WO2017058012A1

WO2017058012A1 - Gold(iii) compounds as tartrate resistant acid phosphatase inhibitors, and therapeutic uses thereof

Info

Publication number: WO2017058012A1
Application number: PCT/NL2016/050666
Authority: WO
Inventors: Barbro Natascha MELGERT; Angela Casini
Original assignee: Rijksuniversiteit Groningen
Current assignee: Rijksuniversiteit Groningen
Priority date: 2015-09-29
Filing date: 2016-09-29
Publication date: 2017-04-06
Anticipated expiration: 2018-03-29

Abstract

The invention relates to gold (III) compounds as Tartrate Resistant Acid Phosphatase (TRAP) inhibitors, and to uses thereof, like in the treatment of pulmonary diseases like COPD and asthma. The inhibitory compounds have the general formula (A), (B), (C), (D) or (E).

Description

Title: Gold(III) compounds as Tartrate Resistant Acid Phosphatase inhibitors, and therapeutic uses thereof.

The invention relates to the fields of organometallic chemistry, enzymology, and medicine. In particular, it relates to gold (III) compounds as Tartrate Resistant Acid Phosphatase (TRAP) inhibitors, and to uses thereof, among others in the treatment of respiratory /pulmonary diseases like chronic obstructive pulmonary disease (COPD) and asthma.

COPD is a common but relatively unknown disease in which airways are chronically inflamed (chronic bronchitis) and lung tissue is destroyed (emphysema). Patients are characterized by varying contributions of both phenotypes. It affects around 10% of the population worldwide and is caused by cigarette smoking, air pollution and/or indoor biomass cooking. COPD is the fourth cause of mortality and morbidity in the world and is projected to be the third by 2030. Currently no effective therapy exists and transplantation is the only option for end-stage disease.

The pathogenesis of emphysema is incompletely understood but macrophages have been shown to be important in the process (6). Exposure to cigarette smoke and air pollution induces inflammation in the lungs with infiltration of macrophages and neutrophils and, after prolonged exposure, development of emphysema (7). Macrophages are considered to be the more relevant effector cells because deletion studies in mice have shown that these were indispensable to developing emphysema, while neutrophils were not (8). Macrophages can produce a host of proteolytic enzymes like matrix metalloproteins (MMPs) and cathepsins that have the ability to degrade extracellular matrix (ECM) and therefore lead to lung tissue destruction. In addition, alveolar macrophages are one of the few macrophage subsets in the body that highly express the enzyme TRAP, which in bone is involved in ECM turnover and may have a similar function in the lung (1). Asthma is a chronic inflammation of the airways after becoming allergic to airborne allergens and affects 5-20% of the people in the Western world. Treatment consists of anti -inflammatory drugs and bronchodilators. However, a substantial group of patients with severe asthma are not treated effectively with the currently available drugs and are particularly

susceptible to acute worsening of their disease.

An important clinical problem for patients with lung diseases like COPD or severe asthma is their susceptibility to exacerbations. These exacerbations are an acute worsening of symptoms mostly caused by viral and/or bacterial infections. Exacerbations are the major cause of morbidity and mortality in COPD and asthma. Dysfunction of alveolar macrophages has been presumed to be an important cause of impaired responses to infections (9).

A study by Capelli et al. (10) reported that the enzyme TRAP was found to be upregulated in alveolar macrophages of smokers as compared to nonsmokers. Vuillenemot et al. (11) reported upregulation of TRAP gene ACP5 in an animal model of COPD.

The present inventors hypothesized that TRAP expressed by macrophages in the lung may be involved in the pathogeneses of asthma and COPD. Indeed, in both diseases a higher expression of TRAP was found in lung tissue of patients as compared to controls (Figure 1A-B), suggesting TRAP may be involved in alveolar macrophage dysfunction. In addition, increased TRAP expression was found in mouse models of asthma and COPD (Figure 1C and D).

ACP5 encodes the enzyme tartrate-resistant acid phosphatase

(TRAP; EC3.1.3.2), which belongs to the family acid phosphatases and distinguishes itself by its resistance to inhibition by tartrate. It is a metalloenzyme and contains two iron atoms at its active site. It is primarily found in lysosomes, and similar organelles, of osteoclasts, alveolar macrophages, and activated macrophages and dendritic cells. Its function is unknown but two main functions have been proposed based on the two enzymatic activities the protein harbours. Its only known substrate is the heavily phosphorylated protein osteopontin (OPN). OPN can be present extracellularly as part of lung ECM and intracellularly as a signalling molecule. One of TRAP's proposed functions is the dephosphorylation of extracellular OPN to inhibit adhesion of macrophages to ECM and promote migration of macrophages (12,13). TRAP has also been shown to promote the metastasizing behaviour of tumors, confirming the idea it may be involved in cell migration (14).

In addition, TRAP may be involved in impaired antimicrobial responses by dephosphorylating intracellular OPN. For instance, viral stimulation of TLR9 induces phosphorylation of OPN and subsequently leads to increased IFNa production to combat the virus. In patients lacking TRAP expression, IFNa levels have been found to be increased (15).

Conversely, in patients with COPD and asthma lower levels of IFNa have been found, suggesting overproduction of TRAP, and explaining their impaired responses to viral infections (16, 17).

Thus, from a therapeutic point of view, ACP5/TRAP is an interesting molecular target. Unfortunately, the only known inhibitors of TRAP are the toxic metal compounds molybdate, vanadate, lead acetate, mercuric acetate and gold(III) chloride. The in arthritis therapeutically active gold(I) compounds Aurothioglucose and Aurothiomalate inhibit TRAP only at high concentration (mM range) (20). Gold chloride has been shown to be able to inhibit TRAP (Hayman et al, Cell Biochem Funct 2004, 22L 275- 280), but is too unstable to be used therapeutically since it is prone to undergo reduction to colloidal gold, which may also result in toxic effects. No other substances that are capable of inhibiting TRAP and suitable for therapeutic or diagnostic applications have been reported so far.

Recognizing the potential of ACP5/TRAP as therapeutic target, the present inventors therefore set out to search for novel inhibitors that can be used in vivo to inhibit TRAP. In particular, they aimed at providing non-toxic inhibitory compounds showing an IC50 value in the nanomolar range.

This goal was met by the surprising finding that various gold(III) coordination compounds bearing N-donor ligands are potent TRAP inhibitors in vitro with IC50s in the nM range. Moreover, these compounds are not cytotoxic to macrophages in the concentration range showing an inhibitory effect. Interestingly, TRAP was shown to be involved in

macrophage migration and this migration could be inhibited using the novel gold-based inhibitors. Lastly, it was demonstrated that inhibition of TRAP modulates the responses of macrophages to microbial compounds.

Accordingly, in one embodiment the invention provides a gold(III) coordination compound bearing N-donor ligands, the compound having the general formula A, B, C, D or E

wherein dotted lines can be absent or present; each of Ri through Rn is independently selected from the group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH2, aliphatic amines -R-NH2); halogens (e.g. chloride, iodide); moieties with hydroxyl functional groups (e.g. -OH or-Y-OH); ether containing moieties of general formula -Y-O-Y'; carbonyl containing moieties (-Y- C(O)OH); or of amide bonds (-Y-C(O)N-Y-); sulfonamidic groups; nitrile/nitro groups, and peptide moieties, wherein Y and Y are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms;

L and L' are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thiol -bearing groups (e.g. thio-sugars, cysteine and methionine groups); and

Z is a cyclic moiety selected from the group consisting of homocyclic and heterocyclic aromatic/aliphatic moieties, preferably 6,6-, 5,6- or 6,5-fused bi-homocyclic and bi- heterocyclic aromatic/aliphatic moieties, wherein the heterocyclic moieties may include nitrogen, oxygen and/or sulfur atoms, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating and/or alleviating the symptoms of a disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity.

Some gold (Ill)-compounds for use in the present invention are known in the art. For example, Messori et al. (J Med Chem 2000, 43, 3541-3548) discloses gold complexes wherein the gold (III) center is coordinated to a polydentate ligand with nitrogen donors, such as the compound [Au(terpy)Cl]Cl2 that belongs to the general formula B shown herein above. Discussed is the use of the gold complexes as cytotoxic and antitumor drug. Casini et al. (Dalton Trans., 2010, 39, 2239-2245) disclose the synthesis, characterization and biological properties of square planar gold(III) compounds with modified bipyridine and bipyridylamine ligands according to the general formula A. The complexes showed cytotoxicity in vitro towards the A2780 human ovarian carcinoma cell line and a cisplatin resistant variant thereof.

WO2013/005170 relates to metal-based modulators that selectively bind to cell transmembrane proteins aquaglyceroporins AQPs, leading to its inhibition. The selective inhibition of AQP channels was accomplished among others by compounds tetracoordinated to gold (III) complexes according to the general formula A, B, C or E as shown herein above. Also disclosed is the use thereof in manufacturing pharmaceuticals, cosmetics and chemical reagents for diagnostic, treatment, prophylaxis and prevention of clinical conditions directly or indirectly related to aquaglyceroporins AQPs functions.

Hayman et al. (2014, Cell. Biochem. Funct. 22: 275-280)

investigated whether gold (III) compounds mediate their effect on

osteoclastic bone resorption by modification of TRAP activity. It was found that gold chloride was a powerful TRAP inhibitor whereas aurothioglucose or aurothiomalate had no effect.

Thus, the art fails to teach or suggest the use of the gold (III) compounds as claimed herein for the inhibition of TRAP or for the treatment of a disease associated with TRAP activity, like respiratory/ or pulmonary diseases such as COPD and asthma.

In a gold(III) compound for use according to the invention, each of Ri through Rii on the N-donor ligands is independently selected from the group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cyclo aliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH2, aliphatic amines -R-NH2); halogens (e.g. chloride, iodide); moieties with hydroxy! functional groups (e.g. -OH or -Y-OH); ether containing moieties of general formula -Y-O-Y; carbonyl containing moieties (-Y-CO- OH); or of amide bonds (-Y-CO-N-Y-); sulfonamidic groups; nitrile/nitro groups, and peptide moieties, wherein Y and Y are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic- aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and

heterocycloaliphatic groups comprising up to four C-atoms.

In a preferred embodiment, each of Ri through Rn (also referred to herein as "R substituents" or "R groups") is independently selected from the group consisting group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C- atoms. For example, each of Ri through Rn is independently selected from H, C1-C4 alkyl and C1-C4 alkoxy.

In another embodiment, at least one of the R substituents is selected from the group consisting of amines (e.g. NH2, aliphatic amines -R-NH2);

halogens (e.g. chloride, iodide); moieties with hydroxyl functional groups (e.g.-OH; -Y-OH); ether containing moieties of general formula -Y-O-Y; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y-); sulfonamidic groups; fluorophores; nitrile/nitro groups, and peptide moieties, wherein Y and Y are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic,

heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms. In some embodiments, one or more of Ri though Rn is F, -CH₃, -CH2CH3, -OH, -OCH3, or -OCH2CH3. In some embodiments, at least one of Ri through Rn is -CH3 or -OCH3. In one embodiment, at least one of Ri through Rn is other than H, so that the (hetero)aromatic rings contain one or more substituents. The number of R substituents other than H can vary. In one embodiment, the compound contains up to 6, up to 5, or up to 4 R-groups other than H. For example, suitable compounds for use according to the invention are those having one, two, three or four R substituents selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines; halogens; moieties with hydroxyl functional groups; ether containing moieties of general formula -Y-O-Y'; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y'-);

sulfonamidic groups; nitrile/nitro groups, and peptide moieties, while the remaining R groups are H. In another embodiment, all of the R groups are H, i.e. the gold(III) coordination compound bears unsubstituted N-donor ligands.

L and L' are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thiol-bearing groups e.g. thio-sugars, cysteine and methionine groups. In a preferred embodiment of the invention, L and L' are halogen, preferably chloride or iodide. Compounds according to any one of formula A, B, C, D or E can be screened for their efficacy using methods known in the art. For example, US

6,451,548 discloses an assay employing (recombinant) TRAP that has been activated with a cysteine protease and a phosphotyrosine or phosphoserine containing test compound. Disclosed herein below is a method using recombinant TRAP or a cell lysate as source of TRAP, and para- nitrophenylphosphate (PNPP) as enzyme substrate.

In one aspect, the invention provides a compound for use as TRAP inhibitor according to formula A, being an [Au(substituted-2,2'-bipyridine)LL']ⁿ⁺ with n = 1 or 2 or an [Au(2-phenylpyridine)LL'] ⁿ⁺ with n = 0 or 1. In a preferred aspect, the invention provides the use of a compound of formula A based on a bipyridine i.e. wherein X is N.

In an inhibitor according to formula A, all substituents Ri through Rs can be H. In one aspect, at least one, two, three or four of Ri through Rs is other than H. The substituents Ri through Rs can be symmetric or asymmetric. Symmetrically substituted formula A compounds include those having substituents other than H at positions Ri and Rs; R2 and R₇; R3 and R6; R4 and R5; Ri, R2, R7 and Rs; R3, R4, R5 and R6, and so forth. In one

embodiment, the substitutions are (only) at positions 3,3'; 4,4'; 5,5' or 6,6' of the bipyridine or phenylpyridine ring. Preferably, the substituents other than H are (solely) at positions 4, 4'. For example, provided is a TRAP inhibitor according to formula A wherein X is N, and wherein the

substitutions are at positions 4, 4 of the bipyridine ring.

In another embodiment, the formula A compound is asymmetrically substituted. Exemplary asymmetric compounds include those having a single substituent other than H, and those having substituents other than H at positions Ri and R₇; R2 and Rs; R3 and R5; R4 and Rs; Ri, R2, R6 and R₇; R3, R4, R7 and Rs, and so forth. In a specific aspect, a compound of formula A is symmetric having two aliphatic or heteroaliphatic substituent at positions R3 and R6, such as those wherein R3 and R6 are methoxy or ethoxy.

In one aspect, each of substituents Ri through Rs of a formula A inhibitor compound is independently selected from the group consisting of H, C1-C4 alkyl and C1-C4 alkoxy. An exemplary Formula A compound is

[Au(bpOMe)Cl₂][PF₆] (bpOMe = 4,4'-dimethoxy-2,2'-bipyridine] (Aubipy-O- Me). Useful variants of Aubipy-O-Me include compounds having different substituents at the same positions, and compounds having one or more Ci- C4 alkoxy substituent(s) at different positions of the bipyiridine ring. In a further embodiment, the inhibitor is a substituted-2,2',2"- terpyridine compound of formula B, wherein preferably up to four of substituents Ri through Rii are other than H. In one aspect, it is an [Au(substituted-2,2',2"- terpyridine)L]ⁿ⁺ with n = 2 or 3. An exemplary formula B compound is

[Au(2,2':6',2"-terpyridine)Cl]Cl (Auterpy).

In a still further embodiment, the compound for use according to the invention is a polypyridyl compound of formula C. The polypyridyl moiety depicted by Z is a cyclic moiety selected from the group consisting of homocyclic and (hetero)cyclic aromatic and aliphatic moieties. The heterocyclic moiety may include nitrogen, oxygen and/or sulfur atoms. In one embodiment, Z is a fused ring system comprising one, two or three (hetero)cyclic moieties. Preferably, Z is a 6,6-, 5,6- or 6,5-fused bi-homocyclic or bi-heterocyclic aromatic/aliphatic moiety. Preferably, Z is a heterocyclic aromatic moiety, more preferably a heterocyclic aromatic moiety comprising two nitrogen atoms. In one aspect, the compound is an

[Au(polypyridyl)LL']ⁿ⁺ with n = 1 or 2.

The polypyridyl scaffold can for example be based on dipyrido [3, 2-f:2', 3'-h] quinoxaline (DPQ), dipyrido [3, 2-a:2', 3'-c] phenazine (DPPZ), or dipyrido

[3,2-a :2',3^*-c] (6,7,8, 9-tetrahydro) phenazine (DPQC).

DPQ DPPZ DPQC Accordingly, exemplary inhibitory compounds according to formula C include those wherein the polypyridyl moiety is dipyrido[3,2-f:2',3'- h]quinoxaline, dipyrido[3,2-a:2',3'-c]phenazine, or dipyrido[3,2-a:2',3'- c] (6 , 7, 8 , 9 -tetr ahy dro)phenazine . In a further embodiment, the inhibitory compound has a structure according to formula D. The dotted lines in the general formula D can be present or absent. For example, the compound may bear bidentate or tridentate nitrogen donor ligands selected from 2-(2-pyridyl)imidazole, 2- phenylimidazole, 2,6-bis(benzimidazol-2-yl)pyridine, and l-methyl-2-[2- pyridyl]-lH-benzo[d]imidazole). Very good results were observed with compounds wherein up to four of Ri through R9 are other than H.

Preferably, at least R5 is other than H, for example R5 is C1-C4 alkyl or C1-C4 alkoxy. In a specific embodiment, only R5 is other than H, for example R5 is C1-C4 alkyl or C1-C4 alkoxy. For example, the inhibitory compound is [Au(l- methyl-2-[2-pyridyl]-lH-benzo[d]imidazole)Cl2]Cl (AuPblmMe). A compound according to formula D has not been disclosed or suggested in the art.

Hence, the invention also provides a compound according to formula D and its use as medicament. Preferably, the compound is [Au(l-methyl-2-[2- pyridyl]-lH-benzo[d]imidazole)C12]Cl (AuPblmMe) or functional equivalent thereof. Also provided is a pharmaceutical composition comprising a compound according to formula D, and a pharmaceutical carrier, diluent or excipient.

Still further aspects of the invention relate to inhibitory compounds according to formula E, being based on a phenantroline scaffold. Preferably, the compound is an [Au(substituted-l, 10-phenantroline)L2]ⁿ⁺ with n= 1, 2 or 3. Good results can be observed with compounds wherein up to four of Ri through Re are other than H.

An inhibitor compound for use in the present invention can be synthesized using methods known in the art. See for example Casini et al., Dalton Trans. 2010, 39, 2239- 2245 or HoUis et al, J. Am. Chem. Soc. 1983, 105, 4293 -4299. Compounds of formula D can be synthesized adapting the conditions from Casini et al., Dalton Trans. 2010, 39, 2239- 2245, and also from Serratrice et al., Inorg. Chem. 2012, 51, 3161-3171.

A gold (III) compound disclosed herein is suitably used as in vitro or in vivo inhibitor of tartrate-resistant acid phosphatase (TRAP). In addition, it can be modified to serve as (chemical) probe for detecting TRAP activity. For example, a probe can be a fluorescent derivative of a gold(III) compound of the invention. The probe finds its use for example in assessing the role of TRAP activity in a certain physiological process or cellular pathway. For example, the role of TRAP in the interplay between macrophages and cancer stem cells can be determined. In one aspect, the probe compound contains in at least one of the R positions a fluorophore such as an anthracenyl or coumarin moiety. Compounds containing 1, 10-phenantroline are also fluorescent per se.

Specifically, one of the R groups is an anthracenyl or coumarin moiety.

R = coumarin moeity In a further aspect, the gold (III) inhibitory compound is used, e.g. for experimental, scientific and/or drug-screening purposes, as in vitro inhibitor of TRAP.

In a preferred embodiment, an inhibitor compound finds it use in a method of treating and/or alleviating the symptoms of a respiratory disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity. Respiratory disease is a medical term that encompasses

pathological conditions affecting the organs and tissues that make gas exchange possible in higher organisms, and includes conditions of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, and the nerves and muscles of breathing. Respiratory diseases range from mild and self -limiting, such as the common cold, to life-threatening entities like bacterial pneumonia, pulmonary embolism, acute asthma and lung cancer. The study of respiratory disease is known as pulmonology. In a specific embodiment of the present invention, an inhibitor compound finds it use in a method of treating and/or alleviating the symptoms of a pulmonary disease associated with increased expression of TRAP, like asthma, COPD, smoke-induced pulmonary inflammation (e.g. cigarette smoke, wood smoke, biomass smoke, fuel combustion smoke) or (pulmonary) sarcoidosis.

In a preferred embodiment, the disease is a pulmonary disease such as chronic obstructive pulmonary disease (COPD) or asthma. Without being bound by theory, upregulation of ACP5 / TRAP in pulmonary diseases like COPD assists in degradation of collagen by macrophages and, therefore, aids parenchymal lung tissue destruction and assists in stimulation of macrophage migration through the lung. This enhanced migration could magnify the areas in which lung tissue can be destroyed. In addition, we propose that increased expression of ACP5/TRAP impairs antimicrobial responses in both asthma and COPD by yet unknown mechanisms, causing enhanced disease and an increased susceptibility to exacerbations. The inventors hypothesize that inhibition of TRAP in emphysema will slow down lung tissue destruction, and in COPD and asthma it will improve responses to infections. Accordingly, also provided is a pharmaceutical composition formulated for respiratory or pulmonary administration and/or inhalation, comprising one or more TRAP inhibitory compounds as disclosed herein. The inhalable formulation is a solution, suspension, emulsion, colloidal dispersion, or dry powder, wherein the formulation is suitable for administration to the lungs of a mammal.

In a specific aspect, the TRAP inhibitor compound is formulated for use or contained in a medical device called a metered dose inhaler (MDI) or a dry powder inhaler (DPI). A metered dose inhaler is a handheld device that delivers a specific amount of medication in aerosol form. The MDI consists of a pressurized canister inside a plastic case, with a mouthpiece attached. Its portability makes it easy to use anywhere, anytime. MDIs use a chemical propellant to push medication out of the inhaler. In a preferred embodiment, the compound is in the form of a dry powder and suitable for use in a dry powder inhaler, which is also a handheld device. A DPI delivers medication to the lungs as you inhale through it. It doesn't contain propellants or other ingredients.

To give the most effective dry powder aerosol, therefore, the particles should be large while in the inhaler, but small when in the respiratory tract. In an attempt to achieve that situation, one type of dry powder for use in dry powder inhalers may include carrier particles to which the fine active particles comprising the TRAP inhibitor adhere whilst in the inhaler device, but which are dispersed from the surfaces of the carrier particles on inhalation into the respiratory tract to give a fine suspension. In a specific aspect of the invention, the TRAP inhibitory compound is administered by the multidose breath-actuated dry powder inhaler (DPI) known under the tradename Novolizer.

A further embodiment of the invention relates to a method of treating and/or alleviating in a subject the symptoms of a respiratory disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity, comprising administering to the subject an effective amount of a TRAP inhibitor as herein disclosed. In a specific aspect, the method comprises administering at least one compound selected from the group consisting of [Au(bpOMe)Cl2][PF₆] (bpOMe = 4,4'-dimethoxy-2,2'-bipyridine] (Aubipy-O-Me); an [Au(substituted-2,2',2"- terpyridine)L]ⁿ⁺ with n = 2 or 3; [Au(2,2':6',2"-terpyridine)Cl]Cl (Auterpy); a compound of formula C, being an [Au(polypyridyl)LL']ⁿ⁺ with n = 1 or 2.; or a compound of formula C, based on dipyrido[3,2-f:2',3'-h]quinoxahne, dipyrido[3,2-a:2',3'-c]phenazine, or dipyrido[3,2-a:2',3'-c](6,7,8,9-tetrahydro)phenazine. In another embodiment, the method comprises administering a compound of formula D, bearing bidentate or tridentate nitrogen donor ligands selected from 2-(2-pyridyl)imidazole, 2-phenylimidazole, 2,6-bis(benzimidazol-2- yl)pyridine, and l-methyl-2-[2-pyridyl]-lH-benzo[d]imidazole; [Au(l-methyl- 2-[2-pyridyl]-lH-benzo[d]imidazole)Cl2]Cl (AuPblmMe); or a compound of formula E, being an [Au(substituted-l, 10-phenantroline)L2]ⁿ⁺ with n= 1, 2 or 3.

Exemplary diseases to be treated include those mentioned herein above, in particular respiratory diseases associated with increased expression of TRAP such as asthma, COPD, smoke-induced inflammation and sarcoidosis.

Also provided herein is a method of inhibiting TRAP activity in a mammal, comprising administering to the mammal an inhalable formulation described herein. In one aspect, the mammal has at least one symptom of a TRAP -dependent or TRAP-mediated (respiratory) disease or condition.

LEGEND TO THE FIGURES

Figure 1:

A: Numbers of macrophages showing TRAP activity are higher in

parenchymal lung tissue of patients with fatal asthma as compared to controls dying of nonpulmonary causes.

B: TRAP gene ACP5 is a major upregulated gene in lung tissue of COPD patients compared to non-COPD controls.

C: Numbers of macrophages showing TRAP activity are higher in

parenchymal lung tissue of mice exposed to house dust mite to induce experimental asthma than in healthy control mice.

D: Numbers of macrophages showing TRAP activity are higher in

parenchymal lung tissue of mice exposed to cigarette smoke to induce experimental COPD than in control mice exposed to air.

Figure 2:

Structures of exemplary compounds showing TRAP inhibitory activity. Figure 3:

Recombinant human TRAP was used to assess the TRAP inhibitory capacity of the gold (III) compounds Aubipy-O-Me, Auterpy, Auoxol and

Aurothiomalate as compared to NaAuCU (see also figure 4). TRAP can catalyze the formation of p-nitrophenyl (PNP) from the colorimetric substrate p-nitrophenyl phosphate, which can be measured

photospectometrically. Increasing concentrations of the gold compounds were used to study their inhibiting potential of this conversion. NaAuCU and Aubipy-O-Me were the most potent inhibitors (n=3). Figure 4:

Cytotoxicity of the gold compounds of the gold (III) compounds Aubipy-O- Me, Auterpy, Auoxol and Aurothiomalate as compared to NaAuCl₄ was measured by a proliferation assay using 3H-thymidin.

Figure 5:

Cell lysates of murine alveolar macrophages (MPI, kind gift of G. Fejer) were used to assess the TRAP inhibitory capacity of our gold compounds Aubipy-O-Me, AuPblmMe, (dipyNH)AuCl2 and (py^b-H)AuCl2 as compared to NaAuCl₄. PNP formation was assessed in the presence of tartrate to eliminate the contribution of other phosphatases to the dephosphorylation of PNPP. NaAuCl₄, Aubipy-O-Me and AuPblmMe were the most potent inhibitors (n=3).

Figure 6:

Pooled tissue lysate of lung tissue obtained from human COPD patients was used to assess the TRAP inhibitory capacity of the gold compound Aubipy-O- Me as compared to NaAuCl₄. PNP formation was determined in the presence of tartrate to eliminate the contribution of other phosphatases to the dephosphorylation of PNPP.

Figure 7:

A. In a transwell set-up, RAW 264.7 macrophages were allowed to migrate over an osteopontin-coated membrane with or without RANKL stimulation (200ng/ml). RANKL-stimulated macrophages migrated significantly more through an osteopontin-coated membrane as compared to vehicle-stimulated macrophages. B. The effect of RANKL-induced TRAP expression on migration behaviour was specific for osteopontin, as no effects of RANKL or Aubipy-O- Me were seen on macrophages cultured on collagen-coated membranes.

The number of cells that had migrated from the insert to the bottom well was calculated relative to the number of unstimulated cells that had migrated. Each experiment was performed in quadruplicate and performed 6 times.

C. Live cell tracking of macrophages in an osteopontin-coated well revealed that macrophage migratory behavior was higher in the presence of RANKL (200ng/ml) as compared to the vehicle -treated control and

AubipyOMe inhibited this migration. Differences were tested with a

Kruskal-Wallis test and a Dunn's post test, *P<0.05.

Figure 8:

Human monocyte-derived macrophages (n=3) have higher expression of several pro-inflammatory genes when stimulated with 100 ng/ml

hpopolysaccharide (LPS) as compared to control macrophages. Inhibition of TRAP during this LPS stimulation with 300 nM or 1 μΜ Aubipy-O-Me lowered the expression of these pro-inflammatory genes.

EXPERIMENTAL SECTION Materials and methods Cell culture

The Murine RAW 267.4 macrophage cell line was obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, Penicillin/ Streptavidin (Invitrogen, Carlsbad, CA) and L-glutamine (Invitrogen, Carlsbad, CA) at 37°C under 5% CO2 and humidified conditions.

Gold inhibitors

The following compounds were tested for their ability to inhibit TRAP activity: [AuCl₄]-, Authiomalate , AubipyOMe , Auterpy, Auoxol,

AuPblmMe, (dipyNH)AuCl2 and (py^b-H)AuCl2, wherein dipyNH means dipyridin-2-ylamine and py^b means benzylpyridine. AubipyOMe and

(dipyNH)AuCl2 were prepared and characterized using the procedure described by Casini et al. (Dalton Trans. 2010 Mar 7;39(9):2239-45). Auoxol was prepared according to reported procedure by Casini et al. (J Med Chem. 2006 Sep 7;49(18):5524-31). (py^b-H)AuCl₂ was prepared following the procedure reported by Cinellu et al. (J. Chem. Soc, Dalton Trans., 1996, 4217-4225). Auterpy was synthesized according to Hollis et al. (J. Am.

Chem. SOC. 1983, 105, 4293-4299). [AuCl₄]- and Authiomalate are commercially available from Sigma-Aldrich. AuPblmMe was synthesized according to conditions adapted from Casini et al. (Dalton Trans. 2010, 39, 2239- 2245). TRAP activity assay

TRAP activity was assessed by incubating either recombinant TRAP

(1.25ng/ml, R&D, Minneapolis, USA) or cell lysate (1:2 ratio) with L para- Nitrophenylphosphate (PNPP) solution [100 mM PNPP, 200 mM sodium citrate, 200 mM sodium chloride, 80 mM sodium tartrate (L+), pH 4.5]. Cell lysate was prepared by collecting cells directly from a culture flask in acetate buffer (pH 5.0) at a density of 500.000 cells/ml and sonication of cells. Subsequently, recombinant TRAP or cell lysate was incubated with increasing concentrations of AubipyOMe (range 0-40 μΜ). Samples were incubated with PNPP solution at a 1: 1 ratio up to an hour at 37°C. The reaction was stopped using 1M NaOH and the absorption at 410 nm was measured using a spectrophotometer. The inhibitory effect was calculated from the ratio of absorbance between the treated and untreated cells. The IC50 value was calculated as the concentration of inhibitor caused a 50% reduction in TRAP activity. Data was presented as mean ±SEM and consisted of at least three independent experiments.

Proliferation assay

Cytotoxicity of the compounds was assessed using 3H-thymidin. In short, RAW macrophages were grown in 96-well plates (Corning Incorporated, NY) in a medium volume of lOOul, at a density of 5000 cells/well and grown for 48h. Cells were exposed to various concentrations of inhibitor were prepared from a freshly made stock solution (lOmM) in DMSO and diluted in medium including relevant negative controls (range 31-3934nM). After 28h hours, tritium thymidine (0.25uCi/ml) (Perkin Elmer, Waltham, USA) was added for 10 hours, after which excess of thymidine was washed away. Cells were subsequently incubated with ΙΟΟμΙ of 10.5% TCA solution at 4°C for 30 min and lysed in ΙΟΟμΙ of 1M NaOH. The lysate was transferred to a scintillation vial with 4 ml of scintillation fluid (Perkin Elmer). Samples were measured using the LS 6500 Multi-purpose Scintillation Counter (Perkin Elmer). Macrophage motility

Motility of RAW macrophages was assessed by either live cell tracking using a confocal microscope or a Corning transwell culturing system (Sigma- Aldrich, St. Louis, USA). For the live cell tracking experiments, RAW cells were plated on osteopontin (OPN, K^g/ml) coated Lab-tek chamber slides (Nunc, Hatfield, USA) at a density of 7500 cells/well. Cells were stimulated with RANKL (200ng/ml, kindly provided by dr. R.H. Cool, University of Groningen, The Netherlands.) for two days to increase TRAP expression, followed by CFSE labeling (Invitrogen, Life Technologies Europe BV, Bleiswijk, The Netherlands), over night tracking in the presence of 1% zymosan solution (Sigma-Aldrich, St. Louise, USA), and presence or absence of 128 nM AubipyOMe. Video material was analyzed using Imaris software.

In addition to live cell imaging, migration ability was assessed using a transwell culturing system with inserts (Sigma-Aldrich, St. Louis, USA) coated with osteopontin (R&D, Minneapolis, USA) or collagen

(Advanced Biomatrix, Delta, Canada) to determine macrophage migration over the membrane. Briefly, RAW macrophages, pre-stimulated with or without 200ng/ml RANKL for 72h to increase TRAP expression, were seeded with 250,000 cells/well on 8 μιη pore inserts coated with 10μg/ml

osteopontin or collagen. Cells were cultured for 16h in the presence or absence of 128 nM AubipyOMe. The next day, total cell numbers in the lower compartment, including dead cells, were counted. The number of cells migrated was calculated relative to cells not stimulated with RANKL. Generation of monocyte-derived macrophages

Monocytes were isolated from buffy coats of healthy blood donors (NHS Blood and Transplant, UK) by Lymphoprep (Axis-Shield, Oslo, Norway) density gradient centrifugation followed by plastic adherence in IMDM (Lonza, Basel, Switzerland) containing 10% human pool serum, 1% penicillin/streptomycin (Invitrogen, San Diego, CA), and 0.25% ciprofloxacin (Bayer, Leverkusen, Germany). PBMCs were incubated, and nonadherent cells were removed after 2 h incubation by extensive washing with PBS. Subsequently, adherent monocytes were detached using PBS containing 10 mM EDTA at room temperature and plated for macrophage maturation at a density of 1 x 10⁵ cells/cm² in IMDM containing 10% human pool serum, 1% penicillin/streptomycin, and 0.25% ciprofloxacin.

TRAP-dependent macrophage responses to lipopolysaccharide (LPS)

For microarray analysis LPS -responses, MDMs were cultured for 7 days. At day 7, LPS (100 ng/ml; Salmonella minnesota R595; Alexis Biochemicals, Lausen, Switzerland), and 300 nM , or 1 μΜ Aubipy-O-Me were added 6 h prior to harvesting the cells (n = 3). Control cells received vehicle. RNA extraction and analysis

RNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. After extraction, the sample was incubated with Turbo DNase at 37 °C for 30 minutes and subsequently re-purified using RNeasy clean-up protocol. Expression of he following genes were

subsequently analyzed as compared to the expression of β-actin: Interferon- regulatory factor- 1 (IRF 1), Serpin Peptidase Inhibitor, Clade G (C 1

Inhibitor), Member 1 (SERPINGl), Apolipoprotein L3 (APOL3), interleukin- 6 (IL6), tumor necrosis factor alpha (TNF), Chemokine (C-C motif) ligand 20 (CCL20), Interleukin-12 p40 (IL12B), ndoleamine 2,3-dioxygenase 1 (IDO l), and Chemokine (C-C Motif) Ligand 4 (CCL4).

Animals

Male A/JOlaHsd mice and male and female BALB/c mice (8-10 weeks old) were obtained from Harlan (Horst, The Netherlands). Animals were kept in a temperature-controlled room with a 12h dark/light cycle and with permanent access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee. The animal

experiment was performed in the animal facility of the University of

Groningen according to strict governmental and international guidelines on animal experimentation.

Smoke-induced lung inflammation in mice

To model COPD, we exposed male A/JOlaHsd mice nose-only to mainstream cigarette smoke for 9 months in an experimental setup as described before by us (Van der Strate, 2006). In short, mice were exposed daily to mainstream smoke from four 2R1 Reference Cigarettes (University of Kentucky, KY). This protocol was continued for 5 days/week for 9 months. Control mice were sham-exposed to room air under similar conditions, following the same duration of exposure as the smoke-exposed group. The method used to check the delivery of total particulate matter by the smoking equipment has previously been described by Griffith and Hancock. The smoking equipment was calibrated before every smoking session to ensure accurate and standardized smoke exposure. Carboxyhaemoglobin levels were measured to determine actual smoke exposure.

Allergic lung inflammation

To model asthma, we exposed male and female BALB/c mice intranasally to whole body house dust mite (HDM) extract (Dermatophagoides

pteronyssinus, Greer laboratories, Lenoir, USA) in 40 μΐ phosphate-buffered saline (PBS) according to a protocol we have described before (Draijer med inflame 2013). In short, mice were exposed to HDM extract under isoflurane anesthesia: one time to a high dose of HDM (100 μg) in the first week, 5 times to a low dose (10 μg) in the second week and were sacrificed on day 21. Control animals (n=8) were exposed to 40 μΐ PBS according to this same schedule. Mice were sacrificed on day 24, three days after the last HDM exposure and the right lung was inflated with 0.5 ml 50% Tissue-Tek® O.C.T.™ compound (Sakura, Finetek Europe B.V., Zoeterwoude, The

Netherlands) in PBS and formalin-fixed for histological analyses. Other parameters of allergic lung inflammation of these animals are described in detail by Draijer et al {Draijer 2013}.

Enzyme histochemistry

TRAP activity was measured via enzyme histochemistry. The staining was performed on 3μιη sections of mouse lung tissue imbedded in paraffin.

Deparaffinated, rehydrated sections were incubated over night in a zinc- buffer solution (0.1M Tris, pH 7.4 complemented with 30 mM calcium acetate, 23 mM zinc acetate and 37 mM zinc chloride), following preincubation in a 0.2 M acetate buffer (0.2 M sodium acetate, 50 mM L(+) tartaric acid, pH 5.0). Slides were then incubated for 2 hours at 37°C with the reaction solution (0.2 M acetate buffer containing 0.5 mg/ml Naphtol AS- MX phosphate (Sigma-Aldrich, St. Louis, USA) and 1.1 mg/ml fast red TR salt (Sigma-Aldrich, St. Louis, USA). A hematoxylin counterstaining was applied and stained sections were imbedded in DEPEX mounting medium (VWR, Murarrie, Australia). The number of positive cells (#/mm2) was counted manually with the aid of ImageScope software.

Patient material

COPD

Gene expression data of TRAP was obtained from 311 patients with COPD and 270 non-COPD controls who were part of the Lung eQTL dataset from three academic sites. Details of this population as well as a detailed description of the whole genome mRNA profiling has been previously published by Brandsma et al. {Thorax 2015}. Data was corrected for the following potential confounders: age, gender, pack-years and smoking status. Lung tissue samples were collected from patients undergoing lung tumor resection or lung transplantation. In case of tumor resections, macroscopically normal lung tissue was taken far distant from the tumor and histology of all samples was checked for abnormalities using standard haematoxylin and eosin staining. Lung samples were obtained in accordance with local ethical guidelines.

Asthma

Post mortem lung tissues from subjects with fatal asthma or death from nonpulmonary causes (controls) were retrieved from the Department of Pathology of Sao Paulo University (Sao Paulo, Brazil). A detailed clinical and demographic description of this population has been previously published by Mauad et al. { Rev Panam Salud Publica 2008; 23: 418-423} For this study we investigated the presence of TRAP activity in paraffin- embedded peripheral lung tissue samples of 10 asthma patients and 10 controls as described below.

EXAMPLE 1: Gold(III) compounds inhibit TRAP activity The inhibitory capacity of four different Gold(III) coordination compounds on TRAP was assessed by using recombinant TRAP in combination with increasing concentrations of candidate inhibitors and these were compared to the positive control NaAuCL (figure 3). Two out of four compounds were able to reduce TRAP activity within the used concentration range, namely Aubipy-O-Me and Auterpy. Aubipy-O-Me was almost as potent as positive control NaAuCL in inhibiting TRAP activity. Its IC50 value was 476 nM as compared to 280 nM for NaAuCL (table 1). Notably, the lowest

concentration of Aubipy-O-Me already gave a significant reduction of TRAP activity compared to vehicle. The inhibitory effect of Aubipy-O-Me further increased in a dose-dependent manner and the highest concentration Aubipy-O-Me reduced TRAP activity significantly. Auterpy was less potent than Aubipy-O-Me and had an IC50 value of 1643 nM.

Cytotoxicity of these gold compounds was measured by a proliferation assay using 3H-thymidin (figure 4). None of the compounds except for Auterpy showed severe cytotoxicity in the concentration range needed for successful inhibition of TRAP activity. The IC50 value for inhibiting cell proliferation was lower than the value for inhibiting TRAP (1024 nM for proliferation inhibition versus 1643 nM for inhibition of TRAP), rendering Auterpy unsuitable for use of TRAP inhibition in cells (table 1 and 2).

EXAMPLE 2: Inhibition of cell-derived TRAP

The inhibitory capacity of three other different Gold(III) coordination compounds on TRAP in comparison to Aubipy-O-Me and NaAuCU was assessed by using cell lysates of high TRAP-expressing macrophages in combination with increasing concentrations of the candidate inhibitors (figure 5). AuPblmMe was equally effective as Aubipy-O-Me in this system, while the other two, (py^b-H)AuCl2 and (dipyNH AuC , were less potent. In this more complex cell mixture Aubipy-O-Me was less effective in inhibiting cell-derived TRAP as compared to using recombinant TRAP. The IC50 value increased 6-fold to 3 μΜ (table 3).

To test whether the Gold (III) compounds could also inhibit TRAP in lung tissue of COPD patients, pooled lung tissue lysates of COPD

patients were incubated with increasing concentrations of Aubipy-O-Me or NaAuCl₄ (see figure 6). Aubipy-O-Me could inhibit human TRAP in lung tissue of COPD patients with an IC50 of 8 μΜ. Tables 1-3 herein below summarize the IC50 data obtained. Table 1. IC50 of recombinant TRAP inhibition.

Recombinant

TRAP inhibition IC50 (nM)

Authiomalate No inhibition found

Auterpy 1643

Auoxo 1 No inhibition found

Aubipy-O-Me 476

[AuCU]- 280

Table 2. IC50 of the cytotoxicity measured with proliferation

Cytotoxicity

Proliferation IC50 (nM)

Authiomalate No toxicity found

Auterpy 1024

Auoxo 1 No toxicity found

AubipyOMe 7494

[AuCU]- No toxicity found

Table 3. IC50 of cell-derived TRAP inhibition.

TRAP isolated

from cells IC50 (nM)

[AuCU]- 470

AubipyOMe 2951

AuPblMe 3559

(dipyNH)AuCl₂ 4093

(py^b-H)AuCl₂ 10396 EXAMPLE 3: Macrophage motility depends on TRAP activity and is inhibited by AubipyOMe

Macrophage motility is suggested to be TRAP-dependent. Macrophages may require TRAP activity to detach and enable cell movement when attached to an osteopontin surface. Therefore, macrophages were seeded on osteopontin- coated surfaces when macrophage motility was assessed using live tracking of macrophages by confocal microscopy or using a trans well-incubation system in the absence or presence of the TRAP inhibitor AubipyOMe.

Videos of live-tracked macrophages (movies not shown, but representative tracking plots are shown in figure 7C)), indicated little movement of macrophages incubated with the vehicle, but a significant amount of movement in the presence of RANKL, a cytokine that induces the

expression of TRAP in these macrophages. Following incubation with the inhibitor, macrophages' movement is less, suggesting that TRAP activity is necessary for macrophage movement. These results were quantified in a transwell-migration assay, in which more RANKL-stimulated macrophages migrated through an osteopontin-coated membrane compared to vehicle- stimulated cells (figure 7A), while this migration was suppressed in the presence of Aubipy-O-Me. This effect was specific for an osteopontin-coating, because migration through a coating of collagen was not affected by RANKL stimulation and/or Aubipy-O-Me incubation (figure 7B). EXAMPLE 4: Human macrophage responses to the bacterial compound lipopolysaccharide (LPS) are altered when co-incubated with AubipyOMe.

Macrophage responses to micro-organisms such as bacteria and viruses is suggested to be influenced by TRAP. For instance intracellular TRAP was shown to inhibit production of interferon alpha (15). To investigate if TRAP influences the responses to Gram-negative bacteria, human macrophages were stimulated with LPS in the presence or absence of 2 concentrations of Aubipy-O-Me. LPS clearly induced the expression of genes associated with inflammation such as IRFl, SERPINGl, APOL3, IL6, TNF, CCL20, IL12B, IDO l, and CCL4 (figure 8). Aubipy-O-Me was found to inhibit these responses.

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Claims

1. A gold(III) coordination compound bearing N-donor ligands, the compound having the general formula A, B, C, D or E

B

or C)

each of Ri through Rn is independently selected from the group consisting of H; aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; amines (e.g. NH2, aliphatic amines); halogens (e.g. chloride, iodide); moieties with hydroxyl functional groups (e.g. -OH or -Y-OH); ether containing moieties of general formula -Y-O-Y'; carbonyl containing moieties (-Y-CO-OH); or of amide bonds (-Y-CO-N-Y-);

sulfonamidic groups; fluorophores; nitrile/nitro groups, and peptide moieties, wherein Y and Y are independently selected from aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups comprising up to four C-atoms; L and L' are independently selected from the group consisting of halogen, hydroxyl, acetate, phosphane, and thiol -bearing groups (e.g. thio-sugars, cysteine and methionine groups); and

Z is a cyclic moiety selected from the group consisting of homocyclic and heterocyclic aromatic/aliphatic moieties, wherein the heterocyclic moieties may include nitrogen, oxygen and/or sulfur atoms; dotted lines can be absent or present; or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating and/or alleviating the symptoms of a respiratory disease associated with increased tartrate-resistant acid phosphatase (TRAP) activity.

2. Compound for use according to claim 1, wherein L and L' are halogen, preferably chloride or iodide.

3. Compound for use according to any one of the preceding claims, wherein each of Ri through Rn is independently selected from the group consisting of H, C1-C4 alkyl and C1-C4 alkoxy.

4. Compound for use according to any one of the preceding claims according to formula A, being an [Au(substituted-2,2'-bipyridine)LL']ⁿ⁺ with n = 1 or 2 or an [Au(2-phenylpyridine)LL']ⁿ⁺ with n = 0 or 1.

5. Compound for use according to any one of the preceding claims according to formula A, wherein the substitutions are (only) at positions 6,6'; 3,3'; 4,4'; or 5,5' of the bipyridine or phenylpyridine ring.

6. Compound for use according to claim 4 or 5, wherein X is N, preferably with the substitutions of the bipyridine ring at positions 4, 4'.

7. Compound for use according to any one of claims 4-6, wherein each of Ri through R₇ is independently selected from the group consisting of H, C1-C4 alkyl and C1-C4 alkoxy.

8. Compound for use according to claim 7, being [Au(bpOMe)Cl2][PF6] (bpOMe = 4,4'-dimethoxy-2,2'-bipyridine] (Aubipy-O-Me).

9. Compound for use according to any one of claims 1-3 of formula B, preferably wherein up to four of Ri through Rn are other than H.

10. Compound for use according to claim 9, being an [Au(substituted- 2,2',2"- terpyridine)L]ⁿ⁺ with n = 2 or 3;

11. Compound for use according to claim 9, being [Au(2,2':6',2"- terpyridine)Cl]Cl (Auterpy).

12. Compound for use according to any one of claims 1-3 of formula C, being an [Au(polypyridyl)LL']ⁿ⁺ with n = 1 or 2.

13. Compound for use according to any one of claims 1-3 of formula C, based on dipyrido[3,2-f:2',3'-h]quinoxahne, dipyrido[3,2-a:2',3'-c]phenazine, or dipyrido[3,2-a:2',3'-c](6,7,8,9-tetrahydro)phenazine.

14. Compound for use according to any one of claims 1-3 of formula D, bearing bidentate or tri dentate nitrogen donor ligands selected from 2-(2- pyridyl)imidazole, 2-phenylimidazole, 2,6-bis(benzimidazol-2-yl)pyridine, and l-methyl-2-[2-pyridyl]-lH-benzo[d]imidazole).

15. Compound for use according to any one of claims 1-3 of formula D, wherein up to four of Ri through R9 are other than H.

16. Compound for use according to claim 15, being [Au(l-methyl-2-[2- pyridyl]-lH-benzo[d]imidazole)Cl₂]Cl (AuPblmMe).

17. Compound for use according to any one of claims 1-3 of formula E, being an [Au(substituted- l, 10-phenantroline)L2]ⁿ⁺ with n= 1, 2 or 3

18. Compound for use according to any one of the preceding claims, wherein said disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), smoke-induced

inflammation and sarcoidosis.

19. Compound for use according to claim 18, wherein said disease is COPD or asthma.

20. Compound as recited in any one of claims 1 to 17, for use as in vitro inhibitor of tartrate-resistant phosphatase (TRAP).

21. A pharmaceutical composition formulated for pulmonary administration / inhalation, comprising one or more compounds as recited in any one of claims 1-17.

22. Compound according to formula D as defined in any of claims 1-3, 14 and 15.

23. Compound according to claim 22, for use as medicament.

24. Pharmaceutical composition comprising a compound according to claim 22, and a pharmaceutical carrier, diluent or excipient.

25. A medical device comprising a TRAP -inhibitory compound as recited in any one of claims 1 to 17.

26. Medical device according to claim 25, being a metered dose inhaler (MDI) or a dry powder inhaler (DPI).

27. Medical device according to claim 25 or 26, wherein said TRAP- inhibitory compound is present in the form of a dry powder.