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Title:
COORDINATION COMPOUNDS AND FORMULATIONS, PREPARATION METHODS AND USE THEREOF AS ANTIPHLOGISTIC AGENTS
Document Type and Number:
WIPO Patent Application WO/2018/100560
Kind Code:
A1
Abstract:
The present invention concerns Au and Cu-based mononuclear coordination compounds, drug formulations thereof, the relating synthesis and encapsulation method in macromolecules or supramolecular aggregates or nanostructures, as well as their application in the treatment of inflammations, particularly those associated to osteoarthritis and rheumatoid arthritis or other chronic and non-chronic rheumatic (or systemic) diseases. Said compounds and formulations present a promising toxicological profile and an high anti-inflammatory activity, as well as a remarkable stability and solubility in physiological means. Formula (I):

Inventors:
NARDON, Chiara (Via Ceroni 49, Montegalda, 36047, IT)
FREGONA, Dolores (Via Reoso 49, San Pietro Viminario, 35020, IT)
Application Number:
IB2017/057592
Publication Date:
June 07, 2018
Filing Date:
December 02, 2017
Export Citation:
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Assignee:
NARDON, Chiara (Via Ceroni 49, Montegalda, 36047, IT)
FREGONA, Dolores (Via Reoso 49, San Pietro Viminario, 35020, IT)
International Classes:
A61P35/00; A61K31/27; A61K31/30; A61K31/401; A61K31/7008; C07D207/16; C07F1/12; C07H15/22; C07K1/107
Other References:
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BENIGNO MACIAS ET AL: "Copper complexes with dithiocarbamates derived from natural occurring amino acids. Crystal and molecular structure of [Cu(en)(EtOH)(H 2O) 3][Cu(dtc-pro) 2]", POLYHEDRON, 1 January 2002 (2002-01-01), pages 1899 - 1904, XP055366149, Retrieved from the Internet DOI: 10.1016/S0277-5387(02)01098-7
DIAZ A ET AL: "Interactions of nitric ocide with copper (II) dithiocarbamates in aqueous solution", JOURNAL OF INORGANIC BIOCHEMISTRY, ELSEVIER INC, US, no. 95, 1 January 2003 (2003-01-01), pages 283 - 290, XP002335489, ISSN: 0162-0134, DOI: 10.1016/S0162-0134(03)00126-0
ROBERTO CAO ET AL: "Gold Nanoparticles Enhancing Dismutation of Superoxide Radical by Its Bis(dithiocarbamato)copper(II) Shell", INORGANIC CHEMISTRY, vol. 50, no. 11, 6 June 2011 (2011-06-06), EASTON, US, pages 4705 - 4712, XP055366148, ISSN: 0020-1669, DOI: 10.1021/ic101770h
SUZUKI Y ET AL: "The origin of an EPR signal observed in dithiocarbamate-loaded tissues - Copper(II)-dithiocarbamate complexes account for the narrow hyperfine lines", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - GENERAL SUBJECTS, ELSEVIER, AMSTERDAM, NL, vol. 1335, no. 3, 6 June 1997 (1997-06-06), pages 242 - 245, XP004275984, ISSN: 0304-4165, DOI: 10.1016/S0304-4165(97)00027-5
FRAGOSO ALEX ET AL: "Esterase activity of cyclodextrin dithiocarbamates", TETRAHEDRON LETTERS, vol. 45, no. 21, 2004, pages 4069 - 4071, XP085010797, ISSN: 0040-4039, DOI: 10.1016/J.TETLET.2004.03.156
ALEX FRAGOSO ET AL: "Influence of Electrostatic Interactions and Hydrogen Bonding on the Activity of Cyclodextrin-based Superoxide Dismutase Models", SUPRAMOLECULAR CHEMISTRY., vol. 13, no. 5, 1 September 2001 (2001-09-01), US, pages 619 - 625, XP055374831, ISSN: 1061-0278, DOI: 10.1080/10610270108039795
MUHAMMAD ALTAF ET AL: "Synthesis, characterization and in vitro cytotoxicity of gold(III) dialkyl/diaryldithiocarbamato complexes", RSC ADVANCES: AN INTERNATIONAL JOURNAL TO FURTHER THE CHEMICAL SCIENCES, vol. 5, no. 99, 1 January 2015 (2015-01-01), GB, pages 81599 - 81607, XP055366145, ISSN: 2046-2069, DOI: 10.1039/C5RA15123F
VEYSEL T YILMAZ ET AL: "Metal Complexes of Phenylpiperazine-Based Dithiocarbamate Ligands. Synthesis, Characterization, Spectroscopic, Thermal, and Antimicrobial Activity Studies", SYNTHESIS AND REACTIVITY IN INORGANIC AND METALLORGANICCHEMISTRY, MARCEL DEKKER INC. NEW YORK, US, vol. 33, no. 4, 1 January 2003 (2003-01-01), pages 589 - 605, XP008184283, ISSN: 0094-5714, [retrieved on 20070215], DOI: 10.1081/SIM-120020326
LUCA RONCONI ET AL: "Latest Insights into the Anticancer Activity of Gold(III)-Dithiocarbamato Complexes", ANTI-CANCER AGENTS IN MEDICINAL CHEMISTRY, BENTHAM SCIENCE PUBLISHERS LTD, NL, vol. 10, no. 4, 1 January 2010 (2010-01-01), pages 283 - 292, XP008184285, ISSN: 1871-5206, DOI: 10.2174/187152010791162298
CHIARA NARDON ET AL: "Gold Complexes for Therapeutic Purposes: an Updated Patent Review (2010-2015)", CURRENT MEDICINAL CHEMISTRY : THE NEW INTERNATIONAL JOURNAL FOR TIMELY IN-DEPTH REVIEWS IN MEDICINAL CHEMISTRY, vol. 23, no. 29, 1 August 2016 (2016-08-01), NL, pages 3374 - 3403, XP055366360, ISSN: 0929-8673, DOI: 10.2174/0929867323666160504103843
M. NEGOM KOUDOM, J. INORG. BIOCHEM, 2012
DIAZ; COWORKERS, INORG BIOCHEM, 2003
SIMILARLY, SUZUKI ET AL., BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1335, 1997, pages 242 - 245
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J. SUPRAMOLEC. CHEMISTRY, 2001, . SUPRAMOLEC. CHEMISTRY, 2001
NARDON ET AL., CURRENT MEDICINAL CHEMISTRY, vol. 23, 2016, pages 3374 - 3403
"Remington's Pharmaceutical Science", MACK PUBLISHING COMPANY
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Claims:
CLAIMS

What is claimed is:

1 . A mononuclear coordination compound comprising a neutral or ionic complex and optionally at least one counter-ion G, said compound being represented by the general formulas l(a) and l(b):

1(a) 1(b)

wherein any form of isomerism exhibited by said compound is included, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof, either as racemes or in various ratios, and wherein:

- the arc connecting the two sulfur atoms represents a first dithiocarbamato ligand (DTC);

- M is a metal center selected between Au(lll) or Cu(ll) or Cu(lll) and is tetracoordinate in a coordination geometry preferably selected from the group consisting of: square- planar, tetrahedral, pyramidal, or any distorted geometry thereof;

X is independently selected from M, and X≠S is a monoatomic ionic ligand selected between CI, Br, I, or X=S and it is a donor atom part of a second dithiocarbamato ligand (DTC) equal to or different from said first dithiocarbamato ligand,

and wherein:

- said at least one counter-ion G is selected between:

- a pharmaceutically acceptable ion, or;

- an ion produced from the synthesis of said compound;

d is the electric charge of said counter-ion represented by an integer number comprised between -4 and +4;

- e is the stoichiometric coefficient of said counter-ion, represented by an integer number whose absolute value is equal to n/d or equal to zero;

- n is the electric charge of said complex represented by an integer number comprised between -4 and +4, wherein n=0 corresponds to a neutral complex having e=0. The coordination compound according to the preceding claim wherein said first or second dithiocarbamato ligand (DTC) is represented by the formula:

wherein:

R is selected from: -OH, -OCH3, -OC(CH3)3, -NH2, a substituted amine, a natural or synthetic aminoacid residue, a glucide or a carbohydrate, an OTEG (wherein OTEG is a triethyleneglycol-monomethyl ether), salt forms thereof, esterified forms thereof, forms functionalized with protecting groups thereof, or a combination thereof;

it is included any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof either as racemes or in various ratios.

The coordination compound according to the preceding claim wherein R is a glucide or a carbohydrate, forming a bond to the carbonyl group of said first or second dithiocarbamato ligand (DTC) in the C1 or C2 position of said glucide or carbohydrate, directly or by means of a unit A consisting of an atom or a functional group or a spacer, said unit A comprising single or double or triple bonds, said unit A being:

- an atom selected from: H, C, O, N, S, P, Se, or

- a first group selected from: -CH, -CH2, -CH3, -C(CH3)3, -NH, -NH2, -NHRS, -NRs2, -S- S-, -SH, SeH, -PH, -OH, -COOH, -CH-Br, -CHCH2NH2, -CHCH2OH, -CHCH2NH-, - CHCH20-, (-CN), -CF3, -C2H5, -C2H4, -C4H9, -C3H7, -C3H6, -C3H6OH, -C4H8, -N02, - CH2OH, -C2H4OH, -C4H8OH, -C(OH), -C(OH)H2, -C(OH)H; -S02, -COO-, -N(CH)3-, - CHN(CH), -NN(CH),-NCHNCH-, -N(CH)2N-, -CHNHCH-, -NH(CH), -NHCHN-, - NHNCH-; -CONH-, -CONH2, -CONHRs2, -CONRs2, (-0-C(0)-Rs), or a combination thereof, wherein Rs are substituent groups of the type aliphatic, alkyl, halo-alkyl, cycloalkyl, alkene, alkyne, alkynyl, aryl, hetero-aryl, heteroaliphatic, aromatic, hetero- aromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and hetero-cycloaliphatic or

- a second group selected from: ester, amide, sulfonamide, carbonyl, acetate, ethyl, propyl, butyl, isopropyl, acyl, ureidic, thioureidic, thiolate, imine, halogen, ether, nitro groups, nitrile groups, aryl, benzyl, sulphonamide groups, C1 -C18 alkyl saturated linear or branched optionally substituted with one or more Rs groups, or comprising one or more unsaturated bonds, or a combination thereof, or - a third group of the kind -(CH2)m, -(CH20)m, -(CH2CH2S)m, (CH2CH2NH)m or of the kind (CH2CH20)m, (CH2CH2N)m wherein m represents an integer number higher than 1 , or a combination thereof, or

- a combination of said atom or said first group or said second group or said third group,

said unit A optionally being in a salt form comprising a pharmaceutically acceptable counter-ion, said unit A optionally being substituted with one or more of said atoms or said groups in any combination and position, including any form of isomerism, preferably coordination isomers, structural isomers, conformational isomers, optical isomers such as enantiomers and/or diastereoisomers, mixtures thereof either as racemes or in various ratios.

4. A composition comprising:

- at least one coordination compound according to one or more of the claims 1 to 3;

- one or more entities selected from: macromolecules, supramolecular aggregates, nanostructures, or a combination thereof, said entities having constituent units equal to or different from each other, and said supramolecular aggregates optionally consisting of molecules, polymers or oligomers, natural or synthetic, having composition equal to or different from each other, and

wherein at least one of said coordination compound:

establishes intermolecular interactions with one or more of said entities, or

- is encapsulated in one or more of said entities.

5. The composition according to the preceding claim wherein said macromolecules or said supermolecular aggregates or said nanostructrures:

- are carriers binding, preferably encapsulating, said at least one coordination compound to improve the pharmacological profile, preferably in terms of: solubility, stability, bioavailability, controlled release, articular lubrication, or a combination thereof;

have dimensions between 0.2 and 1200 nm and have neutral, positive or negative superficial charge, conveniently balanced by pharmaceutically acceptable counter-ions; and

- are selected from the group consisting of: polymeric micelles, non-polymeric micelles, dendrimers, liposomes, cyclodextrines, proteins, organic nanoparticles, polymeric nanoparticles, inorganic nanoparticles, nanotubes, nanohorns, polymers, polymeric aggregates, metal oxides, semimetal oxides, hyaluronic acid, or a combination thereof.

6. A pharmaceutical formulation for treating human or animal diseases comprising:

- at least one compound according to one or more of the claims 1 to 3 in combination with pharmaceutically acceptable excipients and/or additives and optionally at least another active agent or another drug,

or - the composition according to one or more of the claims 4 to 5 in combination with pharmaceutically acceptable excipients and/or additives and optionally at least another active agent or another drug,

said excipients and/or additives being selected from the group consisting of: diluents, solvents, bulking agents, rheology modifiers, stabilizers, pH stabilizers, binders, buffers, disaggregating, preservatives, elasticizing, emulsifiers, chelating agents, lubricants, top sweeteners, sweeteners, dyes, contrast agents and flavoring, alone or in combination thereof.

7. Use of the pharmaceutical formulation according to claim 6, for treating inflammatory disorders.

8. A process for the preparation of the mononuclear coordination compound according to one or more of the preceding claims 1 to 3, comprising the following steps:

a) synthesis of one or more of said dithiocarbamato ligand (DTC) in water or in an organic solvent, preferably methanol or tetrahydrofuran (THF), by reaction between carbon disulfide (CS2), an amine precursor and optionally a base. After at least 5 minutes, preferably in a protective atmosphere according to the choice of said precursor amine, optionally to carry out:

- reduction of the water volume or of said solvent volume and optionally,

- isolation of one or more DTC ligands by means of standard separation techniques, preferably by co-precipitation with addition of organic solvent and optionally washing and drying;

b) in organic solvent, preferably in a protected atmosphere, or in water, synthesis of the coordination compound of formula l(a) and/or l(b) through the coordination of one or more of said DTC, synthetized and optionally isolated in the step a), to a metal center selected from Cu (II, III), Au (I, III), starting from the corresponding precursors with the same oxidation state, preferably chlorides or halide salts, in anhydrous or hydrated form, or alternatively said corresponding precursors are derivatives wherein the metal center occurs in a lower or higher oxidation state, preferably said derivatives being organometallic precursors, amines, thioethers, phosphine derivatives, or alternatively, said corresponding precursors may be some of said coordination compounds, said coordination being carried out for at least 5 minutes, said process optionally including the following step:

c) isolation of the compound synthesized in the preceding step b) by means of standard separation techniques, in order to obtain the coordination compound of formula l(a) and/or l(b) or a mixture containing at least one of said coordination compounds.

9. The process according to the preceding claim further comprising at least one of the following steps:

- a step wherein said at least one counter-ion G is exchanged; - one or more steps to protect one or more of said forms functionalized with protecting groups or of said glucide or carbohydrate or of said precursor amine or of said aminoacid residue or of said unit A, by means of the functionalization with protecting groups;

- one or more deprotection steps to deprotect one or more of said protecting groups present in said glucide or carbohydrate or in said amine precursor or in said aminoacid residue or in said unit A, wherein said one or more deprotection steps are carried out by chemical or biochemical methods, including the use of enzymes or pseudo-enzymes.

10. The process according to one or more of claims 8 or 9, for the preparation of said mononuclear coordination compounds further comprising the following phases I) or II):

I) in formula l(a) selecting a metal center M between Au(lll) or Cu(lll) and alternatively one of the following schemes a), b) or c):

a) with Cu(lll) and X=Br or CI, alternatively one of the following schemes:

- dissolution of said compound of general structure l(b) with M=Cu(ll) in an organic solvent, preferably halogenated, followed by the addition of at least 1 eq. of halogenating agent, preferably thionyl halide (SOCI2 o SOBr2), or

- dissolution of said compound of general structure l(b) with M=Cu(ll) in an organic solvent, preferably CS2, followed by the addition of an excess of solid Cu (at least 2 eq.) to form a suspension stirred for at least 2 hours. The resulting mixture is filtered and an intermediate of the type [Cu'(DTC)] is isolated, for example by precipitation with an organic solvent, preferably ethyl ether. Said intermediate is then dissolved in an organic solvent, preferably halogenated, and reacted with at least 1 eq. of halogenated oxidizing agent, preferably bromine Br2 or Cl2 chlorine,

or alternatively

b) with Au(lll) and X=Br or CI, alternatively one of the following schemes:

- oxidative addition to the corresponding precursor complex of the type Au(l)- DTC [Au2(DTC)2] of the halogen in the form of Br2 or Cl2 or halogenating reagents in situ, said oxidative addition preferably carried out at reflux for at least 5 minutes in water or organic solvent, preferably halogenated, said precursor complex being obtained:

- in saturated sodium chloride (NaCI) solution at a temperature comprised between -10 and 10 °C, after in situ reduction of a reagent salt of Au(lll), preferably NaAuC 2H20, to Au(l) with reductant agents, preferably Na2S03, by addition of 0.5-2.0 eq. of said dithiocarbamato ligand (DTC) until a precipitate is formed,

or alternatively, - by means of two consecutive reactions, the first reaction between at least 1 eq. of a ligand having sulfur donor atoms, preferably thioether ligands, such as tetrahydrothiophene (THT) or dimethylsulfide, or alternatively phosphorus-based ligands, preferably phosphine ligands, such as triphenylphosphine, and 1 eq. of reagent of Au(lll), preferably KAuX4 (X=CI, Br) in alcoholic medium, preferably methanol or ethanol, under stirring for at least 5 minutes; the second reaction based on the Au(l) product obtained from said first reaction, said product after reaction with at least 1 eq. of dithiocarbamato ligand (DTC) results in a precipitate, said precipitate being optionally dried, preferably in vacuum in the presence of phosphoric anhydride (P205), till said precursor complex is obtained;

- coordination in water or organic solvent, preferably at reflux for at least 5 minutes, of at least 1 eq. dithiocarbamato ligand (DTC), dissolved in water or organic solvent, preferably methanol, to the precursor complex [AuX3(am)] (wherein X=CI, Br and "am" is an amine, preferably pyridine, hereinafter also "py") dissolved in an organic solvent, preferably dichloromethane (DCM), said precursor complex being obtained:

- by reaction in water between at least 1 eq of amine and a halogenated reagent of Au(lll), preferably AuX3 or the salt KAuX4 or HAuX4 or the salt NaAuX4, to obtain a precipitate, said precipitate washed with water and subsequently dried, preferably in vacuum in the presence of phosphoric anhydride (P205), till said precursor complex is obtained,

or,

c) with Au(lll) and X=Br, I, heterogeneous reaction between at least 2 eq. of KX (X=Br, I), dissolved in water, and the coordination compound of formula l(a) [AuY2(DTC)] (Y=Br, CI), dissolved in organic solvent, preferably dichloromethane (DCM). At the end of said reaction, separation of the organic phase and subsequent anhydrification, preferably by addition of sodium sulphate.

in formula l(b) selecting a metal center M between Au(lll) or Cu(ll) or Cu(lll) and carrying out alternatively one of the following schemes a) or b):

a) with M=Cu(ll) coordination in water or organic solvent, preferably methanol, for at least 5 minutes, of at least 2 eq. of said dithiocarbamato ligand (DTC) to the metal precursor, until a precipitate is obtained;

b) with M=Au(lll) or Cu(lll), saturation of the coordination sphere of the metal center with sulfur donor atoms of said coordination compound of formula I (a), preferably dissolved in dichloromethane (DCM) or water, by addition of 0.1 -2.0 eq. of said second dithiocarbamato ligand (DTC), dissolved in water or an organic solvent, preferably methanol, said second DTC ligand being equal to or different from said first ligand DTC.

A process for preparing the composition according to one or more of claims 4 or 5, comprising at least the following steps:

a) obtaining one or more coordination compounds according to one or more of claims 1 to 3;

b) optionally, evaluating the partition coefficient n-octanol/water (logP) of said one or more coordination compounds;

c) choosing one or more of said entities able to establish intermolecular interactions with one or more of said compounds or able to encapsulate one or more of said coordination compounds;

d) choosing said constituent units or said molecules, polymers, oligomers;

e) carrying out one of the following schemes:

- co-dissolving, in the desired stoichiometric ratio, one or more of said compounds and one or more of the elements selected in the step d) in at least one organic solvent, preferably dichloromethane (DCM), methanol or chloroform; removing said organic solvent, preferably at reduced pressure, and optionally drying, preferably in vacuum, so to obtain a dry residue; hydrating said dry residue by addition of water or a saline solution or a buffer, preferably a phosphate buffer, so to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d),

or, alternatively,

co-dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent, preferably dichloromethane (DCM), methanol or chloroform, or in a mixture thereof; removing said organic solvent, preferably at reduced pressure, optionally drying, preferably in vacuum, so to obtain a dry residue; addition of an aqueous solution of one or more of said compounds, so to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);

or, alternatively,

dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in water or in a mixture glycerol/ethanol, or a combination thereof, followed by addition of an aqueous solution of one or more of said compounds, to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d),

or, alternatively,

dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent; removal of the solvent and optionally drying so as to obtain a film; hydration of said film with an aqueous solution and treatment of the obtained suspension by mechanical processes, preferably sonication and/or membrane extrusion, dialysis against aqueous solution; addition and incubation with the solution of said compound under continuous stirring and optionally, freeze-drying followed by hydration, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);

or, alternatively,

dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d) in an organic solvent; removal of the solvent and optionally drying so as to obtain a film; hydration of said film with an aqueous solution and treatment of the obtained suspension by mechanical processes, preferably sonication and/or membrane extrusion, addition and incubation with the solution of said compound under continuous stirring, optionally in the presence of cryopreservatives, and optionally freeze-drying followed by hydration, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d);

or, alternatively,

dissolving, in the desired stoichiometric ratio, one or more of said elements selected in the step d), in an organic solvent, for example chloroform, followed by addition of the aqueous solution of said compound and formation of an emulsion; stirring of the obtained mixture and evaporation of said solvent; addition of an aqueous solution, preferably a buffer, so as to obtain a mixture of one or more of said compounds and of one or more of said elements selected in the step d); f) subjecting said mixture to at least one of the following treatments: sonication, stirring, freezing/thawing cycles preferably in liquid nitrogen and water bath; extrusion through a porous membrane, centrifugation;

g) optionally, purifying the mixture obtained at the end of the step f) by a process comprising filtration, dialysis and sterilization;

h) optionally, obtaining from the mixture obtained at the end of the step f) or of the step g) a powder consisting of one or more of said entities and one or more of said coordination compounds.

Description:
TITLE: COORDINATION COMPOUNDS AND FORMULATIONS, PREPARATION METHODS AND USE THEREOF AS ANTIPHLOGISTIC AGENTS

TECHNICAL FIELD

This invention concerns Au and Cu-based mononuclear coordination compounds, drug formulations thereof, the relating synthesis and encapsulation method in macromolecules or supramolecular aggregates or nanostructures, as well as their application in the treatment of inflammations, particularly those associated to osteoarthritis and rheumatoid arthritis or other chronic and non-chronic rheumatic (or systemic) diseases, including: psoriatic arthritis, ankylosing spondylitis, erythematosus lupus, scleroderma, Sjogren's syndrome and also crystalline arthropathy (gout).

BACKGROUND ART

The incidence of inflammatory-related pathologies is notably increasing in developed countries, also due to a higher life expectancy of the population in the world today. Such pathologies include osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, erythematosus lupus, scleroderma, Sjogren's syndrome and crystalline arthropathy (gout). Amidst the rheumatic diseases (chronic or degenerative inflammatory diseases), rheumatoid arthritis and osteoarthritis are associated with an undermined quality of life and occasionally to disability, with significant social and economic implications for the patients, their families and healthcare. Osteoarthritis, the most common form of rheumatic disease, has become the largest cause of disability in the elderly population.

In recent years, biomedical research has made it possible to better understand the pathophysiological mechanisms underlying these diseases. Free radicals present in inflammations represent a crucial factor of these mechanisms. Amidst these, those derived from oxygen and considered biologically relevant are called "Reactive Oxygen Species" (ROS): hydroxyl radical, superoxide anion (0 2 ' " ) and hydrogen peroxide. These highly reactive species can degrade hyaluronic acid, modify collagen, alter and interact with immunoglobulins, activate enzymes, and inactivate their inhibitors. In particular, the (0 2 '" ) radical can also damage endothelial cells and increase the microcirculation permeability favouring the migration of neutrophils to the foci of the inflammation, releasing pro-inflammatory cytokines.

Drug therapies currently known for the treatment of rheumatoid arthritis are based on: FANS (non-steroidal anti-inflammatory drugs), corticosteroids, hydroxychloroquine, sulfasalazine, cytotoxic drugs and immunosuppressants {e.g. azathioprine, methotrexate and cyclosporine) and compounds based on gold in oxidation state +1 . Furthermore, in the last years biotechnological drugs based on innovative platforms have added to these traditional drugs. With regards to the treatment of osteoarthritis, in particular the one involving the hip and knee, both non-pharmacological strategies, aimed at reducing changeable risk factors {e.g., lifestyle) or the biomechanical dysfunction as well as drug therapies which include corticosteroid and hyaluronic intra-articular injections, topical or oral analgesics, NSAIDs, dietary supplements {e.g., Condroitin ® sulphate and glucosamine) are currently being used. In this context, despite the steadily-increasing focus of the pharma industry on biotechnological drugs (due to the potential therapeutic benefits that these molecules have compared to traditional ones), biotechnology drugs are still characterized by severe limitations and also long-term effects that are not known yet.

Finally, it is worth noting that a number of coordination compounds based of Au or Cu are indeed reported in the scientific literature, but they are intended as potential anti-inflammatory agents, or they present a totally different chemical structure with respect to this patent specification as it will be apparent in the following.

For instance, M. Negom Koudom (J. Inorg. Biochem, 2012) reported the synthesis of Au(lll)/DTC derivatives containing oligopeptides (tri-, tetra and pentapeptide binders) and their anticancer activity, but he didn't investigate their anti-inflammatory activity. B. Macias et al. obtained a "double complex salt" type containing as anion an omoleptic compound of Cu(ll) based on dithiocarbamic-derived proline derivatives. In his work (Polyhedron, 2010), they characterized the magnetic and spectroscopic properties of said complex but they investigate its antiflogistic activity. Diaz and coworkers (Inorg Biochem, 2003) synthesized homoleptic Cu (II) complexes containing certain proline-based ligands for the purpose of studying their interaction with the NO molecule. Similarly, Suzuki et al. (Biochimica et Biophysica Acta 1335 1997. 242- 245) used a proline-dithiocarbamate derivative but studying the potential anti-inflammatory properties was out of the scope of his work. Cao et al. (Inorg Chem, 201 1 ) reported a series of heteroleptic complexes containing DTC derivatives derived from proline in order to functionalize gold nanoparticles with mimetic capacity of the enzyme superoxidodismutase. In his paper no homoleptic complexes containing DTC derivatives were considered. In three different papers, Fragoso et. (Tetrahedron Letters 45 (2004) 4069-4071 , J. Supramolec. Chemistry, 2001 , . Supramolec. Chemistry, 2001 ) reported the use of complexes containing ligands based on cyclodextrins functionalized with a DTC group: anyhow, Fragoso research were not addressed to new anti-inflammatory active agents. Finally, in a review by Nardon et al. (Current Medicinal Chemistry, 2016, 23, 3374-3403), Au(l) and Au(lll) complexes with oligopeptides functionalized with sulfur donors having anti-tumor or anti-inflammatory activity are described. Nevertheless, the structure does not include a dithiocarbamic binder based on proline and furthermore exhibit anti-tumor activity rather than anti-inflammatory.

Technical Problem

The intense research activity conducted over the last few decades has attempted to mitigate the known side-effects but it has only partially achieved the desired results.

The chronic nature of many degenerative articular pathologies such as osteoarthritis or inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, crystalline arthropathy (gout) requires the development of safe drugs suitable for chronic administration.

Therefore, in view of the above drawbacks of the prior art, the present invention intends to overcome the existing disadvantages related to coordination compounds used to treat inflammations.

DISCLOSURE OF INVENTION

Object/scope of the invention

Accordingly, it is a first and main object of the present invention to provide stable coordination compounds with distinguish properties which are valuable as anti-inflammatory agents.

In particular, it is a second object of this invention to identify coordination compounds characterized by new metal centers and/or oxidation states that can operate as a "scavenger" or as "mimetic SODs" against ROS radicals and/or that can coordinate to other pro-inflammatory mediators in order to inactivate inhibit or hinder the development of the flogistic process, and possibly to prevent tissue destruction. Furthermore, it is a third object of this invention to identify coordination compounds characterized by low or negligible toxicity over known drugs, high stability and bioavailability, intrinsic or achieved by encapsulation in macromolecules or supramolecular aggregates such as micelles, liposomes, polymers such as hyaluronic acid, peptides, proteins or cyclodextrins.

In addition, a fourth object of this invention is to provide a stable drug formulation with one or more of said coordination compounds which can be used as an anti-inflammatory agent and which can be administered preferably intra-articularly or orally. This formulation would preferably allow a prolonged release and maintain the therapeutic concentration of an intraarticular drug for a period of a few weeks.

Within the above-mentioned main tasks, the fifth object of the present invention is to develop a method for synthesizing said Au-based and Cu-based coordination compounds as well as a method for encapsulating said compounds within macromolecules or supramolecular aggregates, constituted by one or more biocompatible polymers or oligomers.

Still, a further object of this invention is to produce coordination compounds through a synthesis process which can provide intrinsically high-pure compounds and, at the same time, can be scaled-up on an industrial scale with well-known and cost-effective technologies compared to state-of-the-art solutions.

Finally, within the main tasks outlined above, the last object of the present invention is to disclose the use of said coordination compounds as anti-inflammatory agents, particularly in the treatment of inflammations associated with osteoarthritis, rheumatoid arthritis or other rheumatic diseases (chronic or degenerative inflammation). 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 antiinflammatory agents. These studies were mainly directed to address the well-known instability of coordination compounds having metallic centers with high oxidation states.

After long terms of practice the inventors found a new class of Au-based and Cu-based mononuclear coordination compounds. Said compounds are described by one of the following general formulae 1(a) or 1(b):

1(a) 1(b)

wherein M is a metal center chosen between Au(lll) or Cu(ll, III) and the arch connecting the two S-atoms represents a first dithiocarbamato (DTC) ligand. In the case of the l(a) structure, X is a monoatomic ion ligand selected between CI, Br, I, while in the case of the l(b) structure, X is a donor atom which is part of a second dithiocarbamato ligand (DTC) which may be equal to or different from said first dithiocarbamato ligand. In the case the complex presents an n charge, different from zero, there is a counter G ion with a d charge and a stoichiometric coefficient e, to ensure the neutrality of the coordination compound.

The coordination compounds of structure I (a) and / or I (b) are capable of accomplishing a new mechanism of anti-inflammatory action that directly intervenes on the mediators of inflammation, as it will be evident to the skilled expert through the following description and some examples which will be provided herein by way of non-limiting example of this invention.

Advantageous Effects of Invention

The coordination compounds of structure l(a) and/or l(b) 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.

Particularly, the coordination compounds according the present invention accomplish their anti- inflammatory activity due to the peculiar chemical properties of their metallic centers which, despite having high oxidation states, are stabilized by the presence of at least one dithiocarbamic ligand (DTC). Such compounds are also characterized by low or negligible toxicity over known drugs and surprisingly, in some cases, unexpected solubility in physiological media in spite of the presence of hydrophobic groups in the structure. In addition, they are easily encapsulated in supramolecular aggregates, such as liposomes or cyclodextrins, acting as nanocarriers to further increase solubility in aqueous media and stability under physiological conditions as well as being bioavailable. The aforementioned properties are potentially usable in a pharmaceutical formulation administrable intra-articularly and comprising said compound and at least one oligomeric or polymeric carrier agent (e.g., hyaluronic acid) capable of further promoting mobility and articular lubrication..

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 Drawings

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

- Figure 1 depicts the biochemical principle of SOD (superoxide dismutase), in which superoxide radicals are produced by an enzyme system (xanthine and xanthine oxidase) and detected by the NBT spectrophotometer probe.

- Figure 2 shows the "scavenging" activity of superoxide radicals of said compounds, expressed as a percentage of inhibition of NBT probe reduction.

Figure 3 presents data obtained in vivo. In letter A), the body weights of mice treated with the encapsulated compound [AuBr 2 (ProO©uDTC)] and those treated with the carrier alone; in letter B), the average "paw score" recorded for the different groups during the treatments.

- Figure 4 presents the UV-Vis spectra recorded over time in saline for the coordination compound [Au(ProOtBuDTC) 2 ] Br of general structure (V) encapsulated in dipalmitoylphosphatidylcholine (DPPC) liposomes (at 37° C).

- Figure 5 represents the UV-Vis spectra of the coordination compound [Cu(ProOMeDTC) 2 ], of general structure (IV). At letter A) said compound is dissolved in an aqueous medium consisting of a pH 7.4 phosphate buffer 94.5-5% v/v human serum, with a final DMSO concentration of 0.5% v/v, an organic solvent used to pre-dissolve the compound (kinetics recorded at 37° C for 72 hours), while in letter B) it is encapsulated in Pluronic ® PF127 (5 mg/mL) micelles, dissolved in aqueous medium consisting of a pH 7.4 phosphate buffer, human serum 95-5% v/v.

Figure 6 shows the UV-Vis kinetics recorded over 72 hours for the complex [Cu(ProOMeDTC) 2 ] encapsulated in an ΗΡ-β-CD in a phosphate / cell culture medium buffer 9: 1 v / v (at 37 ° C).

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

Coordination compounds

It is a first object matter of the present invention a new class of mononuclear coordination compounds of Au or Cu, whose features are defined in the appended independent claim. Such compounds are described by one of the following general formulae (I) or (II):

1(a) 1(b)

wherein M represents the metal center of the compound and it is selected among Au (III), Cu (II) or Cu (III), briefly also Cu (II, III) while the arch connecting the two S atoms represents a first dithiocarbamato (DTC) ligand, in accordance with the usual chemical notation. In the case of the l(a) structure, X is a monoatomic ion ligand selected among CI, Br, I, while in the case of the l(b) structure, X is a donor atom which is part of a second dithiocarbamato ligand (DTC) which may be equal to or different from said first dithiocarbamato ligand. In the l(b) Formula, the arch connecting X with X represents a second DTC ligand.

When the complex presents an n charge that is different from zero, a counter G ion with a d charge and a stoichiometric coefficient e is included to ensure the neutrality of the coordination compound. In particular, the integer number n can range from -4 to +4 where the case n = 0 corresponds to a neutral complex, and e = n /d or is zero (in the latter case the counter-ion is evidently not present and even n = 0). G is selected among pharmaceutically acceptable ions. By way of example, but not limitation, G is preferably selected from CI " , , nitrite and nitrates (e.g., N0 2 " , N0 3 " ), acetates, phosphates (e.g. hexafluorophosphate, H 2 P0 4 " , P0 4 3" ), sulfates (e.g., triflate, HS0 4 " ), carbonates, stearates, lactates, malate, pyruvate, citrate, ascorbate, palmitate and maleate. Other chemically equivalent salts can, however, be used. For example, those disclosed in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company. Alternatively, G represents at least one counter ion generated by the synthesis of the coordination compound and it may be a halide or a complex itself and preferably selected among CI " , I " , Br " , AuX 2 " , AuX 4 " (X = CI Br); or other chemically equivalent ones.

Depending on the choice of the metallic center and the donor atoms as well as the ligands (as it is well known, for example, in the chloride ligand the donor atom coincides with the ligand itself), the compounds according to this invention have different coordinate geometries, namely: square-planar, tetrahedral, pyramidal, or one of the previously distorted structured geometries. second dithiocarbamic ligand (DTC) is represented by the following formula

wherein R is selected among -OH, -OCH 3 , -OC(CH 3 ) 3 , -NH 2 , substituted amide, an amino acid residue, a glucide or a carbohydrate, OTEG (where OTEG is triethylenglycol monomethylether), salified forms of the foregoing, esterified forms of the foregoing. "Substituted amide" means R = -NHR' or -NR 2 ', where R ' is an organic substituent.

By way of example, but not limitation, if R is a natural or synthetic amino acid residue this may be glycine, serine, tryptophan, AIB (alpha-amino-butyric acid) or other chemically-equivalent residues. Furthermore, as a non-limiting example of this invention, if R is a glucide or a carbohydrate this may be glucose or glucosamine. Optionally, the hydroxyl groups of carbohydrates or the C-terminus end of amino acids can be protected with protecting groups such as esters. Alternatively, R is a glucide or carbohydrate that binds to the carbonyl group of said first or second dithiocarbamato (DTC) ligand in the C1 or C2 position of said glucide or carbohydrate, either directly or through a unit A consisting of an atom or a functional group or a spacer. The unit A can include single, double, or triple bonds and, depending on the needs, it can be variedly designed.

Synthesis and characterization of coordination compositions

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

In the preferred embodiment of the present invention, hereby described by way of example, but not limitation, the coordination compounds of general formula l(a) and l(b) can conveniently be synthesized by a process comprising at least the following steps:

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

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

c) Isolation of the coordination compound synthesized in step b).

Optionally, this process may include a further step d) i.e. purification and drying of the coordination compound obtained at step c).

a) Preparation and optional isolation of Dithiocarbamate (DTC)

The synthesis of dithiocarbamate ligands (DTC) is carried out at the temperature range -30 to 60 °C in water or methanol by reaction between a precursor amino and carbon disulfide (CS 2 ), optionally in the presence of a base such as KOH or sodium tert-butoxide or an excess of said precursor amine. In particular, said precursor amine is the proline residue present in Formula 2. Said proline, even in an esterified form, can be used as such in the synthesis of the DTC ligand or alternatively it may be functionalized, for example with an amino acid residue or TEG or a glucide or carbohydrate prior to the CS 2 reaction. The ligand can be optionally isolated by precipitation or co-precipitation with the addition of an organic solvent, preferably ethyl or n- hexane. In the case of some amines, it is preferable to work in a protected atmosphere using known equipment and techniques.

Using this synthesis strategy, which involves the isolation of the pure DTC ligand, the inventors have untrivially achieved a number of remarkable advantages over the state of the art.

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 involves the coordination of the dithiocarbammic ligand (DTC), optionally isolated in the preceding step, to a selected metal center Cu(ll, III) or Au(l, III). The metal center is selected from the corresponding precursors, preferably chlorides or salts of halides, for example copper chloride, in the case the selected metal center is Cu(ll, III). Alternatively, precursors are derived in which the metal exhibits a lower oxidation state, for example in organometallic amine, organometallic thioether, phosphine derivatives or alternatively, some of said coordination compounds may in turn be precursors for the synthesis of other complexes.

c) Isolation of the coordination compound synthesized in step b).

Isolation of the compound synthesized in the above step b) by conventional separation techniques, preferably filtration followed by reduction of the volume of water or of said solvent, and precipitation with ethyl ether, or by evaporation of water or said solvent in conditions of reduced pressure to obtain the coordination compound of general structure l(a) and/or (b) or a mixture containing said coordination compound.

d) Purification and drying of the coordination compound.

At this stage, purification is carried out by techniques known to those skilled in the art, for example by chromatography, precipitation from organic solvent, washing with water or organic solvents and the drying of said coordination compound.

The above-described synthetic process may include further steps as outlined below by way of example but not limitation of this invention.

Concerning the coordination compounds of Formula l(a) and Formula l(b) containing dithiocarbamato ligands functionalized with amino acid residues, well-known peptide-coupling techniques are used which are conducted in solid phase or in solution, with particular care to avoid racemization phenomena. By way of example, the synthesis of the dipeptide (precursor of the DTC ligand) HCI L- ProAibOtBu was conducted in solution starting from the single protected amino acids and using suitable coupling agents. The general procedure is described in the diagram below.

Scheme 1: Synthetic route for the preparation of HCI L-ProAibOtBu (R = OtBu; AAi = L / D-Pro; AA 2 = Aib). Briefly, the C-terminal amino acid residue is esterified first, for example as terbutyl ester (obtained by reaction of the Z-protected C-terminal amino acid residue with isobutane for one week). Subsequently, the N-terminal end is deprotected by catalytic hydrogenation (Pd/C (10%) in organic solvent) and then coupled with the Z-protected proline residue. After removing the Z group from the proline residue, the esterified dipeptide is transformed into its chlorohydrate form by reaction with HCI in ether.

Similarly to the Z-Aib-OH esterification with a terbutyl group, Z-Pro-OH is functionalized with TEG by coupling with agents such as EDC/HOBt, where EDC designates 1 -ethyl-3- (3- dimethylaminopropyl) carbodiimide hydrochloride, while HOBt, 1 -hydroxy-1 H-benzotriazole. The coordination compounds of Formula l(a) and Formula l(b) contain dithiocarbamato ligands, which can be functionalized with carbohydrates in order to modulate the solubility in the physiological media of said compounds.

With reference to Formula 2, the glycoconjugation process involves a series of synthetic steps aimed at obtaining the proline-based amine precursor to be converted into the corresponding dithiocarbamato ligand (DTC) through the step a) of the process, for the subsequent complexation of the metallic center (Au, Cu). In general, the synthesis provides for the protection of the hydroxyl groups of carbohydrate with orthogonal protector groups at the reaction conditions contemplated for the subsequent synthetic steps. The deprotection of protecting groups can be carried out by chemical or biochemical methods that include the use of enzymes.

By way of example, but not limitation, the Scheme 2 below shows the synthesis of a dithiocarbamato ligand (a-g) derivative of a glucose functionalized in position 1 (β-amido- glycoside). In the h-i steps, the complexation of a center of Au(lll) occurs, followed by deprotection of the silyl protecting groups.

Scheme 2. Example of functionalization in position CI: a) NaN 3 , acetone, 56 ° C, crystallization, 60%; b) H 2 / Pd, MeOH / EtOAc, 98%; c) Z-Pro-OH, NMM, isobutyl chloroformate, THF-dry, -15 ° C, 83%; d) NaOMe, MeOH-dry, resin acid, rt, 100%; e) TMS-CI, Esamethyldisilazane, pyridine, CH 2 CI 2 , 0 ° C, flash chromatography, 85%; f) H 2 / Pd, MeOH / EtOAc, 90%; g) CS 2 , KOH, MeOH, 100%; h) AuX3py (X = CI, Br; py = pyridine), CH 2 CI 2 , 42 ° C, flash chromatography, 40%; i) acid resin, CH 2 CI 2 , rt, 95%

Without loss of generality, the Scheme 3 below illustrates the synthesis of a dithiocarbamato ligand (steps c-g) derived from a glucose functionalised in position 2 (glucosamide namely an amide derivative of glucosamine). In h-i steps, the complexation to a Cu (II) center occurs, followed by deprotection of the silyl protecting groups.

With reference to the step b) of the synthesis process of the compounds according to this invention, various synthetic approaches are possible based on the metal center M selected between Au or Cu, and depending on the oxidation state (+2 or +3) and the whether or not ionic ligands such as halides are present. Based on these choices, neutral or ionic complexes are obtained (further details on the synthetic process are defined by the enclosed claims).

Scheme 3. Functionalization example in C2 position : c) Z-Pro-OH, NMM, isobutyl chloroformate, THF-dry, -15 ° C, 83%; d) NaOMe, MeOH-dry, resin acid, rt, 100%; e) TMS-CI, Esamethyldisilazane, pyridine, CH 2 CI 2 , 0 ° C, flash chromatography, 85%; f) H2 / Pd, MeOH / EtOAc, 90%; g) CS2, KOH, MeOH, 100%; h) CuCI 2 , MeOH, flash chromatography; i) acid resin, CH 2 CI 2 , rt.

For the synthesis of the coordination compounds obtained through formula 1(b) with M = Au (III) or Cu(lll) and X = S, the coordination sphere of the general formula 1(a) is saturated with S- donor atoms. Advantageously, the counter ion G can be chosen or replaced using techniques known to the skilled in the art to optimize the drug profile. Indeed, cationic complexes of the [M(DTC) 2 ] + type were prepared with different counter ions (CI, Br) starting from the [MX 2 (DTC) complex with X = CI, Br (dissolved in dichloromethane) and adding an equivalent of DTC ligand (previously dissolved in methanol).

Instead, cationic complexes of the [M (DTC) 2 ] + type having, for example, M = Au (III) and AuCI 4 " or AuBr 2 " as counterion, are synthesized starting from the Au(lll) precursor [AuPyX 3 ] (X = CI , Br and Py = pyridine), adding respectively 0.5 eq. or 1 .5 eq. of DTC ligand.

The process may further and optionally include one or more protecting steps of one or more functional groups (e.g., hydroxy groups, amines and thiols) present in the precursor amine or in the carbohydrate or glucide or amino acid or said unit A and one or more deprotection phases of one or more of the said protecting groups in which deprotection is conducted by chemical or biochemical methods; but it may also include the use of enzymes or pseudoenzymes. Pseudoenzymes generally refer to proteins such as HSA (Human Serum Albumin) which may hydrolyze, for example, ester substrates due to the presence of numerous nucleophilic residues (e.g., lysine) on their surface, which yet do not return to their native state after the hydrolysis reaction.

Advantageously, the syntheses described herein allow to intrinsically obtain pure compounds and therefore potentially usable in the pharmaceutical field. Examples of coordination composites

By way of example, but not limitation, some specific examples of coordination compounds with formula 1(a) or 1(b) are reported below. The compounds were characterized by various techniques, including elemental analysis, NMR spectroscopy, FT-IR spectrophotometry and ESI-MS mass analysis.

Examplel: [AuCI 2 (ProOMeDTC)J

Appearance: solid orange; yield: 89%

R.f. (silica gel, CH 2 CI 2 ):Anal. Calc. for C 7 H 10 AuCI 2 NO 2 S 2 (MW = 472.16 g mole 1 ): C, 17.81 ; H 2.13; N, 2.97; S 13.58. Found: C, 17.86; H 2.22; N 2.90; S 13.65. 1 H-NMR (DMSO-d 6 , 300.13

MHz): 5(ppm) 2.20-3.98 (m, H (5) + 0-CH 3 ) 2.20 (m, H (3) + H (4) 5.38 (m, H (2) ).

Medium FT-IR (KBr) v(cm "1 ) = 2951.38 (v a , C-H); 1746.56 (v, C = O); 1559.95 (v a , N-CSS);

1173.93 (v a , C-OMe); 979.24 (v a , CSS). Far FT-IR (nujol): v (cm 1 ) = 546.77 (v s , CSS); 381.91

(v a , Au-S); 359.01 (va, Au-CI); 339.07 (v s , Au-S); 318.79 (v s , Au-CI).

Example 2: [AuBr 2 (ProOtBuDTC)]

Appearance: orange, needle shaped; Yield: 90%

R.f. (silica gel, CH 2 CI 2 ):Anal. Calc. for Ci 0 H 16 AuCI 2 NO 2 S 2 (MW = 603.14 g mole 1 ): C 19.91 ; H, 2.67; N 2.32; S 10.63. Found: C 20.02; H, 2.70; N 2.29; S 10.71.

1H NMR (DMSO-d 6 , 300.13 MHz): δ (ppm) = 1.41-2.26 (m, H (3) + H (4) + OC (CH 3 ) 3 ), 3.21-4.02 (m, Hp ) ), 4.36-5.30 (m, H (2) ). Medium FT-IR (KBr): v (cm 1 ) = 2977.36 (v a , C-H); 1736.46 (v, C=0); 1559.96 (v a , N-CSS); 1146.69 (v a , C-OtBu); 952.67 (v a , CSS).

Far FT-IR (nujol): v (cm 1 ) = 540.70 (v s , CSS); 379.35 (v a , Au-S); 344.53 (v s , Au-S); 238.34 (v a , Au-Br); 219.02 (v s , Au-Br).

Example 3: [Cu(ProOMeDTC) J

COOMe Appearance: solid dark green;

Yield: 80%

R.f. (silica gel, CH 2 CI 2 ) : 0.50

Anal. Calc. for Ci 4 H 2 oCuN 2 0 4 S4 (MW = 472.13 g mole 1 ): C 35.62; H, 4.27; N, 5.93; S 27.17. Found: C 35.83; H 4.13; N, 5.81 ; S 27.47.

1 H-NMFt (CDCI 3 , 300.13 MHz): δ (ppm) = 2.1 1 -2.93 (m, 8H, H (3) + H (4) ), 3.94 (m, 6H, 0-CH 3 ). Medium FT-I R (KBr): v (cm 1 ) = 2951 .87 (v a , C-H); 1750.79 (v, C = O); 1471 .56 (v a , N-CSS); 1 153.76 (v a , C-OMe); 939.75 (va, CSS).

Far FT-I R (nujol): v (cm 1 ) = 565.71 (vs, CSS); 342.87 (va, Cu-S); 279.96 (vs, Cu-S).

ESI-MS m / z, [M + ] - found (Calc): 470.97 (470.96).

Example 4: [CufProOtBuDT J

COOtBu

Appearance: solid brown; Yield: 74%

R.f. (silica gel, CH 2 CI 2 ): 0.72

Anal. Calc. for C 2 oH 32 CuN 2 0 4 S4 (MW = 556.29 g mole 1 ): C 43.18; H 5.80; N 5.04; S 23.06.

Found: C, 43.42; H, 5.92; N, 4.90; S 23.21 .1 H-NMR (CDCI 3 , 300.13 MHz): δ (ppm) = 1 .57 (s,

18H, 0-C(CH 3 ) 3 ), 1 .99-2.83 (m, 8H, H (3) + H (4) ). Medium FT-IR (KBr): v (cm 1 ) = 2974.81 (v a , C-

H); 1733.37 (v, C = O); 1469.68 (v a , N-CSS); 1 148.53 (v a , C-OtBu); 930.08 (v a , CSS).

Far FT-IR (nujol): v (cm 1 ) = 568.57 (v s , CSS); 340.1 1 (v a , Cu-S); 276.1 1 (v s , Cu-S).

ESI-MS m / z, [M + ] - found (Calc): 555.06 (555.05).X-ray structure shown in Figure 2 (C).

Example 5: [AuBr 2 (tetraAc-glicosilammido-ProDTC)]

Appearance: solid orange; Yield: 70%

R.f. (silica gel, EtOAc): 0.65 Anal. Calc. for C 20 H 27 AuBr 2 N 2 O 10 S 2 (MW = 876.34 g mor 1 ):C, 27.41 ; H, 3.1 1 ; N, 3.20; S, 7.32; Found: C 27.48; H, 3.28; N, 3.31 ; S 7.55.

1 H-NMR (CDCI 3, 300.13 MHz): δ (ppm) = 2.03, 2.08, 2.09, 2.17 (12H, s); 2.24 - 2.52 (4H, m); 3.78 - 3.88 (2H); 3.94 (1 H, m); 4.06-4.10 (1 H, ddd); 4.28-4.33 (1 H, ddd); 4.48-4.51 (1 H, ddd); 4.90 (1 H, t); 5.08 (1 H, t); 5.18 (1 H, t); 5.32 (1 H, t); 6.79 (1 H, m).

FT-IR (KBr): v(cm "1 ) = 1748 (v, C = O); 1542 (v, C = O); 1664 (va, N-CSS)

Example 6: [AufProOtBuDTQrfBr

Appearance: solid orange; Yield: 84%

Anal. calc. for C 2 oH 32 AuBrN 2 0 4 S4 (MW = 769.61 g mole-1 ): C, 31 .21 ; H, 4.19; N, 3.64; S, 16.67;Found: C, 31 .17; H, 4.20; N, 3.37; S, 16.47.

ESI-MS [M] + : 689.09 m / z;

1 H NMR (CD 2 CI 2 , 400.13 MHz, TMS, δ / ppm): 1 .49 (s, 9H, C (CH 3 ) 3 ) 2.25, 2.52 (m, 4H, CH 2 pos.3.4); 3.98 (m, 2H, CH 2 pos.5); 4.78 (dd, 1 H, CH pos. 2);

1 3 C NMR (CD 2 CI 2 , 100.67 MHz, TMS, δ / ppm): 196.25 (CSS);

FT-MIR (KBr,v max / cm 1 ): 2975 (v a , C-H); 1733 (v, C = O); 1535 (v a , N-CSS); 1221 (v, C-0 (tBu)); 1 149, 837 (v a,s , (OtBu)); 1003, 586 (v a,s , CSS);

FT-FIR (Nujol, v max / cm 1 ): 370 (v a,s , Au-S).

Example 7: [CuftetraAc-glicosilammidoProDT rf

Appearance: solid brown; Yield: 97% Anal. Calc. for C40H54C11N4O20S4 (MW = 1 102.68 g mole-1 ): C 43.57; H, 4.94; N, 5.08; S 1 1 .63. Found: C, 43.81 ; H 4.82; N, 5.15; S 1 1 .94.

1 H-NMR (CDCI 3, 300.13 MHz): δ (ppm) = 2.03, 2.08, 2.09, 2.17 (12H, s); 2.18 - 2.36 (4H, m);

3.21 -3.24 (2H); 3.78 (1 H, m); 4.06-4.10 (1 H, ddd); 4.28-4.33 (1 H, ddd); 4.48-4.51 (1 H, ddd);

4.90 (1 H, t); 5.08 (1 H, t); 5.18 (1 H, t); 5.32 (1 H, t); 6.79 (1 H, m).

FT-IR (KBr): v(cm "1 ) = 1749 (v, C = O); 1540 (v, C = O); 1632 (v, N-CSS)

ESI-MS m / z, [M + ] found: 1 101 .19

Example 8: [Au(DTC-L-Pro-Aib-OtBu)JCI

+

Appearance: solid orange; Yield: 68%

Anal. Calc. for C 28 H 46 AuCIN 4 0 6 S4 (MW = 895.37 g mole 1 ): C, 37.56; H 5.18; N, 6.26; S 14.32. Found: C, 37.74; H, 5.14; N 6.51 ; S 14.44.1 H-NMR (acetone-d 6 , 300.13 MHz): δ (ppm) = 8.0 (s, br, 1 H, NHAib), 4-5 (Ctf + CH 2 5 Pro), 2.2-2.7 (CH 2 4 + CH 2 3 Pro), 1 ,4-1 .5 (CH 3 fBu; CH 3 Aib) Medium FT-IR (KBr): v(cm "1 ) = 3342 (v, N-H); 2978/2932 (v, C-H); 1731 (v, C = O esters); 999 cm 1 (v a , S-C-S). Far FT-IR (nujol): v (cm 1 ) = 534 (vs, S-C-S); 413 (v a , S-Au-S); 375 (v s , S-Au- S).

Example 9: [AuCI 2 (DTC-ProOTEG)J

Appearance: solid brown; Yield: 75%

Anal. Calc. for C 13 H 22 AuCI 2 N0 5 S 2 (MW = 604.32 g mole 1 ): C 25.84; H, 3.67; N 2.32; S 10.61 . Found: C 25.88; H 3.62; N, 2.38; S 10.72.

1 H NMR (DMSO-d 6 , 300.13 MHz): δ (ppm) = 5.1 -5.3 (m, H (2) ), 3.3-3.9 (m, H (5) ), 3.22 (s, OCH 3 TEG), 2.15 -2.28 (m, H (3) + H (4) ), 4.10-4.1 1 (s, br, 2H, -CH 2 TEG), 3.30-3.65 (m, 10H, CH 2 TEG) Preparation of supramolecular aggregates encapsulating antiflogistic 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 of which are set forth in the enclosed independent claim.

By way of example, but not limitation, convenient supramolecular systems encapsulating coordination compounds with structures 1(a) and 1(b) may be micelles, vesicles (liposomes), cyclodextrins, organic polymer nanoparticles or inorganic nanoparticles {e.g., silica, zirconia, titania). In turn, such supramolecular architectures can be made with different polymers, natural and synthetic, as well as proteins or other organic or phospholipid molecules, such as chitosan, polyethylene glycol (PEG), mPEG, polyglycolic co-glycolic acid (PLGA), Pluronic ® , cholesterol, phosphatidylcholine and phosphatidylethanolamine derivatives, Cremophor ® , pullulan, hyaluronic acid, ferritin, human serum albumin (HSA).

The nanocarrier (e.g., liposome, micelle) can be conveniently coated with a hydrophilic and biocompatible coating to improve the pharmacokinetic profile of the "naked" supramolecular aggregate itself. In practice, by exploiting the so-called "stealth effect", known to date for polymers such as PEG and polyamino acids, the surface of the supramolecular aggregate is hidden from blood components, including opsonine proteins, responsible for the recognition and attack of the nanocarrier by fagocytes (monocytes, macrophages), thus eluding the natural biotransformation/elimination processes of exogenous constituent substances or entities. This masking strategy, among other things, results in an increased bioavailability of the loaded compound and in an increase in circulation times compared to supramolecular aggregates (incorporating the coordination compounds of this invention) which are not coated by said hydrophilic and biocompatible coating. The application of the "stealth effect" to the coordination compounds accordingly with this invention may also limit or eliminate any physiological problems such as hemolysis or immunogenicity of the active principle. In addition, the nanocarrier hydrophilic coating with polymers such as PEG reduces the tendency to aggregate by steric stabilization with an immediate impact on the freshly-prepared formulation for hospital injection and storage.

Alternatively, and to achieve the same benefits, the surface of the nanocarrier may be adsorbed or conjugated with human serum albumin (HSA) to conveniently increase the stability of the formulation, its circulation time and make it more biocompatible, "anticipating" the adsorption of HSA present in the blood while avoiding the adsorption of the abovementioned opsonines. In order to engineer the most suitable encapsulation system (e.g., micelle, liposome), the n- octanol/water partition coefficient (P) was expressed in the logarithmic (logP) form for some coordination compounds of general structure l(a) and l(b), described below. Examples of nano-formulations

As it will be evident from the examples provided below, by way of example, but not limitation of the present invention, using various biocompatible polymers and oligomers, the inventors have shown the possibility of making nano-formulations characterized by different hydrodynamic diameters (Dl). Advantageously, the size of said nano-formulations falls within the range of 10 ÷ 100 nm, being ideal for pharmaceutical applications.

LogP Assessment Procedure

By way of example, we describe the procedure for assessing the n-octanol/water partition coefficient (P), expressed in logarithmic form (logP), of some coordination compounds with general structure l(a) and l(b). As it is well known, this procedure is useful in selecting the most suitable supramolecular system for a particular type of compound and/or application. The concentration of the compound in the organic phase first (C 0 ) and after separation (d) was evaluated by UV-Vis spectrophotometry. The values obtained for some compounds were used in the calculation of the n-octanol/water partition coefficient (P) logP = logf^/CCo - C ] as shown in the following table:

Table 1. Experimental values of logP = log[C 1 /(C 0 — C^] of coordination compounds according to this invention.

Given the results shown in Table 1 , it is evident that the inventors have engineered coordination compounds according to this invention having differentiated logP values. Some examples of supramolecular systems that the inventors have developed based on the determined logP value are reported below.

Encapsulation in vesicles

Without loss of generality of this invention, we describe below a procedure for loading some compounds accordingly to this invention having logP <0 into nanolipidic systems, or vesicles known as liposomes. Among the nanolipid systems, the inventors have advantageously prepared 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocoline vesicles (DPPCs) as they are highly biocompatible, biodegradable, low in toxicity and able to self-assembly to form vesicular structures (CMC = 0.46 nM). Liposomes were prepared by dissolving DPPC (4 mg) and a compound (0.4 mg) in chloroform. The solvent was slowly evaporated through nitrogen stream to obtain a homogeneous DPPC/[Au (ProOtBuDTC) 2 ] Br film, which was further dried under reduced pressure. Subsequent hydration took place at temperatures above the critical temperature for the phospholipids (about 50 °C) by adding 1 mL of saline solution (NaCI (aq) 0.9% w/v). The obtained suspension was then agitated for 30 min and then subjected to fast freeze/thaw cycles in liquid nitrogen and water bath at 37°C to obtain large polydispersed unilamellar vesicles, then subjected to an extrusion process through a porous polycarbonate membrane having an average pore diameter of 100 nm to advantageously isolate small unilamellar vesicles. The liposomes thus obtained were subjected to a dialysis process for two hours against saline to eliminate traces of non-encapsulated coordination compound and free phospholipid.

The stability of the compound was determined by UV-Vis spectrophotometry in saline (NaCI (aq) 0.9% w/v) at a compound concentration of about 50 μΜ (at 37 ° C for 72 h). With reference to the enclosed Figure 4, here given merely as an example, the spectra recorded over time for the DPPC vesicle-encapsulated [Au(ProOtBuDTC) 2 ]Br compound exhibit the stability of the compound and the hyperchromic effect of the absorption bands is attributable to a diffused light increase due to the formation of larger size aggregates in the solution. This was then confirmed by sample DLS measurements after 72 hours, recording a Dl that varies from 90 ± 17 to 1799 ± 480 nm.

Although this example refers to liposomes of dipalmitoylphosphatidylcholine derivatives (DPPCs), with trivial modifications to the skilled in the art, the procedure is practically similar for other classes of amphiphilic polymers useful for the production of vesicles. The inventors have also identified some alternative processes for the preparation of liposomes encapsulating hydrophilic coordination compounds accordingly to the invention, in which an aqueous solution of the compound itself is used instead of an organic solvent. By way of non-limiting example of this invention, a first process involves: the dissolution of the biocompatible polymer or more than one in an organic solvent such as chloroform; removal of the solvent; hydration of the lipid film with an aqueous solution and treatment of the suspension obtained by ultrasounds and/or sonication; extrusion through membranes having a certain cut-off; dialysis against aqueous solution such as saline or a phosphate buffer; incubation with the solution of the compound at a T>Tc in water bath under continuous stirring for at least 20 minutes; lyophilization (in the presence of cryoprotector); hydration and extrusion. The cryoprotector may be, for example, mannitol or lactose or sucrose or trealose or glucose or maltose, ethanol or a combination of them. Alternatively, a second process involves: dissolution of the biocompatible polymer in water or a glycerol/ethanol mixture or alternatively the dissolution of several polymers in an organic solvent, such as chloroform, followed by the removal of the solvent; the addition of the aqueous solution of the compound; agitation of the obtained mixture and multiple freeze/thaw cycles in liquid nitrogen and water bath at 37 °C; extrusion.

Finally, a third procedure involves the dissolution of the biocompatible polymer (or polymers) in an organic solvent, for example chloroform; adding the aqueous solution of the compound to form an emulsion; agitation of the obtained mixture and evaporation of the solvent medium; the addition of a buffer and purification by extrusion.

Encapsulation in micelles

A procedure for loading the compounds accordingly to this invention having logP> 0 into micellar systems is described below. By way of example, but not limitation, the inventors have developed an encapsulation system using Pluronic ® F127 (PF127) as a polymer substrate. However, the procedure is practically analogous to any amphiphilic polymer, for example mPEG, with trivial modifications for the skilled in the art.

The encapsulation of the complexes having Formula l(a) and/or l(b) in micelles of PF127 was obtained by a process comprising the following steps described herein by way of non-limiting example of this invention: co-dissolution, in the desired stoichiometric ratio, of the compound to load and of the polymer (e.g., 0.5 mg and about 500 mg respectively) in an organic solvent, preferably chloroform; evaporation of the organic solvent under reduced pressure and drying of the obtained powder, preferably under vacuum; next hydration by adding deionized water; purification to remove the non-encapsulated compound, bacteria and other impurities, for example by membrane filtration having a pore size of 0.20 μηι; freeze in dry ice/acetone bath at -78 °C and cryo-drying to remove aqueous solvent residues and get a ready-to-use formulation to be stored for first use.

The amount of 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 determined by the law of Lambert-Beer, after experimental determination of the molar extinction coefficient ε in DCM (for "matrix effect" in the presence of the same polymer used for the preparation of micelles) for some absorption bands. The results obtained are shown in the following table.

Table 2. Amount of encapsulated compound in terms of drug loading (mol of encapsulated compound per mg of formulation) and Encapsulation Ratio (ER), i.e., the percentage amount of encapsulated compound compared to the amount of nominally-loaded compound

Advantageously, the encapsulation in micelles of metallic compounds accordingly to this invention increases their stability in the physiological media and makes them soluble in water for at least 72 hours. Indeed, with reference to the enclosed Figure 5, illustrated herein by way of example and not limiting this invention, the electronic spectra collected over time surprisingly show no significant change if compared to the same non-encapsulated compound. It is evident that in this way, the inventors have obtained compositions comprising the coordination compounds of this invention characterized by total solubility and stability in the physiological media compared to the corresponding non-encapsulated compounds, thus achieving a further scope of this invention.

Table 3: Structural parameters of the micellar systems shown here by DLS analysis.

It is evident, given the results presented in the table, that micellar systems having 25 nm ca hydrodynamic diameters are particularly advantageous for pharmaceutical applications. As mentioned, the use of PF127 does not limit the scope of this invention, and other polymeric substrates can be conveniently used.

Encapsulation into cvclodextrines

By way of example, but not limitation, a procedure for loading a Cu(ll) coordination compound having logP>0, into cyclodextrin is described below. The inventors hereby provide a formulation based on 2-hydroxypropyl^-cyclodextrin (ΗΡ-β-CD), a cyclic oligomer consisting of seven a- (1 ,4)-D- (+) -glucopiranoside with a 0.8 degree of molar substitution for hydroxypropyl groups. ΗΡ-β-CD cyclodextrins are a nanocarrier with a high biocompatibility and biodegradability and are already used in clinics in formulations administered parenterally.

Samples are prepared by dissolving the cyclodextrin and the coordination compound [Cu (ProOMeDTC) 2 ] (molar ratio 1 :1 ) in DMSO and shaking the system for 15 hours. The organic solvent is then removed under reduced pressure, the residue dissolved in water, centrifuged, and the filtered liquid phase (0.22 μηι filter) is lyophilized to form a ready-to-use formulation. As shown in Figure 6, By way of example, but not limitation, the UV-Vis curves collected over time do not show significant spectral variations, demonstrating the high stability of the formulations presented herein.

EVALUATION OF THE BIOLOGICAL ACTIVITY SOD in vitro test (superoxide dismutase).

As mentioned above, the production of ROS species increases in articular diseases such as osteoarthritis and rheumatoid arthritis. Among ROS species, the superoxide 0 2 ~ is the main one and its increased production results in tissue damage associated with inflammation.

Therefore, in order to assess the "scavenging" activity of superoxide radicals of the claimed compounds, an enzymatic method based on xanthine oxidase (XO, an enzyme belonging to the class of oxidoreductase, was utilized, which, as illustrated in Figure 1 , catalyzes the following reaction: xanthine + H 2 0 + 0 2 ^ uric acid + H 2 0 2 .

The anti-inflammatory activity was assessed over time by spectrophotometry at 595 nm using 96-well plates and recording the in situ reduction of the NBT colorimetric probe (nitroblue ditetrazolium chloride) to the corresponding blue formazan derivative by reaction with superoxide radicals. The latter are produced by oxidation of HX (Hypoxanthine) mediated by xanthine oxidase enzyme (XO).

The conditions adopted were as follows: V T0T = 200 μΙ_; C X = 452 μΜ; C XO = 0.26 U / ml_; C NB T = 500 μΜ; C SO D = 328 U / ml_; C Co mposto = 50 μΜ.

With reference to the enclosed Figure 2 by way of example, but not limitation of this invention we report data collected for 4 compounds and, for comparison purposes, the data related to the reference drug for the treatment of rheumatoid arthritis (Auranofin/Ridaura ® ). The pure superoxide dismutase enzyme (SOD, from bovine liver) served to check the production of superoxide radicals in situ and then to compare it with said compounds.

It is evident that the compounds tested at 50 μΜ exhibit significant scavenging capacity towards superoxide radicals compared to the SOD enzyme. In addition, these compounds exhibit a marked activity in this inflammatory model, which is higher than the one for the reference drug, which has been recorded at a higher concentration of 200 μΜ.

In vivo anti-inflammatory activity

An in vivo therapeutic efficacy study on an animal model of collagen-induced experimental arthritis (called CIA) was carried out. CD-1 mice were immunized with a collagen bovine solution emulsified with a complete Freund adjuvant. Animals were monitored to assess the onset of experimental arthritis. Twenty-one days after immunization with bovine collagen, the administration of the nanoformulation and empty carrier (control) administration was performed twice per week by the intraperitoneal route. 8 mice were taken for treatment with the encapsulated compound [AuBr 2 (ProOtBuDTC)] (the dosage was 5 mg/kg) and 8 with the empty carrier (solvent medium: phosphate buffer). Mice were assessed beginning from the induction of arthritis according to a number of parameters: the weight of the animal, the "paw score" and the diameter of the ankle joint and the femoral swelling. In particular, in order to assess the progression of the disease, a standard score of 0-5 was used, in which 0 means healthy mice while 5 is associated with severely ill animals and close to death. The score level was defined by assessing the vitality of the animals, their posture, the presence of orchitis, signs of pain, swelling of the lower and upper limbs as well as the presence of alopecia and sores. Most treatments began at a 2 score stage of the pathology, while some at stage 3 or 4. The onset of arthritis is occasionally very rapid and disabling. Control mice (ill and treated with the empty nanocarrier) reached score 5 quickly and were sacrificed. With reference to the enclosed Figure 3(a), during the 8 treatments the weights of the animals increased similarly to control. The data given in the enclosed Figure 3 (b) show how animals treated with [AuBr 2 (ProOtBuDTC)] survived until the end of the study with a general betterment in vitality and overall mobility. Notably, those treated starting with an overall 2 score showed a decrease in the severity of the disease over time, whereas those who started treatment at a 3 or 4 score remained stable. It will therefore be evident to the skilled in the art, through the in vivo tests carried out on a derivative of Au(lll), as herein exemplified, how the coordination compounds accordingly to this invention perform an anti-inflammatory activity and effectively stall the progression of the disease by reducing the severity of tissue inflammation.

Acute toxicity test in mice

Some compounds were tested in vivo to assess the acute toxicity by intravenous administration of a single dose to male mice (provided by Charles River Laboratorio Italia, Calco, Lecco, age at the beginning of the study: 6 weeks). This route of administration was chosen for its reduced barrier to absorption of substances; it provides a more stringent measure for the toxicity compared to other routes used in 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 on this species and strain, given the ample bibliography on it.

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

The compounds tested are: [Cu(tetraAc-glycosylammideProDTC) 2 ], [AuBr 2 (tetraAc- glycosylammide-ProDTC)]. The vehicle was chosen to be DMSO-EtOH-RL 50:10:40% v/v where RL stands for Ringer lactate (Eurospital ® ). Each experimental group consisted of 6 mice + 4 control animals (treated with the only vehicle).

Each dose was prepared by dissolving a calculated amount of compound in DMSO; this volume was then diluted with ethanol and lactate Ringer (RL) to obtain the desired final concentration. The administration of the compounds examined was carried out by intravenous injection into the caudal artery of each experimental mouse. Each treated animal received an accurate injected volume of 100 μί, containing the amount of test substance as described above, i.e. 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 the injection, during the first hour and then onward in the following days for 7 days of experimentation, together with their body weight.

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 animal groups survived until the end of the study, and during the post-treatment period, they did not show signs of toxicity. Macroscopic necroscopy did not show any obvious signs of toxicity on the organs examined: the brain, the lungs, the spleen, the testes, the heart, the liver and the kidneys.

Table 4.Treated and control mice body weight

Table 5. Clinical and behavioural observations during the experimental study. TRADEMARKS

Ridaura ® is a trademark of Prometheus Laboratories Inc;

Pluronic ® is a trademark of BASF AG;

Cremophor ® is a trademark of BASF AG;

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

Eurospital ® is a trademark of Eurospital S.p.A.;

Infliximab ® is a trademark of Hospira UK Limited;

Condroitin ® is a trademark of Labyes do Brasil Ltda;

Salazopirina ® is a trademark of Pharmacia AB;

Etanercept ® is a trademark of Immunex Corp.;

HUMIRA ® is a trademark of AbbVie Biotechnology Ltd;

Kineret ® is a trademark of Amgen Inc.;

Hyalgan ® is a trademark of Fidia Farmaceutici S.p.A.;

ARTZ ® is a trademark of Seikagaku Corp.;

Myocrisin ® is a trademark of Sanof i S.p.A;

Solganal ® is a trademark of Schering-Plough Canada Inc.