"INHIBITORS OF AQUAGLYCEROPORINS, METHODS AND USES THEREOF"

Field of the Invention

The present invention relates to the field of selective inhibition of transmembrane glycerol transport through aquaglyceroporins , in general, and to respective modulator metal-based compounds, in particular. The present invention further relates to methods and uses of said modulator compounds in healthcare and cosmetic industries.

Background and Prior Art

Aquaporins (AQPs) belong to a highly conserved group of membrane proteins called the major intrinsic proteins (MIPs) present in all types of organisms and involved in the transport of water and small solutes such as glycerol, nitrate and urea [1] .

The 13 human AQP isoforms (AQPO-12) are differentially expressed in many types of cells and tissues in the body and can be divided into two major groups: 1) those strictly selective for water (called orthodox aquaporins) and 2) those that besides water are also permeable to small solutes including glycerol (called "aquaglyceroporins" ) [2] . Both groups of channels serve in many physiological functions [ 3 ] .

There is considerable potential for transferring knowledge of AQP structure, function and physiology to the clinic, and certainly there is great translational potential in aquaporin-based therapeutics. AQP-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases such as kidney diseases, cancer, obesity, glaucoma, brain edema and epilepsy [4] .

Specifically in cancer, AQPs have been reported to be overexpressed in many tumour types and to be associated with increased risk for metastasis and with unfavourable outcome, and forced expression of AQPs was shown to promote tumour cell proliferation [5] and invasion [6] . Notably, AQPs are also essential for angiogenesis [7] including tumour angiogenesis [8] .

There are at present very few reported AQP inhibitors that are suitable candidates for clinical trials. Though various AQPs are inhibited by mercurial compounds such as HgCl ₂ [9], these substances are non-selective in their action and are extremely toxic. Other inorganic salts such as Ag 03 and HAuCl ₄ , that are prone to interact with sulfhydryl groups of proteins as mercurials, have been also shown to inhibit water permeability in plasma membrane from roots, and in particular Ag 03 has been reported to efficiently inhibit water permeability in human red blood cells (hRBC)

(EC ₅ o = 3.9 μΜ) [10] . Various other candidate blockers of AQP1 have been also reported, including tetraethylammonium [11], acetazolamide [12] and DMSO [13]; however, other studies indicated little or no AQP1 inhibition by tetraethylammonium salts or acetazolamide [14] and inhibition by DMSO results from an osmotic clamp effect rather than true inhibition [15] . Recently, several papers reported AQP4 inhibition by a series of arylsulfonamides (Patent application US 2007/0281978 Al), antiepileptic drugs and related molecules, with strong inhibition at low micromolar concentrations [16]; however, these results could not be confirmed, with no inhibition activity found even at high concentrations of any of the putative AQP4 inhibitors [17] . Very recently, Yool et al . reported on AQP1 and AQP4 inhibition by an analogue of the sulfonamide Burnetamide [18] .

Summary of the Invention

The present invention describes metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins that bind selectively with at least one aquaglyceroporin, preferably AQP3, AQP7 and AQP9.

A preferred embodiment of the present invention provides metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins that consist of compounds tetracoordinated to gold(III) complexes.

In another embodiment of the present invention, metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins comprise inorganic compounds where Au(III) and Au ( I ) derivatives bears monodentate, bidentate or tridentate nitrogen donor ligands, phosphane groups and sodium triphenylphosphine trisulfonate.

A preferred embodiment of the present invention provides metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins comprising organogold compounds, where the organic ligands are preferably 6- ( 1 , 1-dimethylbenzyl ) - 2 , 2 ' -bipyridine ) , substituted-2-phenyl-pyridine or 2- [[ (dimethylamino ) methyl ] phenyl ] moiety and 1 , 3-bis (pyridin- 2-ylmethyl ) benzene is preferable, together with phosphine groups and thiolate ligands.

In another embodiment of the present invention, metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins comprising Au(III) dithiocarbamate complexes, Au ( I ) and Au(III) with N-heterocyclic carbenes (NHC) ligands, multinuclear gold complexes, preferably dinuclear Au compounds .

A preferred embodiment of the present invention provides metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins comprising metal centres such as Pt(II), Pd(II) and Cu(III) and preferably heteronuclear complexes such as Au/Ti, Au/Ru, Au/Pt, Au/Pd.

In another embodiment of the present invention, metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins are preferably selected from cysplatin, NAMI-A, silver sulfadiazine, aurothioglucose and auphen.

A preferred embodiment of the present invention provides metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins used in treatment, prophilaxys and prevention of clinical conditions, such as wound healing, tumours and cancer growth, angiogenesis , pathological skin conditions, obesity, kidney disorders, salivary gland disorders, allergic diseases, glaucoma, brain edema and epilepsy or any clinical conditions are related to AQP3 function .

In another embodiment of the present invention, metal-based inhibitory modulators of cellular transmembrane aquaglyceroporins used to manufacture pharmaceutical compounds and diagnostic reagents kits wherein the active substrate is selected based in similar biophysiological properties of any of the aforementioned inhibitory modulators of aquaglyceroporins.

A preferred embodiment of the present invention provides diagnostic kits used in the detection of aquaglyceroporin activity . General Description of the Invention

Aquaporin gold-based modulator drugs have broad potential utility in the treatment of edematous states, cancer, obesity, wound healing, epilepsy and glaucoma due to the significant specificity and efficacy of the binding and inhibition mechanism involved.

In the present invention metal-based compounds are considered to be possible AQP inhibitors, and we report here the highly specific inhibitory effect of a series of metal complexes based on different transition metals, comprising the following: the anticancer drug cis- [PtCl ₂ (NH ₃ ) ₂ ] (cisplatin) , the antimetastatic trans- [Ru(dmso) (Him)Cl ₄ ] (dmso = dimethylsulfoxide, Him = imidazole, NAMI-A) (Patent application WO 98/00431), the antibacterial Ag ( I ) sulfadiazine (AgSDZ) [19], the antirheumatic agent aurothioglucose (AuTG) [20], and the anticancer gold(III) compound [Au (phen) CI ₂ ] CI (phen = 1,10- phenatroline, Auphen) [21,22,23] (Figure 1) on the glycerol permeability of AQP3. The effect of the compounds was tested by a stopped-flow method on hRBC that specifically express large amount of AQP1 and AQP3 [24,25] and on stably transfected PC12 cell lines with overexpression of either AQP1 or AQP3.

The objective of the present invention was to identify metal-based modulators that selectively bind to aquaglyceroporins , including AQP3, and respective uses in methods for development and/ or production and/ or therapeutic use of pharmaceutical compounds and/ or cosmetics and diagnostic kits. The aforementioned objective is attained according to the present invention by means of certain gold-based compounds as described hereunder.

Description of the Drawings

The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

Figure 1 - Metal compounds used in the present invention; Figure 2 - (A) Water and glycerol permeabilities (% of control) in hRBC after 5 min treatment with the compounds used and with HgCl ₂ (100 and 500 μΜ) . A marked effect of Auphen (100 μΜ) is depicted.

Representative traces of water (B) and glycerol (C) permeability of hRBC (control and after incubation with 5 μΜ Auphen at room temperature) . (D) Urea permeability (% of control) after treatment with Auphen (5 and 100 μΜ) .

Figure 3 - Time dependent inhibition of glycerol transport by 5 Μ Auphen. The inset shows the progressive decrease of glycerol permeability observed in the activity assays where the increase in cell volume due to glycerol entrance decreases drastically with the incubation time.

Figure 4 - Concentration dependent inhibition of glycerol transport in hRBC by Auphen (compound concentrations were in the range 0.5-10 μΜ; IC ₅₀ = 0.78 ± 0.08 μΜ) .

Figure 5 - Inhibition of glycerol permeability (% of control) of hRBCs after Auphen treatment (2μΜ), and reversibility by washing with PBS or incubation with 2- mercaptoethanol (1 mM for 30 min) . Figure 6 - (A) Permeability to water and glycerol of wild type PC12 cells and PC12 transfected with either AQP1 or AQP3. Significant differences were found for the PC12-AQP3 clones with respect to PC12-wt. (B) Effect of Auphen on glycerol permeability of PC12-AQP3 transfected cells.

Figure 7 - (A) Structures of the Au(III) complexes Audien and Aucyclam, and of the ligand Phen. (B) Effect of Auphen, Phen, Aucyclam and Audien on glycerol permeability (% of control) . (B) Dose response curve of Audien (IC ₅₀ = 16.62 ±

1.61 μΜ) .

Detailed description of the invention

Methods

Chemistry

Gold compounds and NAMI-A were prepared according to literature procedures (see references throughout the text). The purity of the compounds was confirmed by elemental analysis, and all of them showed purity greater than 98%. Cisplatin, silver sulfadiazine, aurothioglucose and 2- Mercaptoethanol were from Sigma.

Erythrocyte sampling and preparation

Venous blood samples, collected in citrate anticoagulant (2.7 % citric acid, 4.5 % trisodium citrate and 2% glucose) , were obtained from healthy human volunteers (Faculdade de Farmacia, Universidade de Lisboa) . Fresh blood was centrifuged at 750 xg for 5 min at 4°C and plasma and buffy coat were discarded. Packed erythrocytes were washed three times in PBS (KC1 2.7 mM, KH ₂ P0 ₄ 1.76 mM, Na ₂ HP0 ₄ 10.1 mM, NaCl 137 mM, pH 7.4), diluted to 0.5% haematocrit and immediately used for experiments. Cell Culture and Transfections

To obtain stable clones of PC12 (cell line derived from a pheochromocytoma of rat adrenal medulla) that overexpress either rat AQP1 or rat AQP3, twenty micrograms of pcDNA3- AQP1 or pcDNA3-AQP3 were transfected into wild type PC12 by electroporation . After seletction with geneticin sulphate (GIBCO) 40 clones were analyzed for levels of expression of either AQP1 or AQP3. Out of 20 positive clones, with variable levels of AQPs expression, we select for each AQP those with higher expression. PC12 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% foetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin (Invitrogen) in a CO ₂ (10%) incubator at 37 °C. Geneticin at 0,2 mg/ml was added to culture AQP-overexpressing clones. Levels of AQP overexpression were determined by Northern blot analysis and expressed relative to wild type PC12. RNA analysis and functional characterization of these clones have been described in detail previously [32] .

Cell volume measurements

hRBC mean volume in isotonic solution was determined using a CASY-1 Cell Counter (Scharfe System GmbH, Reutlingen, Germany) and was calculated as 82 fL . Equilibrium volumes of PC12 cells were obtained by phase contrast microscopy on an inverted microscope (Axiovert Zeiss 100M) equipped with a digital camera. Plated cells were dislodged by mechanical aspiration with pipette, washed and ressuspended in PBS. For each measured set of data, an aliquot of cell suspension was placed on a microscope slide and an average of 6 pictures with 4-6 cells each were taken and analysed using NIH ImageJ software. Cells were assumed to have a spherical shape with a diameter calculated as the average of the maximum and minimum dimensions of each cell. Calculated volumes were 1076 ± 123 μηι ³ for all the clones measured .

Stopped-flow light scattering experiments

Stopped-flow experiments were performed on a HI-TECH Scientific PQ/SF-53 apparatus, with 2 ms dead time, temperature controlled and interfaced with a microcomputer. Experiments were performed at temperatures from 10 °C to 37 °C. For each experimental condition, 5-7 replicates were analysed. For measuring the osmotic water permeability (P _f ) , 100 of a suspension of fresh erythrocytes (0.5%) or PC12 cells (1.5 x 10 ³ to 3.5 x 10 ³ cells/mm ³ ) was mixed with an equal volume of PBS containing 200 mM sucrose as a non-permeable osmolyte to produce a 100 mM inwardly directed sucrose gradient. The kinetics of cell shrinkage was measured from the time course of 90° scattered light intensity at 400 nm until a stable light scatter signal was attained .

Pf was estimated by Pf = k (V ₀ /A) (1/V„ (osm _out ) ) , where V„ is the molar volume of water, V ₀ /A is the initial cell volume to area ratio and (osm _out )∞ is the final medium osmolarity after the applied osmotic gradient and k is the single exponential time constant fitted to the light scattering signal of erythrocyte shrinkage. For PC12 cells, a double exponential function was used instead and the weighted averaged rate constant k _de = (AIiki+Al2k2 ) / ΔΙ1+ΔΙ2) , where ΔΙι and ΔΙ ₂ correspond to the signal changes with either a slow rate constant ki or a fast one k ₂ , was alternatively used to calculate P _f [38] .

For glycerol permeability {P _g i _y ) , 100 of erythrocyte or

PC12 cell suspension was mixed with an equal volume of hyperosmotic PBS containing 200 mM glycerol creating a 100 mM inwardly directed glycerol gradient. After the first fast cell shrinkage due to water outflow, glycerol influx in response to its chemical gradient was followed by water influx with subsequent cell reswelling. P _g i _y was calculated as Pgiy = k (V ₀ /A) , where k is the single exponential time constant fitted to the light scattering signal of glycerol influx in erythrocytes [27] . For PC12 cells, the rate of reswelling due to glycerol influx was measured as the slope of a linear regression fit.

For inhibition experiments cells were incubated with different concentrations of complexes, from freshly prepared stock aqueous solutions, for various times at room temperature before stopped flow experiments. The inhibitor concentration necessary to achieve 50% inhibition (IC ₅ o) was calculated by nonlinear regression of dose-response curves (Graph Pad Prism, Inc) to the equation: y=y _m in+ (ymax- y _min ) / (1+10 ^{((LogIC5CMIrih])H))} , where y is the percentage inhibition obtained for each concentration of inhibitor [Inh] and H is the Hill slope. The activation energy (E _a ) of water and glycerol transport was calculated from the slope of the Arrhenius plot (lnP _f or lnP _gJy as a function of 1/Γ) multiplied by the gas constant R. All solution osmolarities were determined from freezing point depression on a semi- micro osmometer (Knauer GmbH, Berlin, Germany) using standards of 100 and 400 mOsM.

Statistic analysis

Data were presented as mean ± standard error of the mean of at least four independent experiments, and were analysed with either paired Student's t test or the one-way analysis of variance (ANOVA) followed by Tukey test. A value of P < 0.01 was considered to be statistically significant.

Results The effect of different metal complexes based on Pt(II), Ru(III), Ag ( I ) , Au(I,III) (Figure 1) was tested on water and glycerol permeability of hRBCs. For this purpose hRBCs incubated in isotonic PBS buffer were challenged with hypertonic sucrose solution (impermeant solute, inducing cell shrinkage) or hypertonic glycerol solution (permeant, cells shrink due to the hyper-osmotic gradient and re-swell due to glycerol entrance) . Since hRBCs were shown to express a large amount of AQP1 and AQP3 accountable for membrane permeability to water and glycerol [25,26,27], this experiment allows the direct evaluation of these aquaporins activity and is thus a promising screening assay for modulators of aquaporin function. From the rate of cell volume changes (shrinkage and re-swelling) after imposed osmotic shocks the membrane permeability for water and for glycerol can be calculated [27] .

Figure 2A shows the effect induced by the metallodrugs on AQP1 and AQP3 in comparison to HgCl ₂ , a well-known inhibitor of aquaporin activity. The obtained results demonstrated that the Au(III) complex Auphen is the most effective of the series on glycerol permeability, and far more effective than the mercurial compound (P<0.5) . Figure 2B and C shows representative traces of stopped-flow experiments with control and Auphen treated hRBCs where the inhibition of the osmotic water flux (mainly through AQP1, Figure 2B) or glycerol flux (through AQP3, Figure 2C) is illustrated. For control hRBCs, the osmotic water permeability (P _f ) values at 10 °C were (4.17 ± 0.39) x 10 ^~2 cm s ^_1 (n=5) and the glycerol permeability (P _g i _y ) values at 23 °C were (1.84 ± 0.23) x 10 ^~5 cm s ^_1 (n=5) . As shown in Figure 2A, Auphen showed a modest effect on water permeability (ca. 20% inhibition), while being able to drastically reduce glycerol transport with a residual permeability of ca. 11% (90% inhibition) . The smaller effect obtained for water permeability points to a specific effect on AQP3, which itself is also a water-transporting channel, [28] but with a smaller contribution to the total bulk of water flow through hRBC membranes where AQPl is the main water channel.

To assure the compound selectivity, the effect of Auphen was also tested on urea transport as described in the detailed description section. Notably, no significant effect could be observed for both concentrations tested in comparison to the controls (P>0.05) (Figure 2D) .

Following these promising results we further investigated the inhibition of glycerol transport through AQP3 evaluating the effect of the incubation time of hRBCs with Auphen. Figure 3 shows the inhibitory effect of a fixed concentration of Auphen (5 Μ) where a maximum inhibition, after an exponential decay of activity, could be observed after 30 min incubation of the samples at room temperature (r.t) . It is worth mentioning that no cell haemolysis was detected even after 3 hours incubation with the compound ensuring a non-toxic inhibitory effect.

Subsequently, the concentration dependent inhibition of glycerol transport in hRBC by Auphen incubated 30 min at r.t. was assessed (Figure 4) . According to the obtained results the IC ₅ o value for Auphen was calculated as 0.78 ± 0.08 μΜ.

The activation energy (E _a ) for water and glycerol transport, a valuable parameter indicating the contribution of protein channels to permeation, was also estimated from an Arrhenius plot. Upon treatment of hRBC with 5 μΜ Auphen, similar E _a values for water transport were obtained for control (3.90 ± 0.35 kcal mol ^-1 ) and Auphen treated hRBCs

(4.12 ± 0.98 kcal mol ^-1 ) . However, the E _a for glycerol permeation increased ca. 54 % when Auphen was present (8.52 ± 0.81 to 13.15 ± 1.12 kcal mol ^-1 ). Since the Auphen concentration used in the assay was higher than the IC ₅ o (corresponding approximately to 80% inhibition) , the increase in E _a is in accordance with a partial blockage of the AQP3 channel. Regarding water transport, the observed variation on the E _a was not significant (P>0.05); indeed, in hRBC the contribution of the channel pathway to the total water permeability is usually considered to be 90% while the bilayer adds the remaining 10% [29] . The total 20% inhibition observed with Auphen (Figure 2) would only reduce the contribution of the AQP1 pathway to 87.5%, thus accounting for the equivalent value (to the control) of measured Ea. AQP1 being still active, it may account for the total bulk of water flow maintaining a E _a for water permeation unaltered.

In order to assess the reversibility of inhibition by Auphen, hRBCs pre-treated with 2 μΜ Auphen for 30 min r.t. were subsequently washed with PBS or with the reducing agent 2-mercaptoethanol (MeOH, 0.1 mM) . As seen in Figure 5, washing the sample twice with PBS had a limited effect in the recovery of Auphen inhibition of glycerol permeability. Conversely, incubation of the Auphen-treated hRBC sample with MeOH for 30 min produced an almost complete recovery of glycerol permeability (ca. 90%), suggesting that MeOH is competing with Auphen for the thiol groups .

The results obtained with MeOH, as well as the known affinity of gold ions for binding to sulfhydryl groups of proteins suggest that AQP3 inhibition by Auphen might involve direct protein binding of the Au centre to Cys residues, as it has already been reported for HgCl ₂ . Indeed, mercury inhibition is likely to occur both via covalent modification to Cys 189 located immediately after the extracellular entrance of the water pore of hAQPl [30] and also to other regions of the protein, causing either blockage or conformational changes with a resultant inhibition of water transport [31] .

To further confirm the specific effect of Auphen on AQP3 glycerol transport, the inhibition of glycerol permeation by Auphen was also assessed on PC12 cells stably transfected with rat AQPl or AQP3 (Table 1) [32] .

Table 1. Folds of expression and permeabilities of PC12 cells. Folds of mRNA expression were determined by Northern blot analysis and are normalized relative to the clone PC12wt.

For this purpose, cells were plated on flasks and after reaching confluence they were mechanically detached by pipetting in and off into the cell culture medium. Cells were washed twice with PBS and were suspended at a density of 1.5 x 10 ³ to 3.5 x 10 ³ cells/mm ³ . Their permeabilities for water and glycerol were analysed by stopped-flow experiments similar to those described before for hRBC and results are depicted in Figure 6A. The enhancement of water permeability for AQPl overexpressing cells (2.7 -folds) and that of glycerol permeability for AQP3 overexpressing cells (1.8 -folds) correlates well with their respective protein level of expression (Table 2) . When treated with Auphen (10 to 1000 μΜ) a decrease in glycerol permeability was only observed for the PC12-AQP3 cell line (Figure 6B) ; none of the other two cell lines (wild type and PC12-AQP1) were affected regarding glycerol or water permeability (data not shown) .

In order to evaluate our mechanistic hypothesis that sees the gold centre as responsible for AQP3 inhibition, the ligand Phen, as well as the Au(III) complexes [Au (dien) CI ] CI2 [22] (dien = diethylentriamine, Audien) and [Au (cyclam) ] (CIO ₄ ) ₂ C1 [22] (cyclam = 1,4,8,11- tetraazacyclotetradecane, Aucyclam) (Figure 7A) were tested for glycerol transport inhibition in hRBCs . It must be noted that Auphen can undergo hydrolysis of the chlorides in aqueous environment and react with biomolecules upon ligand substitution reactions. Instead, while Audien presents an AuN ₃ Cl core which can also undergo activation via release of the chloride ligand, Aucyclam is a gold(III) complex with a AuN ₄ chromophore which lacks of chemical activation resulting into poor reactivity and scarce biological (e.g. anticancer) effects [22]. The obtained results, plotted in Figure 7B, confirm that while Audien at a concentration of 50 μΜ reaches an inhibition of 80% (significantly lower than the 90% inhibition observed for Auphen at the same concentration) , its IC ₅₀ is 20-fold higher than observed for Auphen (IC ₅₀ = 16.62 ± 1.61 μΜ, Figure 7C) . On the other hand, Aucyclam and Phen are completely inactive.

While AQP1 is a selective water channel, AQP3 permeates both water and glycerol on human erythrocytes. Besides being also a water transporting channel [28], its contribution to the total bulk of water flow through hRBC membranes is minimal compared to AQP1 [25] . Conversely, it has been shown to mediate most of the glycerol movements across RBCs membranes [25,27] . In addition to RBCs, AQP3 has a wide tissue distribution in the epithelial cells of kidney, airways and skin and in immature dendritic cells, suggesting a role in water reabsorption, mucosal secretions, skin hydration, allergic diseases, and cell volume regulation [33] .

In the present invention we report on the screening of different metallodrugs for the inhibition of AQP1 and AQP3 in hRBC. Among the various compounds tested, the potent and selective inhibition of the glycerol permeability through AQP3 in hRBC by gold (III) metal complexes, namely Auphen and Audien, was observed. Both compounds are tetracoordinated gold(III) complexes with square planar geometry in which the Au(III) oxidation state, at variance with the case of NaAuCl<i, is stabilised by the presence of nitrogens on the phenantroline and diethylentriamine ligands. Interestingly, both compounds have been previously reported to possess anticancer properties in vitro [22,23]. Thus, Auphen and Audien resulted to inhibit glycerol transport in hRBC with an IC ₅₀ = 0.78 ± 0.08 μΜ and IC ₅₀ = 16.62 ± 1.61 μΜ, respectively, while having only a very modest inhibitory effect on water permeability.

The observed increase in the E _a for glycerol transport in hRBC with no concomitant E _a change for water transport upon treatment with Auphen, points to a specific blockage of the AQP3 channel without affecting AQP1. Notably, the Au(III) compounds are much more effective on AQP3 than the non ^¬ specific inhibitor of aquaporins HgCl ₂ , and most importantly they are not toxic for the cells in the entire range of investigated concentrations. Indeed, during the time span of the experiments, no haemolysis was detected even after 4 hours incubation with the Au(III) compounds.

The specificity of Auphen towards AQP3 was further confirmed by assessing glycerol transport on PC12 cell lines transfected with either AQP1 or AQP3 from rat. The marked inhibitory effect of the compound exclusively for the PC12-AQP3 cell clone denotes its specific interaction with residues that cannot otherwise be assessed in AQP1. However, a higher concentration of Auphen was needed to produce the same inhibitory effect observed in hRBCs; besides the fact that Auphen might have a lower affinity for rat AQP3 than for human AQP3, the possibility of Auphen binding to other reactive groups within the whole cell membrane decreasing its effective concentration in the media and therefore leading to an underestimation of the IC ₅ o should not be disregarded. The PC12 cells are much larger than RBCs; hence, the presence of a larger number of these bias reactive groups may eventually contribute to a higher decrease in the effective Auphen concentration available for AQP3 blockage.

The time-dependent inhibition of AQP3 by Auphen is in accordance with the typical reactivity pattern of this gold(III) complex in aqueous solution. In fact, it is worth mentioning that, as commonly found for several other metallodrugs , gold (III) compounds behave as "prodrugs" [34] . In other words they require a "chemical activation" process i.e. a specific chemical transformation (e.g. ligand substitution, redox processes, hydrolysis) before they can react with biomolecular targets; only the "activated species" are able to bind the target and produce the pharmacological effects. In the case of Auphen activation is most likely achieved through release of at least one halide ligand from the tetracoordinated gold(III) chromophore Au ₂ Cl ₂ and substitution with water /hydroxide ligands [22] prior possible direct metal coordination to AQP3. Similarly, Audien can bind AQP3 after hydrolysis of the unique chloride ligand, but slightly less efficiently than Auphen as demonstrated by its higher IC ₅ o value. Conversely, the chemically stable Au(III) complex Aucyclam shows lack of inhibition properties supporting the hypothesis that the gold centre is essential for inhibition and most likely is involved in protein binding.

Auphen also resulted to be ineffective as inhibitor of urea transport on hRBC . The ability of AQP3 to transport urea has been debated [35, 36] . In the erythrocyte the urea transporter UT-B [37] accounts for a very high urea permeability reducing osmotic shrinkage of RBCs while passing through the kidney. Therefore, compared to urea transporters, urea permeability through AQP3 may have only a negligible contribution and thus its inhibition would not be enough to observe decreased urea permeability. Since Auphen did not affect urea permeability, this result indicates that this metallodrug is specific for AQP3 not having any effect on UT-B. The mechanism of gold inhibition is most likely due to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteins. This hypothesis is partly confirmed by the almost complete recovery of AQP3 activity upon treatment of hRBC with MeOH. However, other modes of Auphen binding might also occur (e.g. with histidine groups) .

Aquaporin metal-based modulator drugs have broad potential utility in the treatment of edematous states, cancer, obesity, wound healing, epilepsy and glaucoma. There are at present no reported aquaglyceroporins inhibitors that are suitable candidates for clinical development. Here we reported on the selective and potent inhibition of AQP3 channels by gold (III) complexes screened on hRBC and PC12 cells. The obtained results suggested the use of gold(III) complexes with nitrogen-based ligands as possible aquaglyceroporin inhibitors, specifically of AQP3, that could be exploited in the prevention, prophylaxis and treatment of phatophysiological states directly or indirectly related to aquaglyceroporins (e.g. AQP3), or used as biological tools to assess AQPs function.

According to a first aspect of the invention, it is proposed an inhibitory modulator of aquaglyceroporins regarding its transmembrane glycerol transport properties, wherein the modulator selectively binds to aquaglyceroporins, including AQP3, AQP7 and AQP9.

According to another aspect of the invention, it is proposed an inhibitory modulator of aquaglyceroporin within the context of the first mentioned aspect, comprising inorganic compounds of general formula: [Au ( substituted- 1 , 10-phenantroline ) X2 ] X (X = CI, OH) of the type presented below (I), where R can be proton or is selected from a group consisting of aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups; or of amines (e.g. NH ₂ , aliphatic amines -R-NH ₂ ) ; or of alogens (e.g. chloride, iodide); or of ligands with hydroxyl functional groups (e.g. -R-OH) ; or of ether containing ligands (of general formula -R-O-R'); or of carbonyl containing ligands (-R-CO-OH) ; or of sulfonamidic groups, or of nitrile/nitro groups, or of peptide moieties. In addition the phenantroline scaffold can include one or more substituting nitrogen, oxigen and/or sulfur atoms.

According to a further aspect of the invention, it is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising an inorganic compound of formula: [Au ( substituted-2 , 2 ' - bipyridine ) CI ₂ ] PF ₆ of the type presented below (II), where the substitution of the bipyridine ring is in 6,6' (a), or 4,4' (b) , or 5, 5 '(c), and where R is selected from a proton or from a group consisting of aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic-heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups; or of amines (e.g. NH ₂ , aliphatic amines -R-NH ₂ ) ; or of alogens (e.g. chloride, iodide) ; or of ligands with hydroxyl functional groups (e.g. -R-OH) ; or of ether containing ligands (of general formula -R-O-R'); or of carbonyl containing ligands (-R-CO- OH) ; or of sulfonamidic groups, or of nitrile/nitro groups, or of peptide moieties. In addition the bipyridine scaffold can include one or more substituting nitrogen, oxigen and/or sulfur atoms.

(ID According to a further aspect of the invention, it is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising an inorganic compound of formula: [Au ( substituted-2 , 2 ' , 2 " - terpyridine ) X] X ₂ (X =C1, OH) of the type presented below (III), where R is selected from a proton or from a group consisting of aliphatic, heteroaliphatic, aromatic, heteroaromatic, aliphatic-aromatic, heteroaliphatic- heteroaromatic, cycloaliphatic, and heterocycloaliphatic groups; or of amines (e.g. N¾, aliphatic amines -R-NH ₂ ) ; or of alogens (e.g. chloride, iodide); or of ligands with hydroxyl functional groups (e.g. -R-OH) ; or of ether containing ligands (of general formula -R-O-R'); or of carbonyl containing ligands (-R-CO-OH) ; or of sulfonamidic groups, or of nitrile/nitro groups, or of peptide moieties. In addition the terpyridine scaffold can include one or more substituting ni fur atoms.

(III)

According to a further aspect of the invention, it is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising an inorganic compound of formula: [Au (polypyridyl ) X2 ] PF6 (X = CI, OH) of the type presented below (IV), where the polypyridyl moiety can be for example dipyrido [ 3 , 2-f : 2 ' , 3 ' -h] quinoxaline (DPQ) , dipyrido [ 3 , 2-a : 2 ' , 3 ' -c] phenazine (DPPZ), dipyrido [3, 2-a: 2 ', 3 '-c] ( 6 , 7 , 8 , 9-tetrahydro ) phenazine (DPQC) ) . In addition, the polypyridyl main scaffold can include 6,6-, 5,6 or 6,5-fused bicyclic aromatic groups with or without one or more substituting nitrogen, oxigen and/or sulfur atoms.

DPQ DPPZ DPQC

(IV)

According to a further aspect of the invention, there is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising an inorganic compound of formula:

[Au (diethylenetriamine ) CI ] CI2 of the type presented below (V) .

(V)

According to a further aspect of the invention, there is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising inorganic compounds where Au(III) and Au ( I ) derivatives bears monodentate, bidentate or tridentate nitrogen donor ligands such as 2- ( 2-pyridyl ) imidazole (a), 2- phenylimidazole (b) , and, 2 , 6-bis (benzimidazol-2- yl)pyridine (c) , respectively, as well as phosphane groups (e.g. triphenylphosphine, 1 , 3 , 5-triaza-7-phosphaadamantane, 3, 7-diacetyl-l,3, 7-triaza-5-phosphabicyclo [3.3.1] nonane, and sodium triphenylphosphine trisulfonate ) (representative examples provided in (VI) ) . Ligands:

Gold(in) derivatives:

Gold(I) derivatives:

(VI)

According to a further aspect of the invention, there is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising organogold compounds including those of the type reported in (VII), where the organic ligands are for example 6- (1,1- dimethylbenzyl ) -2,2' -bipyridine) , substituted-2-phenyl- pyridine, or a 2- [[ (dimethylamino ) methyl ] phenyl ] moiety. In addition 1 , 3-bis (pyridin-2-ylmethyl ) benzene as Au(III) ligand could also be considered, together with phospine groups and thiolate ligands.

R = H, amine, alogen, alkyl, aryl,

carboxy, alkoxy etc.

X = alogen, OH, CH ₃ COO, etc.

(VII)

According to a further aspect of the invention, there is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising Au(III) dithiocarbamate complexes, Au ( I ) and Au(III) with N- heterocyclic carbenes (NHC) ligands, multinuclear gold complexes (e.g. dinuclear Au compounds).

According to a further aspect of the invention, there is proposed an inhibitory modulator of the aquaglyceroporin within the context of the first aspect, comprising metal complexes bearing similar organic ligands as reported above, but with different metal centres such as Pt(II), Pd(II) and Cu(III). In this context, heteronuclear complexes (eg. Au/Ti, Au/Ru, Au/Pt, Au/Pd etc.) are also considered .

According to a further aspect of the invention, there is provided for the use of any of the aforementioned inhibitory modulators of aquaglyceroporin, in a method including a selective binding of any of said inhibitory modulators with at least one respective aquaglyceroporin. According to a further aspect of the invention, said methods include the use of pharmaceutical compounds and/ or cosmetic and of diagnostic kits.

Pharmaceutical compounds and/ or cosmetic for the prevention, prophylaxis and/ or treatment of disease pathology directly or indirectly related to AQP3 function comprising and inhibitory modulator of AQP3 according to any of the previous description and details.

Pharmaceutical compounds and/ or cosmetic according to the previous description and details, for the treatment and prophylaxis of the group of pathologies including wound healing, tumours and cancer growth, angiogenesis , pathological skin conditions, obesity, kidney disorders, salivary gland disorders and allergic diseases, wherein the active substrate is selected based in having similar biophysiological properties of any of the aforementioned inhibitory modulators of aquaglyceroporins .

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