomeric Pt-n-BP derivative). No traces of Pt-BP were observed after 24 h due to formation of new species as shown in Fig. 6B and 6D such as monomeric Pt-n-BP (M). Finally, the second Pt(en) moiety was released only after 96 h as confirmed by the appearance of free BP (singlet at 17.10 ppm, indicated BP in Fig. 6B).

To simulate the acidic environment in the vicinity of cancer tissue, we explored the stability of Pt-BP complexes at pD = 5.25 at 37 °C by ³¹ P-NMR spectroscopy (Fig. 6C). In contrast to our observations at pD 7.4, hydrolysis of Pt-BP leading to formation of monomeric Pt-n-BP derivative (M in Fig. 6C) was slower at acidic pD. However, at pD 5.25, hydrolysis of the monomeric Pt-n-BP derivative with concomitant formation of free bisphosphonate (BP) was faster than at pD 7.4 since Pt-BP and BP were detected after 48 h (Fig. 6C). Thus, fast release of a single Pt(en) moiety Pt-BP was observed at pD 7.4 within 24 h, whereas faster release of both Pt(en) moieties were observed at pD 5.5 after 48 h.

Example 10: In vivo biodistribution of cold Pt-species and quantification of Pt accumulation The Pt-BP and Pt(NC>3)2(en) complexes were administered intravenously in the tail vein of C57BL/6N mice (n = 5 per each platinum complex). Sterile saline solution (0.9% NaCI) was used to dissolve platinum complexes. The concentration of injected Pt-BP or Pt(NC>3)2(en) solutions was 2.5 mM platinum, calculated based on the maximum tolerated dose (MTD) for cisplatin of 6 mg per kg body weight of the mice (52). The mice were euthanized with CO224 h after injection, after which blood (approximately 600 mg per mouse), liver, spleen, kidneys, heart, lungs, and bones (femur, humerus, tibia, and spine) were harvested. Approximately half of the tissues were prepared for inductively coupled plasma-mass spectrometry (ICP-MS) analysis by digestion in 65% (v/v) nitric acid at 75 °C for approximately three days until the tissues were digested completely. Each tissue was cut into three samples and the samples were weighed prior to digestion in nitric acid. The digested solutions were diluted with up to 6 ml of Milli-Q water to obtain 2% (v/v) nitric acid in order to measure platinum concentrations by ICP-MS (X series I, Thermo Electron Corporation). The detection limit for determining platinum concentration with ICP-MS was 1 ppb. The standard solutions were prepared from 1000 mgT ¹ platinum ICP standard Certipur ( 1.70341.0100, Merck) ranging from 1 ppb to 2500 ppb. The measured isotopes for platinum were ¹⁹⁴ Pt, ¹⁹⁵ Pt, ¹⁹⁶ Pt, and ¹⁹⁸ Pt. Furthermore, scandium ( ⁴⁵ Sc) was added as an internal standard, which was prepared with 1000 mgT ¹ scandium standard Certipur (1.19513.0100, Merck) to correct for matrix effects and long-term fluctuations of the measurement signal. The platinum concentrations in the various tissues were calculated relative to 1 mg of the specific tissue and represented as ng Pt/mg _tissue . Three replicates of Pt-BP and Pt(NC>3)2(en) solutions with volumes and platinum concentrations identical to the solutions injected to the mice (200 pi and 2.5 mM platinum, respectively) were also measured for their platinum content using ICP-MS as control measurements. The percentage of injected dose (%ID) of Pt-BP and Pt(NC>3)2(en) in each mouse tissue 24 h after injection was calculated by dividing the total amount of platinum per tissue through the total amount of platinum detected in the control solutions.

Example 11: Pt-DNA adducts quantification using High Resolution ICP-MS The remaining half of the tissues from all mice were dissected and weighed for DNA extraction. DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen, USA) for soft tissue and ChargeSwitch® gDNA Plant Kit (Thermofisher, USA) for bone samples. The DNA was digested with DNase I Solution (Thermofisher, USA) and filtered using 0.2 pm acrodisc GHP before analysis for Pt content using high resolution-ICP-MS (Element2, Thermo Finnigan).

The detection limit for determining the platinum concentration was 2 ng/l. The standard solutions were prepared from 1000 mg·! ¹ platinum ICP standard Certipur ( 1.70341.0100, Merck) ranging from 5 ppt to 10 ppb. The measured isotopes for platinum were ¹⁹⁴ Pt and ¹⁹⁵ Pt. Furthermore, thallium (Tl) was added as an internal standard and measured at a molecular mass of 205 Da, which was prepared using a 1000 mg·! ¹ thallium standard (CGTL- 1 , Inorganic Ventures) to correct for matrix effects and long-term fluctuations of the measurement signal. The Pt-DNA adduct concentration is represented as percentage of Pt involved in adduct formation to the total amount of Pt accumulated in the specific tissue.

Example 12: In vivo biodistribution of radioactive Pt-species and micro-SPECT quantification of ^195m pt biodistribution

The ^195m pt-BP and ^195m Pt(N0 ₃ ) ₂ (en) complexes were reconstituted in sterile saline solution (0.9% NaCI) and administered intravenously in the tail vein of C57BL/6N mice (n = 5 per each platinum complex). All mice received an intraveous injection with a dose of approximately 10 MBq ^195m pt. Immediately after injection and 1, 3, and 7 days after injection, images were acquired with U-SPECT-ll/CT(MILabs) (76). Mice were scanned under general anesthesia (isoflurane/02) for 15 to 60 minutes using the 1.0-mm diameter pinhole mouse high sensitivity collimator tube, followed by a CT scan (spatial resolution 160 mm, 65 kV, 615 mA) for anatomical reference. Scans were reconstructed with Ml Labs reconstruction software using an ordered-subset expectation maximization algorithm, with a voxel size of 0.4 mm. SPECT/CT scans were analyzed and maximum intensity projections (MIP) were created using the Inveon Research Workplace software (IRW, version 4.1). A 3D volume of interest was drawn using CT threshold to differentiate soft tissue from skeletal tissue and uptake was quantified as the percentage injected dose per gram (%l D/g), assuming a tissue density of 1 g/cm ³ . The hot spot in the skeletal tissue region of interest (ROI) was chosen with the location of the edge of the ROI contour representing 75% of maximum intensity. The mice from ^i95m pt(N0 ₃ )2(en) group were euthanized with CO2 after 3 days due to excessive loss of body weight, whereas mice from treated with ^195m pt-BP were sacrificed at the end of experiments. After sacrifice, blood (approximately 600 mg per mouse), liver, spleen, kidneys, heart, lungs, and bones (femur, humerus, tibia, and spine) were harvested and analyzed with a gamma counter (1470 Wizard, Perkin Elmer).

Fig. 3. ^195m Pt radioactivity in mice. Percentage of injected dose (%ID) of ^195m pt-BP and ^i95m pt(N0 ₃ ) ₂ (en) in mice, measured using an ionization chamber. ** P < 0.01; ****P < 0.0001 as determined by two-way ANOVA with a Bonferroni (multiple comparisons) post-hoc test.

Example 13: Laser ablation ICP MS imaging of ^195m pt biodistribution in mice tibia The tibias of mouse were incubated in neutral buffered formaldehyde for 36 h. Tibias were dehydrated in ascending grades of 70% to 100% ethanol and embedded in poly(methyl methacrylate) (pMMA) resin, freshly prepared by mixing 600 ml_ of methyl methacrylate monomer (Acros Organics BVBA, Geel, Belgium), 60 ml dibutyl phthalate (Merck KGaA, Darmstadt, Germany) and 1.25 g perkadox ^® 16 (Aldrich, Netherlands). The polymerization was followed by cutting serial horizontal sections (perpendicular to the tibia sample) of 5 pm thickness within the trabecular region of interest using an RM2155 microtome with a TC 65 blade (Leica Microsystems GmbH, Wetzlar, Germany). Microscopic images were obtained using an inverted fluorescence/ bright field microscope (BZ-9000, Keyence Deutschland GmbH, Neu-lsenburg, Germany). For image recording and processing, the software BZ-II Viewer and BZ-II Analyzer were used, respectively. To calculate the percentage of platinum co-localized with calcium, the background of the image and all pixels without hard bone tissue were excluded from the calculations. A pixel is considered to be background if its calcium intensity is below 15%. The platinum concentrations of the remaining pixels were then added up and divided by the sum of the entire platinum image to yield the percentage of platinum co-localized with calcium.

Example 14: Pt-BP favors bone-specific delivery of Ptwith reduced Pt-DNA adduct formation The bone-seeking properties of Pt-BP and its bisphosphonate-free precursor Pt(N0 ₃ Men) were evaluated by quantitatively analyzing Pt biodistribution using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) in different tissues (e.g. tibia, femur, humerus, spine, blood, heart, lung, kidney, liver, and spleen) as shown in Fig. 7A. To this end sterile saline solutions containing Pt-BP or Pt(N0 ₃ Men) with identical Pt concentrations were , intravenously injected into the tail vein of mice, which were sacrificed 24 h after injection. The harvested tissues were weighed and digested in acid for subsequent ICP-MS analysis of Pt content. Fig 7B shows the Pt concentrations in different tissues 24 h after injection, normalized for specific tissue weight. Pt-BP clearly showed bone-seeking properties as evidenced by higher amounts of Pt accumulation (~3 ng Pt/mg tissue) in hard tissue (bone) compared to BP-free control Pt(NC>3)2(en) complexes (~1 ng Pt/mg tissue).

Fig. 7. shows the biodistribution profile of Pt-BP and Pt(NC>3)2(en) in vivo. (A) Schematic representation of Pt biodistribution study in C57BI/6N mice. Pt-BP or Pt(NC>3)2(en) compounds were administered intravenously in mice (2.5 mM Pt concentration) followed by sacrifice after 24h. Hard tissues (tibia, femur, humerus, spine), soft tissues (heart, lungs, kidney, liver, spleen) and blood were collected. Collected tissues were subjected to nitric acid digestion and genomic DNA extraction for Pt quantification using ICP-MS and High-resolution ICP-MS, respectively. (B) Pt concentration in different tissues normalized for specific tissue weight 24 h after injection. Data from 5 mice per group are represented as ng Pt/mg tissue ± SD. (C) Percentage of Pt uptake in different tissues 24 h after injection. Data from 5 mice per group are represented as % I.D./g ± SD of specific tissue type. (D) Percentage of Pt-DNA adduct formation relative to total Pt uptake in specific tissue as quantified by High Resolution- ICP-MS. Data from 4-5 mice per group are presented. ** P < 0.01; ***P < 0.001; ****p < 0.0001 as determined by two-way ANOVA with a Bonferroni (multiple comparisons) post-hoc test.

The specificity of the two different Pt-species towards bone tissue relative to other soft tissues is depicted as the percentage of injected dose per gram of tissue (%l D/g). Higher uptake of the Pt-BP was observed in hard tissue (12.18 ± 0.56 %l D/g) compared to excretory organs such as the kidney (5.70 ± 0.15 %ID/g), liver (2.34 ± 0.17 %ID/g) and spleen (1.22 ± 0.12 %l D/g). On the other hand, Pt(N0 ₃ ) ₂ (en) showed higher uptake in kidney (3.38 ± 0.28 %l D/g) compared to hard tissue (2.69 ± 0.26 %ID/g), liver (1.54 ± 0.33 %l D/g) and spleen (0.38 ± 0.04 %l D/g). Overall, Pt-BP exhibit a 4.5-fold higher affinity for bone compared to Pt(N0 ₃ ) ₂ (en)).

The efficacy of Pt-based drugs for cancer treatment relates to the formation of Pt-DNA adducts, which hinder mitotic processes and halt cell division. We measured the extent of Pt- DNA adduct formation by extracting genomic DNA from different tissues and quantifying the amount of Pt using high-resolution ICP-MS. Fig. 7D shows the relative Pt uptake within specific tissues leading to Pt-DNA adduct formation 24h after injection. Pt-BP formed a low amount of Pt-DNA adducts in all tissues (<0.5%) except for the kidney (2.8%) and spleen (1.4%). In contrast, the bisphosphonate-free Pt complexes showed a much higher extent of Pt-DNA adduct formation, especially in the kidneys (4.8%) and spleen (9.8%).

Example 15: ^195m Pt-BP accumulates specifically in metabolicallv active bone Radioactive ^195m pt-BP was synthesized using ^195m Pt(NC>3)2(en) as the precursor for ^195m pt as reported above. To compare the biodistribution of ^195m pt-BP with the precursor ^i95m pt(N0 ₃ ) ₂ (en), a dose of approximately 10MBq ^195m Pt was administered intravenously via the tail vein in C57BL/6N mice. Though the administered Pt dose was below the Pt toxicity dose of 6 mg/Kg for mice (52), injection of ^195m Pt(NC>3)2(en) evoked a significant body weight loss within 3 days, leading to premature sacrifice of these animals which reached a humane end point. On the other hand, no behavioral or body changes were observed for ^195m pt-BP treated mice.

Micro-SPECT/CT was used to visualize ^195m Pt-BP uptake upon intravenous administration in a time window between 1 h to 7 days (Fig. 8A). The micro-SPECT/CT scans clearly demonstrated effective targeting of ^195m pt-BP to growth plates in long bones, whereas ^i95m pt(N0 ₃ ) ₂ (en) showed specific accumulation in soft tissues. Generally, growth plates are metabolically active regions in bones of young mice since they are responsible for longitudinal bone growth. After excision of tissues, we used a gamma counter to quantify uptake of radioactive ^195m pt-BP complexes (Fig. 8A). Bone-specific uptake of ^195m pt-BP was highest in the femur (3.1 ± 0.37%ID/g) followed by the tibia (2.86 ± 0.46%ID/g) and humerus (2.53 ± 0.33%ID/g), whereas ^19m Pt(NC>3)2(en) showed limited uptake in these bones (1.4 ±

0.15%l D/g). ^195m Pt uptake was highest in soft tissue for ^195m Pt(NC>3)2(en) with significantly higher uptake compared to ^195m Pt-BP in kidneys (3.95 ± 0.24%ID/g), liver (3.22 ± 0.28%ID/g), spleen (2.76 ± 0.67%ID/g), and lungs (1.08 ± 0.16%ID/g). Conversely, ^195m pt-BP showed relatively low uptake in soft tissues (< 0.11 % I D/g) , except for kidneys (0.43 ± 0.16%l D/g).

Fig. 8 shows a biodistribution profile of ^195m pt-BP and ^195m Pt(N03)2(en) in vivo.

Representative whole-body micro-SPECT/CT images of biodistribution of ^195m pt-BP (A) and ^195m Pt(N03)2(en) (B) in nude mice 24 hours after systemic administration. Rectangles show specific biodistribution of these compounds in long bones (femur-tibia) 24 h, 72 h and 168 h after systemic administration.

Next, we quantitatively assessed micro-SPECT/CT images for uptake of radioactive ^195m pt-BP complexes. ^195m pt-BP showed rapid and strong uptake in bone (1.8 ± 0.25%I.D/g) at 1 h, which remained constant (1.0 ± 0.12%l. D/g) from 24h until day 7. ^195m pt-BP uptake was significantly lower in soft tissue compared to hard tissue (0.93 ± 0.27%I.D/g at 1 h and 0.18 ± 0.03%I.D/g at 24h). In contrast, ^195m Pt(NC>3)2(en) showed equal uptake in both hard (1.5 ± 0.1 %l . D/g) and soft tissue (1.43 ± 0.21 %l. D/g) at 1 h, and the biodistribution remained unchanged until day 3 as shown in Fig. 9B. Subsequently, the quantification of the hot spots in bone represents a twofold increased uptake of ^195m pt for ^195m pt-BP compared to ^i95m pt(N0 ₃ ) ₂ (en) for all time points (Fig. 9C). The hard-to-soft tissue uptake ratio of ^195m pt-BP increased periodically from 3.3 at 1h, 6 at 24h and 6.7 after 72h. On the other hand, the hard- to-soft tissue uptake ratio of ^195m Pt(NC>3)2(en) remained almost constant at 1 at 1 h, 1.2 at 24h and 1.2 at 72h (Fig. 9D).

Fig. 9. Quantification of ^195m pt-BP and ^195m Pt(N03)2(en) biodistribution in vivo.

(A) Percentage of injected dose (%ID/g) of ^195m pt-BP (after 7 days) and ^195m Pt(N0 ₃ ) ₂ (en)

(after 3 days) in mice measured using gamma counting. (B) Percentage of injected dose (%ID/g) of ^195m pt-BP and ^195m Pt(N0 ₃ ) ₂ (en) in mice as quantified from the micro-SPECT/CT images in soft and hard tissues (C) Percentage of injected dose (%l D/g) of ^195m pt-BP and ^i95m pt(N0 ₃ ) ₂ (en) in mice as quantified from the micro-SPECT/CT images in the hard tissue region of interest (ROI) with the location of the edge of the ROI contour representing 75% of maximum intensity. (D) ^195m pt hard-to-soft tissue uptake ratio excluding bladder uptake at 1h. Data from 4-5 mice per group are presented. ** P < 0.01; ****P < 0.0001 as determined by two-way ANOVA with a Bonferroni (multiple comparisons) post-hoc test. For the ratios, paired t-test was used to determine the differences among the two groups where **P < 0.01 was considered as significantly different.

Example 16: Spatial co-localization of Pt with Ca in bone

Fig. 10. Spatial distribution of Pt in metabolically active bone. (A) Representative elemental mapping of calcium (Ca), phosphorus (P) and Pt in the proximal tibia of mice upon systemic administration of ^195m pt-BP and ^195m Pt(N03)2en. The bottom pictures shown an overlay of calcium (red) and platinum (green) mapping where co-localization of platinum and calcium is indicated in yellow. (B) Percentage of platinum co-localized with calcium in ^195m pt-BP and ^i95m pt(N0 ₃ ) ₂ (en) treated mice. ****P < 0.0001 , two-tailed student’s test. (C) Total amount of Pt co-localized with Ca (in hard tissue, Pt ^« Ca) and Pt not co-localized with Ca (in soft tissue, Pt ¹ Ca). Data are presented from 10 sections from three tibia per group. ****P < 0.0001 as determined by two-way ANOVA with a Bonferroni (multiple comparisons) post-hoc test.

The spatial distribution of Pt, Ca and P as determined using Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is depicted in Fig. 10. The calcium and phosphorus distribution (Fig. 10A) clearly conformed to the structure of trabecular bone as observed in the dark-colored features in the microscopic image (Fig. 10A). The overlay of Ca and Pt distinctly differentiates co-localization of Pt with Ca (yellow) from Pt surrounding the trabecular structure (green). ^195m pt-BP showed predominant Pt uptake along the trabecular bone and inner region of the cortical bone, as reflected by an increased density of Pt co localization with calcium. Conversely, ^195m Pt(N0 ₃ ) ₂ (en) showed the highest Pt density in the soft tissue surrounding the cortical bone.

To quantitatively examine the co-localization of Pt with Ca, we calculated the percentage of Pt co-localized with calcium. This calculation (Fig. 10B) shows a co-localization of Pt with calcium of 73.5% for ^195m pt-BP and only 37.3% for ^195m Pt(N0 ₃ ) ₂ (en). Relative to the extent of co-localization of Pt with Ca as observed for ^195m pt-BP, co-localization of ^195m Pt(NC>3)2(en) was only 27% (Fig. 10C). The total amount of Pt not co-localized with Ca refers to Pt in soft tissue of the proximal tibia where 10% and 20% of Pt was detected in soft tissue for ^195m Pt(NC>3)2(en) and ^195m pt-BP, respectively. ^195m pt-BP showed almost a fourfold increased accumulation of Pt in bone compared to ^195m Pt(NC>3)2(en) as shown in Fig 10C. These results confirm again that ^195m Pt-BP binds specifically to the bone and effectively penetrates the trabecular structure of mouse tibiae.

The inventors synthesized a Pt-BP compound comprising two ^195m pt moieties and a bone seeking bisphosphonate group to target the compound specifically to bone. This synthesis of Pt-BP (40, 42) was modified compared to previously reported synthesis strategies to facilitate introduction of radioactive ^195m pt by using ^195m Pt(NC>3)2(en) as Pt precursor. ^195m Pt(NC>3)2(en) is obtained from H ₂ [ ^195m PtCl ₆ ]-6H ₂ 0, which is the Pt precursor used for synthesis of radioactive cisplatin, thereby allowing for future upscaling and clinical translation of ^195m pt-BP (30).

Example 17: ^195m Pt-BP accumulates specifically in metastatic intratibial bone tumors in mice To compare the biodistribution of ^195m pt-BP with ^195m Pt-Cisplatin in an intratibial bone metastases model, mice bearing metastatic tibial bone tumors were randomly distributed for different treatments, namely intravenous administration of ^195m pt-BP, ^195m Pt-Cisplatin or a placebo/negative control (PBS). A dose of 9 ± 0.5 MBq ^195m pt-BP and a dose of 5 ± 0.3 MBq of ^195m Pt-Cisplatin was administered intravenously via the tail vein in C57BL/6N mice bearing metastatic tibial tumors. The administered Pt dose for ^195m Pt-Cisplatin was below the Pt toxicity dose of 6 mg/kg for mice, in order to prevent a significant body weight loss within the first 3 days post injection (p.i.). On the other hand, no behavioral or body changes were observed for ^195m pt-BP treated mice. Micro-SPECT/CT images were acquired 1 h - 7 days post intravenous administration of ^195m pt. (FIG 11) The micro-SPECT/CT images were quantitatively analyzed to determine the uptake of radioactive ^195m pt complexes in the metastatic tibial bone tumors(volume of interest, VOI) (Fig. 12). ^195m pt-BP showed rapid and strong uptake in the VOI (8.2 ± 1.7%I.D/g) at 1 h p.i., which reduced to 6.2 ± 1.5%I.D/g at 24 h and remained almost constant until day 7 (5.9 ± 1.1 %I.D/g). In comparison, ^195m Pt-Cisplatin exhibited reduced uptake in the VOI at all time points, with the highest value (3.7 ± 0.8%I.D/g) obtained at 1 h. In addition, ^195m pt-BP uptake was significantly higher in tumor-bearing tibias compared to control tibias as shown in Fig. 12B. ^195m pt uptake (%I.D/g) in VOI was normalized to ^195m pt uptake (%l . D/g) in control tibia within individual mice to determine bone tumor-selective uptake of ^195m pt complexes. ^195m pt-BP uptake in VOI was increased by factor of 2.8 and 2.3 at 1 h and 24 h, respectively. In contrast, ^195m Pt-Cisplatin showed equal uptake in both tibia at 1 h and 24 h. Fig. 12. Quantification of ^195m pt-BP and ^195m Pt-Cisplatin biodistribution in vivo in bone-tumor bearing mice. (A) Percentage of injected dose (%ID/g) of ^195m pt-BP and ^195m Pt-Cisplatin in mice as quantified from the micro-SPECT/CT images in metastatic tibial (volume of interest, VOI). (B) ^195m Pt tumor tibia-to-control tibia uptake ratio. Data from 3-5 mice per group are presented. ***P < 0.001 as determined by two-way ANOVA with a Bonferroni (multiple comparisons) post-hoc test. For the ratios, paired t-test was used to determine the differences among the two groups where *P < 0.01 was considered as significantly different.

Example 18: Therapeutic effects of ^195m pt accumulation in metastatic tibial bone tumors in mice

The entire tibia sections were stained with Elastine van Gieson (EvG) to distinguish hard and soft tissue region in tibia. The effect of ^195m pt on inducing DNA double-strand breaks was evaluated by immunohistochemical staining of g-H2AC molecules. Further, FragEL DNA fragmentation detection kit was employed to detect apoptosis. Counterstaining with methyl green was performed for immunohistochemical staining of g-H2AC and FragEL DNA fragmentation detection to aid in the morphological evaluation. The g-H2AC positive tumor cells and apoptotic tumor cells were observed as dark brown.

Fig. 13. Representative images of tumor region in bone metastases mice model.

Based on immunohistochemical staining of g-H2AC molecules, therapeutic effects of ^195m pt-BP on tumor tissue are visualized in Fig. 12 represented by high number of g-H2AC positive tumor cells confirming double-strand breaks in DNA for ^195m pt-BP treated group. Similar results were also observed regarding detection of apoptotic cells for the ^195m pt-BP treated group, which confirms the radiotherapeutic potential of ^195m pt-BP. The amount of Y-H2AX-positive tumor cells and apoptotic cells was lower for mice treated with ^195m Pt-Cisplatin.

Similar experiments are performed with Pt-pyrophosphate complexes and ^193m pt isotopes.

Example 19: Targeted ^195m pt accumulation in tibial lesion promotes radiation-induced double strand DNA breaks and induces apoptosis in metastatic tumor cells

Whole tibia sections treated with various types of Pt-based drugs were stained with hematoxylin and eosin (H&E) to differentiate between bone marrow (dark purple) and tumor regions (light purple or purplish-pink) (Fig. 14A-H). This histological analysis confirmed the presence of tumor cell mass within the bone marrow and surrounding tibial lesions. One specific lesion treated with ^195m Pt-BP showed the presence of a necrotic tumor region (Fig. 14D).

Immunohistochemical staining of g-H2AC molecules specific for double-strand DNA breaks confirmed DNA damage caused by either radiation or interstrand crosslinking of DNA by Pt-based drugs (Fig. 14M-P). Highest numbers of dark-stained Y-H2AX-positive tumor cells were observed upon ^195m pt-BP treatment (Fig. 14P). Moreover, apoptosis within the tumor region was examined by visualization of DNA fragments corresponding to apoptosis (Fig. 14Q- T). Similarly, the highest numbers of dark-stained apoptotic tumor cells within the tumor region were observed upon ^195m pt-BP treatment (Fig. 14T). Subsequently, we quantified the percentage of y-H2AX positive and apoptotic areas within the tumor regions. The y-H2AX- positive tumor cell area within the tumor region for mice treated with ^195m pt-BP (1.6 ± 0.4%) was 4.6-fold higher than ^195m Pt-cisplatin (0.3 ± 0.1%), 11-fold higher than radio-inactive Pt-BP (0.2 ± 0.1%) and 32-fold higher than saline control (0.05 ± 0.04%) (Fig. 14U). Similarly, the apoptotic tumor cell area within the tumor for mice treated with ^195m pt-BP (0.9 ± 0.5%) was 3- fold higher than treatment of mice with ^195m Pt-cisplatin (0.3 ± 0.1%), 3-fold higher than radio inactive Pt-BP (0.3 ± 0.2%) and higher than saline control (0.2 ± 0.02%). (Fig. 14V). Generally, ^195m Pt-BP treatment caused DNA damage and apoptosis in tumor cells more efficiently than all other treatment groups.

Fig. 14. Representative histochemical and immunohistochemical images of tibial lesions in a prostate cancer cell-induced bone metastasis model after treatment. (A-D) Representative overview of H&E stained tibial lesions. The rectangular box within each group (A-D) is magnified in (E-H). (BM: bone marrow stained as dark purple; T: tumor stained as light purple/purplish-pink; NR: necrotic region; B: bone stained as light pink). (M-P) Immunostaining of y-H2AX-positive tumor cells (dark brown) in the tibial lesions correspond to double-strand DNA breaks in tumor cells. (Q-T) Detection of apoptotic tumor cells (dark brown) in the tibial lesions based on DNA fragmentation. (U) Percentage of y-H2AX-positive tumor cell area relative to the total tumor area. (V) Percentage of apoptotic tumor cell area relative to the total tumor area. Scale bars correspond to pm (A-D) and 200 pm (E-T). H&E: hematoxylin and eosin. Data from 3-4 mice per group, 6-9 histological sections analyzed for each mouse. (*P £ 0.05; **P £ 0.01 ; ***P £ 0.001 ; ****p < 0.0001 ; one-way ANOVA with a T ukey posthoc test).

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