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We recently showed that the treatment with Resveratrol (RES) contrasts the effects of ageing on the skeletal muscle (SKM), reduces the appearance of tubular aggregates (TAs), and improves the fatigue resistance. Since fatigue resistance depends on the SKM capillary network, and RES has been described to improve vascularisation, we analysed the SKM capillarization in naturally age- ing C57BL/6J male mice, fed with 0.04% RES in the diet for 6months, which showed a better fatigue resistance in a previous work. Our data show an inverse correlation between the number of capillaries per fibre (CAF) and TAs in both control and treated type IIB fibres, and an increase of CAF in ageing SKM upon RES-treatment. The present work suggests that capillarization is one of the determinants of the development of TAs and fatigue resistance, and that RES can be con- sidered a good candidate to counteract capillary rarefaction in the SKM tissue.

The expansion of metal-based complexes in the last 20 years has been very intense and many metals have been involved. Among the many compounds studied, the ruthenium-based complex NAMI-A embodies the unique paradigm of the ability to selectively inhibiting and preventing the development and the growth of distant metastases originating from solid tumors in all the tumor models on which it has been tested. An activity that can be detected only in vivo since the compound is virtually free of measurable direct cell cytotoxicity in vitro. Recently, a published paper reported on a significant in vitro cytotoxicity against some leukemic cells. The present study was undertaken to reproduce those experiments to further support this novel antileukemic activity that would have put NAMI-A on a new trajectory for development. Our results do not confirm the efficacy of NAMI-A in vitro against the human HL-60 promyelocytic leukemia cell line either using test cultures identical to those reported in the study of reference or in even more stressed conditions, supporting the lack of in vitro direct cell cytotoxicity of NAMI-A. The present study also helps to elucidate that many factors can influence the outcome of in vitro tests of cytotoxicity and suggests caution to speculate on possible therapeutic properties based on the results of simple and reductive in vitro tests of cytotoxicity.

The involvement of viruses and SARS-CoV-2 in autoimmune diseases is well known. The recent demonstration that ChAdOx1 nCoV-19 Covid-19 (AstraZeneca) vaccine (ChA) favors the production of anti-platelet factor 4 (anti-PF4) antibodies, blood clots, and thrombocytopenia raises the question of whether other anti-CoViD-19 vaccines favor the same patterns of events. We assessed the frequency of severe adverse events (SAEs) documented in the EudraVigilance European database up to April 16, 2021 related to thrombocytopenia, bleeding, and blood clots in recipients of ChA compared to that of recipients of the BNT162b2 Covid-19 (Pfizer/BioNTech) vaccine (BNT). ChA administration was associated with a much higher frequency of SAEs in each AE Reaction Group as compared with that elicited by BNT. When considering AEs caused by thrombocytopenia, bleeding and blood clots, we observed 33 and 151 SAEs/1 million doses in BNT and ChA recipients, respectively. When considering patients with AEs related to cerebral/splanchnic venous thrombosis, and/or thrombocytopenia, we documented 4 and 30 SAEs and 0.4 and 4.8 deaths/1 million doses for BNT and ChA recipients, respectively. The highest risk following ChA vaccination is in young people and, likely, women of reproductive age, as suggested by hypothesized scenarios. In conclusion, the immune reaction promoted by ChA vaccine may lead to not only thrombocytopenia and cerebral/splanchnic venous thrombosis but also other thrombotic and thromboembolic SAEs. These events are not favored by BNT vaccine. Our study may help in the evaluation of the benefit/risk profile of the ChA vaccine considering the epidemic curve present in a country.

The ChAdOx1 nCoV-19 (ChA) (AstraZeneca) and Ad26.COV2.S (AD26) (Janssen) vaccines are virus-based coronavirus disease 2019 (COVID-19) vaccines used worldwide. In spring 2021, venous blood clots and thrombocytopenia were described in some vaccine recipients. We evaluated the frequency of severe adverse events (SAEs) documented in the EudraVigilance European database in young adult (18-64 years old) and older (≥65 years old) vaccine recipients up to 23 June 2021 and related them to coagulation disorders and arterial, cardiac, and nervous system events. Comparison between the frequency of SAEs and SAE-related deaths in ChA and AD26 vs. BNT162b2 COVID-19 (BNT) (Pfizer/BioNTech) vaccine recipients demonstrated: 1) ChA and AD26 recipients than BNT recipients had higher frequencies of not only SAEs caused by venous blood clots and hemorrhage, but also thromboembolic disease and arterial events, including myocardial infarction and stroke; 2) a corresponding higher frequency of SAE-related deaths. The frequency was higher in both young adults and older adults. Comparison between the frequency of SAEs and SAE-related deaths in AD26 vs. ChA recipients demonstrated in AD26 recipients: 1) lower frequency of thrombocytopenia; 2) lower frequency of SAEs in young adult recipients; 3) higher frequency of SAEs in older recipients. Interestingly, most of the venous thrombotic SAEs associated with ChA and AD26 vaccines were not associated with thrombocytopenia, suggesting that TTS (thrombosis with thrombocytopenia syndrome) is not the only type of thrombosis observed following virus-based vaccines. In conclusion, both virus-based COVID-19 vaccines show more SAEs than BNT, but the frequency of the SAE type in the different age groups differs, suggesting that the mechanisms responsible of SAEs overlap only partly.

Skeletal muscle aging is accompanied by mass reduction and functional decline, as a result of multiple factors, such as protein expression, morphology of organelles, metabolic equilibria, and neural communication. Skeletal muscles are formed by multiple fibers that express different Myosin Heavy Chains (MyHCs) and have different metabolic properties and different blood supply, with the purpose to adapt their contraction to the functional need. The fine interplay between the different fibers composing a muscle and its architectural organization determine its functional properties. Immunohistochemical and histochemical analyses of the skeletal muscle tissue, besides evidencing morphological characteristics, allow for the precise determination of protein expression and metabolic properties, providing essential information at the single-fiber level. Aiming to gain further knowledge on the influence of aging on skeletal muscles, we investigated the expression of the MyHCs, the Succinate Dehydrogenase (SDH) activity, and the presence of capillaries and Tubular Aggregates (TAs) in the tibialis anterior muscles of physiologically aging C57BL/6J mice aged 8 (adult), 18 (middle aged), and 24 months (old). We observed an increase of type-IIB fast-contracting fibers, an increase of the oxidative capacity of type-IIX and -IIA fibers, a general decrease of the capillarization, and the onset of TAs in type-IIB fibers. These data suggest that aging entails a selective modification of the muscle fiber profiles.

The search for alternatives to antibiotics in aquaculture has focused on the use of vaccines for immune-prophylaxis. The purpose of this study was to examine the feasibility and characteristics of chitosan-alginate microparticles for the oral delivery of immune-prophylactics to finfish. The microparticles, which incorporate fluorescent-labelled lysozyme, were produced by spray-drying method; their structural properties and uptake from the gastrointestinal tract of Tilapia (Oreochromis niloticus) were assessed by microscopy. The main findings show that the microparticles are able to retain their content in an acidic environment and to release it later in slightly alkaline conditions such as those found in the intestines. Moreover, both the microencapsulation procedure and the biopolymers used have no deleterious impact on the lysozyme lytic activity, which is maintained after the protein has been released from the microparticles. Administered in vivo in Tilapia by medicated food, the microparticles transit unaffected through the stomach, and reach the anterior intestines, in particular the villum sectum and the basal lamina of epithelial cells, 2 and 4 h after feeding. Overall, the evidence obtained here supports the potential of these chitosan-alginate microparticles as agents for oral immune-prophylaxis in the management of fish diseases.

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-&alpha, /IL-1&beta, pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA&reg, identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

The oral administration of substances in crustaceans is a growing field of research, and is considered the most effective way to convey elements in their body instead injections or other invasive techniques. It presents huge potentialities, in fact the production of emulsions that preserves the substance to be conveyed until it reaches the target organ finds application in the development of new vaccines, in the convey of substances for aquaculture purposes and in the administration of stressors to contrast the spreading of invasive crustacean species. The red swamp crayfish ( Procambarus clarkii , Girard 1852) is one of the most spread invasive species worldwide, but it is also considered among the most economically important freshwater crustacean species. For these reasons it is considered as a paradigmatic species. The chemical synthesis of the D-Crustacean Hyperglycemic Hormone isoform (D-Phe-CHH) from P. clarkii is here presented, with its species-specificity, verified through the injection of D-Phe-CHH in both the red swamp crayfish and the threatened European species Austropotamobius italicus (Faxon, 1984). The D-Phe-CHH has been successfully oral-delivered in P. clarkii by using a water/oil/water microemulsion entrapped in a jellified matrix. After the administration of CHH, the glycaemia levels significantly increased in eyestalk-less animals at hour 6, and in intact crayfish at hour 5, from the ingestion. These encouraging findings pave the way for the use of the double emulsion method to oral administer different peptides to both handle physiological aspects of P. clarkii life cycle for aquaculture purposes and to create species-specific peptides-based mix to hamper the spreading of invasive crayfish populations. Statement of relevance Through the injection of the de novo synthetized D-Phe-Crustacean Hyperglycaemic Hormone isoform in the haemolymphatic circulation of P. clarkii , we evaluated its action in triggering hyperglycaemia. The peptide has been then successfully orally delivered in P. clarkii by w/o/w microemulsion and its bioactivity has been demonstrated. The formulated emulsion to convey peptides in crustaceans represents a very promising technique to modulate growth and reproduction in aquaculture plans.

NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration.

International audience; Targeting specific tumor metabolic needs represents an actively investigated therapeutic strategy to bypass tumor resistance mechanisms. In this study, we describe an original approach to impact the cancer metabolism by exploiting the redox properties of a ruthenium organometallic compound. This organometallic complex induced p53-independent cytotoxicity and reduced size and vascularization of patients-derived tumor explants that are resistant to platinum drugs. At the molecular level, the ruthenium complex altered redox enzyme activities and the intracellular redox state by increasing the NAD+/NADH ratio and ROS levels. Pathway analysis pointed to HIF-1 as a top deregulated metabolite pathway. Unlike cisplatin, treatment with the ruthenium complex decreased HIF1A protein levels and expression of HIF1A target genes. The rapid downregulation of HIF1A protein levels involved a direct interaction of the ruthenium compound with the redox enzyme PHD2, a HIF1A master regulator. HIF1A inhibition led to decreased angiogenesis in patient-derived xenografted using fragments of primary human colon tumors. Altogether, our results show that a ruthenium compound impacts metabolic pathways acting as anticancer agents in colon cancer via an original mechanism of action that affects redox enzymes differently than platinum-based drugs.

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5β1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5β1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5β1 modulates the expression/activity of another integrin, α2β1, involved in the cell adhesion to collagen I. These results suggest that α5β1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2β1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5β1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.

Clinical pharmacology (CP) is a multidisciplinary scientific field involving all aspects of the relationship between drugs and humans, encompassing professionals with a wide variety of skills including medicine, pharmacology, pharmacy, and biomedical science. The term Bclinical pharmacologist^ is commonly used in the professional sense to refer to medical doctors or PhD-level natural scientists/pharmacists with proven experience in improving patient care by developing new medicines and promoting the safer and more effective use of drugs. Clinical pharmacologists today should have experience even in various phases of drug development in basic and clinical research, regulatory agencies, and/or the pharmaceutical industry.

Platinum drugs are extensively used in the clinic to treat cancer, often leading to a palliative response rather than a cure. While DNA is considered to be the primary target of platinum drugs, there is no clear relationship between cellular platinum accumulation, DNA platination and Pt-DNA adduct removal, and herein we describe new mechanistic insights of platinum drugs related to the hallmarks of cancer and how they interfere with the tumour microenvironment. We then proceed to describe the properties of other metal drugs, including both non-targeted compounds that do not significantly interact with DNA and targeted compounds that interfere more selectively with specific pathways responsible for tumour growth and invasion. Our analysis of the cancer biology and the selected drugs allows us to propose possible routes for future drug development based on metal scaffolds. (C) 2018 Elsevier B.V. All rights reserved.

Tumours are not merely masses of abnormally proliferating cancer cells. Today, we have a clearer view of cancer complexity in which the participation of cancer and host cells leads to a tremendous heterogeneity of neoplastic diseases concerning the genetics, epigenetics, proteomics and biochemistry of the tumour [1]. Such intra-tumour heterogeneity provides the basis for inter-metastatic heterogeneity among different metastatic lesions of the same patient, each originating from a founder cell, or small group of cells, with a very different mutation kit, and likely originating from different and distinct primary tumour areas. This situation has important implications regarding chemotherapeutic sensitivity and responses. In addition, the offspring of the founder cell(s) can generate heterogeneity among the cells of an individual metastasis, affecting the response to systemic therapies and providing the seeds for drug resistance.

Cancer chemotherapy is almost always applied to patients with one or more diagnosed metastases and is expected to impact these lesions, thus providing significant benefits for the patient. The outcome of metastasis is determined by the interplay between the specific subpopulation of metastatic cells and host homeostatic factors in specific microenvironments. In clinical practice, metal-based drugs are represented by platinum compounds, which are constituents of a wide variety of chemotherapeutic regimens, that and are only rarely active against tumour metastases unless they are combined with drugs that target specific pathways characterizing the malignancy of the tested tumour. On experimental grounds, a number of complexes based on ruthenium and other metals have been frequently studied in vitro using models and experimental conditions mimicking one or more steps of the metastatic process, such as invasion and migration. The ruthenium-based drug NAMI-A, is the only one to have been subject to clinical testing for the treatment of metastatic tumours. The capacity of NAMI-A to modulate the relationships established between metastatic cells and their microenvironment suggests that metal-based drugs shall be viewed as an opportunity for the treatment of tumour metastases.

NAMI-A is a ruthenium-based drug endowed with the unique property of selectively targeting solid tumour metastases. Although two clinical studies had already been completed, limited information exists on the behavior of NAMI-A after injection into the bloodstream. PK data in humans informs us of a rather low free drug concentration, of a relatively high half-life time of elimination and of a linear relationship between the administered dose and the corresponding AUC for up to toxic doses. In the present study, we examined the chemical kinetics of albumin binding with or without the presence of reducing agents, and we evaluated how these chemical aspects might influence the in vivo PK and the in vitro ability of NAMI-A to inhibit cell migration, which is a bona fide, rapid and easy way to suggest anti-metastatic properties. The experimental data support the binding of NAMI-A to serum albumin. The reaction is facilitated when the drug is in its reduced form and, in agreement with already reported data, the adduct formed with albumin maintains the biological activity of the ruthenium drug. The formation of the adduct is favored by low ratios of NAMI-A : HSA and by the reduction of the drug with ascorbic acid. The difference in in vivo PK and the faster binding to albumin of the reduced NAMI-A seem to suggest that the drug is not rapidly reduced immediately upon injection, even at low doses. Most probably, cell and protein binding prevail over the reduction of the drug. This observation supports the thesis that the reduction of the drug before injection must be considered relevant for the pharmacological activity of NAMI-A against tumour metastases.

AIM OF THE STUDY: The tumor metastases targeting ruthenium complex NAMI-A synergistically improves the activity of gemcitabine in combination therapies. High-throughput screening was used to identify other potential drug combinations from a library of FDA approved drugs. Doxorubicin was identified as a hit compound and was therefore evaluated in combination with NAMI-A in vitro and in a preclinical in vivo model. RESULTS: High-throughput screening identified eight structurally diverse compounds that synergize with NAMI-A including doxorubicin. The combination index on MCF-7 cells showed synergism as the concentration of NAMI-A increases independent of the doxorubicin concentration. In MCa mammary carcinoma of CBA mice, NAMI-A (35 mg/kg/day i.p. on days 7-12) followed by doxorubicin (10 mg/kg i.p. on day 16), significantly increased the effects of the individual drugs on metastases with 70 % animals resulting free of macroscopically detectable tumor nodules in the lungs at sacrifice. NAMI-A, unlike doxorubicin, cured 60 % of the treated mice but the combination therapy was toxic to the animals. CONCLUSIONS: The combined therapy of NAMI-A with doxorubicin synergizes on lung metastasis in a preclinical mouse model. The combination therapy at the maximum tolerated doses of the two drugs is toxic. Hence, this combination is not suitable for clinical studies using maximum tolerated doses.

To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.

Angiogenesis is considered responsible for the growth of primary tumours and of their metastases. With the present study, the effects of three ruthenium compounds, potassiumchlorido (ethylendiamminotetraacetate)rutenate(III) (RuEDTA), sodium (bis-indazole)tetrachloro-ruthenate(III), Na[trans-RuCl4Ind2] (KP1339) and trans-imidazoledimethylsulphoxidetetrachloro-ruthenate (NAMI-A), are studied in vitro in models mimicking the angiogenic process. The ruthenium compounds reduced the production and the release of nitrosyls from either healthy macrophages and immortalized EA.hy926 endothelial cells. The effects of NAMI-A are qualitatively similar and sometimes quantitatively superior to those of RuEDTA and KP1339. NAMI-A reduces the production and release of nitric oxide (NO) by the EA.hy926 endothelial cells and correspondingly inhibits their invasive ability; it also strongly inhibits the angiogenesis in matrigel sponges implanted subcutaneously in healthy mice. Taken together, these data support the anti-angiogenic activity of the tested ruthenium compounds and they contribute to explain the selective activity of NAMI-A against solid tumour metastases, the tumour compartment on which angiogenesis is strongly involved. This anti-angiogenic effect may also contribute to the inhibition of the release of metastatic cells from the primary tumour. Investigations on the anti-angiogenic effects of NAMI-A at this level will increase knowledge of its pharmacological properties and it will give a further impulse to the development of this class of innovative metal-based drugs.

The study of metal complexes for the treatment of cancer diseases has resulted in the identification of some unique properties of ruthenium-based compounds. Among these inorganic-based agents, two of them, namely the ruthenium(III) drugs NAMI-A and KP1019 have undertaken with some success the clinical evaluations of phase I and preliminary phase II trials in patients. Here we highlight the strategies that have led to the discovery of metal-based (NAMI-A and KP1019) and of organometallic (RM175, RAPTA-T, RDC11 and DW1/2) ruthenium-based complexes, and we report their main biological/pharmacological characteristics and expectations for further development.

We have reported the synthesis and characterization of two new trans platinum complexes in the phosphane series, where the phosphane ligand is triphenylphosphine [P(Ph)3] and the group in the trans configuration is represented by chiral aliphatic amines, (racemic in complex 1 and S–NH2CH2CH(CH3)CH2CH3 in complex 2). The anti-proliferative activity detected in tumor cells treated with the two new complexes is more pronounced when the aliphatic amine is racemic compared to the S-enantiomer. Moreover, for both compounds, the activity is fast after cell exposure and, unlike that of cisplatin, virtually independent of the duration of cell challenge.

Both metal complexes and organic molecules are widely used for the treatment of various diseases including cancer - in addition to surgery and radiotherapy. Recent years have witnessed a surge of interest in the application of organometallic compounds to treat cancer and other diseases. Indeed, the unique properties of organometallic compounds, intermediate between those of classical inorganic and organic materials provide new opportunities in medicinal chemistry. In this review, based on the award lecture at ICBOMC'10, we describe a class of ruthenium(II)-arene complexes that are weakly cytotoxic in vitro, but show selective antimetastatic activity in vivo. These compounds, [Ru(eta(6)-p-arene)Cl-2(pta)] termed RAPTA, interact strongly with proteins, with the ability to discriminate binding to different proteins, but show a relatively low propensity to bind DNA, which is considered to be the main target of many metal-based drugs. The basic RAPTA structure is quite stable in physiological environments, and studies have shown that aquation of the chloride bonds occurs, it may not be an essential step for anticancer drug activity - direct substitution with biomolecular targets is also possible. Based on the favorable physicochemical properties of RAPTA compounds, combined with their highly promising pharmacological properties, the structure represents an ideal scaffold for rational drug design. Thus far, strategies to overcome drug resistance, by interference with critical enzymes responsible for drug deactivation, and tumor targeting, by tethering to human serum albumin via hydrolyzable linkers, have been demonstrated. However, many more approaches can be envisaged. In any case, the net result are a type of hybrid compounds, that occupy a niche somewhere between classical cisplatin-type anticancer agents that are widely applied to many tumor types and targeted therapies based on organic structures used to inhibit specific enzymes. As such, should these compounds prove themselves in the clinic it is not inconceivable that they could be rapidly refined to form personalized chemotherapies. (C) 2010 Elsevier B.V. All rights reserved.

We report here two novel "extended-arms" porphyrins, TetbpyPP and TedabpyPP, in which four peripheral bpy fragments are connected to the meso positions of the macrocycle through flexible linkers of different length and hydrophilicity. We describe also the new, water-soluble, tetracationic conjugate [TedabpyPP{Ru([9]aneS3)Cl}(4)][Cl](4) (6). Compound 6 belongs to the series of cationic Ru-porphyrin conjugates 1-5, each bearing four peripheral Ru(II) half-sandwich coordination compounds, that we recently prepared as potential photosensitizing chemotherapeutic agents. The in vitro cell growth inhibition of conjugates 1-6 toward MDA-MB-231 human breast cancer cells and HBL-100 human nontumorigenic epithelial cells are reported, together with the phototoxic effects of 1, 4, and 6 on MDA-MB-231 cells. All conjugates have IC(50) values in the low micromolar range that decrease by 1 order of magnitude upon irradiation of cell cultures with visible light. The most promising compounds 1 and 6 are phototoxic at low light and drug doses.

Gene expression profiling has been introduced into drug development to understand the activity of chemical entities in pre-clinical settings. The present study investigates the changes induced in gene expression by the ruthenium-based compound NAMI-A. The genes differentially expressed by NAMI-A are evaluated through whole-transcriptome analysis and RNA-sequencing in the metastatic MDA-MB-231 mammary carcinoma cells, in comparison to the non-tumorigenic HBL-100 mammary gland cells. NAMI-A treatment rapidly induces a relevant gene up-regulation that quickly returns to normal values. These changes differ between MDA-MB-231 and HBL-100 cells, highlighting the selectivity of the NAMI-A induced transcriptional perturbation in the invasive rather than in the non-tumorigenic cells. The transcriptional response, in the invasive MDA-MB-231 cells, comprises a set of early-response transcription factors and reveals a pharmacological signature in good agreement with the most peculiar NAMI-A behavior as a metastasis inhibitor such as cell cycle regulation and ECM remodeling. Globally, the results of this study indicate some transcription factors influencing the expression and activity of many downstream genes and proteins fundamentally involved in the functional effects of NAMI-A in vitro and in vivo.

The ruthenium-based drug imidazolium trans-imidazoledimethylsulphoxidetetrachlorido ruthenate (NAMI-A) is a novel antitumour drug under clinical evaluation. In this study, NAMI-A is tested on aortic rings in vitro and on the systolic blood pressure in vivo with the aim of evaluating its effects on smooth muscle cells and, more in general, on the vascular system. Pre-incubation of aortic rings with 10 µM NAMI-A for 10 min potentiates the contraction induced by phenylephrine (PE). The reduction of the B max value of [(3)H]-prazosin bound to NAMI-A-treated aortic rings and the ability of NAMI-A to displace [(3)H]-prazosin and [(3)H]-IP3 binding by 25 and 42%, respectively, suggest the involvement of α1-adrenoceptor in mediating the effects on smooth muscle cells. NAMI-A also decreases the number of maximal sites of [(3)H]-prazosin bound to kidney membrane preparation from 34 to 24 fmol/mg proteins. A single i.p. dose (105 mg/kg) or a repeated treatment for 6 consecutive days (17 mg/kg/day) in Wistar rats increases the systolic blood pressure, respectively, 1 h and 3 days after treatment, and the responsiveness of rat aortic rings to PE. Atomic absorption spectroscopy confirms the presence of ruthenium in the aortic rings excised from the treated rats. These findings suggest monitoring the cardiovascular parameters when the drug is used in humans for treating cancer patients, particularly if the drug is associated with chemicals that are potentially active at the cardiovascular level.

Ten complexes of general formula [Ru(η6-arene)Cl2(L)], [Ru(η6-arene)Cl(L)2][X], and [Ru(η6-arene)(L)3][X]2 (η6-arene = benzene, p-cymene; L = imidazole, benzimidazole, N-methylimidazole, N-butylimidazole, N-vinylimidazole, N-benzoylimidazole; X = Cl, BF4, BPh4) have been prepd. and characterized by spectroscopy. The structures of five representative compds. have been established in the solid state by single-crystal X-ray diffraction. All the new compds. were assessed by the same in vitro screening assays applied to [imidazole-H][trans-RuCl4(DMSO)(imidazole)] (NAMI-A) and [Ru(η6-arene)Cl2(1,3,5-triaza-7-phosphaadamantane)] (RAPTA) compds. It was found that the new compds. show essentially the same order of cytotoxicity as the RAPTA compds. toward cancer cells. Several of the compds. were selective toward cancer cells in that they were less (or not) cytotoxic toward nontumorigenic cells that are used to model healthy human cells. Thus, two of the compds., [Ru(η6-p-cymene)Cl(vinylimid)2][Cl] (vinylimid = N-vinylimidazole) and [Ru(η6-benzene)(mimid)3][BF4]2 (mimid = N-methylimidazole), have been selected for a more detailed in vivo evaluation.

All-trans retinoic acid (ATRA) represents the therapy of choice for patients with acute promyelocytic leukemia (APL). However, patients often relapse due to ATRA-resistance. The molecular basis of APL alterations indicates that addition of a histone deacetylase inhibitor to ATRA may restore the sensitivity to retinoids. We explored the in vitro and in vivo effects of a novel retinoic/butyric hyaluronan ester (HBR) on a retinoic acid (RA)-sensitive human myeloid cell line, NB4, and on its RA-resistant subclone, NB4.007/6. In vitro, HBR induced growth arrest and terminal differentiation in RA-sensitive NB4 cells (as confirmed by an increased expression of CD11 family members and nitroblue tetrazolium assay), whereas it inhibited the growth of RA-resistant cells by apoptosis, paralleled by an increase in the levels of caspase 3 and 7. In vivo, HBR treatment of NB4-inoculated severe combined immunodeficient mice resulted in a statistically significant increase in survival time (P

The coordination of the antimetastatic agent NAMI-A, [H(2)im][trans-RuCl(4)(dmso-S)(Him)], (Him=imidazole; dmso=dimethyl sulfoxide), to the DNA model base 9-methyladenine (9-MeAde) was investigated in water. NMR spectroscopy was first applied for the study of the molecular stability and hydrolysis of NAMI-A in aqueous solution over a range of pH (3.0-7.4) and chloride ion concentrations (0-1 M) at 37.0 degrees C. In physiological conditions (phosphate buffer, pH 7.4) NAMI-A disappears from the solution in 15 min due to chloride and dmso hydrolysis, leading to uncharacterised poly-oxo Ru species. Conversely, at lower pH (3.0-6.0) and in water (pH approximately 5.5), only a partial dmso hydrolysis occurs, slowly forming the [trans-RuCl(4)(H(2)O)(Him)](-) complex. This latter species coordinates to 9-MeAde (via the N7 of 9-MeAde), forming the [trans-RuCl(4)(9-MeAde)(Him)](-) complex. NAMI-A and [trans-RuCl(4)(H(2)O)(Him)](-) give comparable intracellular ruthenium concentrations and accumulate in KB cells (human mouth carcinoma) and accumulate these at the G(2)/M phase, while poly-oxo Ru species do not, and their cell uptake is reduced to 50%. On the contrary, G(2)/M arrest and protein content in the murine metastatic cell line metGM, are not influenced by NAMI-A hydrolysis. Hydrolysed NAMI-A species apparently are easier taken up by the metGM cells, showing intracellular ruthenium concentrations one order of magnitude greater than those of intact NAMI-A. Therefore, it is proposed that the selective antimetastatic activity of NAMI-A during in vivo experiments can be attributed to its hydrolysed species.

Ruthenium compounds have gained large interest for their potential application as chemotherapeutic agents, and in particular the complexes of the type (X)[trans-RuCl4(dmso-S)L] (X = HL or Na, NAMI-A or NAMI, respectively, for L = imidazole) are under investigation for their antimetastatic properties. The NAMI(-A)-like compounds are prodrugs that hydrolyze in vivo, and the investigation of their hydrolytic properties is therefore important for determining the nature of the potential active species. The NAMI-A-type Ru(III) complex 1, (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)] (dmtp is 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine), and the corresponding sodium analogue 2, (Na)[trans-RuCl4(dmso-S)(dmtp)], were synthesized. The hydrolyses of 1 and 2 in water as well as in buffered solutions were studied, and the first hydrolysis product, [mer-RuCl3(H2O)(dmso-S)(dmtp)]·H2O (3), was isolated and characterized. The molecular structures of 1 and 3 were determined by single-crystal X-ray diffraction analyses and prove the ...

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium-NO adducts was documented by electronic absorption, FT-IR spectroscopy and (1)H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-Br-cGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Imidazolium-trans-dimethylsulfoxideimidazoletetrachlororuthenate (NAMI-A) is a ruthenium compound effective on solid tumor metastases. In this study, we evaluated the effects of different routes of administration of NAMI-A on the distribution to primary tumor, lungs and kidneys in BD2F1 hybrids with Lewis lung carcinoma or in CBA inbred mice with MCa mammary carcinoma. NAMI-A concentration and the percentage of cumulative dose (%Dtot) retained in these tissues is independent of the animal strain and of the tumor model used. Also the presence of the tumor does not change the distribution of NAMI-A in the lungs and in the kidneys. A dose-dependent antimetastatic effect is evident with intraperitoneal (i.p.) treatments at three different doses. Treatment of tumor bearing mice with NAMI-A administered i.p., per os or by aerosol showed a similar effect on lung metastases, although the concentration of ruthenium reached in the lungs was markedly different. On the basis of the data obtained, we can conclude that the antimetastatic effects are related to the amount of NAMI-A administered, rather than to the lung's concentration of the compound.

Imidazolium trans-tetrachloro(dimethylsulfoxide)imidazoleruthenium(III) (NAMI-A) is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in the Netherlands. Pharmaceutical quality control of NAMI-A drug substance and lyophilized product warranted the development of an assay for determination and quantification of NAMI-A and degradation products. A high performance liquid chromatography (HPLC) method was developed, consisting of a C18 column with 0.50 mM sodium dodecylsulfate in 3% methanol at pH 2.5 (acidified using trifluoromethanesulfonic acid) as the mobile phase and UV-detection at 358 nm. The HPLC method was proven to be linear, accurate and precise. Stress testing showed that degradation products were separated from the parent compound. By combining results of nuclear magnetic resonance (NMR) and HPLC experiments, one degradation product was identified as the mono-hydroxy species of NAMI-A. HPLC analysis with off-line detection of the eluate with flameless atomic absorption spectrometry (F-AAS) showed that under most conditions, all ruthenium-containing compounds show a peak in the HPLC chromatogram and that all ruthenium applied to the column is recovered quantitatively. For completely degraded solutions of NAMI-A some ruthenium is retained on the column. Suitability of the HPLC method for the pharmaceutical quality control of NAMI-A lyophilized product was demonstrated.

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5β1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5β1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5β1 modulates the expression/activity of another integrin, α2β1, involved in the cell adhesion to collagen I. These results suggest that α5β1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2β1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5β1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

Two fullerene derivatives (fullerenes1and2), bearing a hydrophilic chain on the pyrrolidinic nitrogen, were developed with the aim to deliver anticancer agents to solid tumors. These two compounds showed a significantly different behaviour on human neoplastic cell linesin vitroin respect to healthy leukocytes. In particular, the pyrrolidinium ring on the fullerene carbon cage brings to a more active compound. In the present work, we describe the effects of these fullerenes on primary cultures of human monocytes and macrophages, two kinds of immune cells representing the first line of defence in the immune response to foreign materials. These compounds are not recognized by circulating monocytes while they get into macrophages. The evaluation of the pronecrotic or proapoptotic effects, analysed by means of analysis of the purinergic receptor P2X7 activation and of ROS scavenging activity, has allowed us to show that fullerene2, but not its analogue fullerene1, displays toxicity, even though at concentrations higher than those shown to be active on neoplastic cells.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

NAMI-A is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in The Netherlands. The photostability of this new agent in solid state and in solution has been investigated utilizing a stability-indicating reversed-phase high performance liquid chromatographic (HPLC) assay and ultraviolet/visible (UV/VIS) light spectrophotometry. In solid state, NAMI-A proved to be photostable. In solution, however, the compound degraded rapidly, in a pH-independent manner in the pH range of 2–5. At alkaline pH, the degradation rate was higher than at acidic pH. The type of buffer species had little influence. NAMI-A concentration was inversely related to the photostability. Addition of photostabilizers (5% DMSO, 2% benzyl alcohol, 0.001% curcumin) marginally increased the half-life. NAMI-A's photostability in solution was influenced to the greatest extent by addition of an alcohol, with the least polar solvent system (50% propylene glycol) providing the most stable medium. Based on the presented results, it is recommended to store NAMI-A solutions in the dark.

Nanoscale secondary ion mass spectrometry (NanoSIMS) combined with transmission electron microscopy (TEM) can be a powerful approach to visualize the exact distribution of drugs at the sub-cellular level. In this work, we exploit this approach to identify the distribution and localisation of the organometallic ruthenium(II)-arene drug Ru((6)-C6H5Me)(pta)Cl-2, termed RAPTA-T, in MDA-MB-231 and MCF-7 human breast cancer cells. These cell lines have been chosen because the former cell lines are highly invasive and resistant to most chemotherapeutic agents and the latter ones are very sensitive to hormonal-based therapies. In the MDA-MB-231 cells, RAPTA-T was found to predominantly localise on the cell membrane and to a lesser extent in the nucleolus. These findings are consistent with the previously reported anti-metastatic properties of RAPTA-T and the observation that once internalized RAPTA-T is associated with chromatin. RAPTA-T shows a lack of membrane accumulation on the non-invasive MCF-7 cells, which correlates well with its selective anti-metastatic properties on invasive cell lines.

A novel class of dianionic Ru(III) dimers of formula Na2[[trans-RuCl4(Me2SO)]2(mu-L)], with L = pyrazine (pyz, 1), pyrimidine (pym, 2), 4,4'-bipyridine (bipy, 3), and 1,2-bis(4-pyridine) ethane (etbipy, 4), was developed by us with the specific aim of assessing their antitumor properties. The dimers are in fact structurally related to the antimetastatic mononuclear compound (ImH) [trans-RuCl4(Me2SO)(Im)] (NAMI-A, Im = imidazole). Preliminary results concerning the antineoplastic activity of 1-4 against the murine MCa carcinoma model, a tumor which spontaneously metastasizes in the lungs, are reported. Similarly to what is normally observed with NAMI-A, the treatment with the dimeric complexes was scarcely effective against the growth of the primary tumor. However, dimers 1, 2, and 4 reduced very effectively the number and, in particular, the weight of lung metastases (to about 5% with respect to controls); in particular, Na2[[trans-RuCl4(Me2SO)]2(mu-etbipy)] (4) was as effective as NAMI-A in reducing the spontaneous metastases at a dosage which, in terms of moles of ruthenium, is about 3.5 times lower compared to that normally used for NAMI-A. Furthermore, in vitro tests showed that dimers 1-4 are capable of forming interstrand cross-links with linearized plasmidic DNA in a time-dependent manner. All the dimeric species are more active in inducing cross-links compared to NAMI-A, and the dimer bridged by the etbipy ligand (4) is the most effective among those tested.

The compounds [Pt(Me(2)phen)(acy)(2)](NO(3))(2) (1), [Pt(Me(2)phen)(pen)(2)](NO(3))(2), [Pt(phen)(acy)(2)](NO(3))(2) (2), and [Pt(phen)(pen)(2)](NO(3))(2), containing the bidentate 1,10-phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (Me(2)phen, neocuproine) and the antiviral agents acyclovir (acy) or penciclovir (pen), show different in vitro toxicity, the Me(2)phen complexes being appreciably more toxic than the phen complexes. To explain the different behavior, we investigated the reaction of complexes 1 and 2 with glutathione (gamma-glutamylcysteinylglycine, GSH), a peptide believed to play an important role in driving the cellular effects of platinum drugs. The reaction led to different products, the phen complexes forming a stable binuclear mu-thiol-bridged species still containing the phenanthroline and the Me(2)phen complexes releasing the neocuproine ligand and forming an insoluble material. In vitro tests confirmed that the greater cell toxicity of complex 1 is due to the displacement of the neocuproine ligand by GSH. The results highlight the great dependence of the glutathione reactivity upon relatively small changes in the platinum coordination sphere.