Pt(IV) antitumor prodrugs: dogmas, paradigms, and realities.

Platinum(II)-based drugs are widely used for the treatment of solid tumors, especially in combination protocols. Severe side effects and occurrence of resistance are the major limitations to their clinical use. To overcome these drawbacks, a plethora of Pt(IV) derivatives, acting as anticancer prodrugs, have been designed, synthesized and preclinically (often only in vitro) tested. Here, we summarize the recent progress in the development and understanding of the chemical properties and biochemical features of these Pt(IV) prodrugs, especially those containing bioactive molecules as axial ligands, acting as multi-functional agents. Even though no such prodrugs have been yet approved for clinical use, many show encouraging pharmacological profiles. Thus, a better understanding of their features is a promising approach towards improving the available Pt-based anticancer agents.

The Strange Case: The Unsymmetric Cisplatin-Based Pt(IV) Prodrug [Pt(CH3COO)Cl2(NH3)2(OH)] Exhibits Higher Cytotoxic Activity with respect to Its Symmetric Congeners due to Carrier-Mediated Cellular Uptake.

The biological behavior of the axially unsymmetric antitumor prodrug (OC-6-44)-acetatodiamminedichloridohydroxidoplatinum(IV), 2, was deeply investigated and compared with that of analogous symmetric Pt(IV) complexes, namely, dihydroxido 1 and diacetato 3, which have a similar structure. The complexes were tested on a panel of human tumor cell lines. Complex 2 showed an anomalous higher cytotoxicity (similar to that of cisplatin) with respect to their analogues 1 and 3. Their reduction potentials, reduction kinetics, lipophilicity, and membrane affinity are compared. Cellular uptake and DNA platination of Pt(IV) complexes were deeply investigated in the sensitive A2780 human ovarian cancer cell line and in the corresponding resistant A2780cisR subline. The unexpected activity of 2 appears to be related to its peculiar cellular accumulation and not to a different rate of reduction or a different efficacy in DNA platination and/or efficiency in apoptosis induction. Although the exact mechanism of cell uptake is not fully deciphered, a series of naïve experiments indicates an energy-dependent, carrier-mediated transport: the organic cation transporters (OCTs) are the likely proteins involved.

Unsymmetric Cisplatin-Based Pt(IV) Conjugates Containing a PARP-1 Inhibitor Pharmacophore Tested on Malignant Pleural Mesothelioma Cell Lines.

Cisplatin is widely employed as a first-line chemotherapeutic agent for many solid tumors, including malignant pleural mesothelioma (MPM). However, its clinical use is limited by heavy side effects and acquired resistance, the latter being mainly related to enhanced DNA repair. Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPis) have been carried out, with the hope that such combinations might lead to improved therapeutic efficacy against tumors. Here, the synthesis and efficacy in reducing MPM cell viability of four cisplatin-based Pt(IV) prodrugs containing the PARPi 3-aminobenzamide (3-ABA) fragment are described. The most promising conjugate is more effective than cisplatin or cisplatin/3-ABA combination, administered in equimolar doses, in inhibiting PARP-1 activity and inducing apoptosis in BRCA1/2 wild type MPM cells, grown as monolayer or as multicellular spheroids.

Can the Self-Assembling of Dicarboxylate Pt(IV) Prodrugs Influence Their Cell Uptake?

The possibility of spontaneous self-assembly of dicarboxylato Pt(IV) prodrugs and the consequences on their uptake in cancer cells have been evaluated in different aqueous solutions. Four Pt(IV) complexes, namely, (OC-6-33)-diacetatodiamminedichloridoplatinum(IV), Ace, (OC-6-33)-diamminedibutanoatodichloridoplatinum(IV), But, (OC-6-33)-diamminedichloridodihexanoatoplatinum(IV), Hex, and (OC-6-33)-diamminedichloridodioctanoatoplatinum(IV), Oct, have been dispersed in i) milliQ water, ii) phosphate buffered saline, and iii) complete cell culture media (RPMI 1640 or DMEM) containing fetal bovine serum (FBS). The samples have been analyzed by dynamic light scattering (DLS) to measure the size and distribution of the nanoparticles possibly present. The zeta potential offered an indication of the stability of the resulting aggregates. In the case of the most lipophilic compounds of the series, namely, Oct and to a lesser extent Hex, the formation of nanosized aggregates has been observed, in particular at the highest concentration tested (10 µM). The cell culture media had the effect to disaggregate these nanoparticles, mainly by virtue of their albumin content, able to interact with the organic chains via noncovalent (hydrophobic) interactions. For Oct, at the highest concentration employed for the uptake tests (10 µM), the combination between passive diffusion and endocytosis of the self-assembled nanoparticles makes the cellular uptake higher than in the presence of passive diffusion only. During the study of cellular uptake on A2780 ovarian cancer cells pretreated with cytochalasin D, a statistically significant inhibition of endocytosis was observed for Oct. In these experimental conditions, the relationship between uptake and lipophilicity becomes almost linear instead of exponential. Since Oct anticancer prodrug is active at nanomolar concentrations, where the aggregation in culture media is almost abolished, this phenomenon should not significantly impact its antiproliferative activity.

Cis,cis,trans-[PtIVCl2(NH3)2(perillato)2], a dual-action prodrug with excellent cytotoxic and antimetastatic activity.

Two Pt(iv) conjugates containing one or two molecules of perillic acid (4-isopropenylcyclohexene-1-carboxylic acid), an active metabolite of limonene, were synthesized both with traditional and microwave-assisted methods and characterized. Their antiproliferative activity was tested on a panel of human tumor cell lines. In particular, cis,cis,trans-[PtIVCl2(NH3)2(perillato)2] exhibited excellent antiproliferative and antimetastatic activity on A-549 lung tumor cells at nanomolar concentrations. A number of in vitro biological tests were performed to decipher some aspects of its mechanism of action, including transwell migration and invasion as well as wound healing assay.

Can an Elusive Platinum(III) Oxidation State be Exposed in an Isolated Complex?

Platinum(IV) complexes are extensively studied for their activity against cancer cells as potential substitutes for the widely used platinum(II) drugs. PtIV complexes are kinetically inert and need to be reduced to PtII species to play their pharmacological action, thus acting as prodrugs. The mechanism of the reduction step inside the cell is however still largely unknown. Gas-phase activation of deprotonated platinum(IV) prodrugs was found to generate products in which platinum has a formal +3 oxidation state. IR multiple photon dissociation spectroscopy is thus used to obtain structural information helping to define the nature of both the platinum atom and the ligands. In particular, comparison of calculations at DFT, MP2 and CCSD levels with experimental results demonstrates that the localization of the radical is about equally shared between the dxz orbital of platinum and the pz of nitrogen on the amino group, the latter acting as a non-innocent ligand.

A multi-methodological inquiry of the behavior of cisplatin-based Pt(IV) derivatives in the presence of bioreductants with a focus on the isolated encounter complexes.

The study of Pt(IV) antitumor prodrugs able to circumvent some drawbacks of the conventional Pt(II) chemotherapeutics is the focus of a lot of attention. This paper reports a thorough study based on experimental methods (reduction kinetics, electrochemistry, tandem mass spectrometry and IR ion spectroscopy) and quantum-mechanical DFT calculations on the reduction mechanism of cisplatin-based Pt(IV) derivatives having two hydroxido (1), one hydroxido and one acetato (2), or two acetato ligands (3) in axial position. The biological reductants glutathione and ascorbic acid were taken into consideration. The presence of a hydroxido ligand resulted to play an important role in the chemical reduction with ascorbic acid, as verified by 15N-NMR kinetic analysis using 15N-enriched complexes. The reactivity trend (1 > 2 > 3) does not reflect the respective reduction peak potentials (1 < 2 < 3), an inverse relationship already documented in similar systems. Turning to a simplified environment, the Pt(IV) complexes associated with a single reductant molecule (corresponding to the encounter complex occurring along the reaction coordinate in bimolecular reactions in solution) were characterized by IR ion spectroscopy and sampled for their reactivity under collision-induced dissociation (CID) conditions. The complexes display a comparable reduction reactivity ordering as that observed in solution. DFT calculations of the free energy pathways for the observed fragmentation reactions provide theoretical support for the CID patterns and the mechanistic hypotheses on the reduction process are corroborated by the observed reaction paths. The bulk of these data offers a clue of the intricate pathways occurring in solution.Graphic abstract.

New Platinum-Based Prodrug Pt(IV)Ac-POA: Antitumour Effects in Rat C6 Glioblastoma Cells.

Gliomas are the most frequent primary tumours of the nervous system, characterised by high degree of malignancy, widespread invasion and high-rate proliferation. Cisplatin and analogue are currently employed in clinical trials as active chemotherapeutic agents for the systemic treatment of this type of malignancy. Despite therapy benefits, clinical use of these agents is hampered by severe side effects including neurotoxicity. Therefore, the aim of the present study was to analyse the effect of a new compound of platinum(IV) conjugate, named Pt(IV)Ac-POA, which can generate a synergistic antineoplastic action when released along with cisplatin, after a specific reduction reaction within tumour cells. To assess the effects of the novel compound on rat C6 glioma cells, cell cycle and cell death activation analyses were carried out using flow cytometry. Morphological changes and activation of different cell death pathways were evaluated by both transmission electron microscopy and immunofluorescence microscopy. Protein expression was investigated by western blotting analysis. The novel compound Pt(IV)Ac-POA, bearing as axial ligand (2-propynyl)octanoic acid (POA), which is a histone deacetylase inhibitor (HDACi), acts as a prodrug in tumour cells, inducing cell death through different pathways at a concentration lower than those tested for other platinum analogues. The current results showed that Pt(IV)Ac-POA could represent a promising improvement of Pt-based chemotherapy against gliomas, either inducing a chemosensitisation and reducing chemoresistance.

Antiproliferative Activity of Pt(IV) Conjugates Containing the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Ketoprofen and Naproxen †.

Cisplatin and several non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to act synergistically or at least additively on several tumor cell lines. Dual-action cisplatin-based Pt(IV) combos containing ketoprofen and naproxen offer good antiproliferative performance on a panel of human tumor cell lines, including a malignant pleural mesothelioma (MPM) one, a very chemoresistant tumor. The main reason of the increased activity relies on the enhanced lipophilicity of these Pt(IV) conjugates that in turn promotes increased cellular accumulation. A quick Pt(IV)→Pt(II) reduction generates the active cisplatin metabolite. The NSAID adjuvant action seems to be almost independent from cyclooxygenase-2 (COX-2) expression in the tumor cells under investigation (lung A-549, colon HT-29, HCT 116, SW480, ovarian A2780, and biphasic MPM MSTO-211H), but it seems to rely (at least in part) on the activation of the NSAID activated gene, NAG-1 (a member of the transforming growth factor beta, TGF-ß, superfamily), which has been suggested to be involved in NSAID antiproliferative activity.

Elusive Intermediates in the Breakdown Reactivity Patterns of Prodrug Platinum(IV) Complexes.

Kinetically inert platinum(IV) complexes are receiving growing attention as promising candidates in the effort to develop safe and valid alternatives to classical square-planar Pt(II) complexes currently used in antineoplastic therapy. Their antiproliferative activity requires intracellular Pt(IV)-Pt(II) reduction (activation by reduction). In the present work, a set of five Pt(IV) complexes has been assayed using mass spectrometry-based techniques, i.e., collision-induced dissociation (CID), and IR multiple photon dissociation (IRMPD) spectroscopy, together with ab initio theoretical investigations. Breakdown and reduction mechanisms are observed that lead to Pt(II) species. Evidence is found for typically transient Pt(III) intermediates along the dissociation paths of isolated, negatively charged (electron-rich) Pt(IV) prodrug complexes.

Pt(IV) Bifunctional Prodrug Containing 2-(2-Propynyl)octanoato Axial Ligand: Induction of Immunogenic Cell Death on Colon Cancer.

The synthesis, characterization, and in vitro activity of a cyclohexane-1 R,2 R-diamine-based Pt(IV) derivative containing the histone deacetylase inhibitor rac-2-(2-propynyl)octanoato, namely, ( OC-6-44)-acetatodichlorido(cyclohexane-1 R,2 R-diamine)( rac-2-(2-propynyl)octanoato)platinum(IV), are reported together with those of its isomers containing enantiomerically enriched axial ligands. These Pt(IV) complexes showed comparable activity, of 2 orders of magnitude higher than reference drug oxaliplatin on three human (HCT 116, SW480, and HT-29) and one mouse (CT26) colon cancer cell lines. In vivo experiments were carried out on immunocompetent BALB/c mice bearing the same syngeneic tumor. The complex ( OC-6-44)-acetatodichlorido(cyclohexane-1 R,2 R-diamine)( rac-2-(2-propynyl)octanoato)platinum(IV) showed higher tumor mass Pt accumulation than oxaliplatin, due to its higher lipophilicity, with negligible nephro- and hepatotoxicities when administered intravenously. A remarkable tumor mass invasion by cytotoxic CD8+ T lymphocytes, following the Pt(IV) treatment, indicated a strong induction of immunogenic cell death.

Wine evolution during bottle aging, studied by 1H NMR spectroscopy and multivariate statistical analysis.

The study of wine evolution during bottle aging is an important aspect of wine quality. Ten different red wines (Vitis vinifera) from Piedmont region were analysed 3 months after bottling and after a further 48 month conservation in a climate controlled wine cellar kept at a constant/controlled temperature of 12 °C. Two white wines (Vitis vinifera) were included in this study for comparison purposes. White wines were analysed 3 months after bottling and after further 24 months of bottle aging in the same climate controlled wine cellar. Metabolite changes during this period were evaluated using 1H NMR spectroscopy combined with statistical analysis. Metabolite variations due to wine aging were minimal compared to those that resulted from a different wine type and wine geographical origin. Therefore, it was necessary to remove this source of variability to discriminate between fresh and refined samples. The storage at low and controlled temperature for 2 or 4 years permitted a slow but progressive evolution of all wines under investigation. 1H NMR spectroscopy, implemented with statistical data analysis, allowed identifying and differentiating wine samples from the two aging stages. In most wines, a decrease in organic acids (lactic acid, succinic acid and tartaric acid) and an increase in esters (ethyl acetate and ethyl lactate) was observed. Catechin and epicatechin decreased during aging in all wines while gallic acid increased in almost all red wines.

Hybrid inorganic (nonporous silica)/organic (alginate) core-shell platform for targeting a cisplatin-based Pt(IV) anticancer prodrug.

Nonporous silica nanoparticles with an external shell containing the 3­aminopropyl arm (SiNP) were further decorated with alginic acid (SiNP-ALG) as a potential biocompatible delivery system for Pt antitumor agents. Such particles were coupled with the prodrug (OC­6­44)­acetato(ß­alaninato)diamminedichloridoplatinum(IV), 1, through the formation of amide bonds between the pendant carboxylate groups on SiNP-ALG and the free amino group of the complex. Cytosol extracted from tumor cells was able to quickly and efficiently reduce the Pt(IV) prodrug, and produces the active metabolite cisplatin. SiNP-ALG-Pt conjugate was more active than both cisplatin and 1, due to its more efficient cell uptake, whereas the SiNP-ALG unplatinated nanoparticles were deprived of any nonspecific toxicity.

Isolation and characterization of a photosystem II preparation from thylakoid membranes of the extreme halophyte Salicornia veneta Pignatti et Lausi.

Salicornia veneta (Pignatti et Lausi) is an extreme halophyte living in salt marsh where NaCl concentration may be as high as 1 M. Here we report on the isolation and characterization of a PSII preparation obtained by Triton X-100 solubilisation of the thylakoid membrane. By a combination of gel electrophoresis, immunoblotting and mass spectrometry, the depletion of a number of PSII proteins such as PsbQ, PsbM and PsbT was highlighted. Moreover, the requirement of Cl- and Ca2+ for optimal oxygen evolution was determined, showing that in absence of PsbQ a higher level of these ions are required. At high Cl- concentrations, oxygen evolution was inhibited in the same way in Salicornia veneta and spinach. Reconstitution of Salicornia veneta PSII preparation with partially purified spinach PsbP and PsbQ restored oxygen evolution activity at low Cl- and Ca2+ concentrations. Adaptation to high salt makes several PSII proteins dispensable.

A new platinum-based prodrug candidate: Its anticancer effects in B50 neuroblastoma rat cells.

AIMS: Neuroblastoma is a rare cancer that affects children, mostly under the age of 5. This type of cancer starts in very early forms of immature nerve cells or developing cells found in embryo or fetus. To date cisplatin represents one of the most potent antitumor agent known, however, the onset of systemic side effects and the induction of drug resistance limit its use in the clinic for long-term treatment. In the present study we have analysed the effects of a new compound of platinum(IV) conjugates, named Pt(IV)Ac-POA, which is able to generate a synergistic antineoplastic action when released along with cisplatin upon intracellular Pt(IV) → Pt(II) reduction. MAIN METHODS: To assess the growth inhibition of the compounds under investigation, a cell viability test, i.e. the resazurin reduction assay was used on the B50 neuroblastoma rat cells. Further analysis on the cell cycle and metabolic alterations were carried out through flow cytometry. Morphological changes and activation of different cell death pathways after treatment, were observed at transmission electron microscope and by immunocytochemistry at fluorescence microscopy. Protein expression was examined by western blot analysis. KEY FINDINGS: This compound bearing bioactive axial ligand, such as the active histone deacetylase inhibitor (HDACi) (2-propynyl)octanoic acid (POA), induced cell death through different pathways at a concentration ten times lower than cisplatin. SIGNIFICANCE: The results showed that Pt(IV)Ac-POA could represent a promising improvement of Pt-based chemotherapy against neuroblastoma.

The cisplatin-based Pt(iv)-diclorofibrato multi-action anticancer prodrug exhibits excellent performances also under hypoxic conditions.

Multi-action cisplatin-based mono- (1) and di-clofibric acid (2) Pt(iv) "combo" derivatives were synthesized via both traditional and microwave assisted procedures. The two complexes offered very good performances (IC50 values in a nanomolar range) on a panel of human tumor cell lines, including the highly chemoresistant malignant pleural mesothelioma ones. Moreover, both 1 and 2 bypass the cisplatin resistance. Indeed, cisplatin and clofibric acid, the metabolites of the Pt(iv) → Pt(ii) intracellular reduction, proved to act synergistically. The adjuvant action of clofibric acid relies on the activation of peroxisome proliferator-activated receptor α (PPARα) that, in turn, decreases the level of Hypoxia-Inducible Factor-1α. Both compounds induced extensive apoptosis in tumor cells, also via oxidative stress. Finally, 2 exhibited excellent performances also under the hypoxic conditions typical of solid tumors, where cisplatin is less effective.

JQ1, a BET Inhibitor, Synergizes with Cisplatin and Induces Apoptosis in Highly Chemoresistant Malignant Pleural Mesothelioma Cells.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an asbestos-associated tumor with poor prognosis and few therapeutic options. JQ1, a selective antagonist of BRD4, modulates transcription of oncogenes, including MPM chemoresistance-associated c-Myc and Fra-1. OBJECTIVE: We investigated if JQ1 could enhance the efficacy of cisplatin against MPM. METHODS: The antiproliferative activity of cisplatin in combination with JQ1 was assessed on MPM cell lines representative of the cellular phenotypes of this tumor (epithelioid, sarcomatoid and biphasic), and on one cisplatin resistant sub-line. The combination schedule was optimized adopting a 3Dspheroid model. Drug combination effects were correlated with cell cycle distribution and senescence- associated ß-galactosidase positive cells. The expression of c-Myc and Fra-1 proteins and some apoptosis markers was assessed by immunoblotting and RT-qPCR. DNA damage and repair were evaluated by means of alkaline comet assay. RESULTS: JQ1 in combination with cisplatin elicited additive or synergistic (superadditive) antiproliferative effects on MPM cells, depending on the cell line. The combination showed tumor regression on the 3D-spheroid model. It induced increased apoptosis, along with decreased c-Myc and, sometimes, Fra-1 expression. JQ1 decreased cisplatin-induced DNA breaks in all MPM cells and increased senescence even in less proficient cells, thus enhancing the DNA Damage Response (DDR). CONCLUSION: The superadditive effect is due to c-Myc repression. The consequent DDR enhancement triggers to apoptosis induction and/or permanent growth arrest (senescence), depending on the MPM cellular context, leading to tumor regression. Thus, the pharmacological modulation of BET activity could represent a promising tool for future MPM therapy.

An unsymmetric cisplatin-based Pt(iv) derivative containing 2-(2-propynyl)octanoate: a very efficient multi-action antitumor prodrug candidate.

The design, synthesis, characterization and biological properties of a Pt(iv) complex containing the very active inhibitor of histone deacetylase (2-propynyl)octanoic acid, POA, as an axial ligand are reported here. The title complex, namely (OC-6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(iv), 1, containing POA in racemic or in enantiomeric forms, was one/two orders of magnitude more active than cisplatin, depending on the chemo-sensitivity of the cancer cell lines. Moreover, 1 exhibited similar or even better antiproliferative activity than (OC-6-33)-diamminedichloridobis(2-propylpentanoato)platinum(iv), 2, containing two molecules of the well-known histone deacetylase inhibitor 2-propylpentanoic (valproic) acid. The high potency of 1 is likely due to its high cellular accumulation and to the synergism between the DNA-damaging cisplatin and the histone deacetylase inhibitor POA, both released upon the intracellular reduction of 1. Prodrug 1, after oral administration, caused an impressive reduction of the tumor mass (94%) in a model of solid tumor (murine Lewis lung carcinoma), compared to that of the control, whereas (intraperitoneal) cisplatin induced a tumor regression of 75% only. A good accumulation of 1 was observed in the tumor mass. The time course of the body weight attested that cisplatin induced elevated anorexia, whereas treatment with 1 did not induce significant body weight loss throughout the therapeutic experiment.