THEREOF IN ONCOLOGY.

TECHNICAL FIELD

The present invention regards mononuclear and dinuclear Ru-based and Ga-based coordination compounds, pharmaceutical formulations thereof, the related synthesis and encapsulation method in macromolecules, supramolecular aggregates or nanostructures, as well as their use in the diagnosis and/or the treatment of neoplasms. Advantageously, such coordination compounds and/or their formulations are glycoconjugated with carbohydrates which act as cancer-targeting moieties, thus increasing the therapeutic selectivity.

BACKGROUND ART

Cisplatin is a milestone among the anticancer compounds and, since its introduction in pharmacopoeia in 1978, has shown to be one of the most effective drugs, particularly in the treatment of testicular, ovarian, lungs, bladder and uterine cervix cancer, mesothelioma and head and neck carcinoma. Despite its therapeutic efficacy, the clinical use of cisplatin is characterized by severe side effects. The most serious one is the high toxicity, especially to kidneys, to auditory apparatus and to bone marrow. In addition, cisplatin demonstrates intrinsic or acquired resistance to certain tumors. Finally, it is characterized by a very limited intrinsic selectivity, so that only a small percentage (around 2 - 5% in moles) of cisplatin reaches the tumor cell DNA, which is currently recognized as its most important target. Further limitations to the clinical use of cisplatin are the poor solubility and the high reactivity of the platinum(ll) center which limit the bioavailability and, therefore, the mode of administration.

The intense research activity carried out in the recent decades has attempted to moderate these effects and limitations but has only partially achieved the desired effects. In fact, the numerous cisplatin-like compounds, such as carboplatin and oxalilplatin, still have a lower antitumor activity or a poor toxicological profile. The main reason for this failure is due to the fact that this metal has a high affinity for sulfur-containing residues of biomolecules.

As mentioned above, the oncological research in recent years has considered alternative approaches that will be described briefly here.

A first strategy aims to synthesize new coordination compounds with metal centers other than platinum that can maintain the antitumor activity but, at the same time, are characterized by a better toxicological profile. For example, in a first patent Fregona et al., were able to synthesize a class of Au(lll) dithiocarbamato complexes, described in ITMI20030600 which proved intrinsically less toxic than the Pt(ll) counterparts. Moreover, in a second document, WO2010105691 A1 , Fregona et al. have claimed a new class of Au(lll) dithiocarbamato compounds functionalized with a peptide moiety designed to be internalized by tumor cells by PEPT transporters. However, the syntheses therein proposed do not permit to obtain the compounds in pure form, but as a mixture of two different complexes having the same empirical formula. This result was not evident from the elemental analysis. This aspect represents a serious limitation, as in the oncology field, syntheses that lead to pure compounds or to mixtures of easily purified molecules are needed in order to favor both administration to humans and the industrial-scale production. In addition, for some types of cancer, PEPT transporters do not represent the optimal target to favor selectivity as they are not adequately overexpressed in tissues.

The ruthenium-based complexes represent another example of compounds reported in the literature. For instance, H. Fasal et al. report that (J. Coord. Chem., 2016, 70 (2), 279-295), ionic Ru(ll) mononuclear complexes are known, and they contain DMP (2,9-dimethyl-1 ,10- phenanthroline) and dithiocarbamates derived from piperazine (with the nitrogen atom in position 4 functionalized with a para- or meta-methoxyphenyl group) as ligands. Such complexes have been studied as DNA (both purified and derived from calf thymus) intercalants. Other authors reported on some Ru (II, III) complexes with cytotoxic properties: Nardon et al., (Future Med. Chem., 2016), Nagy et al. (Chem. Eur. J, 2012 and Dalt. Trans. 201 1 ), Giovagnini et al. (Dalt. Trans, 2008). In particular, these compounds are mono- and dinuclear complexes with DTCs of the type DMDT (dimethyl-DTC), PDT (pyrrolidine-DTC) and RSDT (DTCs derived from sarcosine; R = tert-butyl, ethyl, methyl). Another class of coordination compounds with potential biomedical applications consists of mononuclear Ga(lll) complexes with alkyl- dithiocarbamato ligands, wherein the nitrogen atom is bound to a methyl and a 2-methyl-1 ,3- dioxolane or a CH ₂ -CH-(OMe) ₂ group, alternatively (Ferreira et al., J. Coord. Chem., 2014, 67 (6), 1097-1 109).

Along with the development of new coordination compounds, a second and promising strategy in the oncological research is associated with the exploitation of the so-called Warburg effect. In this regard, most cancer cells trigger an accelerated metabolism sustained by an high carbohydrate consumption, if compared to healthy cells. In particular, tumor cells produce energy mainly through glycolysis, followed by the reduction of pyruvate to lactic acid in the cytoplasmic matrix, rather than oxidation of pyruvate within the mitochondria, which is the favorite ATP-producing mechanism in almost all the normal cells. The enclosed Figure 1 clearly highlights that the expression levels of the GLUT1 transporter generally increase during the neoplasia development, as well as with the degree of malignancy of the tumor itself.

Nowadays, this phenomenon is exploited in imaging-based diagnostic techniques and in the oncological patient follow-up via the administration of the tracer FDG (2-[ ¹⁸ F] fluoro-2-deoxy-D- glucose). Accordingly, glucose and other carbohydrates are potentially useful biomolecules to selectively target antitumor agents. According to this approach, a variety of glyco-conjugates made up of known cytotoxins or chemotherapeutics linked to glucose (or other carbohydrates) have been synthesized to increase their selectivity to and absorption by the tumor cells. For example, the glyco-conjugate glufosfamide is currently in clinical trials for the treatment of ovarian and pancreatic cancer, glioblastoma multiforme and non-small cell lung carcinoma. Other examples of metal and non-metal glyco-conjugates derivatives are cited in various works published in recent years, including that of Emilia C. Calvaresi and Paul J. Hergenrother "Glucose conjugation for the specific targeting and treatment of cance , the paper of S. Tabassum and coworkers "Exploration of glycosylated-organotin(IV) complexes as anticancer drug candidates", and the work of P. M. Abeysinghe and M. M. Harding "Antitumour bis(cyclopentadienyl) metal complexes: titanocene and molybdocene dichloride and derivatives".

Concerning the patent literature, for example, Lippard et al. attempted to overcome the above- mentioned limits of platinum-based drugs through the conjugation of one or more metal- coordinated ligands with different cancer-targeting moieties, including carbohydrates. However, these documents {e.g., the patent US/ 2014/034313), claim but do not describe synthetic procedures useful for an expert in order to conjugate a coordination compounds to carbohydrates, to be used just as cancer-targeting moieties.

To sum up, according to the best knowledge of the present inventors, only a limited number of coordination compounds coniugated to sugars, their synthetic processes, optimized for a specific metal, are known up to date.

Lastly, a further strategy adopted to mitigate the side effects (toxicity) and the limitations (low solubility and bioavailability) of cisplatin, as well as of other drugs used in oncology, is the recourse to encapsulation techniques in supramolecular systems, such as liposomes. However, if these techniques are already mature in the oncological field for organic compounds, the encapsulation of coordination compounds is still in the early stages due to inherent difficulties when combining highly- reactive metal-based compounds with biocompatible polymers. For these reasons, the encapsulation of coordination compounds is a still poorly explored research field in literature, except for some studies related to platinum derivatives. Among these, at present the formulation of cisplatin in liposomes composed of cholesterol and phosphatidylcholine, known with the LipoPlatin ^® is in phase III clinical trials. In addition, cisplatin modified to bond by a linker (polyglutamic acid) the PEG (Nanoplatin ^® , NC -6004) has received the authorization to start the clinical phase III of test on pancreatic cancer in combination with gemcitabine. Among the formulations based on cisplatin substitutes, oxalilplatin encapsulated in liposomes (known under the trade term of Lipoxal ^® ) is currently in an advanced stage of development, while the same oxalilplatin, encapsulated in transferrin-conjugated liposomes (MBP-426), has completed the Phase I. A modified oxalilplatin (NC-4016) wherein a PEG is linked to the platinum(ll) metal center via a polyglutamic acid linker is about to start the phase I.

TECHNICAL PROBLEM

The intense research activity conducted over the last few decades has attempted to mitigate the side effects (toxicity) and the limitations (low solubility and bioavailability) of cisplatin, but the strategies adopted so far have only partially achieved the desired results. Due to the peculiar reactivity preferably against neoplastic cells, drugs based on coordination compounds still represent one of the most promising anti-cancer option, but there is a need to make available a large number of new compounds with metal center other than platinum, which may replace cisplatin in the future, considering that most candidate compounds do not pass the advanced preclinical or clinical tests and therefore do not become drugs.

There is also a need for other coordination compounds with metals other than platinum and conjugated with cancer-targeting moieties, represented by carbohydrates, potentially useful in oncology and characterized by high selectivity (limited or negligible "off-target activity") and effectiveness towards different types of tumors and ultimately low or negligible toxicity. Lastly, it is necessary to have coordination compounds with metals other than platinum encapsulated in supramolecular aggregates, such as micelles, liposomes or cyclodextrins, which are conjugated or not to cancer-targeting moieties represented by carbohydrates.

DISCLOSURE OF INVENTION OBJECT/SCOPE OF THE INVENTION

Through the present invention, the present inventors intend to overcome the existing limits in the state-of-the-art related to coordination compounds useful as antitumor agents.

Accordingly, it is a first and main object of the present invention to design and synthesize Ru- based and Ga-based mononuclear and dinuclear coordination compounds to be used as antitumor agents. Furthermore, a second object of the present invention is to identify mononuclear and dinuclear Ru-based and Ga-based coordination compounds with carbohydrate-functionalized ligands which act as selective cancer-targeting moieties.

Particularly, the present invention intends to disclose the advantageous use of carbohydrates as selective cancer-targeting moieties. In addition, carbohydrates could provide the additional advantage of increasing the solubility of the final compound in aqueous media.

Still, a third important object of the present invention is to identify coordination compounds endowed with low or negligible toxicity with respect to other metal-based compounds, such as cisplatin and subsequent compounds. Furthermore, a fourth object of the present invention is to identify coordination compounds characterized by high solubility in aqueous media, high stability and bioavailability, inherent or achieved by encapsulation in macromolecules, nanostructures or supramolecular aggregates, such as micelles, liposomes, proteins or cyclodextrins. In particular, such structures may or not be functionalized with carbohydrates on the outer surface.

Moreover, a fifth object of the present invention is to provide a stable pharmaceutical formulation including one or more of said coordination compounds, usable as an antitumor agent and preferably administrable intravenously or orally. Such formulations may optionally include additional anticancer drugs.

Within the above-mentioned main tasks, a further object of the present invention is to provide a method for synthesizing said mononuclear and dinuclear Ru-based and Ga-based coordination compounds, bioconjugated or not with carbohydrates, as well as a method for encapsulating said compounds in macromolecules, supramolecular aggregates made up of biocompatible polymers, functionalized or not with carbohydrates.

In addition, a further object of the present invention, is to disclose the use of said coordination compounds as antitumor agents, particularly in the treatment of "orphan tumors", such as the "triple negative" breast cancer (TNBC), the castration-resistant prostate cancer (CRPC), head and neck cancer, the NSCLC, the melanoma, the mesothelioma, the lung, pancreas and liver carcinoma. Last but not least, a final object of the present invention is to produce coordination compounds which, besides possessing the necessary chemical-biological properties, are stable and obtainable through a synthesis process both able to intrinsically yield high-purity compounds and being industrially applicable with known and cost-effective technologies compared to state-of-the-art solutions.

TECHNICAL SOLUTION

In view of the above disadvantages or drawbacks of the prior art, the present inventors have made a lot of studies related to the preparation of coordination compounds useful as anti-cancer agents.

After long terms of practice the inventors found a new class of Ru-based and Ga-based mononuclear and dinuclear coordination compounds, which are described by one of the following eneral formulae I (a) or l(b):

1(a) 1(b)

wherein , M ₂ , M ₃ identify the metal center of the coordination compound which can be Ga(lll), Ru(ll), Ru(lll). The mononuclear and dinuclear coordination compounds herein disclosed are neutral or ionic complexes whose charge is neutralized by at least one counter- ion G and have different coordination geometries, for example regular octahedral or distorted octahedral.

Further features and advantages of the new coordination compounds and the corresponding synthesis and encapsulation methods thereof, as well as the use of said compounds, will become apparent to those skilled in the art via the detailed description of its four preferred embodiments, herein provided below by way of example but not limitation. In a first preferred embodiment of the present invention, the coordination compounds of formula 1(a) or 1(b) are not bioconjugated with a cancer-targeting moiety and act as antitumor agents due to the inherent reactivity of the Ru and Ga metal centers.

In a second preferred embodiment, the coordination compounds of formula 1(a) or 1(b) are bioconjugated with a carbohydrate which acts as a selective cancer-targeting moiety, advantageously exploiting the so-called "Warburg effect". Surprisingly, the presence of the cancer-targeting moiety, in combination with the physico-chemical properties of the final compound {e.g. ionic nature), can result in a very high solubility in physiological media.

In the third preferred embodiment of the present invention, the coordination compounds of formula I (a) or l(b) are encapsulated in macromolecules, nanostructures or in supramolecular aggregates, such as liposomes or cyclodextrins, acting as carriers to carry out a passive- targeting mechanism mediated by the Enhanced Permeability and Retention (EPR) effect that characterizes tumor districts. Said compounds may be or not conjugated with carbohydrates. In a fourth preferred embodiment, the coordination compounds described in the formula l(a) or l(b) may be addressed to the tumor site by encapsulating such compounds in macromolecules, nanostructures or supramolecular aggregates, for example micelles, in turn functionalized with carbohydrates to achieve an active-targeting approach ("Warburg effect"), which accompanies and strengthens the passive-targeting mechanism of the previous embodiment. Even in this case, the encapsulated compounds can be or not conjugated with carbohydrates.

ADVANTAGEOUS EFFECTS OF INVENTION

The coordination compounds of structure I (a) and/or l(b) according to four preferred embodiments have a number of remarkable advantages which cannot be achieved by prior art compounds as it will be apparent to those skilled in the art.

First of all, the present invention discloses Ru-based and Ga-based coordination compounds, glycoconjugated or not, which, according to experimental tests, possess remarkable anti-tumor properties. Furthermore, various synthesis processes of said coordination compounds have been disclosed, characterized by high yields: they allow the expert to choose the most advantageous scheme depending on the metal center and type of desired ligands, including those containing a cancer-targeting moiety represented by a specific carbohydrate. Said cancer targeting moiety and metal centers can in turn be chosen based on the molecular profile of the patient's neoplasm according to the paradigm of personalized medicine.

The Ru-based and Ga-based coordination compounds according to the present invention can be loaded into supramolecular aggregates or macromolecules, consisting of a wide range of biocompatible polymers so to obtain stable nanoformulations. In other words, said compounds do not react nor establish interactions with other loaded complexes, and with the polymeric carrier neither. Advantageously, the coordination compounds and nanoformulations allow to realize a both active and passive cancer-targeting mechanism. These compounds are characterized by optimal LiPE values for a promising pharmaceutical development {"druglikeness").

Moreover, said nanoformulations not only make the hydrophobic active principles soluble in the aqueous media but also mask the active principles herein described, both hydrophobic and hydrophilic, from reactions/interactions with the cellular and molecular components of the blood. Additional objects and advantages of the invention will be set forth in part in the detailed description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

DESCRIPTION OF PR A WINGS

The present invention will be more fully understood by reference to the following drawings which are for illustrative purposes only:

- Figure 1 depicts the levels of expression of the glucose transporter (Glutl ) in different cell lines. In letter A), Northern analysis of the Glutl mRNA levels evaluated in healthy human prostate samples and in three different human prostate cancer cell lines with decreasing differentiation. LNCaP is a hormone-sensitive cancer cell line, whereas the DU-145 and PC3 lines represent poorly-differentiated tumors (source: P. Effert et al., Anticancer Research 24: 3057-3064, 2004). In letter B), Western analysis of Glutl protein levels evaluated in samples derived from 4 different cell lines: cervix adenocarcinoma (HeLa), lymphoblast T (Jurkat), epidermoid skin cancer (A431 ), human embryonic kidney (HEK293) (from Abeam pic). Finally, in letter C), Western analysis of the Glutl protein levels evaluated (cell lysate 20 μg/lane, anti-Glut-1 ab 2 g/mL, anti-β- actin ab 0.5 g/mL, secondary antibody 1 :1000) in samples derived from 3 different human cell lines: triple negative breast cancer (MDA-MB-231 ), pulmonary adenocarcinoma (A549), colon carcinoma (Caco2).

Figure 2 shows the UV-Vis spectra of the coordination compound [Ru ₂ (PipeDTC) ₅ ]CI with (VII) general structure. In panel A) said compound is dissolved in phosphate buffer (pH 7.4, 0.5% v/v DMSO, spectra recorded at 37° C over 24 hours), whereas in panel B), it is encapsulated in PF127 micelles (5 mg/mL) dissolved in phosphate buffer - cell culture medium 9:1 v/v (spectra recorded at 37 °C for 72 hours).

- Figure 3 displays the UV-Vis spectra of [Ru ₂ (PipeDTC) ₅ ]CI encapsulated in PF127 micelles dissolved in acetate buffer (pH 5.5), recorded over a period of 72 hours at 25 °C.

- Figure 4 shows the DLS analysis of [Ru ₂ (PipeDTC) ₅ ]CI encapsulated in PF127 micelles with 10% of the polymer conjugated to glucose via glycosylation (C1 position).

- Figure 5 collects TEM images of the formulations according to the third and fourth embodiments of the present invention, wherein PF127 micelles encapsulate the complex [Ru ₂ (PipeDTC) ₅ ]CI. These figures illustrate and demonstrate various features and embodiments of the present invention, and of the manufacturing method thereof, but are not to be construed as limiting the invention.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

For the purpose of understanding the specification and the appended claims, in the following description the chemical elements are defined by means of the respective symbols as reported in a common Periodic Table of Elements, such as that present in the "Handbook of Chemistry and Physics, 93rd ed ". In addition, unless otherwise indicated, the chemical symbol includes all isotopes and ions. Therefore, the chemical symbols C, F, Cu, Ga , Au and I include, as an example, their respective isotopes ¹¹ C, ¹⁸ F, ⁶⁴ Cu, ⁶³ Cu, ⁶⁵ Cu, ⁶⁸ Ga, ¹⁹⁸ Au, ¹³¹ 1, ¹²⁷ l, ¹²⁹ l. Moreover, the claimed and described structures are intended to include all possible isomers such as coordination isomers, structural isomers, and conformational isomers. In particular, wherever one or more chiral centers are present, the claimed and described structures are designed to include all possible optical isomers, such as enantiomers and/or diastereoisomers, their mixtures, either as racemic mixture or in various ratios. These chiral centers may be present in the coordination compound or already present in the involved chelating ligands, for instance in the molecular fragments comprising a carbohydrate T.

In the description and claims, the reference to classes of chemical compounds or functional groups, for example, an ester or an amide, has to be interpreted in the broadest meaning of the term in the chemical field, and, in particular it includes all possible types of substituents.

For the sake of brevity, the functional groups or solvents reported in the present invention may be indicated by acronyms or abbreviations widely used in the context of reference of the present invention (or defined in the aforementioned "Handbook"). Finally, for the sake of clarity, in the general formulae the chelating dithiocarbamato ligand (also indicated with the acronym DTC) is represented through a stylized arch that connects two sulfur atoms, in accordance with the usual chemical notation.

In some preferred embodiments, the coordination compounds having formula l(a) and/or l(b) are bioconjugated to cancer-targeting moieties. This is a well-known term for an expert in the field, and it defines a molecular fragment of the compound (moiety), which has been ad hoc engineered for its recognition by cell-membrane proteins present in tumor cells. Within the scope of the present invention, said "cancer-targeting moiety" is a carbohydrate, and according to this, the terms "glycoconjugation" and "glycoconjugates" will be also used to indicate the "bio- conjugation" and the "bio-conjugates", respectively. The synthetic processes of the first and second embodiments involve amino functional groups for the preparation of the dithiocarbamato ligand. These groups may be ad hoc introduced in a specific stage of the process, or alternatively they can be generated by subsequent reactions during the previous steps. On the other hand, a suitable amine reagent can be purchased and used as such. Anyway, in the context of the present patent, these amino groups and amine reagents will be defined "precursor amine(s)" for brevity.

Some embodiments of the present invention involve the term "pharmaceutical formulations" to define preparations made up of a therapeutically-effective amount of the coordination compounds of Formula 1(a) and/or 1(b) together with "pharmaceutically acceptable" additives and/or diluents and/or excipients. These additives, diluents, and excipients are intended to be compounds that, in contact with animal or human tissues, in light of their composition, dosage and administration route or anything else, do not cause any toxicity, irritation, allergy, and other complications considered excessive or unacceptable by a specialized physician based on a reasonable risk/benefit ratio.

Said compounds may optionally be encapsulated in "pharmaceutically acceptable" macromolecules or supramolecular structures or nanostructures. For the sake of clarity, the term "encapsulation" means a process by which a therapeutically-effective amount of the coordination compounds of Formula l(a) and/or l(b), establishes intermolecular bonds {e.g. van der Waals forces, hydrogen bonds) with the single units of a "nanocarrier" or with specific structural domains. As an example, the surface or the hydrophobic core of the nanocarrier (e.g., micelles), or the hydrophilic core {e.g., liposomes), or also the lipidic/polymeric layer.

Finally, within the scope of the present invention, the term "theranostic compound" or more generally "theranostic agent" will refer to the coordination compound according to this invention, the composition containing at least one of said coordination compounds, or also a pharmaceutical formulation made up of the said compound and/or composition, which combines therapeutic properties with regard to a pathology, preferably neoplastic pathologies, along with the property to be detectable by suitable devices/detectors, hence to be usable in association with diagnostic and imaging systems for patient follow-up in therapeutic treatments and, if possible, to take the necessary corrective action.

COORDINATION COMPOUNDS

It is a first object matter of the present invention a new class of mononuclear and dinuclear Ru- based and Ga-based coordination compounds, whose features are defined in the appended independent claim. Such compounds are described by one of the following general formulae (here also referred to as "Formula 1 a)" or "Formula 1(b)"):

1(a) 1(b)

Said mononuclear and dinuclear coordination compounds are neutral or ionic complexes whose charge is neutralized by at least one counter-ion G and have different coordination geometries, for example regular octahedral or distorted octahedral. In light of the Formula 1(a) or Formula 1(b) Mi , M ₂ , M ₃ , X, Y, W, Z, L _{1 5} L ₂ , L ₃ and L ₄ are independently chosen and have the following meaning.

, M ₂ , M ₃ identify the metal center of the coordination compound and they are selected from Ga(l ll), Ru(l l), Ru(l l l), also defined in short as Ru (11,111). The stylized arch connecting the two S- atoms represents a first ligand of dithiocarbamic nature (DTC);

X, Y, W, Z, I_2, L ₃ and L ₄ are selected from the group consisting of: N, S, O, P, C and Se, and represent donor atoms being the same or different from each other and they belong to one or more bidentate chelating ligands.

The integer n represents the charge of the complex, ranging from -4 to +4, where the case with n = 0 corresponds to a neutral complex.

The G symbol identifies at least one counter-ion having the total charge d, being an integer ranging from -4 to +4. Such counter-ion is present with a stoichiometric coefficient e, identified by the integer n/d (in absolute value) or zero (in the latter case of course the counter-ion is not present and also n=0). G is pharmaceutically acceptable ion, which, by way of example and not limitation, is selected among : CI ^" , I ^" , F ^" , Br ^" , CH ₃ C0 ^2" , PF ₆ ^" , BF ₄ ^" , SbF ₆ ^" , [B{C ₆ H ₃ (CF ₃ ) ₂ } ₄ ] ^" , [B(C ₆ F ₅ ) ₄ ] ^" , OH ^" , 0 ₃ SNH ₂ ^" , nitrites and nitrates {e.g., N ₂ 0 ^" , N0 ₃ ^" ), acetates, phosphates {e.g., hexafluorophosphate, H ₂ 0 ₄ P ^" , P0 ₄ ^3" ), sulfates {e.g., triflate, HS0 ₄ ^" ), carbonates, perchlorates, acetylacetonates {e.g., hexafluoroacetylacetonate), propionate, glycolate, stearate, lactate, malate, pyruvate, tartarate, citrate, ascorbate, palmitate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulphonate, ethane-1 ,2-disulfonate, trifluoromethanesulfonate, oxalate, malonate, succinate, glutarate, adipate, pimelate or isethionate, Na ⁺ , K ⁺ , Mg ²⁺ . However, the text "Remington's pharmaceutical Sciences, 17 th ed." (Mack Publishing Company) reports other chemically equivalent salts that can be used.

For the sake of clarity, in the Formula l(a) and Formula l(b), the stylized arch connecting the S- donor atoms indicates a bidentate chelating dithiocarbamato ligand (DTC), in compliance with the usual chemical notation. The number of chelating ligands in the structures l(a) and l(b) is 3 and 5, respectively. Said chelating dithiocarbamato ligands (DTC) may have a closed (cyclic) or a open {e.g., linear or branched) structure.

In some embodiments, said first or second DTC ligand has a closed structure such as that depicted below, which is presented by way of example but not limitation of the present invention:

Such structure comprises groups R _{1 5} R ₂ , R ₃ and R ₄ and optionally one R ₅ group. The group and - if present - the R ₅ group, are bound to the dithiocarbamic nitrogen atom and to the R ₂ and R ₄ groups, respectively; the remaining R, groups are bound to the next and the previous group in the cyclic structure. If R ₅ is absent, R ₄ is directly bound to the dithiocarbamic nitrogen atom. All the bonds involving the R, groups, where i is an integer ranging from 1 to 5, may be single or double.

Optionally, the DTC ligand with the closed structure herein described comprises a glucide or carbohydrate T, which is bound to at least one of said R, groups directly or through a unit A by respectively T-R, bonds or T-A-R, bonds, which can be single, double or triple. Depending on the situation, the nature of said bonds may be: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C. The unit A is made up of an atom or a functional group or a spacer, meaning with this term a molecular fragment properly designed to join the glucidic moiety (T) with the cyclic structure described above, for example -COOCH ₂ CH ₂ 0-CH ₂ CH ₂ 0-CH ₂ CH ₂ 0-CH ₂ CH ₂ 0- or - CH ₂ CH=CHCH ₂ CH ₂ - or also -COOCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ . However, for the unit A, other chemically-equivalent molecular fragments may be selected by the expert of the branch.

In other embodiments, said DTC ligands have an open structure of the type depicted below by way of example, but not limitation:

In this case, the structure comprises a terminal group R linked to the dithiocarbamic nitrogen atom, and optionally one or more R ₂ , R ₃ , R ₄ and R ₅ groups.

If R ₂ is present, it represents a terminal group which can be bound to R ₃ or R ₄ or R ₅ or directly to the dithiocarbamic nitrogen atom, depending on which groups R, (i = 3, 4 or 5) are absent or not. If R ₂ is present, it represents a terminal group which may be linked to R ₃ , R ₄ , or R ₅ or directly to the said dithiocarbamic nitrogen atom, depending on which R, groups (i = 3, 4 or 5) are absent or not. If present, the R ₃ group is bound to R ₂ , or alternatively R ₃ is a terminal group when R ₂ is absent. In both cases, R ₃ is bound to R ₄ or R ₅ , or to said nitrogen atom, according to which group (among R ₄ or R ₅ ) is absent or not. Similarly, the R ₄ group, if present, is a terminal group and is bound to R ₅ or to said nitrogen atom; alternatively, R ₄ is bound to R ₅ or to said nitrogen atom and is bonded to R ₃ or R ₂ , depending on whether the R ₃ or R ₂ groups are absent or not. Finally, the R ₅ group, when present in the structure, is a terminal group and is linked to said nitrogen atom or alternatively it is bound to R,, according to which R, (i = 2, 3 or 4) groups are absent or not. All the bonds involving the R, groups, where i is an integer from 1 to 5, can be single, double or also triple.

Optionally, the DTC ligand with the open structure herein described comprises at least one glucide or a carbohydrate T. Said unit T is linked to said dithiocarbamic nitrogen atom or to at least one of the said R, groups (where i is an integer from 1 to 5) directly with respectively T-N or T-R, bonds, or alternatively via a unit A, by T-A-N or T-A-R, bonds. Depending on the cases, said bonds may be single, double or triple, and of the type: C-C, C-O, O-C, C-N, N-C, C-S, S-C, C-P, P-C, C-Se, Se-C. Also in this case, the unit A may be an atom or a functional group or a spacer, as well as the unit A is made up of other chemically-equivalent molecular fragments. In both types of structures (closed or open) of said DTC ligands, said unit A or said R, group (where i is an integer ranging from 1 to 5) are preferably selected from: an atom such as H, C, O, N, S, P, Se or a group selected among -CH, -CH ₂ , -CH ₃ , -C(CH ₃ ) ₃ , -NH, -NH ₂ , -NHR _S , -NR _s2 , -S-S-, -SH, -PH, -OH, -COOH, -CH-Br, -CHCH ₂ NH ₂ , -CHCH ₂ OH, -CHCH ₂ NH-, -CHCH ₂ 0-, (- CN), -CF ₃ , -C ₂ H ₅ , -C ₂ H ₄ , -C ₄ H ₉ , -C ₃ H ₇ , -C ₃ H ₆ , -C ₃ H ₆ OH, -C ₄ H ₈ , -N0 ₂ , -CH ₂ OH, -C ₂ H ₄ OH, - C ₄ H ₈ OH, -C(OH), -C(OH)H ₂ , -C(OH)H; -S0 ₂ , -COO-, -N(CH) ₃ -, -CHN(CH), -NN(CH),-NCHNCH- , -N(CH) ₂ N-, -CHNHCH-, -NH(CH), -NHCHN-, -NHNCH-; -CONH-, -CONH ₂ , -CONHR _s , - CONR _s2 , (-O-C(O)-Rs), where R _s indicates substituent groups of the type: aliphatic, alkyl, halo- alkykl, cycloalkyl, alkenyl, alkynyl, aryl, etero-aryl, etero-aliphatic, aromatic, etero-aromatic, aliphatic-aromatic, eteroaliphatic-eteroaromatic, cycloaliphatic, eterocycloaliphatic, esteric, amidic, sulphonamidic, carbonylic, acetyl, ethyl, propylic, butilyc, isopropylic, acylic, ureidic, thioureidic, thiolate, imine, halogens, ethers, nitro groups, nitrile, aryl, benzyl, sulphonamidic, saturated linear or branched C1 -C18 alkylic optionally substituted with one or more R _s groups or including one or more unsaturated bonds, a group of the type -(CH ₂ ) _m , -(CH ₂ 0) _m , -(CH ₂ CH ₂ S) _m , (CH ₂ CH ₂ NH) _m or (CH ₂ CH ₂ 0) _m , (CH ₂ CH ₂ N) _m where m represents an integer greater than 1 , or it is a combination thereof.

Depending on the needs, as unit A or groups R, it is possible to use molecular fragments being chemically-equivalent to those herein reported by way of example, but not limitation of the present invention. In addition, in some embodiments, combinations of the indicated species are possible. Furthermore, said DTC ligands may contain in the unit A or in the R, groups chemical groups in salified form comprising pharmaceutically acceptable counterions {e.g., -NH ₃ ⁺ CI ^" ). It will be apparent to the skilled in the art that the unit A or the various R, groups may be variously substituted, in any combination and position, with one or more of said atoms or said groups. Similarly to the coordination compound, said DTC ligands may a priori have different isomeric forms. Hence, the herein disclosed formulae are intended to include any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, as racemic mixtures or in various ratios.

Again with reference to both closed or open structures of said chelating dithiocarbamato (DTC) ligands, said glucide or carbohydrate T is preferably a monosaccharide or a deoxy- monosaccharide, in particular a those, a tetrose, a pentose, an esose, an eptose. By way of example, but not limitation, said glucide or carbohydrate T is selected from the group consisting of: glucose, galactose, mannose, xylose, rhamnose, arabinose, glucosamine, galactosamine, mannosamine, fructose, fructosamine, ribose, ribulose, sedoheptulose, erythrose, threose, erythrulose, allose, altrose, lixose, gulose, idose, talose. Alternatively, said glucide or carbohydrate T is a natural or synthetic polysaccharide, also in deoxy-carbohydrate form, for example maltose, cellobiose, lactose, trealose, sucrose, amylose, amylopectin.

In the case of the closed-structure DTC ligands, the T-R, or T-A-(R,) bond (where i is an integer ranging from 1 to 5) takes preferably place in at least one of the carbon positions of the T moiety, preferably in the positions C1 or C2 or C3 or C4 or C6, for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.

In the case of the open-structure DTC ligands, the T-N or T-R, bond, or T-A-N or T-A-R, bond (where i is an integer ranging from 1 to 5) occurs preferably in the C1 or C2 or C3 or C4 or C6 positions for the hexoses, and preferably in the C1 or C2 or C3 or C5 positions for pentoses.

Said chelating DTC ligands are symmetrical bidentate ligands whose chelating function is generally characterized by a negative q charge {q = -1 ). In light of the choice of the R, groups, of the unit A and the glucide T, the ligand may have an overall neutral {e.g., zwitterionic), positive or negative charge.

Moreover, taking into account both closed and open structures of said chelating dithiocarbamato (DTC) ligands, and with reference to compounds of both the first embodiment and the second embodiment (i.e. containing carbohydrates T in the DTC ligand), one or more functional groups can be suitably protected during the synthetic process. Well-known groups are available for the protection of hydroxyl, amine, amide and carboxylic acid groups, as well as the conditions in which protection and deprotection can occur, even in so-called orthogonal conditions. In particular, protecting groups are introduced during the synthesis to avoid undesirable side-reactions and may be removed or not at the end of the synthetic procedure. In the second case, the new herein disclosed compounds may contain protective groups, even mutually different, in order to modulate the in vivo therapeutic profile, for example in terms of reactivity, solubility, stability and bioavailability. For example, one or more of the hydroxyl groups (also known as alcoholic) of one or more cancer-targeting moieties T of the second embodiment can be functionalized with equal or different protecting groups chosen, for example, from silyl ethers {e.g., trimethylsilyl ether or dimethyltertbutylsilylether) or esters {e.g., acetate, pivalate, propionate, carbonate, phosphate, butyrate). These concepts are in summarized in the structure of the hexose shown below only by way of example but limitation of the present invention.

In particular, the abovementioned protecting groups, identical or different, are indicated here by the notation R ₆ ; the substituent in position C1 may have an a or β conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C2 (as indicated by the black rectangle). For a pentose, depicted below only by way of example, but not limitation of the present invention, the protecting groups, equal or different, are again indicated by the notation R ₆ ; the substituent in position C1 may have an a or β conformation, and this monosaccharide binds, directly or through the unit A, to said R, groups or to the dithiocarbamic nitrogen atom in position C5 (as indicated by the black rectangle).

From the above description, it is clear that the first and the second chelating dithiocarbamato (DTC) ligand may be either equal to or different from each other. In the first case, the coordination compound is referred to as homoleptic, while in the latter it is heteroleptic.

SYNTHESIS AND CHARACTERIZATION OF THE COORDINATION COMPOUNDS

It is another subject matter of the present invention a process for the synthesis of the coordination compounds of general formula I (a) and l(b), whose features are set forth in the enclosed independent claim.

In the first preferred embodiment, herein described by way of example, but not limitation of the present invention, the coordination compounds having the general formula I (a) and l(b) may be conveniently synthesized by a process including at least the following steps:

a) Preparation and optional isolation of the dithiocarbamato ligand (DTC);

b) Coordination of the DTC ligand to one specific metal center;

c) Isolation of the coordination compound synthesized during step b). This process may optionally include a further step d) related to the purification and drying of the coordination compound obtained at the end of the step c).

a) Preparation and isolation of the dithiocarbamato ligand (PTC)

The synthesis of the dithiocarbamato ligands (DTC) is carried out at a temperature ranging from -30 and 60 °C in water, or methanol, or dry THF via reaction between an amine precursor and carbon disulfide (CS ₂ ), optionally in the presence of a base, such as KOH or sodium tert- butoxide, or an excess of said amine precursor. In the case of some amines, it is preferable to work under inert atmosphere, using well-known equipment and techniques. Subsequently, the volume of the solvent is reduced and the DTC ligand can be isolated via precipitation or co- precipitation by adding diethyl ether, washed with diethyl ether and dried under vacuum in presence of P ₂ 0 ₅ .

By means of this synthetic strategy, which yields the isolation of pure DTC ligands (for example, as sodium salt, potassium salt or with the precursor amine under the form of ammonium), the present inventors have inventively achieved a number of remarkable advantages over the state of the art. First of all, this allows an improvement in the yield of the subsequent complexation reaction. Moreover, the DTC salt can be tested in vitro for its biological activity, in order to demonstrate that the antitumor activity is mediated by the coordination compound as a whole and not just by the ligand (or the metal center).

b) Coordination of the DTC ligand to a specific metal center

The synthesis of the coordination compound of general formula I (a) and/or l(b) is carried out in water or in an organic solvent, and it involves the coordination of the DTC, which has been optionally isolated in the previous step, to a selected metal center among Ru (II, III), and Ga (III). In the case of some compounds, it is preferable to work under inert atmosphere, using well- known equipment and techniques.

The metal center is selected starting from the corresponding precursors, preferably chlorides or metal-halide salts, or complex salts. Alternatively, the precursors are metal derivatives with the metal center presenting a lower or higher oxidation state, such as organometallic-, amino-, thioether- precursors, or phosphine derivatives, or alternatively some of said coordination compounds can be themselves precursors, useful for the synthesis of other complexes.

During this step, contrast agents can be advantageously integrated in the coordination compound according to the invention in the form, for example, of radioisotopes such as ¹¹ C, ¹⁸ F, ⁶⁸ Ga, ¹²⁷ l, ¹²⁹ l, ¹³¹ 1. In particular, these isotopes can be included in the DTC ligand, or they may coincide with the metal center or also introduced into the counter-ion. As it will be more clearly described in following, these contrast agents allow to combine the treatment of the neoplastic diseases with the diagnosis.

c) Isolation of the coordination compound synthesized during the step b)

The isolation of the compound synthesized in the previous step b) proceeds via usual separation techniques, preferably filtration followed by reduction of the volume of water, or of said solvent, and by precipitation with ethyl ether. Alternatively, water or said solvent is evaporated under reduced pressure, in order to obtain the coordination compound of general formula 1(a) and/or 1(b), or a mixture containing said coordination compound.

d) Purification and drying of the coordination compound

During this step, the purification is optionally carried out using techniques well-known to the skilled in the art, for instance by chromatography, precipitation from organic solvent, washing with water or organic solvents and drying of said coordination compound.

The following sections describe further details of the process described in the step b), related to the coordination of the DTC ligand to a specific metal center.

Preparation of the coordination compounds with M = Ga (III) or Ru (III)

For the mononuclear coordination compounds having the general formula l(a) with M ₁ =Ru(lll) or Ga(lll), L _{1 =} L2=S and X=Y=S, the coordination of the DTC ligand (3 eq., preferentially dissolved in methanol) to the metal, preferably ruthenium(lll) chloride or gallium (III) chloride, occurs in water or THF or methanol. In the case of Ru(lll), despite of the stoichiometric 1 :3 metal-to- ligand ratio, the resulting product is actually a mixture of the neutral mononuclear complex [Ru(DTC) ₃ ] and the dinuclear ionic derivative of the general formula l(b). The reaction mixture is stirred for at least 1 h at room temperature. Subsequently, the solvent is removed and the solid obtained is taken up with chloroform. After filtration, the obtained solution is precipitated with diethyl ether, leading to the isolation of a solid.

Preparation of the coordination compounds with M = Ru (II, III)

Concerning the ionic coordination compounds containing the cation [Ru ₂ (DTC) ₅ ] ⁺ having formula l(b) with M ₂ =M ₃ =Ru(lll), L^L^L^L^S and X=Y=W=Z=S, the synthesis is carried out in water where the ruthenium(lll) precursor RuCI ₃ -3H ₂ 0 is mixed with the selected DTC ligand according to a proper stoichiometric ratio. This reaction leads to a mixture of products consisting of the ligand dimer (DTC) ₂ , the neutral mononuclear derivative [Ru(DTC) ₃ ] and the dinuclear ionic complex [Ru ₂ (DTC) ₅ ]CI (as a mixture of its a and β isomers). The [Ru(DTC) ₃ ] and the α,β- [Ru ₂ (DTC) ₅ ]CI complexes are separated by chromatography. The mixture containing α,β- [Ru ₂ (DTC) ₅ ]CI is refluxed in DCM or isopropanol for 8 hours, in order to convert the residual a- isomer to the thermodynamically more stable β-[Ρυ ₂ (ϋΤΰ) ₅ ]ΰΙ. Then, the purified compounds are washed with n-hexane and dried under vacuum in presence of P ₂ 0 ₅ .

The counter-ion G can be advantageously chosen or replaced, using techniques well-known to the skilled in the art, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target" toxicity, solubility, and stability.

Concerning the dinuclear coordination compounds obtained from the formula l(b) with M ₂ =Ru(lll) and M ₃ =Ru (II) or M ₂ =Ru(ll) and M ₃ =Ru(lll), it is possible to perform the reduction starting from the a or β isomer, or their mixture, using an appropriate reducing agent. Preparation of heteroleptic coordination compounds with M = Ru (III)

Concerning the ionic heteroleptic coordination compounds containing the cation [Ru ₂ (DTC) ₄ (DTC ^* )] ⁺ having formula 1(b) with M ₂ =M ₃ =Ru(lll), and X=Y=W=Z=S, where four DTC ligands are the same whereas the fifth is different, the synthesis was performed according the following novel and inventive scheme.

1) Br ₂ CH ₂ CI ₂ reflux, 10 min

2) hexane, 30 min

3) DTC*, l eq, CH ₂ CI ₂ / MeOH

The first synthetic step is related to the preparation of the organometallic Ru(ll)-NBD complex, synthetized from RuCI ₃ -3H ₂ 0 at reflux with the NBD ligand, according to a procedure reported in literature (J. Organomet. Chem., 1966, 5, 275-282). Successively, the Ru(ll)-NBD derivative is treated with 2 eq. of DTC to form the [Ru"(NBD)(DTC) ₂ ] complex. This step leads to the formation of the "building blocks" of the final heteroleptic complex, and hence the introduced DTC ligands are prevalent in the dinuclear compound (4 units among the 5 ligands). The synthesis of the Ru(ll)-DTC derivative is conducted in hot DMF (100 °C) by adding a DMF solution of the dithiocarbamato ligand (2 eq.) to the Ru(ll)-NBD precursor. The precipitation with an appropriate solvent leads to the isolation of the precursor. Finally, the Ru(ll)-DTC is treated with bromine, to achieve the oxidation of the ruthenium center to Ru(lll), and, after the cleavage of the olefin ligand with hexane, the addition of the DTC ^* ligand results in the formation of the [Ru ₂ (DTC) ₄ (DTC ^* )]Br coordination compound. In most cases, the final product is purified via silica gel chromatography with an appropriate eluent, according to the nature of the DTC ligands.

The counter-ion G can be advantageously chosen or replaced, using well-known techniques, in order to optimize the pharmaceutical profile of the coordination compound in terms of antitumor activity, "off-target" toxicity, solubility, and stability.

Concerning the heteroleptic dinuclear coordination compounds related to the formula l(b) with M ₂ =Ru(lll) and M ₃ =Ru (II) or M ₂ =Ru(ll) and M ₃ =Ru(lll), it is possible to perform the reduction of the a or β dinuclear isomer, or their mixture, using an appropriate reducing agent. EXAMPLES OF COMPLEXES ACCORDING TO THE FIRST EMBODIMENT

By way of example, but not limitation of the present invention, some specific examples of coordination compounds having formula 1(a) or 1(b) are reported below. In the first preferred embodiment of the present invention said compounds do not contain a cancer-targeting moiety in the form of carbohydrate, and contain, for example, dithiocarbamato ligands derived from: piperidine, L-proline methyl ester, and L-proline ferf-butyl ester and which for brevity are named as PipeDTC, ProOMeDTC, and ProOtBuDTC, respectively.

The compounds were characterized using several techniques, including elemental analysis, NMR spectroscopy, FT-IR spectrophotometry, and ESI-MS mass analysis.

Example 1 : [Ga(ProOM

Appearance: white solid; Yield: 75%

Anal. Calc. for C ₂₁ H ₃₀ GaN ₃ O ₆ S ₆ (MW = 682.59 g-mol ^"1 ): C 36.95; H 4.43; N 6.16; S 28.19. Found: C 36.87; H 4.29; N 6.18; S 28.10.

1 H-NMFt (CDCI ₃ , 300.13 MHz): δ (ppm) = 2.05-3.31 (m, 12H, H ₍₃₎ + H ₍₄₎ ), 3.70 (s, 9H, + 0-CH ₃ ), 4.01 (m, 6H, H ₍₅₎ ), 5.02-5.06 (m, 3H, H ₍₂₎ ).

Medium FT-IR (KBr): v (cm ¹ ) = 2965.1 1 , 2831 .24 (v _a , C-H); 1738.19 (v, C=0); 1 165.1 1 (v _a , N- CSS); 1 147.1 1 (v _a, C-OfBu); 999.54 (v _a , CSS).

Far FT-IR (nujol): v (cm ¹ ) = 550.16 (v _s , CSS); 336.29 (v, Ga-S).

Example 2: fi-[Ru ₂ (PipeDTC) ₅ ]CI

Appearance: brown solid; Yield: 29 %

R.f. (silica gel, CH ₂ CI ₂ /MeOH 9:1 ): 0.39

Anal. Calc. for C ₃ oH5oCIN ₅ Ru ₂ Sio (MW = 1039.00 g-mol ^"1 ): C 34.68; H 4.85; N 6.74; S 30.86. Found: C 34.58; H 4.80; N 6.81 ; S 30.88.

1 H-NMFt (CDCI ₃ , 300.13 MHz): δ (ppm) = 1 .50 (m, 10H, H ₍₄₎ ), 1 .83 (m, 20H, H ₍₃₎ + H ₍₅₎ ), 3.03- 4.65 (m, 20H, H ₍₂₎ + H ₍₆₎ ).

Medium FT-IR (KBr): v (cm ¹ ) = 2933.43 (v _a , C-H); 1503.23, 1441 .03 (v _a , N-CSS); 1001 .63 (v _a , CSS).

Far FT-IR (nujol): v (cm ¹ ) = 544.23 (v _s , CSS); 406.62 (v _a , Ru-S); 321 .88 (v _s , Ru-S).

ESI-MS m/z, [M-CI ⁺ ] - found (calc): 1003.93 (1003.94)

Example 3: β -[Ru ₂ (ProOtBuDTC) ₅ ]CI

Appearance: dark red solid; Yield: 32 %

R.f. (silica gel, CH ₂ CI ₂ /MeOH 94:6): 0.52

Anal. Calc. for C ₅ oH ₈ oCIN ₅ Oi ₀ Ru ₂ Sio (MW = 1469.64 g-mol ^"1 ): C 40.87; H 5.49; N 4.77; S 21 .82. Found: C 40.80; H 5.39; N 4.60; S 21 .88.

¹ H-NMR (CDCI ₃ , 300.13 MHz): δ (ppm) = 1 .41 -2.26 (m, 65H, H ₍₃₎ + H ₍₄₎ + 0-C(CH ₃ ) ₃ ), 3.21 -4.02 (m, 10H, H ₍₅₎ ), 4.36-5.30 (m, 5H, H ₍₂₎ ).

Medium FT-IR (KBr): v (cm ¹ ) = 2975.16 (v _a , C-H); 1735.39 (v, C=0); 1481 .10, 1450.06 (v _a , N- CSS); 1 148.87 (v _a, C-OfBu); 935.12 (v _a , CSS).

Far FT-IR (nujol): v (cm ¹ ) = 551 .30 (v _s , CSS); 471 .19 (v _a , Ru-S); 325.69 (v _s , Ru-S).

ESI-MS m/z, [M-CI ⁺ ] - found (calc): 1434.16 (1434.12).

Example 4: fi-[Ru ₂ (PipeDTC) ₄ (DEDT)]Br

By way of example, but not limitation, the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the first preferred embodiment of the present invention does not contain a "cancer-targeting moiety" in the form of carbohydrate. Such compound contains, for example, dithiocarbamato ligands derived from piperidine (DTC= PipeDTC), and ethylenediamine (DTC ^* = DEDT). The compound was characterized using ¹ H-NMR spectroscopy, and elemental and ESI-MS mass analysis.

Appearance: brown solid; Yield: 16 %

R.f. (silica gel, CH ₂ CI ₂ /MeOH 9:1 ): 0.31

Anal. Calc. for C ₂ 9H ₅ oBrN ₅ Ru ₂ Sio (MW = 1071 .45 g-mol ^"1 ): C 32.51 ; H 4.70; N 6.54; S 29.93.

Found: C 32.68; H 4.83; N 6.44; S 30.1 1 .

ESI-MS m/z, [M-Br ⁺ ] - found (calc): 991 .92 (991 .94)

SYNTHESIS AND CHARACTERIZATION OF GLYCO-CONJUGATED COMPLEXES

It is another object of the present invention a process for the glyco-conjugation of the coordination compounds according to the present invention, whose features are set forth in the enclosed independent claim.

In the second preferred embodiment, the coordination compounds of Formula I (a) and Formula l(b) contain dithiocarbamato ligands functionalized with carbohydrates which act as a selective cancer-targeting moiety towards cancerous cells in vivo, advantageously exploiting the Warburg effect.

The glycoconjugation process involves a number of synthetic steps for the functionalization of a specific amino precursor with an likewise specific carbohydrate. Such amino precursor is subsequently converted to the corresponding dithiocarbamato (DTC) ligand, through the phase a) of the procedure of the first embodiment. In particular, the synthesis involves the protection of the hydroxyl groups of carbohydrate with protecting groups which are orthogonal to the reaction conditions of the subsequent synthetic steps. The carbohydrate {e.g., glucose, mannose) is functionalized {e.g., in position 1 or 2) with a molecular fragment containing an amine, preferably secondary, which is able to react with CS ₂ to form a DTC ligand, ready for the subsequent complexation to the metal center (Ru, Ga). Alternatively, such amine group or other functional group, for example -OH, is not directly used to prepare the dithiocarbamato ligand, but its reactivity could be exploited to bind a molecular fragment which, depending on the molecular design, may have different length and chemical composition. However, said molecular fragment must contain, after one or more synthetic steps, an amine group (that is, the precursor amine, as an example a proline) on which the CS ₂ -based reaction will be carried out to form the DTC ligand. The deprotection of protecting groups can be performed by means of chemical or biochemical methods including the use of enzymes or pseudoenzymes. Pseudoenzymes are generally referred to proteins, such as the HSA (Human Serum Albumin), which may hydrolyze, for example, ester substrates due to the presence of several nucleophilic residues (such as lysine) on their surface, but which do not return to the native state after the hydrolysis reaction.

Scheme 1. Example of functionalization in position CI: a) NaN ₃ , acetone, 56°C, crystallization, 60%; b) H ₂ /Pd, MeOH/EtOAc, 98%; c) Z-Sar-OH, N-methyl morpholine, isobutylchloroformiate, TH F-dry, -15°C, 83%; d) NaOMe, MeOH-dry, acid resin, rt, 100%; e) TMS-CI, Hexamethyldisilazane, pyridine, CH ₂ CI ₂ , 0°C, flash chromatography, 85%; f) H ₂ /Pd, MeOH/EtOAc, 90%; g) CS ₂ , KOH, MeOH, 100%.

During the synthetic process, in addition to the glyco-conjugation, various types of reactions can be conducted. For example: nucleophilic substitution, peptide coupling, protection or deprotection processes, redox reactions, formation of iminic or nitrile intermediates, reduction with H ₂ , hydrolysis. By way of example, but not limitation, the Scheme 1 reported below depicts the synthesis of a dithiocarbamato ligand (a-g), bearing a glucose moiety functionalized in position 1 of the pyranosic ring (β-amido-glycoside).

Scheme 2. Example of functionalization in position C2 : c) Z-Pro-OH, N-methyl morpholine, isobutylchloroformiate, THF-dry, -15°C, 83%; d) NaOMe, MeOH-dry, acid resin, rt, 100%; e) TMS-CI, Hexamethyldisilazane, pyridine, CH ₂ CI ₂ , 0°C, flash chromatography, 85%; f) H ₂ /Pd, MeOH/EtOAc, 90%; g) CS ₂ , KOH, MeOH, 100%. The Scheme 2 reported below shows the synthesis of a dithiocarbamato ligand {c-g), being a glucose derivative functionalized in position C-2 (glucosamide, namely an amide derivative of theglucosamine). By way of example, but not limitation, the schemes 3, 4 and 5 show the syntheses of carbohydrate derivatives having an amino function (-NH ₂ ) in C3, C4 and C6 positions, respectively. Such amine function can in turn be functionalized with a molecular fragment on which the dithiocarbamato group is synthesized, or alternatively, such amine group can be directly converted to dithiocarbamate by reaction with CS ₂ or may be advantageously modified in a different functional group, for example a secondary amine or an isothiocyanate, in order to conduct subsequent reactions for the bioconjugation of the metal.

Scheme 3. Example of fu nctionalization in position C3 : i) Ac ₂ 0/py, -20°C; ii) Tf ₂ 0/py, -15°C, iii) NaN ₃ , rt; iv) H ₂ /Pd/C, rt.

On the basis of the reaction Scheme 3-4 herein disclosed, it will be apparent to those skilled in the art that only obvious changes to the procedure are required in case the functionalization involves other carbohydrates, or other positions (depending on the type of carbohydrate), or also in case the synthesis is designed to obtain different amino precursors.

The synthesis of the organic part, the amino-precursor of the carbohydrate-functionalized dithiocarbamato ligand, is particularly challenging. In fact, some synthetic approaches have not been successful.

Some examples of coordination compounds having Formula I (a) or l(b) containing glucides or carbohydrates in one or more dithiocarbamato ligands are reported below, by way of example, but not limitation.

Scheme 4. Example of functionalization in C4: i) BnBr/py, rt; ii) NaCNBH ₃ /TFA, 0°C; iii) MsCI, rt; iv) NaN ₃ 60-7CTC; v) H ₂ /Pd/C, rt.

Scheme 5. Example of functionalization in C6: i) Ph ₃ CCI, BnBr, AICI ₃ , rt; ii) MsCI, rt; iii) NaN ₃ , 60°C; iv H ₂ /Pd/C, rt. EXAMPLES OF COMPLEXES ACCORDING TO THE SECOND EMBODIMENT

Example 5: [Tris(1,3,4,6-tetraacetyl-2-glucosammido-L-prolineDTC)ruthen ium(lll)]

Appearance: dark green solid; Yield: 22%

Anal. Calc. for C ₆₀ H ₈₁ N ₆ 0 ₃ oRuS ₆ (MW = 1659.77 g-mol ^"1 ): C 43.42; H 4.92; N 5.06; S 1 1 .59. Found: C 43.53; H 4.99; N 4.98; S 12.01 .

¹ H-NMFt (CDCI ₃ , 300.13 MHz): δ (ppm) = 0.65-2.24 (m, 12H, broad), 2.03, 2.08, 2.09, 2.17 (36H, s); 4.06-4.10 (3H, ddd); 4.26-4.30 (3H, dd); 4.48-4.51 (3H, dd); 4.91 (3H, t); 5.10 (3H, t); 5.18 (3H, t); 5.40 (3H, t); 6.79 (3H, m). 23.61 -33.1 (6H, broad), 40.07-44.32 (3H, broad).

Medium FT-IR (KBr): v (cm ¹ ) = 1720 (v, C=0); 2975.22, 2872.34 (v _a , C-H); 1440.10 (v _a , N- CSS); 933.50 (v _a , CSS).

Far FT-IR (nujol): v (cm ¹ ) = 547.60 (v _s , CSS); 470.56 (v _a , Ru-S); 320.98 (v _s , Ru-S).

By way of example, but not limitation, the Example 4 refers to a heteroleptic coordination compound having Formula l(b) which in the second preferred embodiment of the present invention contains a "cancer-targeting moiety" in the form of carbohydrate. Such compound contain, for example, dithiocarbamato ligands derived from: piperidine (DTC= PipeDTC), and (DTC ^* = 1 -0-(A/-methylethanolamine)^-D-glucopyranoside dithiocarbamate = MAEGIuOHDTC) The compound was characterized using ¹ H-NMR spectroscopy, and elemental and ESI-MS mass analysis.

Example 6:

Appearance: brown solid; Yield: 1 1 %

R.f. (silica gel, acetone/H ₂ 0 1 :1 ): 0.56

Anal. Calc. for C ₃ 4H ₅₇ BrN ₅ Ru ₂ Sio (MW = 1234.55 g-mol ^"1 ): C 33.08; H 4.65; N 5.67; S 25.97. Found: C 32.90; H 4.73; N 5.54; S 25.81 .

ESI-MS m/z, [M-Br ⁺ ] - found (calc): 1 154.94 (1 154.97)

EVALUATION OF THE BIOLOGICAL ACTIVITY

Antitumor activity in vitro

The human tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) were cultured in RPMI-1640 and Hams-F12 medium, respectively. The cells (8x10 ³ / mL) were seeded in 96-well plates in the growth medium previously mentioned (100 μΙ_) and then incubated at 37 °C in a controlled atmosphere of carbon dioxide. After 24 hours, the cell culture medium was removed and replaced with fresh one containing the tested compound, previously dissolved in DMSO (0.1 % v/v, freshly prepared solution and keeping in the dark), or saline solution, at various concentrations. A total of four independent experiments were conducted and, after 24 hours of treatment, the medium was aspirated, and each well was treated with 100 μΙ_ of a MTT (Methylthiazolyldiphenyl tetrazolium bromide) solution (1 mg/mL in PBS) and the plate was incubated at 37 °C for 4 hours. The MTT solution was then removed and 100 μΙ_ of DMSO were added to each well. The plate was shaked for 10 minutes and then analyzed by spectrophotometry (560 nm).

The cytotoxic activity of each compound was evaluated as a percentage of vital cells in the treated sample compared to the cells treated with the vehicle only (control). From the obtained dose-response curves, the IC ₅₀ values for each compound were calculated {i.e. the concentration expressed in μΜ, which inhibited 50% growth in tumor cells compared to the control).

All the above mentioned culture media and the human tumor cell lines can be easily purchased on the market. By way of example, but not limitation of the present invention, the following table collects the IC ₅₀ data of some compounds of the first and second embodiments, the corresponding dithiocarbamato ligands (DTC) and metal precursors.

Table 1: In vitro cytotoxic activity (IC _50, expressed in μΜ) of some compounds and DTC ligands against the h uman tumor cell lines MeWo (malignant melanoma) and LoVo (colon adenocarcinoma) after 24 hours of treatment; against the human tumor cell lines: cervix adenocarcinoma (HeLa), colon neoplasia (HCT116), HepG2 : epithelial cells of human liver hepatoma and its more aggressive cou nterpart HepG2/SB3, overexpressing the anti-apoptotic protein SerpinB3; NSC lung carcinoma (A-549); acute T cell leukemia (Jurkat); AGS: gastric adenocarcinoma; triple negative breast cancer (crl2335) after 72 hours of treatment. The inorganic reference-drug cisplatin (Sigma-Aldrich) was tested under the same experimental conditions . IC ₅₀ : concentration expressed in μΜ able to inhibit the 50% of the cancer cell growth compared to the control (cells treated with the vehicle). The data represent the mean ± SD of at least four independent experiments.

From the exemplary and non-limiting data shown in Table 1 , it is clear that the inventors have demonstrated that metal precursors alone do not induce any significant antitumor activity. In fact, they show an IC ₅₀ value > 15 μΜ. Similarly, the dithiocarbamato ligands (DTC) alone do not possess remarkable cytotoxic activity. Conversely, the coordination compounds obtained from such precursors, in which the metal is covalently bound to specific DTC ligands, are characterized by a remarkable cytotoxic activity.

Thus, through the present invention, the inventors have shown that such antitumor activity is due to the combination, in a single compound, of a properly-designed DTC ligand with a metal center endowed with specific chemical (oxidation state, reduction potential, geometry coordination, kinetic and thermodynamic properties) and biochemical properties.

It will be apparent to the skilled in the art that a further important object of the present invention has been achieved.

From the data shown in Table 1 , it is also clear that the present inventors have obtained a number of coordination compounds according to the present invention which exhibit higher anti- tumor activity than the reference drug cisplatin. Indeed, as it is well known, the greater the antiproliferative activity, the lower the IC ₅₀ value for a specific compound. In this way, another important aim of the present invention has been achieved.

Acute toxicity test in mouse model

One compound was tested in vivo to evaluate the acute toxicity by intravenous administration of a single dose to male mice (provided by Charles River Laboratory Italy, Calco, Lecco; 6-week- old mice). This administration route has been chosen because it presents a reduced barrier to substance absorption, provides a more stringent toxicity measure compared to other in vivo assays and it is a typical route of administration in humans. The species/strain mouse/CD-1 ^® was chosen because many regulatory authorities accept and indicate that preclinical acute toxicity tests are performed with this species/strain, in light of the large amount of related bibliography.

The administered dose (10 mg/kg) was selected based on the dosages clinically used on humans {e.g., 2 mg/kg) in order to highlight potential signs of toxicity.

The tested compound was: β-[Ρυ ₂ (ΡίρβΟΤΟ) ₅ ]ΟΙ. The chosen vehicle was DMSO-EtOH-RL 50:10:40 % v/v in which RL stands for Ringer lactate (Eurospital ^® ). Each experimental group consisted of 6 mice + 4 control animals (vehicle-treated only).

Each dose was prepared by dissolving a calculated amount of compound in DMSO; this volume was subsequently diluted with ethanol and Ringer lactate (RL) to obtain the final desired concentration. The administration of the antitumor compound under study was performed by intravenous injection into the caudal artery of each mouse. Each treated animal received an accurate injected volume of 100 μί, containing the amount of test substance as described above, that is the equivalent of 10 mg/kg. All animals were treated with a single dose at T ₀ after detecting the body weight of each mouse. Clinical observations were recorded at the time of injection, during the first hour and then in the following days for total 7 experimental days, the same for the body weight.

Table 2. Body weights report of the treated and control mice. 2day 3day 4day 5day 6day 7day

Group ID sex 1 h 4h 1day

s s s s s s

1 M S P P P P P P P P

2 M P P P P P P P P P

Control

3 M P P P P P P P P P

4 M P P P P P P P P P

1 M s P P P P P P P P

2 M P P P P P P P P P p-[Ru ₂ (PipeDTC) ₅ ]CI 3 M P P P P P P P P P 10 mg/kg/iv 4 M P P P P P P P P P

5 M s P P P P P P P P

6 M P P P P P P P P P

Legend:

ID: mice identification; Grade code: P = Present; S = low; M = Moderate; V = Severe; D = Death. Sex: F = Female; M = male.

Table 3: Clinical and behavioral observations during the experimental study .

At the end of the study period, animals were sacrificed by C0 ₂ asphyxiation. All animals were subjected to autopsy examination including the opening of the cranial, thoracic and abdominal cavities.

All animals survived until the end of the study, and during the post-treatment period they showed no sign of toxicity. The macroscopic necroscopy did not reveal any evident sign of toxicity on the examined organs: brain, lungs, spleen, testes, heart, liver and kidneys.

PREPARATION OF SUPRAMOLECULAR AGGREGATES ENCAPSULANTING ANTICANCER COORDINATION COMPOUNDS

It is another matter of the present invention a process for encapsulating in supramolecular aggregates the coordination compounds according to this invention, whose features are set forth in the enclosed independent claim. Particularly, in the third and fourth preferred embodiment of this invention, the coordination compounds having the general formula l(a) and l(b) are encapsulated in supramolecular aggregates.

By way of example, but not limitation, advantageous supramolecular systems encapsulating the active compound can be micelles, vesicles (liposomes), cyclodextrins, dendrimers, organic polymeric nanoparticles, for instance derived from proteins or peptides, and inorganic nanoparticles {e.g., silica, zirconia, titanium dioxide).

Said supramolecular architectures can be in turn produced with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan, polyethylene glycol (PEG), mPEG acid, poly(lactic-co-glycolic acid) (PLGA), Pluronic®, cholesterol, derivatives of phosphatidylcholine and phosphatidylethanolamine, Cremophor®, pullulan, hyaluronic acid, ferritin, human serum albumin (HSA), heparin, dextran, polyaminoacids {e.g., a- poly-L-glutamic acid) and their derivatives {e.g., PHEA, PHEG), polyglycerol, polyacrylamide, polyvinylpyrrolidone, poly(2-oxazoline) and their derivatives. Moreover, even combinations of these polymers or molecules, such as PEG-PLGA or PEG Pluronic®, may be used for the construction of appropriate supramolecular architectures, usable as nanocarriers for the coordination compounds according to the present invention.

In addition, block copolymers, such as those with the A-B-A or A-B architecture, can be advantageously used to prepare supramolecular systems suitable for the delivery of the coordination compounds herein disclosed. In other words, said building blocks, which can be equal or different each other, can be intended both as the monomer (e.g., ethylene oxide) of a polymer such as PEG or PF127, or of an inorganic nanostructure {e.g., Ti0 ₂ ), and the same polymer inside aggregates, for instance micelles.

The terms "supramolecular aggregates", "macromolecules", nanostructures encompasses also for example micelles, liposomes, protein- and peptide-based aggregates and carriers consisting of cyclodextrins or dendrimers.

Moreover, as an example, biocompatible polymers and oligomers, natural or synthetic, identical or different {e.g., phospholipids, pullulan, PEG, PF127, cholesterol) can form micelles or liposomes, in order to modulate the final properties of the composition and therefore of the final formulation. These polymers, identical or different, are the constituent units that aggregate during the formation of the supramolecular system. One of them or all of these polymers can be conveniently functionalized with carbohydrates (by using the functional groups exploitable for this purpose), and in an independent manner, with yields up to 100%. This/these polymer(s) can then be diluted with the same or other polymers to achieve, for example, a micelle or a liposome with a water-exposed surface having a variable percentage of functionalization, ranging from 1 to 50% mol/mol.

Concerning the drug loading, the range 10 ^"5 ÷10 ^"10 mol of compound/mg of formulation can be achieved when preparing compositions of said coordination compound. Furthermore, said compound can be encapsulated in the hydrophobic core of a micelle, or in the hydrophilic counterpart of a liposome, or in their lipidic/polymeric layer.

Such supramolecular architectures can in turn be made with different polymers, natural and synthetic, as well as with proteins or other organic molecules, such as chitosan and polyethylene glycol. The nanocarrier {e.g., liposome, micelle) can be conveniently covered with a hydrophilic and biocompatible coating in order to improve the pharmacokinetic profile of the "naked" supramolecular aggregate itself. In other words, by exploiting the so-called "stealth effect", to date known for polymers such as PEG and poly-amino acids, the surface of the supramolecular aggregate is hidden from blood components, including the opsonin proteins, which are responsible for the recognition and attack of the nanocarriers by phagocytes (monocytes, macrophages). This masking strategy allows the supramolecular systems to sidestep the natural processes of biotransformation/elimination of exogenous constituent entities or substances. Moreover, it determines an increased bioavailability and, hence, a prolonged circulation time of the compound loaded into the carrier, if compared to supramolecular aggregates incorporating the coordination compounds according to the present invention and non-covered with said hydrophilic and biocompatible coating. The application of the "effect stealth" to the coordination compounds according to the first and the second embodiment of the present invention may also limit or eliminate any problem at the physiological level, such as hemolysis or immunogenicity of the active compounds. In addition, the hydrophilic coating of nanocarrier obtained with polymers such as PEG, reduces the self- aggregation of the particles by means of steric stabilization, thus resulting in an immediate impact on the freshly-prepared formulation for the administration at the hospital level and storage.

In an alternative manner, and to achieve the same benefits, the surface of the nanocarrier can be coated, by adsorption or conjugation, with human serum albumin (HSA) in order to conveniently increase the stability, the circulation times, and the biocompatibility of the formulation. This approach "anticipates" indeed the adsorption of HSA present in the blood and, simultaneously, it avoids the absorption of the aforementioned opsonins.

The whole supramolecular aggregate reduces the off-target release of the drug by the so-called EPR effect (Enhanced Permeability and Retention). The EPR effect is based on the anomalous and high permeability of blood vessels in the tumor region, characterized by not-adherent endothelial cell-to-cell junctions, if compared to normal tissue capillaries. Appropriate nanocarrier sizes, ranging from 20 to 100 nm (as reported in the examples of this invention), result in a prolonged circulation of the supramolecular systems in the blood stream, and the consequent selective extravasation into tumor tissues, resulting in a passive targeted therapy, which positively influences the toxicological profile. In addition, the lack of lymphatic drainage of cancerous compartments promotes the selective accumulation of the nanocarriers in the tumor microenvironment.

Advantageously, the functionalization of the nanocarriers with cancer-targeting moieties can also restrict the onset of side effects, thereby improving the chemotherapeutic index in terms of toxicity/activity ratio (active targeting).

In addition, when the nanocarriers are functionalized with carbohydrates as cancer-targeting moieties, these supramolecular or macromolecular systems give the possibility to increase the water-solubility and the stability of the encapsulated compound(s) as well as to improve the bioavailability, and to extend and facilitate the selective release towards cancer cells.

In summary, by means of the encapsulation of one or more coordination compounds according to the present invention, the inventors have advantageously developed two distinct and complementary strategies for the selective delivery of said compounds in the tumor site. This will be clear from the description of some examples reported below by way of example, but not limitation of present invention.

The first cancer-targeting strategy, associated with the third embodiment, is related to the passive targeting mediated by EPR effect described above. On the other hand, the second strategy, presented in the fourth embodiment, takes into account the active targeting via glycoconjugation of the supramolecular nanosystem, thus providing an additional way to achieve a high therapeutic selectivity.

Anyway, the encapsulation allows the loaded compounds to increase their blood circulation times, as well as to improve the chemotherapeutic index of the active compounds (ratio between the lethal dose LD ₅₀ and the effective dose ED ₅₀ ), in other words their pharmacokinetic and pharmacodynamic profiles.

In order to engineer the most suitable encapsulation system (e.g., micelle, liposome), the partition coefficient n-octanol/water (P) was evaluated (and expressed in its logarithmic form, logP) for some coordination compounds with general structure l(a) and l(b), below described as by way of example, but not limitation of the present invention. It is known that the value P expresses the ratio between the concentration of a compound in n-octanol and the concentration of the same compound in water, at equilibrium.

EXAMPLES OF NANOFORMULA TIONS FOR PASSIVE TARGETING

In the third preferred embodiment of the present invention, the coordination compounds having a general Formula l(a) and/or Formula l(b) are encapsulated in supramolecular aggregates, in particular micelles, to achieve a passive targeting mechanism mediated by the EPR effect. Said encapsulated compounds may be those of the second embodiment, and hence conjugated to carbohydrates, or without a cancer-targeting moiety (first embodiment). For pharmaceutical applications, the ideal size of supramolecular systems ranges from 10 nm to 100 nm. In particular, a fast drainage from the injection site and an effective distribution in vivo are obtained with particles characterized by a hydrodynamic diameter (D _H ) ranging from 10 to 70 nm. In addition, once reached the target cells, the nanocarriers with D _H < 100 nm at the cellular level can be absorbed by an endocytic process. In fact, it is well known that the size of the aggregates can significantly affect the circulation times and the bioavailability of the encapsulated compound.

From the description provided above, by way of example but not limitation, it will be clear that, the inventors have advantageously showed the possibility to achieve nanoformulations characterized by different hydrodynamic diameters (D _H ) with an average value of about 25-30 nm, being of extreme interest for pharmaceutical applications .

LogP evaluation procedure

By way of example, but not limitation, the procedure to evaluate the partition coefficient n- octanol/water (P) (expressed in its logarithmic form, logP) of some coordination compounds having general structure l(a) and l(b) is here described. It is known that this procedure is fundamental for the choice of the most suitable supramolecular system for a specific type of compound and/or application. Going into detail, the concentration of the compounds in the two immiscible phases has been determined in n-octanol before and after mixing with a defined volume of water. In particular, n-octanol was pre-saturated with deionized water for 24 hours under stirring, then let to equilibrate for 6 h at 25 °C. A defined amount of compound is weighted and dissolved in a determined volume of the organic phase, and then stirred at 25 °C for two hours after the addition of the same volume of deionized water. Subsequently, the mixture is equilibrated for 30 minutes. The concentration of compound in the organic phase before (C ₀ ) and after the separation (Ci) was evaluated by UV-Vis spectrophotometry. The obtained values were used in the calculation of the partition coefficient n-octanol/water (P) according to the formula logP= log[Ci / (C ₀ - Ci)]. The results are reported for some compounds in Table 4.

Table 4. Experimental logP= log[Ci / (C ₀ - Q)] va l ues of some coordination com pou nds.

Considering the logP values reported in Table 4 and the IC ₅₀ data recorded in vitro (Table 1 , with reference to the first and second embodiments), it appears evident to the skilled in the art that the here claimed coordination compounds are associated with different LiPE values. This parameter is related to the Lipophilic Efficiency and it is defined by the formula LiPE= - log IC ₅₀ - log P. Some of the analyzed derivatives are characterized by LiPE values ranging from 4 to 6, which are considered ideal for the purposes of the so-called "druglikeness".

Encapsulation in micelles

A procedure for the encapsulation (loading) in micelles of some compounds according to the invention having logP > 0 is described below. Without loss of generality, the inventors have developed a loading system using Pluronic® F127 (indicated also as "PF127") as a polymeric substrate. However, this procedure is basically similar for any amphiphilic polymer (e.g., mPEG), with trivial changes to the skilled person.

The encapsulation of the complexes having Formula l(a) and/or Formula l(b) in PF127 micelles was obtained via a process comprising the following steps, described herein by way of example, but not limitation of the present invention: 1 ) Co-dissolution of the compound to be loaded and the polymer in the desired stoichiometric ratio {e.g., 0.5 mg and 500 mg, respectively) in an organic solvent, preferably chloroform; 2) Evaporation of the organic solvent, preferably under reduced pressure, followed by drying of the obtained powder, preferably under vacuum; 3) Hydration of the powder by addition of deionized water; 4) in order to remove the non- encapsulated compound, bacteria and other impurities, purification for instance by filtration with a membrane with a 0.20 μηι cut-off; 5) Freezing of the sample in a dry ice/acetone cooling bath at -78 °C and cryoesiccation to remove the residues of the aqueous solvent, thus obtaining a storable ready-to-use formulation.

The amount of the encapsulated coordination compound was assessed by UV-Vis analysis, after dilution in DCM of a defined amount of lyophilized micellar formulation. In particular, the concentration of the compound was defined using the Lambert-Beer law, after experimental determination of the molar extinction coefficient ε in DCM (based on the "matrix effect", in the presence of the same polymer used for the preparation of micelles) for some absorption bands. The obtained results are reported in Table 5.

Table 5. Amou nt of encapsu lated compou nd in terms of d rug loading (mol of encapsu lated compou nd per mg of formu lation) a nd Encapsu lation Ratio (ER), i. e., the percentage a mou nt of encapsu lated compou nd compa red to the amou nt of nomi nal ly-loaded compou nd .

Advantageously, the encapsulation in micelles of the metal-based compounds according to the present invention increases their stability in physiological media and make them water-soluble for at least 72 hours. In fact, with reference to the appended Figure 2, here illustrated by way of example, but not limitation, the collected electronic spectra surprisingly show no significant change over time for the considered formulations (if compared to the not encapsulated compound) once dissolved in a physiological environment consisting of phosphate buffer/cell culture medium 9: 1 v/v, and phosphate buffer/human serum 95: 5 v/v.

Hence, it is evident that the present inventors have obtained compositions comprising the coordination compounds according to the present invention, characterized by a complete solubility and stability in physiological media compared to the corresponding non-encapsulated compounds, thus achieving a further object of the present invention.

Similarly, these nanoformulations, engineered and finely tailored, are unexpectedly stable also in a chemically aggressive medium, such as that charactering the tumor microenvironment, here mimicked with an acetate buffer (pH 5.5, Figure 3).

The in vitro experiments highlight also that the high stability of these formulations does not prevent the release of the active compound, followed by its cellular uptake, or alternatively, it does not prevent the release of the active compound associated with the absorption of the entire load.

It is clear from the results presented in the following Table 6 that micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus exploiting to the best the EPR effect. As already mentioned, the use of PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.

Table 6: Structura l parameters of the micel la r systems here reported, determined via DLS ana lysis. PDI= polydispersion index.

It is clear from the results presented in the Table 6 that micellar systems with hydrodynamic diameter of about 25 nm are particularly advantageous for pharmaceutical applications, in particular in the oncological field for the intravenous administration, thus exploiting to the best the EPR effect. As already mentioned, the use of PF127 does not limit the scope of the present invention, since other polymeric substrates can be conveniently used.

Through the non-limitative examples of the present invention described above, the inventors have surprisingly demonstrated the possibility to encapsulate the coordination compounds of general Formula l(a) and l(b) in macromolecules or in a supramolecular system, thus overcoming in a non-trivial manner evident limits in the state of the art. In fact, the encapsulation techniques of the metal-based derivatives are still at the beginning, due to the inherent reactivity of the inorganic molecules.

In this way, the encapsulation allows to protect said compounds from the biotransformation, avoiding interactions/reactions with blood stream biomolecules or cells. In addition, the encapsulation allows increasing the stability as well as the solubility of hydrophobic compounds in the physiological media, as the here-presented data demonstrate.

EXAMPLES OF NANOFORMULA TIONS FOR THE ACTIVE TARGETING

In a fourth preferred embodiment, herein illustrated by way of example, but not limitation of the present invention, the coordination compounds having general Formula l(a) and l(b), are advantageously encapsulated in supramolecular aggregates and functionalized with carbohydrates. Therefore, they are directed to the tumor site according to an active-targeting approach which is paralleled with (hence boosting it) the mechanism of passive targeting of the previous embodiment. In other words, in the fourth embodiment the whole supramolecular aggregate acts as a cancer-targeting carrier, conveniently exploiting the Warburg effect, to further improve the therapeutic selectivity. In particular, the inventors aim to implement a mechanism wherein a coordination compound according to the invention is absorbed by the cell by endocytosis along with the whole supramolecular aggregate; alternatively, said compound can be released from the nanocarrier in the extracellular matrix, and consequently diffusing inside the cell if hydrophobic enough, or entering the cell through alternative mechanisms of uptake, or also triggering its antitumor activity at the cell membrane level. In the latter case, compounds of the first embodiment, which by their chemical nature inherently exhibit affinity for the cell membrane, or compounds of the second embodiment that can exploit the presence of a cancer-targeting moiety T in the selective recognition by carbohydrate transporters {e.g., GLUT1 ), are advantageously used, alternately or in combination between them. Such compounds are hence encapsulated exploiting the procedures disclosed by the inventors with reference to the third embodiment so to form a supramolecular aggregate with a variable degree of functionalization, for example ranging from 1 to 50%. By way of example, but not limitation, convenient supramolecular aggregates can be micelles, vesicles (liposomes), cyclodextrins , dendrimers, organic polymeric nanoparticles, for instance of protein or peptide nature, and inorganic nanoparticles {e.g., silica, zirconia, titania). Carbohydrates can be monosaccharides, polysaccharides, pentoses, hexoses, aldoses, ketoses. Such supramolecular aggregates may have a mixed structure, for example being made up of pullulan and PF127, or PLA and PEG.

FUNCTIONALIZA TION OF BIOCOMPA TIBLE POL YMERS

With reference to the scheme reported below, the following non-limitative examples illustrate some synthesis that demonstrate the possibility to functionalize with different chemical groups the hydrophilic terminal end of biocompatible polymers, such as the polyethylene glycol (PEG) and the family of polymers known under the trade name Pluronic® F127. These functionalized derivatives are subsequently bio-conjugated with specific carbohydrates, in turn suitably functionalized, advantageously exploiting known synthetic strategies.

The biopolymer so functionalized with a carbohydrate, after dilution with a non-functionalized biopolymer (equal or different), is used to form a supramolecular aggregate with a degree of functionalization, ranging from 1 % to 50% mol/mol. By way of example, but not limitation, the schemes and the descriptions of some reactions carried out starting from the methoxy polyethylene glycol (mPEG) 5000 polymer are reported.

Synthesis of mPEG5000-Ts

To a solution of mPEG5000 in dry CH ₂ CI ₂ dry at 0 °C, 0.5 eq. of 4-dimethylaminopyridine (DMAP) and 10 eq. of triethylamine (Et ₃ N) were added. Then, after the addition of 10 eq. of p- toluensulfonyl chloride (TsCI) the reaction was stirred for 24 hours. The mixture was washed with HCI 0.1 M and an aqueous solution saturated with NaCI ("brine"). After treatment of the organic phase with diethyl ether, a white product precipitated. The functionalization degree was equal to 100%, considering the integration values of NMR signals corresponding to the aromatic protons of the tosylate and the methyl protons of the terminal methoxy group of mPEG5000 polymer.

I : v (cm ⁴ ) = 2885, 1467, 1344, 1113, 1060, 842, 664.

Synthesis of mPEG5000 -Phta

The polymer mPEG5000-Ts was refluxed in dry DMF for 5 hours in presence of 20 eq. of phthalimide potassium salt (Phta K). Successively, after filtration of byproducts and precipitation with diethyl ether, a light yellow solid was isolated. The functionalization degree was equal to 100%, in light of the integration values of the NMR signals corresponding to the aromatic protons of the phthalimide and methyl protons of the terminal methoxy group of mPEG5000 polymer.

IR: v (cm ¹ ) = 2885, 1716, 1467, 1344, 1 1 14, 1060, 842, 724, 690.

Synthesis of mPEG5000-NH ₂

This Gabriel-like synthesis of amines was carried out according to a modified literature procedure. 50 eq. of hydrazine (NH ₂ NH ₂ ) were added to a solution of mPEG5000-Phta in EtOH at reflux. After 16 hours the crude product was precipitated with diethyl ether, leading to a white solid. The functionalization degree (-NH ₂ terminal group) is equal to 45%, based on the integration values of the NMR signals corresponding to the a-methylene protons with respect to the amino group (CH ₂ -NH ₂ ) and methyl protons of the terminal methoxy group of mPEG5000 polymer.

IR: v (cm ¹ ) = 2884, 1467, 1344, 1 1 13, 1060, 842.

Synthesis of mPEG5000-SC

This reaction allows the conversion of the terminal hydroxyl group to a carbonate which is very reactive towards nucleophiles due to the presence of a succinimido leaving group. The reaction was performed in a Schlenk line. Briefly, mPEG5000 was dissolved in dry 1 ,4-dioxane and was added to a mixture of DMAP (6 eq) and Ν,Ν'-disuccinimmidil carbonate (DSC) in dry acetone. After 6 hours, the by-products were filtered and the solution volume was reduced to precipitate a white solid after the addition of diethyl ether. The functionalization degree is 100%, considering the integration values of NMR signals corresponding to the methylene protons of the succinimido group and the methyl protons of the terminal methoxy group of mPEG5000 polymer.

Synthesis of mPEG5000-GluOAc

The bio-conjugated mPEG5000-GluOAc was obtained via nucleophilic attack of the terminal hydroxyl group of the polymer to the anomeric C1 carbon of 1 ,2,3,4,6-penta-0-acetyl^- glucopyranose in the presence of BF ₃ Et ₂ 0. In particular, 3 eq. of BF ₃ Et ₂ 0 were added to a mixture of mPEG5000 and 1 ,2,3,4,6-penta-0-acetyl^-D-glucopyranose (3 eq.) in dry CH ₂ CI ₂ dry at 0 °C, and the mixture was stirred for 48 hours. Then it was concentrated under vacuum and precipitated with diethyl ether, leading to the isolation of a white solid. The functionalization degree was equal to 20%, based on the integration values of NMR signals corresponding to the methyl protons of the acetate and the methyl protons of terminal methoxy group of mPEG5000 polymer.

IR: v (cm ¹ ) = 2887, 1759, 1467, 1344, 1 1 1 1 , 1060, 842.

Synthesis of mPEG5000-GluOH

The polymer mPEG5000 functionalized with β-D-glucopyranose was obtained from the product of the previous step (mPEG5000-GluOAc) via deprotection of the acetyl groups under basic conditions (NaOMe in dry methanol) under stirring for 15 hours. Then, the reaction mixture was neutralized with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After the filtration of the resin, the product was precipitated with diethyl ether and dried under vacuum. IR: v (cm ¹ ) = 3453, 2886, 1759, 1467, 1344, 1 1 12, 1060, 842.

The obtained polymer results 20% functionalized as O-glycoside (in C1 position), and can be used as such to encapsulate the coordination compounds, thus forming micelles or aggregates.

Alternatively, it may be used in diluted form in combination with other biocompatible polymers to obtain cancer-targeting nanoformulations such as, without loss of generality: mixed micelles, liposomes, HSA nanoparticles. The latter, covered with a carbohydrate-functionalized mPEG, realize a "stealth effect", which is known to be associated with a decreased opsonization.

Similarly, the reaction scheme related to the PF127 polymer herein illiustrated as non-limitative example of the present invention is reported.

Synthesis of PF127-CHO

Accordin to the following reaction scheme:

PF127-CHO

80%

The oxidation of the terminal hydroxyl groups of the PF127 polymer to aldehyde was performed using the Parikh-Doering oxidation method. Briefly, a DMSO solution of 3 eq. of Py S0 ₃ (Py = pyridine) was reacted with a DMSO solution of PF127 (1 eq.) and Et ₃ N (6 eq.) at 20 °C. After 24 hours, the mixture was diluted in CH ₂ CI ₂ and washed with an aqueous solution saturated with NaCI ("brine"). The organic phase was then concentrated and treated with diethyl ether, thus isolating a white solid. The degree of oxidation to aldehyde (evaluated via ¹ H-NMR in CD ₂ CI ₂ ) was equal to 80%, considering the integration values of NMR signals corresponding to the aldehyde proton and the methyl protons of the central PPO unit of the PF127 polymer.

IR: v (cm ¹ ) = 2884, 1630, 146, 1344, 1 1 15, 1061 , 842.

Synthesis of PF127-GlnOAc

According to the following reaction scheme:

PF127-CHO

PF127-GlnOAc has been obtained by reductive amination between the PF127-CHO derivative described above and 1 ,3,4,6-tetra-0-acetyl-2-amino-2-deoxy^-D-glucopyranose HCI. In particular, the first step involves the nucleophilic attack of the glucosamine, reaction carried out in acetonitrile for 2 hours at room temperature. Subsequently, the reduction of the imine to amine was obtained by the addition of sodium cyanoborohydride. The reaction occurs in acetonitrile for 30 minutes at room temperature with a total stoichiometric ratio 1 :1 :3 between PF127-CHO, glucosamine- HCI and NaCNBH ₃ , respectively. Subsequently the solvent was removed under reduced pressure and the residue is taken up in DCM, washed in a separatory funnel with "brine". The organic phase is anhydrified and the product precipitated with diethyl ether.

The degree of functionalization (assessed via ¹ H-NMR in CD ₂ CI ₂ ) was equal to 44%, considering the integration values of NMR signals corresponding to the acetate protons of glucosamine and the methyl protons of the central PPO unit of the PF127 polymer.

¹ H-NMR (600 MHz, CD ₂ CI ₂ ): δ (ppm) = 1 .10 (t, 241 H, CH ₃ PF127), 1 .99 (s, 3H, COCH ₃ ), 2.03

(s, 3H, COCH ₃ ), 2.12 (s, 3H, COCH ₃ ).

IR: v (cm ¹ ) = 2883, 1757, 1467, 1344, 1 1 14, 1060, 842.

Synthesis PF127-GlnOH

The polymer PF127 functionalized with 2-amino-2-deoxy-D-glucose (glucosamine) was obtained from the PF127-GlnOAc of the previous step by deprotection of the acetyl groups under basic conditions (NaMeO in dry methanol) under stirring for 15 h. Subsequently, the mixture was treated with an acid resin (e.g., Amberlite® FT form) for one hour under stirring. After filtration, the product was precipitated with diethyl ether and dried under vacuum.

IR: v (cm ¹ ) = 3432, 2885, 1467, 1344, 1 1 12, 1060, 842.

The obtained polymer results 44% functionalized with glucosamine in C2 position and can be used as such to encapsulate the coordination compounds, forming micelles. Alternatively, it may be used in combination with other biocompatible polymers to obtain cancer-targeting nanoformulations. By way of example, but not limitation, mixed micelles or supramolecular aggregates can be prepared starting from this derivative, after "dilution" with non-functionalized PF127, or another biocompatible polymer, in order to obtain different degrees of functionalization of the supramolecular aggregate, preferably in the range 2 ÷ 18%.

Through the synthetic schemes described above, the present inventors prepared formulations wherein the coordination compounds according to the invention are encapsulated in micelles which are functionalized with carbohydrates (Table 7).

Table 7: Structural parameters of the micellar systems herein described, determined by DLS analysis. Surprisingly, all the synthetic procedures allow achieving a degree of functionalization equal or greater than 20%, as verified through ¹ H-NMR analysis in CD ₂ CI ₂ .

Based on the syntheses herein disclosed, it will be apparent to the skilled in the art that he present inventors have overcome relevant issues in the state of the art in a novel and inventive manner. Indeed, although the functionalization of polyether derivatives such as Pluronic® F127 and mPEG is widely described in literature, the syntheses herein reported are often difficult to reproduce due to the high molecular weight of the involved surfactants as well as the viscosity of their solutions in organic media {e.g., DCM or THF). Consequently, all the synthetic strategies need to be adapted and optimized in every specific case. In addition, the purification is often troublesome due to the high chemical similarity between the polymeric reagents and their products, characterized by minimal changes in the molecular structure (in terms of molecular weight and introduced functional groups). Given the complexity of these systems, also the functionalization/bio-conjugation yields are often low (<50%), and the assessment of the degree of functionalization of the products results difficult because of the abovementioned purification problems and the instrument sensitivity.

Although the examples of functionalization herein described are referred to mPEG and PF127, it will be clear to the skilled in the art that the application of the usual synthetic techniques allows the functionalization of other types of biocompatible polymers.

EVALUATION OF THE BIOLOGICAL ACTIVITY

Antitumor activity in vitro

From the description and from the experimental data provided above, the coordination compounds according to the first and second embodiment of the present invention possess a remarkable anticancer activity. Likewise, said compounds loaded into aggregates, according to the third and fourth embodiment, maintain such antitumor capacity, thus achieving a further advantageous object of the present invention.

Indeed, by its nature the encapsulation does not alter the anticancer properties of said compounds, independently on the functionalization of the aggregate itself. In other words, these supramolecular aggregates play only the role of nanocarriers, in particular increasing the stability and solubility of said compounds in aqueous medium, and anyway allowing the release of the active compound within the cancer cell or in the extra-cellular matrix, or in general, in the bloodstream.

Therefore, the previously presented in vitro results concerning the antitumor activity of the coordination compounds, can be extended also to the described formulations, with reference to the third and fourth embodiments.

By way of example and not limitation, the antitumor activity of IC50 micelles loaded with Ru complexes is presented in the following table. Formulation Nanoformulation HeLa HepG2/CTR HepG2/SB3 description

RuA Pure PF127 0.21 ± 0.6 1 .72 ± 0.1 2.4 ± 0.3

RuC PF127-GluOH 1 0% 0.12 ± 0.03 1 .1 1 ± 0.09 1 .4 ± 0.1

RuE PF127-p-(D)- 0.105 ± 0.009 0.89 ± 0.09 1 .07 ± 0.09 glucopyranose 10%

RuG PF127-GlnOH 1 0% 0.12 ± 0.03 0.8 ± 0.1 1 .3 ± 0.2

Rul PF127-p-maltose 0.12 ± 0.06 0.56 ± 0.07 0.99 ± 0,07

10%

Table 8IC ₅₀ values (μΜ) for PF127-based formulations evaluated after a 72-h treatment. Values are calculated based on the concentration of the encapsulated ruthenium-DTC complex. Lyophilized micelles were dissolved in cell culture medium. Data represent the mean ± SD of at least three independent experiments. THERANOSTIC USE OF THE COORDINATION COMPOUNDS AND THE RELATED COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS

Advantageously, the coordination compounds, the compositions, and the pharmaceutical formulations according to the present invention can be used as "theranostic agents" i.e. not only for the treatment of human or animal diseases, in particular neoplastic diseases, but also for the diagnosis and the patient follow-up.

Within the more general scope of the present invention, such mechanism that combines therapy and diagnosis can be made in many ways, properly including one or more contrast agents in a coordination compound, or in a composition comprising at least one of said coordination compound, or also in a pharmaceutical formulation that includes said compound and/or composition.

By way of example, but not limitation of the present invention, referring to the general Formula l(a) or l(b), the contrast agent can be "intrinsic" to the compound, for example when the metal centers M M ₂ , M ₃ are radionuclides {e.g., ⁶⁸ Ga, previously introduced). Similarly, also the donor atoms X, Y, W, Z, L L ₂ , L ₃ and L ₄ are radioisotopes {e.g., ¹³¹ 1 , ¹²⁷ l, ¹²⁹ l), as well as T can be 2-deoxy-2-[ ¹⁸ F]-fluoroglucose. Finally, the coordination compound, the unit A, the carbohydrate or glucide T and/or T is paramagnetic or diamagnetic.

In addition, the contrast agent can be "exogenous" to the compound, and may be included in the composition that comprises at least one coordination compound according to the present invention, or also in a pharmaceutical formulation, that includes said compound and/or composition. Again, as non-limitative example of the present invention, this category comprises a carrier that encapsulates said compound, such as a up-converting bismuth oxide nanoparticle (e.g. Italian patent 0001419393, in the name of BEP Sri et al.). In this case, the contrast agent is a dual-mode contrast agent that take advantage of the X-ray absorption and the optical emission (through a NIR->NIR or NIR->VIS mechanism) exhibited by the doped bismuth oxide nanoparticles. Other organic and inorganic luminescent systems, such as nanoparticles and compounds, as well as their use in the diagnosis and imaging of biological systems are well- known. Therefore, depending on the necessity, the "exogenous" contrast agent may also be a component of the pharmaceutical formulation, for example, a known contrast agent.

A "combined" contrast agent can be used and obtained mixing one or more "intrinsic" contrast agents with "exogenous" contrast agents. For example, radionuclides such as ¹³¹ 1, ¹²⁷ l, ¹²⁹ l may be previously introduced as donor atoms X, Y, W, Z, L L ₂ , L ₃ and L ₄ in the general Formula l(a) or l(b) ; the coordination compound can be linked to a carrier consisting of a photoactivatable nanoparticle, such as a properly doped bismuth oxide nanoparticle.

Other examples will appear or become evident to the skilled in the art, on the basis of the disclosure herein provided, or through practice of the same, and on the basis of the knowledge at the state of the art in the field of material science, devices and equipment for medical or biomedical imaging and diagnosis.

In the various non-limitative examples illustrated here, with the only aim to show that the coordination compound according to the invention can be advantageously used to obtain a compound or a theranostic agent, it is essential that the contrast agent is detectable by suitable devices or detectors, hence to be usable in association with diagnostic and imaging systems in the medical or biomedical field. In other words, said contrast agent must be able to emit a signal detectable spontaneously, or after interaction with, for instance, irradiation with external particles, or with an external magnetic/electric field.

Contrast agents capable of spontaneously emit a detectable signal are, for example, radionuclides such as ^{1 1} C, ¹⁸ F, and ⁶⁸ Ga (in this case the signal is a radiation and/or a particle), or radiopaque materials (the signal here is related to the X-ray absorption spectrum).

Other contrast agents emit a signal which is detectable after an external stimulus, which is typically the irradiation with particles and suitable electromagnetic radiations (based on the needs). As an example, this category comprises: luminescent photoactivable contrast agents, such as nanoparticles, dye-molecules or luminophores, capable of emitting IR-Vis radiation as a result of laser irradiation with a suitable wavelength ; contrast agents capable of emitting a detectable signal due to the presence of dipoles or magnetic domains within their structure, which interact with an appropriate electromagnetic field; it is also possible to generate radionuclides inside of the compound and/or the composition according to the invention, treating them with particles and electromagnetic radiations with the suitable energy, using known techniques.

It will be evident to those skilled in the art, based on the second and fourth embodiments, described above, by way of example and not limitation, the theranostic applications of the coordination compounds having general Formula l(a) and/or l(b) are very promising in light of the high selectivity and bioavailability, being as above presented, related to the presence of the carbohydrate or glucide T and/or T', that acts as cancer-targeting moieties able to implement a passive and active targeting-mechanism towards the tumor cells.

CONCLUSIONS

In conclusion, it is apparent to those skilled in the art that the present invention fully achieved the intended aim and objects by means of the disclosure provided herein.

The invention thus conceived is susceptible of numerous modifications and variations, without departing from the basic concepts as disclosed herein. Moreover, all the details may be replaced with other technically equivalent elements. Furthermore, the order of the process steps described above is shown by way of example, but not limitation and can be changed according to convenience. The above description and drawings are only illustrative of preferred embodiments which achieve the objects, features and advantages of the present invention, and it is not intended that the present invention be limited thereto.

All structural, chemical, and functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. It will be appreciated that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art in view of this specification and which are all considered within the scope of the claimed invention

Although the description and examples above contains many details, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Therefore, any modification of the present invention which comes within the spirit and scope of the following claims is considered part of the present invention.

In the appended claims, reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." Where the characteristics and techniques mentioned in any claim are followed by reference signs, those reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly, such reference signs do not have any limiting effect on the interpretation of each element identified by way of example, but not limitation by such reference signs.

Trademarks

Pluronic® is a trademark of BASF AG;

Cremophor ^® is a trademark of BASF AG;

CD-1 ^® is a trademark of di Charles River Laboratories, Inc. Corp.;

Eurospital ^® is a trademark of Eurospital Spa;

Lipoxal ^® is a trademark of Regulon A.E;

Amberlite ^® is a trademark of Santa Cruz Biotechnology Inc.