Gold nanoclusters in the treatment of Friedreich's ataxia

Abstract

The present invention relates to ultrastructured gold clusters, Au-pX, consisting of gold atoms and at least one ligand, for use in the treatment of pathologies associated with oxidative stress, wherein the number of gold atoms in the cluster is between 2 and 100 or the cluster dimensions are less than 2 nm. In a preferred embodiment, the Au-pX ultrastructured gold clusters are used in the treatment of Friedreich's ataxia.

Description

[Technical Field]

[0001] Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by an unstable expansion of a GAA triplet located in intron 1 of the FXN gene (9q21.11), which encodes frataxin. The disease is primarily characterized by progressive gait and limb ataxia, dysarthria, dysphagia, oculomotor disorders, loss of deep tendon reflexes, pyramidal signs, scoliosis, and, in some cases, cardiomyopathy, diabetes, blindness, and hearing impairment. [Background technology]

[0002] There is no cure for FRDA, and currently, the symptomatic framework is treated multidisciplinary, typically with the support of physical therapy, pharmacological agents such as baclofen and botulinum toxin for spasticity control, and antiarrhythmic and anticoagulant medications for cardiomyopathy.

[0003] LIM F, et al. Mol Ther. 2007; 15: 1072-1078 (Non-Patent Document 1); Vyas PM, et al. HUM mol Genet. 2012; 21: 1230-47 (Non-Patent Document 2); Jones J, et al. Mol Ther. 2015; 23: 130-138 (Non-Patent Document 3); Perdomini M, et al. Mean NAT. 2014; 20: 542-547 (Non-Patent Document 4) describe preclinical stem cell or gene therapy treatments tested to increase healthy frataxin levels.

[0004] Huntington's disease (HD) is a neurodegenerative disorder caused by an unstable CAG triplet expansion in the gene encoding huntingtin on the short arm of chromosome 4 (4p16.3). The disease affects muscle coordination and leads to cognitive decline and psychiatric problems. To date, available drug treatments have failed to alleviate many of the numerous symptoms.

[0005] Alzheimer's disease (AD) is the most common form of gradually disabling degenerative dementia, occurring primarily in early old age. No cure for Alzheimer's disease is currently available.

[0006] Parkinson's disease (PD) is a neurodegenerative disorder. The disease's characteristic motor symptoms result from the death of cells that synthesize and release dopamine. Although medication, surgery, and multidisciplinary management can alleviate symptoms, no cure for Parkinson's disease is currently available.

[0007] Oxidative stress is a key component of the pathogenesis of FRDA and may explain DNA damage and neuronal degeneration (Yokota T, et al. Proc Natl Acad Sci USA 2001;98:15185-15190 (Non-Patent Document 5)). Oxidative stress has also been shown to be involved in the pathogenesis of HD (Kumara A, Ratana RR, J Huntington's Dis. 2016;5(3):217-237 (Non-Patent Document 6)), AD (Markesbery WR, Free radical Biology and Medicine 1997;23(1):134-147 (Non-Patent Document 7)), and PD (Henchcliffe C, Beal MF, Nature clinical practice 2008;4(11):600-609 (Non-Patent Document 8)).

[0008] However, clinical studies using antioxidants such as idebenone, MitoQ, CoQ10, and vitamin E to reduce cellular oxidative damage have met with limited success.

[0009] Santiago-Gonzales B et al. Science 2016; 353-571-575 (Non-Patent Document 9) discloses a stable superstructure formed by an assembly of gold atoms, called Au-pX. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] LIM F, et al. Mol Ther. 2007; 15: 1072-1078 [Non-Patent Document 2] Vyas PM, et al. HUM mol Genet. 2012; 21: 1230-47 [Non-Patent Document 3] Jones J, et al. Mol Ther. 2015; 23: 130-138 [Non-Patent Document 4] Perdomini M, et al. Mean NAT. 2014; 20: 542-547 [Non-Patent Document 5] Yokota T, et al. Proc Natl Acad Sci USA 2001; 98: 15185-15190 [Non-Patent Document 6] Kumara A, Ratana RR, J Huntington’s Dis. 2016; 5(3): 217-237 [Non-Patent Document 7] Markesbery WR, Free radical Biology and Medicine 1997; 23(1): 134-147 [Non-Patent Document 8] Henchcliffe C, Beal MF, Nature clinical practice 2008; 4(11): 600-609 [Non-Patent Document 9] Santiago-Gonzales B et al. Science 2016; 353-571-575 [Summary of the Invention] ​​​​​The present invention relates to methods for treating conditions caused by excess oxygen radicals, particularly FRDA, that go beyond the limitations of currently available treatments. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1A shows the cyclic voltammograms of PBS electrolyte in the absence of cluster Au8-pX before (solid line) and after (dashed line) the addition of aqueous H2O2 solution. Figure 1B shows the cyclic voltammograms of PBS electrolyte in the presence of cluster Au8-pX before (solid line) and after (dashed line) the addition of aqueous H2O2 solution. [Figure 2] Figure 2 shows the effect of Au8-pX clusters on 1O2 concentration. [Figure 3] Figure 3 shows the effect of Au8-pX clusters on H2O2 concentration. [Figure 4] Figure 4 shows the effect of Au8-pX clusters on O2·− concentration. [Figure 5] Figure 5 shows neuromotor function in YG8R mice injected with Au8-pX. (A) Representative photographs of footprint tests of hind and forelimbs from YG8sR-PX-treated Au8 mice. (B) The footprint test showed an increase in stride length in Au8-pX-injected mice compared with control animals (CTR). (C) The treadmill test revealed that Au8-pX-injected mice had increased exercise tolerance, measured by pulse number (shock number), compared with controls. Values ​​represent mean ± SEM. *P<0.05; **P<0.01; *P<0.001; ns=not significant. CTR=control animals. [Figure 6]Figure 6 shows cardiac function in YG8R mice injected with Au8-pX. (A) Representative M-mode echocardiographic images of mice treated with Au8-pX (injected) and untreated mice (naive). (B) Echocardiographic analysis revealed that Au8-pX-injected mice had increased ejection fraction (EF) and fractional shortening (FS) compared to controls. Values ​​represent mean ± SEM. *P<0.05. [Figure 7] Figure 7 shows the expression levels of antioxidants in the dorsal root ganglia, cortex, cerebellum, and basal ganglia. QRT-PCR analysis revealed statistically significant increases in Prdx2, Gstm1, and Nrf2 expression in the dorsal root ganglia of Au8-pX-injected mice compared with controls. Data were normalized to GAPDH. Values ​​represent mean ± SEM. p<0.05, p<0.001. [Figure 8] Figure 8 shows the analysis of biochemical redox parameters in the cortex, basal ganglia, cerebellum, pancreas, heart, and skeletal muscle of YG8R mice injected with Au8-pX. (A) Mitochondrial ATP levels were significantly increased in all tissues except the cerebellum and pancreas of Au8-pX-injected mice compared to control animals. (B) Mitochondrial ROS analysis revealed no statistically significant differences between injected and control animals, except in the anterior tibialis and basal ganglia. (D) Mitochondrial lipid peroxidation (OH-nonenal) was reduced in Au8-pX-injected mice compared to controls in all tissues tested except the cortex and heart. (E) Mitochondrial DNA damage (8-oxoguanine) was reduced in Au8-pX-injected mice compared to controls in all tissues tested except the cortex, heart, and pancreas (DNA damage). (F) Mitochondrial SOD levels were significantly reduced in the cortex, cerebellum, quadriceps, tibialis anterior, and soleus muscles of Au8-pX-injected mice. Values ​​represent mean ± SEM. [Figure 9]Figure 9 shows the analysis of biochemical redox parameters in the cortex, basal ganglia, cerebellum, pancreas, heart, and skeletal muscle of YG8R mice injected with Au8-pX. (A) GSH concentrations are elevated in all tissues except the pancreas and heart in Au8-pX-injected mice. (B) GSSG levels are significantly decreased in mice injected with Au8-pX in the cortex, basal ganglia, cerebellum, and soleus muscle. (C) GST levels are significantly elevated in the basal ganglia, cerebellum, quadriceps, and tibialis anterior in Au8-pX-treated mice. [Figure 10] Figure 10 shows image analysis: (A) Proliferation of bone marrow-derived MSCs from FRDA or control subjects exposed or not to 5 or 10 μM Au8-pX. (B) ROS generation in bone marrow-derived MSCs from FRDA or control subjects exposed or not to 5 or 10 μM Au8-pX. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to ultrastructured gold clusters, called Au-pX, for use in the treatment of oxidative stress. A cluster is defined as an assembly of atoms consisting of 2 to 100 metal atoms or maintaining a dimension of less than 2 nanometers, according to the definition given by Yamazoe S et al. (Seiji Yamazoe, Kiichirou Koyasu, Tatsuya Tsukuda, Accounts of Chemical Research 2014 DOI: 10.1 021 / ar400209a).

[0014] In a preferred embodiment, the supramolecular gold clusters are obtained according to the method described in Santiago-Gonzalez et al. 2016, 353: 571-575. In a preferred embodiment, the Au-pX is obtained starting from clusters consisting of eight gold atoms (Au8), which are aggregates of eight gold atoms. The gold clusters are the basic building blocks of colloidal supramolecular superstructures (4-5 nanometers in diameter) called Au8-pX, which are cross-linked to each other by hydrogen bonds formed between the blocking ligands.

[0015] In a preferred embodiment, the ligand is 11-mercaptoundecanoic acid, which exposes a carboxyl functional group and binds a gold atom via a thiol group.

[0016] In a preferred embodiment, the Au-pX is used in the treatment of Friedreich's ataxia (FRDA). In a further embodiment, the Au-pX is used in the treatment of Alzheimer's disease (AD), Parkinson's disease (PD) and / or Huntington's disease (HD).

[0017] In a preferred embodiment, the Au-pX superstructured gold clusters are contained in a composition that also includes a pharmaceutically acceptable excipient for intravenous administration.Methods for treating subjects suffering from conditions associated with oxidative stress, such as AD, PD, and HD, are also described.

[0018] The method comprises the intravenous administration of a composition comprising the Au-pX superstructure gold clusters according to the present invention. In a preferred embodiment, the method comprises a single administration.

[0019] The advantage of the method of the present invention lies in the ability of Au-pX to interact with different cell types in multiple tissues, including the nervous, skeletal, and cardiac systems, by inducing long-term effects on mitochondrial activity. Surprisingly, we demonstrated a reduction in oxidative stress and DNA damage, accompanied by increased mitochondrial activity, in the nervous, skeletal, and cardiac tissues of YG8R animals treated with Au8-pX, despite no change in ROS levels. Because Au-PX does not have a sacrificial role, its effects are maintained for a long period of time after a single administration. This allows for the treatment of subjects suffering from oxygen radical excess with a single administration.

[0020] The following examples are intended only to better illustrate the present invention and are not intended to limit it in any way, the scope of which is defined by the claims that follow. [Example]

[0021] Example 1: Synthesis of colloidal superstructures based on Au8 gold clusters (Au8-pX). Superstructures based on Au8 clusters were obtained as described in Santiago-Gonzalez et al. Briefly, the following steps were followed.

[0022] Synthesis of gold nanoparticles (AuNPs): AuNPs were obtained by adding 1 mL of 1 M NaOH (Sigma Aldrich, pellets >98%, anhydrous) to 90 mL of ultrapure water (Chromasolv plus, HPLC grade). Next, 2 mL of a solution of tetrakis, hydroxymethylphosphonium chloride (THCP) (Sigma Aldrich) was added, which was prepared by mixing 24 mL of THCP with 2 mL of water. The mixture was stirred for 5 min, followed by the addition of 3 mL of 0.03 M HAuCl4·3H2O (Sigma Aldrich, 99.999% trace metal base). The brown color of the resulting solution indicates the presence of Au. +3 was reduced to Au°, resulting in the formation of 2-3 nm gold particles.

[0023] Synthesis of Au8 cluster superstructures (Au8-pX): This nanomaterial was obtained by etching the previously synthesized AuNPs. 2 mL of 100 mM sodium phosphate buffer (pH 7) was added to 10 mL of AuNPs (stored at 4 °C). Next, 2 mL of 0.1 M 11-mercaptoundecanoic acid (MUA) containing an equivalent amount of NaOH (0.2 mL of 1 M NaOH was added to a dispersion of 2 mL of water and MUA). The pH of this solution was adjusted to pH 7.5 with 100 mM phosphate buffer at pH 2.5 and pH 9. The mixture was protected from light and allowed to react in a refrigerator for 72 hours. The resulting pale yellow solution was centrifuged at 11,000 g for 30 minutes to remove excess thiol and purified several times by filtering through a Whatman syringe membrane filter (0.22 μm pore size) to remove any remaining particle aggregates from the solution. The resulting product is Au8-pX.

[0024] Example 2: Determination of the catalytic effect of Au8-pX overstructured gold clusters on the dissociation of hydrogen peroxide in aqueous media The purpose of this experiment was to electrochemically verify the non-sacrificial catalytic activity of the clusters by cyclic voltammetry. Measurements were performed under conditions as close to biological conditions as possible, using phosphate buffered saline (PBS), a common solution used to mimic the cellular environment, as the electrolyte, and introducing controlled aliquots of 345 μM hydrogen peroxide (H2O2) and / or Au8-pX clusters into the system.

[0025] Electrochemical measurements were carried out in a three-electrode cell with the following characteristics:

[0026] Working electrode (WE): Select from gold pin electrode, glassy carbon, and FTO glass pin, i.e., a thin film of Au8-pX deposited by drop casting onto glass coated with a conductive transparent oxide film.

[0027] Reference electrode (RE): A saturated calomel electrode (SCE) was used for measurements in aqueous media, while a pseudo-reference of Ag / AgCl was used for organic environments, followed by calibration with ferrocene.

[0028] Counter electrode (CE): A glassy carbon pin was used for measurements in aqueous media, while a platinum mesh electrode was used for organic environments.

[0029] All measurements were performed using a PARSTAT2273 potentiostat / galvanostat (Princeton Applied Research). The volume of starting electrolyte used was 3 mL.

[0030] The results obtained are reported in Figure 1. Panel A shows the cyclovoltogram of the PBS electrolyte before (solid line) and after (dashed line) the addition of 2 mL of aqueous H2O2 solution (0.3% by volume).

[0031] While PBS appears completely inactive, a current peak due to the oxidation of hydrogen peroxide is evident at voltages above 0.1 V. The signal intensity decreases over time and disappears when all the added peroxide is oxidized. Panel B shows the cyclovoltgrams of a PBS electrolyte containing 345 μM cluster Au8-pX before (solid line) and after (dashed line) the addition of 2 mL of aqueous H2O2 (0.3% by volume). The solid line, despite the presence of Au8-pX, does not modify that observed in the presence of PBS alone, indicating that the cluster is inactive and does not exhibit a current peak due to oxidation or reduction reactions. Conversely, the dashed line is significantly modified in panels A and B, indicating that the current peak associated with the oxidation of H2O2 completely disappears in the presence of the Au8-pX cluster. These data indicate that Au8-pX acts as a catalyst in the hydrogen peroxide dissociation reaction, thereby making hydrogen peroxide unavailable for oxidation.

[0032] Example 3: Measurement of the scavenging effect of Au8-pX clusters on different reactive oxygen species (ROS) Singlet oxygen Singlet oxygen 1The effect of the presence of the clusters on O2 was measured in solution by photoluminescence techniques using the commercially available fluorescent sensor Singlet Oxygen Sensor Green (SOSG, Invitrogen™). SOSG is a conjugated organic molecule. 1 When optically excited in the presence of O2, SOSG undergoes photooxidation and emits light. Therefore, the intensity of the photoluminescence signal of SOSG is distributed throughout the solution. 1 In this example, Rose Bengal (RB) was used as a photosensitizer in a controlled manner. 1 Generates O2.

[0033] Dissolve 100 μg of SOSG in 1 mL of methanol. Dilute this solution 1:5 with HPLC water and split into two samples. One of the samples was diluted with RB 10 -5 Gold clusters are prepared in aqueous solution at a concentration of 345 μM.

[0034] Measurements are carried out on four samples prepared as follows: -RB: SOSG aqueous solution 1mL + H2O 1mL. -RB: 1 mL of SOSG aqueous solution + 1 mL of Au8-pX solution. -SOSG 1mL + H20 aqueous solution 1mL. -1 mL of SOSG aqueous solution + 1 mL of Au8-pX solution.

[0035] 1 To generate O2, RB is excited using an unfocused 532 nm CW laser with a power of 0.3 mW. The SOSG detector is energized using an unfocused 473 nm CW laser with a power of 0.3 mW. 1 The intensity of the photoluminescence signal of the SOSG, which is proportional to the O2 concentration, is recorded with a CCD Spec2000 detector (Horiba Jobin-Yvon) coupled to a Triax190 monochromator.

[0036] The results shown in Figure 2 show that in solution 1The percentage change in O2 concentration as a function of time is shown for a series of samples prepared. In the sample without RB (gray line), no significant increase was recorded. In the sample with RB (black line), the O2 concentration increased significantly over the 25-minute period in which measurements were taken. 1 The amount of O2 is increased by 300% (cross lines) due to the presence of gold clusters (circle lines). 1 The O2 concentration was significantly reduced, with a final increase of only 15%, which is completely comparable to that observed in the sample without RB. 1 The O2 scavenging effect is seen to reduce the final concentration of ROS in the sample by up to 20-fold.

[0037] hydrogen peroxide The effect of the presence of clusters on H2O2 hydrogen peroxide was measured in solution by photoluminescence techniques using diphenyl-1-pyrenylphosphine (DPPA, Invitrogen™) as a luminescent sensor. DPPP is a phosphine that does not emit light until it is oxidized by interaction with H2O2. In this form, DPPP exhibits an absorption peak in the near-UV and emission at 380 nm. Therefore, the emission intensity of DPP is proportional to the H2O2 concentration in solution.

[0038] DPPP to 10 -4 It is dissolved in ethanol at a concentration of 1 M. The hydrogen peroxide solution used in the experiment was prepared by diluting 30% by volume of hydrogen peroxide solution 1:100.

[0039] The gold clusters were prepared in aqueous solution at a concentration of 345 μM.

[0040] Measurements were performed on two samples prepared as follows: -DPPPP solution 1.5mL + H2020.25mL. -1.5mL of DPPA solution + 0.25mL of Au8-pX solution.

[0041] Measurements were performed by continuously irradiating the starting solution with a 3 mW unfocused 355 nm laser and monitoring the photoluminescence intensity of DPP as a function of the amount of H2O2 added to the starting solution. The photoluminescence signal of DPPP was recorded using a CCD Spec2000 detector (Horiba Jobin-Yvon) coupled to a Triax190 monochromator.

[0042] The results shown in Figure 3 indicate that in the absence of gold clusters (circles), the addition of 170 mL of H2O2 increases ROS concentration by 530%. The presence of gold clusters (triangles) does not significantly affect the final concentration of H2O2, which exhibits a relative variation of 440%. This data demonstrates that, as shown in Example 2, gold clusters have hydrogen peroxide quenching activity, but it is much less than their activity against singlet oxygen.

[0043] radical oxygen Radical oxygen O2· - The effect of the presence of gold clusters on the activity of O2 was measured in solution using a commercially available luminescent sensor, MITOSOX™ red (Invitrogen™), by photoluminescence technology. MITOSOX™ red is a molecule that selectively emits light upon reaction with superoxide radicals. Therefore, the luminescence intensity of MITOSOX™ red is proportional to the amount of O2· dispersed in the solution. - In this example, ROS are generated by photolysis of hydrogen peroxide, a source of ROS, as described in Environ. Ski. Technol., vol. 41, no. 21, pp. 7486-7490, 2007.

[0044] 50 μg of MITOSOX™ red was dissolved in 0.5 mL of DMSO and 4.5 mL of H2O2 was added. To generate radical oxygen, 10 μL of H2O2 was added dropwise from a 3% (volume) aqueous solution. Gold clusters were prepared in aqueous solution at a concentration of 345 μM.

[0045] Measurements were performed on two samples prepared as follows: -MITOSOXTM red 1mL+H202 solution 0.5mL. -1 mL of MITOSOXTM red solution + 0.5 mL of Au8-pX solution.

[0046] Measurements were performed by monitoring the photoluminescence intensity of MITOSOX™ red as a function of the amount of H2O2 added to the starting solution under continuous irradiation with a 405 nm unfocused laser with a power of 23.5 mW. The light source used was O2· - The photolysis of hydrogen peroxide required to produce O2· - Both the MITOSOX™ red emission, activated by interaction with the molecule, and the photoluminescence signal intensity were recorded with a CCD Spec2000 detector (Horiba Jobin-Yvon) coupled to a Triax190 monochromator.

[0047] The results shown in Figure 4 show that the radical oxygen O2· - The figure shows the rate of change in the concentration of O2· as a function of the amount of photosensitizer H2O2 added to the starting solution. In samples without gold clusters (black circles), the addition of 300 μL of H2O2 increases the ROS concentration by 440%. The presence of gold clusters (triangles) increases the ROS concentration by 440%. - and significantly suppressed the increase in O2· - shows a final relative variation of 45%. In the case of radical oxygen, the cluster removal effect reduces the final concentration of ROS in the sample by a factor of 10.

[0048] Example 4: In vitro evaluation of proliferation of mesenchymal stem cells derived from bone marrow of FRDA patients and production of reactive oxygen species in mesenchymal stem cells Isolation of mesenchymal stem cells (MSCs) from bone marrow of FRDA patients. Samples were obtained from three FRDA subjects after obtaining informed consent. Six milliliters of bone marrow was aspirated from the left posterior iliac crest under local anesthesia under sterile conditions. The collected bone marrow was filtered through a cell filter (100 μm) to remove bone fragments and blood clots. MSCs were extracted using a red blood cell lysis method. The collected sample was transferred to a 50 mL conical centrifuge tube, and red blood cell lysis buffer and ACK solution (150 mM NH₄Cl, 10 mM KHC03, and 0.1 mM Na₂EDTA) were added at a 1:5 (v / v) ratio. The tube was manually vortexed for 1 minute and then centrifuged at 480 g for 5 minutes. The precipitated bone marrow was then diluted 1:1 with the respective culture medium. Bone marrow-derived mononuclear cells (MNCs) were isolated by gradient density centrifugation using Lymphoprep™ (1.077 g / mL). 2.5 mL of Lymphoprep™ was collected in a sterile 15 mL centrifuge tube and layered with 5 mL of diluted bone marrow (1:2 ratio) without mixing with the Lymphoprep layer. The sample was then centrifuged at 1800 rpm for 20 minutes at room temperature (RT). MNCs accumulated in the plasma prep interlymphatic phase and buffy coat layer were carefully isolated by aspiration, transferred to a new 15 mL centrifuge tube, and suspended in culture medium. The total volume of the resuspended pellet was 175 cm. 2 The cells were transferred to a ventilated flask and cultured in DMEM medium containing 10% FBS in 5% CO2 at 37°C under standard conditions for 24 hours in an incubator. After 24 hours, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS) to remove non-adherent cells. Subsequent MSC culture was performed in MSC basal medium (DMEM / F12, 1:1) (Thermo Fisher Scientific, US) containing 10% FBS (Thermo Fisher Scientific, US). The medium was completely replaced every 3–4 days. When the adherent cells became confluent, the MSCs were treated with trypsin-EDTA (Invitrogen, UK), washed twice with PBS, counted, and collected at 2 × 10 6 New 175cm cells / flask density 2 The cells were distributed into flasks and incubated in an incubator under standard conditions (5% CO2, 37°C).

[0049] Image analysis Bone marrow mesenchymal stem cells (BM-MSCs) derived from either healthy donors (ctr) or patients (ftx) were plated in 24-well plates at 75,000 cells / cm for three experimental replicates for each test condition. 2 BM-MSCs were seeded at a concentration of 10 μM. 24 hours after seeding, when the cells reached 70% confluence, three wells of ctr BM-MSCs and three wells of ftx BM-MSCs were treated with 5 and 10 μM Au8-pX. Images were acquired using an IncuCyte Vive Cell Analysis System (Sartorius). The experiment continued for 24 hours, with four photographs taken every four hours for each well using a 10x objective. Results were analyzed using IncuByte software (Sartorius). The instrument was configured to create a cell mask that best fit all the different conditions tested, and the cell area per well was correlated with time.

[0050] The results obtained, shown in Figure 10, graph A, demonstrate that FRDA bone marrow-derived MSCs (MSC ftx) exhibit significantly higher proliferation in the presence of 5 or 10 μM Au8-pX compared to proliferation without treatment. The degree of proliferation achieved is comparable to that observed with MSC ctr.

[0051] ROS Test To assess reactive oxygen species (ROS) potentially generated during culture, cells were seeded in the same manner as in the image analysis study. Analysis was performed 24 hours after adding Au8-pX to the culture medium. The ROS-Glo™ H202 (Promega) assay was used according to the manufacturer's protocol. A lithium-free assay was performed, and relative luminescence units were measured using a plate reader (GloMax discover, Promega).

[0052] The results obtained, shown in graph B of FIG. 10, show that for MSC ftx exposed to 5 or 10 μM Au8-pX, ROS production was lower than that observed in untreated MSC ftx.

[0053] This result indicates that Au8-pX has the ability to reduce ROS levels without inherent cytotoxicity, thereby limiting its cytotoxicity.

[0054] Example 5: Evaluation of the effects of Au8-pX in the FRDA mouse model Animal models Frataxin-YG8R mice (Jackson Laboratory #024113) are a recognized mouse model of Friedreich's ataxia (Anjomani Virmouni et al., 2015 Mol Neurodegener. 10:22). Notably, frataxin-YG8sR mice begin to show signs of cardiomyopathy and motor dysfunction at 9 months of age, as well as defects in glucose and insulin tolerance and histological signs of cell damage in the brain, muscle, and DRG. These animals appear largely normal and are able to feed themselves, but breeding is difficult. These animals were maintained on a mixed genetic background, C57BL6 / J.

[0055] Preparation and injection of Au8-pX For each experimental group, ten 12-month-old frataxin-YG8R mice (five males and five females) were evaluated. All animals received intravenous (IV) injections of Au8-pX into the tail vein using a syringe with a fine needle (to avoid cluster precipitation). Based on in vitro evidence, the therapeutic dose of Au8-pX was estimated to be 10 μM, corresponding to 300 μg of clusters per mouse weighing 20 g. To avoid in vivo aggregation of clusters and lung damage during IV injection, animals received a weekly dose of 100 μg of Au8-pX suspended in 100 μL of saline for three weeks.

[0056] Exercise testing Footprints: To obtain fingerprints, mouse paws were dipped in non-toxic, water-based food dye. Mice were asked to walk along a 40 cm long, 9.5 cm wide (7 cm high sidewalls) walkway with a white paper floor. All mice completed a training run followed by three test runs. Three strides in the center of each run were measured for the length of the rear pitch and front-left pitch, the length of the rear pitch and front-right pitch, the width of the forelimb base (the width between the right and left forelimbs), and the width of the hindlimb base (the width between the right and left hindlimbs), for a total of nine strides per mouse.

[0057] Treadmill: Exercise tolerance was tested using a treadmill. Mice were placed on a transparent treadmill belt (CleverySys Inc.) with a constant 10% incline and gradually increasing rotational speed. The following program was used: 18 cm / s, 0–10 min; 28 cm / s, 10–20 min; 38 cm / s, 20–25 min; and 42 cm / s, 25–30 min. Mice were trained for three 1-week sessions prior to enrollment. Data collection began at 38 cm / s. The cumulative number of errors was recorded at each time point; if obvious physical fatigue was observed before the end of the test, the animal was removed from the apparatus and assigned an arbitrary value based on the total distance traveled. Tests were repeated at weekly intervals.

[0058] Echocardiography Transthoracic echocardiography was performed using a miniature, high-resolution animal imaging system (VeVo2100, VisualSonics, Inc., Toronto, Canada) equipped with a 22-55 MHz transducer (MicroScan transducers, MS500D). Mice were anesthetized with inhaled isourea (2%) and maintained with mask ventilation (isourea 1%). To optimize physiological conditions and minimize hemodynamic fluctuations, mice were placed in a left-sided low-lying position under strict thermoregulation (37 ± 1°C). To obtain clearer images, chest hair was removed by applying cosmetic cream. Echocardiographic parameters were measured at the papillary muscles in the parasternal short-axis view (M-mode). LV fractional shortening was calculated as follows: FS = (LVEDD - LVESD) / LVEDD × 100, where LVFS is the LV fractional shortening; LVEDD is the LV end-diastolic diameter; and LVESD is the LV end-systolic diameter. LV ejection fraction was automatically calculated by the echocardiographic system. All measurements were the average of five consecutive cardiac cycles per experiment, and cardiac function was assessed when the heart rate was between 450 and 500 bpm.

[0059] Real-time qPCR To quantitatively analyze mRNA expression, tissue pieces isolated and sectioned from uninjected control animals were immediately immersed in Trizol reagent (Roche) after injection and at sacrifice, and extraction was performed as directed by the manufacturer. RNA quality, primer efficiency, and correct product size were verified by RT-PCR and agarose gel electrophoresis. Real-time qPCR was performed on a LightCycler (Roche) using FastStart DNA MasterPLUS SYBR-Green I (Roche). 2 μL of cDNA was used for each reaction. All samples were tested in triplicate. The specificity and absence of primer dimers were controlled by denaturation curves; only one denaturation peak was observed for each mRNA examined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization, calculated using LightCycler software 3.5.3.

[0060] Biochemical analysis of cellular metabolism Tissues were sectioned and immediately frozen by immersion in liquid nitrogen, then pulverized and stored at -70°C. To isolate the mitochondrial fraction, the tissue powder was washed twice with ice-cold PBS and lysed in 0.5 mL of mitochondrial lysis buffer (50 mmol / L Tris, 100 mmol / L KCl, 5 mmol / L MgCl2, 1.8 mmol / L ATP, 1 mmol / L EDTA, pH 7.2) supplemented with protease inhibitor cocktail III (Calbiochem, La Jolla, CA, USA), 1 mmol / L PMSF, and 250 mmol / L NaF. Samples were clarified by centrifugation at 650 g for 3 min at +4°C; the supernatant was collected and centrifuged at 13,000 g for 5 min at +4°C. The pellets—containing mitochondria—were washed once with lysis buffer and resuspended in 0.25 mL of resuspension buffer consisting of 250 mmol / L sucrose, 15 mmol / L K2HP04, 2 mmol / L MgCl2, and 0.5 mmol / L EDTA. A 50 μL aliquot was sonicated and used for protein content determination or Western blotting. To confirm the presence of mitochondrial proteins in the extracts, 10 μg of each sonicated sample was subjected to SDS-PAGE and probed with an anti-porin antibody (Abcam, Cambridge, UK).

[0061] ROS levels in whole cells or mitochondrial extracts were measured by labeling samples with the ROS-sensing fluorescent probe 5-(E-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester (DCFDA-AM). Results were expressed as nmol / mg cellular protein or mg mitochondrial protein.

[0062] To measure electron flow from complex I to complex III, a measure of mitochondrial respiratory activity, 50 μg of unsonicated mitochondrial samples were resuspended in 0.2 mL of buffer A (5 mmol / L K2HP04, 5 mmol / L MgCl2, 5 w / v% BSA) and transferred to a quartz spectrophotometer cuvette. 0.1 mL of buffer B (25 w / v% saponin, 50 mmol / L K2HP04, 5 mmol / L MgCl2, 5 w / v% BSA, 0.12 mmol / L c-oxidized cytochrome C, 0.2 mmol / L NaN3) was then added for 5 minutes at room temperature. The reaction was initiated with 0.15 mmol / L NADH and monitored for 5 minutes by reading absorbance at 550 nm using a Packard EL340 microplate reader (Bio-Tek Instruments, Winoski, VT, USA). Results were expressed as nmoles of reduced cytochrome c mitochondrial protein / min / mg. ATP levels in mitochondrial extracts were measured using a bioluminescent ATP assay kit (Sigma Aldrich). Results were expressed as nmol / mg mitochondrial protein.

[0063] The amount of ATP generated by oxidative phosphorylation was measured in 20 μg of mitochondrial protein using the ATP Bioluminescent Assay Kit (FL-AA; Sigma Chemical Co.). Data were converted to nmol / mg mitochondrial protein using a pre-established calibration curve. The amount of oxidative damage was measured in whole tissue extracts and mitochondrial extracts by two independent assays: 1) quantitative measurement of lipid peroxide (OH-nonenal) by ELISA (Abcam, Cambridge, UK), results are expressed as nmol / mg cellular protein or mg mitochondrial protein; 2) quantitative measurement of 8-oxo-deoxy-guanine (DNA damage) by ELISA (Abcam, Cambridge, UK), results are expressed as nmol / μg DNA.

[0064] To measure SOD1 and SOD2 activity, mitochondria were isolated as previously described (Riganti et al., 2013). Cytosolic SOD1 and mitochondrial SOD2 activities were measured using 10 μg of each extract by incubation with 50 μmol / L xanthine, 5 U / mL xanthine oxidase, and 1 μg / mL oxidized cytochrome C.

[0065] The rate of cytochrome c reduction, inhibited by the presence of SOD, was monitored for 5 min by reading absorbance at 550 nm using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, MT). Results were expressed as μmol reduced cytochrome C / min / mg cytosolic or mitochondrial protein.

[0066] Total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) contents were measured colorimetrically using a Packard EL340 microplate reader (Bio-Tek Instruments) as described in detail in Riganti et al., 2006. Results were expressed as pmol glutathione / mg cellular protein. For each sample, GSH was obtained by subtracting GSSG from total glutathione. GST activity was measured using a glutathione S-transferase (GST) test kit (Sigma Chemicals Co.) according to the manufacturer's instructions. Results were expressed as μmol CDNB-GSH adduct / min / mg protein.

[0067] Lipid peroxidation: 100 μg of whole tissue homogenate protein and 50 μg of extracted mitochondrial protein (Riganti et al., 2013) were tested with a lipid peroxidation kit (4-HNE) to determine the amount of 4-hydroxynonenal (4-HNE), an indicator of protein oxidation. Results were expressed as nmol / total protein or mg mitochondrial protein.

[0068] DNA damage: 50 ng of DNA extracted from whole tissue homogenates and 10 ng of mitochondrial DNA extracted from isolated mitochondria (Riganti et al., 2013) were assessed with an 8-hydroxy-2'-deoxyguanosine ELISA kit (Abcam, Cambridge, UK) to detect DNA oxidative damage. Results were expressed as nmol / mitochondrial DNA or μg total DNA, respectively.

[0069] result: Treatment with Au8-pX improves neuromotor and cardiac function in aged YG8R mice.

[0070] Comprehensive motor performance was assessed in untreated aged YG8R mice (n = 10; 5 females, 5 males) and treated mice (n = 10; 5 females, 5 males) by measuring motor coordination using the footprint test and time to exhaustion and endurance using treadmill performance. YG8R mice (n = 10) treated with Au8-pX were injected when they clinically exhibited coordination and motor deficits (12 months of age) and were tested from 2 months of age (asymptomatic) until sacrifice 6 months after injection (18 months of age). YG8R mice showed a progressive decline in locomotor activity and impaired coordination, as previously described (Al-MaHDawi S, et al. Genomics. 2006; 88: 580-590; Virmouni Anjoli S, et al. DIS Model Mech. 2015; 8: 225-235). Compared with untreated mice, treated YG8R mice showed clear improvements in the footprint test for motor coordination (Figure 5A, B). Notably, endurance times measured at time points 1, 2, and 3 were improved in aged YG8R mice treated with Au8-pX, increasing by ~40% compared to the endurance times of untreated YG8R animals (Figure 5C). Results are shown in Figure 6. Echocardiographic analysis in 18-month-old YG8R mice revealed a decrease in LV end-systolic / end-diastolic volumes (n = 10) accompanied by a significant improvement in left ventricular ejection fraction (LV) after Au8-pX treatment (n = 10). (End-systolic LV volume: p = 0.0123; end-diastolic LV volume: p = 0.0362; ejection fraction: p = 0.0130). Furthermore, significant decreases were observed in scale stroke (SV) volume (p = 0.0049), left ventricular internal diameter at diastole (LVID; p = 0.0148), systolic diameter (diameter; s) and diastolic diameter (diameter; d) lengths (p = 0.0309 and p = 0.0394, respectively).

[0071] Au8-pX affects the redox pathway in YG8R mice.

[0072] Accumulation of oxidized proteins and mitochondrial dysfunction have been previously documented in YG8R mice (Shan Y., et al. 2013 10.1089 / ars.2012.4537; Celine J. Rocca, et al. Ski Transl Med. 2017 9:413). Significant changes in the expression of peroxiredoxin, glutaredoxin, glutathione S-transferase, and Nrf2 were observed in the brain, spinal cord, DRG (Figure 7), pancreas, and muscle tissues of Au8-pX-treated YG8R mice. No differences in ROS levels were observed between untreated and treated YG8R mice in both whole tissue extracts and isolated mitochondria (data not shown). In neuromuscular tissue samples (cortex, cerebellum, basal ganglia) and skeletal (TA, VM, and soleus) muscles of Au8-pX-injected mice, cytosolic and mitochondrial lipid peroxidation (OH-nonenal, Figure 8D) and ROS-dependent DNA damage (deoxyguanine backbone, Figure 8E) were reduced, while increased SOD2 and GSH levels and decreased GSSG levels, accompanied by increased GST activity, were observed (Figure 9). In all these tissues and the heart, Au8-pX treatment significantly increased the mitochondrial electron transport chain (Figure 8C) and mitochondrial ATP levels (Figure 8A) compared with untreated YG8R mice. Collectively, these data indicate that Au8-pX injection reduces oxidative damage and improves mitochondrial function in YG8R mice, consistent with improved neuromotor and cardiac function.

Claims

1. A composition comprising a superstructured gold cluster Au-pX consisting of gold atoms and at least one ligand for use in the treatment of diseases associated with oxidative stress, wherein the number of gold atoms in said cluster is 8, and said superstructured cluster is Au ８ -pX or cluster dimensions are less than 2 nm, said ligand is 11-mercaptoundecanoic acid, and said disease is Friedreich's ataxia (FRDA).

2. A composition described in claim 1 for intravenous administration therapy.

3. 3. The composition of claim 2, wherein the intravenous administration treatment consists of a single intravenous administration.

4. A composition described in any one of claims 1 to 3, used in combination with a pharmaceutically acceptable excipient for intravenous administration.

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