Gold nanoclusters for the treatment of Friedreich's ataxia

Abstract

The present invention relates to superstructured gold clusters Au-pX for treating pathologies associated with oxidative stress, which are composed of gold atoms and at least one ligand, wherein the number of gold atoms in the cluster is from 2 to 100, or the size of the cluster is less than 2 nm. In a preferred embodiment, the Au-pX superstructured gold clusters are used for treating Friedreich's ataxia.

Description

Background Art

[0001] Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by the unstable expansion of the GAA trinucleotide in the first intron of the FXN gene (9q21.11) encoding frataxin. The main features of the disease are progressive gait and limb ataxia, dysarthria, dysphagia, oculomotor dysfunction, loss of deep tendon reflexes, pyramidal tract signs, scoliosis, and in some cases cardiomyopathy, diabetes, vision loss, and hearing deficits.

[0002] There is no definitive therapy for FRDA, and the symptomatology framework is currently treated in a multidisciplinary manner, usually with supportive therapy using physical therapy, pharmacological agents for spasticity control such as baclofen and botulinum toxin, and antiarrhythmic and anticoagulant agents for cardiomyopathy.

[0003] LIM F et al. Mol Ther. 2007;15:1072 - 1078; Vyas PM et al. HUM mol Genet. 2012;21:1230 - 47; Jones J et al. Mol Ther. 2015;23:130 - 138; Perdomini M et al. Mean NAT. 2014;20:542 - 547 described preclinical therapies using stem cells or gene therapy, which were tested and found to increase healthy frataxin levels.

[0004] Huntington's disease (HD) is a neurodegenerative disease caused by the unstable expansion of the CAG trinucleotide in the gene encoding huntingtin on the short arm of chromosome 4 (4p16.3). The disease affects muscle coordination and causes cognitive decline and psychiatric problems. To date, existing pharmacological treatments have not been able to alleviate many of the numerous symptoms.

[0005] Alzheimer's disease (AD) is the most common form of degenerative dementia with progressive disability, mainly occurring in the pre - senile stage. There is currently no definitive therapy for Alzheimer's disease.

[0006] Parkinson's disease (PD) is a neurodegenerative disease. The typical motor symptoms of this condition are caused by the death of cells that synthesize and release dopamine. Although pharmacological treatment, surgery, and multidisciplinary management can relieve symptoms, to date, there is no cure for Parkinson's disease.

[0007] Oxidative stress is an important part of the pathogenesis of FRDA and can explain DNA damage and neuronal degeneration (Yokota T et al. Proc Natl Acad Sci USA 2001; 98: 15185 - 15190). 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), AD (Markesbery WR, Free radical Biology and Medicine 1997; 23(1): 134 - 147) and PD (Henchcliffe C, Beal MF, Nature clinical practice 2008; 4(11): 600 - 609).

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

[0009] Santiago - Gonzales B et al. Science 2016; 353 - 571 - 575 disclosed stable superstructures formed by gold atom aggregates, called Au - pX.

[0010] The present invention relates to a method for treating pathologies (especially FRDA) caused by an excess of oxygen free radicals, which goes beyond the limitations of the therapies available to date. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Cyclic voltammograms of PBS electrolyte before (solid line) and after (dashed line) addition of an aqueous H2O2 solution in the absence (A) or presence (B) of the cluster Au8 - pX.

[0012] Figure 2 : Effect of the Au8 - pX cluster on 1 the O2 concentration.

[0013] Figure 3 : Effect of the Au8 - pX cluster on the H2O2 concentration.

[0014] Figure 4 : Effect of the Au8 - pX cluster on the concentration.

[0015] Figure 5: Neuromotor function of YG8R mice injected with Au8-pX. (A) Representative pictures of the hindlimb and forelimb footprint tests in YG8sR mice treated with Au8-pX. (B) This footprint test showed that the pitch length (stride length) of the mice injected with Au8-pX increased compared with that of the control animals (CTR). (C) The treadmill test showed that the exercise resistance of the mice injected with Au8-pX increased as measured by the number of pulses (number of electric shocks) compared with the control. Values represent mean ± SEM. P < 0.05; **P < 0.01; P < 0.001; ns = not significant. CTR = control animals.

[0016] Figure 6 : Cardiac function in YG8R mice injected with Au8-pX. (A) Representative M-mode echocardiogram images of treated (injected with Au8-pX) and untreated (not treated) mice. (B) Echocardiogram analysis showed that the ejection fraction (EF) and fractional shortening (FS) increased in the mice injected with Au8-pX compared with the control. Values represent mean ± SEM. *P < 0.05.

[0017] Figure 7 : Antioxidant expression levels in the dorsal root ganglion, cortex, cerebellum, and basal ganglia. QRT-PCR analysis showed that Prdx2, Gstm1, and Nrf2 had statistically significantly superior expression in the dorsal root ganglion of the mice injected with Au8-pX compared with the control. The data were normalized to GAPDH. Values represent mean ± SEM. P < 0.05, P < 0.001.

[0018] Figure 8: Analysis of biochemical redox parameters on the cortex, basal ganglia, cerebellum, pancreas, heart, and skeletal muscle in YG8R mice injected with Au8-pX. (A) Compared with control animals, mitochondrial ATP levels were significantly increased in all tissues of mice injected with Au8-pX, except for the cerebellum and pancreas. (B) Analysis of mitochondrial ROS showed no statistically significant differences between injected animals and control animals, except for the anterior tibial ganglia and basal ganglia. (C) Mitochondrial electron transport chain levels in the tissues of mice injected with Au8-pX compared with control animals. (D) Mitochondrial lipid peroxidation (OH-nonenal) was decreased in all tested tissues of mice injected with Au8-pX, except for the cortex and heart compared with the control. (E) Damage to mitochondrial DNA (8-oxo-guanine) was reduced in all tested tissues of mice injected with Au8-pX, except for the cortex, heart, and pancreas (for DNA damage) compared with the control. (F) Mitochondrial SOD levels were significantly decreased in the cortex, cerebellum, quadriceps, anterior tibial, and soleus muscles of mice injected with Au8-pX. Values represent mean ± SEM.

[0019] Figure 9 : Analysis of biochemical redox parameters on the cortex, basal ganglia, cerebellum, pancreas, heart, and skeletal muscle in YG8R mice injected with Au8-pX. (A) In mice injected with Au8-pX, GSH levels were increased in all tissues except the pancreas and heart. (B) In mice injected with Au8-PX, GSSG levels were significantly decreased in the cortex, basal ganglia, cerebellum, and soleus muscle. (C) In mice injected with Au8-pX, GST levels were significantly increased in the basal ganglia, cerebellum, quadriceps, and anterior tibial.

[0020] Figure 10 : Image analysis of MSC proliferation from the bone marrow of FRDA or control subjects (A) in the presence or absence of exposure to 5 or 10 μM Au8-pX. (B) Production of ROS in MSCs from the bone marrow of FRDA or control subjects in the presence or absence of exposure to 5 or 10 μM Au8-pX. Summary of the Invention

[0021] The present invention relates to superstructured gold clusters for treating oxidative stress, called Au-pX. According to the definition provided by Yamazoe S et al. (Seiji Yamazoe, Kiichirou Koyasu, Tatsuya Tsukuda, Accounts of Chemical Research 2014 DOI:10.1 021 / ar400209a), a cluster means an atomic aggregate composed of 2 to 100 metal atoms, or an aggregate that remains smaller than 2 nanometers in size.

[0022] In a preferred embodiment, the superstructured gold clusters are obtained according to the method described by Santiago-Gonzalez et al. in 2016, 353:571-575. In a preferred embodiment, the Au-pX is obtained starting from a cluster composed of 8 gold atoms (Au8), and is an aggregate of 8 gold atoms. The gold cluster is a basic component of a colloidal supramolecular superstructure called Au8-pX (with a diameter of 4-5 nanometers), bridged together by hydrogen bonds formed between closed ligands.

[0023] In a preferred embodiment, the ligand is 11-mercaptoundecanoic acid, which binds to gold atoms through a thiol group that exposes a carboxyl functional group.

[0024] In a preferred embodiment, the use of the Au-pX is for treating Friedreich's ataxia (FRDA). In another embodiment, the use is for treating Alzheimer's disease (AD), Parkinson's disease (PD) and / or Huntington's disease (HD).

[0025] In a preferred embodiment, the Au-pX superstructured gold clusters are included in a composition that also contains a pharmaceutically acceptable excipient for intravenous administration. A method for therapeutically treating a subject suffering from a lesion related to oxidative stress such as AD, PD, HD is also described.

[0026] The method includes intravenous administration of a composition containing the Au-pX superstructured gold clusters according to the present invention. In a preferred embodiment, the method includes a single administration.

[0027] The advantage of the method according to the present invention is that Au-pX can interact with different types of cells in various tissues, including the nervous, skeletal muscle, and cardiac systems, by inducing a long-term effect on mitochondrial activity. Although the ROS level remains unchanged, it has unexpectedly been demonstrated herein that in the nervous, skeletal muscle, and cardiac tissues of YG8R animals treated with Au8-pX, oxidative stress and DNA damage are reduced with an increase in mitochondrial activity. Au-PX does not have a sacrificial role, and thus, after a single administration, its effect persists over time. This allows for the treatment of subjects suffering from diseases of excess oxygen free radicals by a single administration. Examples

[0028] The sole purpose of the following examples is to better illustrate the present invention and is in no way a limitation of the present invention. The scope of the present invention is defined by the following claims.

[0029] Example 1: Synthesis of colloidal superstructures based on Au8 gold clusters (Au8-pX)

[0030] As described by Santiago-Gonzalez et al., superstructures based on Au8 clusters were obtained. Briefly, the following procedure was followed:

[0031] Synthesis of gold nanoparticles (AuNP): AuNP 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). Then, 2 ml of a THCP solution prepared by mixing 24 ml of tetrakis(hydroxymethyl)phosphonium chloride (THCP) (Sigma Aldrich) with 2 ml of water was added. The mixture was stirred for 5 minutes, and then 3 ml of 0.03 M HAuCl4·3H2O (Sigma Aldrich, purity 99.999%, trace alkali metals) was added. The brown color of the resulting solution indicated the reduction of Au +3 to Au°, and gold particles of 2 - 3 nm were formed.

[0032] Synthesis of Au8 cluster superstructure (Au8-pX): This nanomaterial was obtained by etching the previously synthesized AuNP. 2 ml of 100 mM sodium phosphate buffer (pH 7) was added to 10 ml of AuNP (stored at 4 °C). Then, 2 ml of 0.1 M 11-mercaptoundecanoic acid (MUA) containing an equal amount of NaOH (0.2 ml of 1 M NaOH was added to 2 ml of water and MUA dispersion) was added. The pH of the 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 reacted in the refrigerator for 72 hours. The resulting pale yellow solution was centrifuged at 11000 g for 30 minutes to remove excess thiol and filtered through a Whatman syringe membrane filter (0.22 μm pore size) to remove any residual particle aggregates from the solution, and thus purified several times. The resulting product was Au8-pX.

[0033] Example 2: Measurement of the catalytic effect of Au8-pX superstructured gold clusters on the dissociation of hydrogen peroxide in an aqueous medium

[0034] The purpose of this experiment was to electrochemically verify the non-consumptive catalytic activity of the clusters by cyclic voltammetry. Measurements were carried out in an environment as close as possible to the biological environment, using PBS as the electrolyte, which is a phosphate buffer solution commonly used to reproduce the cell environment, and introducing a controllable aliquot of hydrogen peroxide (H2O2) and / or Au8-pX clusters at a concentration of 345 μM into the system.

[0035] Electrochemical measurements were carried out in a three-electrode electrolytic cell with the following characteristics:

[0036] Working electrode (WE): Selected from a gold needle electrode, glassy carbon, and FTO glass needle, i.e., glass with a conductive and transparent oxide layer deposited on it, on which a thin film of Au8-pX has been deposited by drop casting.

[0037] Reference electrode (RE): For measurements in an aqueous medium, a saturated calomel electrode (SCE) was used, while for an organic environment, a pseudo-reference of Ag / AgCI was used and then calibrated with ferrocene.

[0038] Counter electrode (CE): For measurements in an aqueous medium, a glassy carbon needle was used, while a platinum mesh electrode was used in an organic medium.

[0039] All measurements were carried out using a PARSTAT 2273 potentiostat / galvanostat (Princeton Applied Research). The initial electrolyte volume used was 3 ml.

[0040] The results obtained are as Figure 1As shown. Small graph A shows the cyclic voltammogram of the PBS electrolyte before (solid line) and after (dashed line) adding 2 ml of an aqueous H202 solution (0.3% by volume).

[0041] Although it was proven that PBS was completely inactive, at voltages above 0.1 V, the peak current generated by the oxidation of hydrogen peroxide was still obvious. Over time, the signal intensity decreased until all the peroxide added to the solution was oxidized and the signal intensity disappeared. Small graph B shows the cyclic voltammogram of the PBS electrolyte containing 345 μM Au8-pX clusters before (solid line) and after (dashed line) adding 2 ml of an aqueous H2O2 solution (0.3% by volume). Although in the presence of Au8-pX, the solid line did not change at all compared to what was observed when only PBS was present, indicating that the cluster was inactive and did not exhibit a current peak due to any oxidation or reduction reaction. Instead, the dashed line changed deeply from small graph A to small graph B, where in the presence of the Au8-pX cluster, the current peak associated with the oxidation of H2O2 completely disappeared. This data indicates that Au8-pX acts as a catalyst in the dissociation reaction of hydrogen peroxide, so hydrogen peroxide is no longer available for the oxidation reaction.

[0042] Example 3: Measuring the scavenging effect of Au8-pX clusters on different reactive oxygen species (ROS)

[0043] Singlet oxygen

[0044] Using the commercial fluorescent sensor Singlet Oxygen Sensor Green (SOSG, Invitrogen TM), the effect of the presence of the cluster on singlet oxygen 1 O2 in solution was measured using photoluminescence technology. SOSG is a conjugated organic molecule that undergoes a photooxidation reaction upon photoexcitation in the presence of 1 O2, causing it to emit light. Therefore, the photoluminescence signal intensity of SOSG is proportional to the concentration of 1 O2 dispersed in the solution. In the context of this example, 1 O2 was generated in a controlled manner using Rose Bengal (RB) as a photosensitizer.

[0045] Dissolve 100 μg of SOSG in 1 ml of methanol. Dilute the solution 1:5 in HPLC water and divide it into two samples. Add 10 -5 M RB to one of them. Prepare the gold clusters in an aqueous solution at a concentration of 345 μM.

[0046] The measurements were carried out on 4 samples, which were prepared as follows:

[0047] -1 ml RB:SOSG aqueous solution + 1 ml H2O.

[0048] -1 ml RB:SOSG aqueous solution + 1 ml Au8-pX solution.

[0049] -1 ml SOSG + 1 ml H2O aqueous solution.

[0050] -1 ml SOSG aqueous solution + 1 ml Au8-pX solution.

[0051] To generate 1 O2, an unfocused 532 nm CW laser with a power of 0.3 mW was used to excite RB. A 473 nm unfocused CW laser with a power of 0.3 mW was used to energize the SOSG detector. A CCDSpec 2000 detector (Horiba Jobin-Yvon) coupled with a Triax 190 monochromator was used to record the photoluminescence signal intensity of SOSG proportional to the concentration of 1 O2 in the solution.

[0052] The results are as Figure 2 shown, showing the percentage change in the concentration of 1 O2 in the solution as a function of time in the prepared sample set. No significant increase was recorded in the sample without RB (gray line). In the sample containing RB (black line), 1 the amount of 1 O2 increased by 300% (the line marked with crosses) within 25 minutes of measurement. The presence of gold clusters (the line marked with circles) significantly reduced the 1 O2 concentration, showing a final increase of only 15%, which is completely comparable to that observed in the sample without RB. This data indicates that the scavenging of

[0053] Hydrogen peroxide

[0054] The effect of the presence of clusters on hydrogen peroxide H2O2 in solution was measured by photoluminescence techniques using diphenyl-1-pyrenylphosphine (DPPA, Invitrogen TM) as the luminescent sensor. DPPP is a phosphine that does not emit light until it is oxidized by interacting with H2O2. In this form, DPPP exhibits an optical absorption peak in the near-ultraviolet region and emits at 380 nm. Therefore, the luminescence intensity of DPPP is proportional to the concentration of H2O2 in the solution.

[0055] DPPP was used at 10 -4The concentration of M was dissolved in an ethanol solution. The hydrogen peroxide solution used in this experiment was prepared by diluting a 30% by volume hydrogen peroxide solution at a ratio of 1:100.

[0056] Gold clusters were prepared in an aqueous solution at a concentration of 345 μM.

[0057] Measurements were carried out on two samples, which were formulated as follows:

[0058] -1.5 ml of DPPP solution + 0.25 ml of H2O2.

[0059] -1.5 ml of DPPP solution + 0.25 ml of Au8-pX solution.

[0060] Measurements were carried out under continuous irradiation of a non-focused 355 nm laser at a power of 3 mW by monitoring the photoluminescence intensity of DPPP as a function of the amount of H2O2 added to the starting solution. A CCD Spec 2000 detector (Horiba Jobin-Yvon) coupled to a Triax 190 monochromator was used to record the photoluminescence signal of DPPP.

[0061] The results are as Figure 3 shown, showing that in the absence of gold clusters (circles), the addition of 170 ml of H2O2 resulted in a 530% increase in the ROS concentration. The presence of gold clusters (triangles) did not significantly affect the final concentration of H2O2, which showed a relative change of 440%. This data demonstrates the fact that although there is also a digestive activity of gold clusters on hydrogen peroxide as shown in Example 2, it is much lower than the digestive activity on singlet oxygen.

[0062] Oxygen free radicals

[0063] MITOSOX TM Red (Invitrogen TM) was used as a commercial luminescent sensor, and the effect of the presence of gold clusters on oxygen free radicals was measured in solution by photoluminescence techniques. MITOSOX TM Red is a molecule that becomes selectively luminescent when reacting with superoxide radicals. Therefore, the luminescence intensity of MITOSOX TM Red is proportional to the concentration dispersed in the solution. In this case, ROS was generated by utilizing the photolysis of hydrogen peroxide as a generator of this species, as described in Environ. Skiing. Technol., Vol. 41, No. 21, pp. 7486-7490, 2007.

[0064] 50 μg of MITOSOX TMThe red was dissolved in 0.5 ml of DMSO, and then 4.5 ml of H2O2 was added. To generate oxygen radicals, 10 μl of H2O2 was added dropwise from a 3% by volume aqueous solution. Gold clusters were prepared in an aqueous solution at a concentration of 345 μM.

[0065] The measurements were carried out on two samples, which were prepared as follows:

[0066] - 1 ml of MITOSOX TM Red + 0.5 ml of H2O2 solution.

[0067] - 1 ml of MITOSOX TM Red solution + 0.5 ml of Au8-pX solution.

[0068] Under continuous irradiation with a non-focused 405 nm laser at a power of 23.5 mW, the measurement was carried out by monitoring the photoluminescence intensity of Red TM as a function of the amount of H2O2 added to the starting solution. The light source used was capable of simultaneously activating the photolysis of the required hydrogen peroxide and the luminescence of Red activated by interaction with the TM photo-generated molecules. A CCD Spec2000 detector (Horiba Jobin-Yvon) coupled to a Triax 190 monochromator was used to record the intensity of the photoluminescence signal.

[0069] The results are as Figure 4 shown, showing the percentage change in the concentration of oxygen radicals as a function of the amount of the photosensitizer H2O2 added to the starting solution. In the sample without gold clusters (black circles), the addition of 300 μl of H2O2 led to a 440% increase in the ROS concentration. The presence of gold clusters (triangles) significantly reduced the increase, which showed a final relative change of 45%. In the case of oxygen radicals, the scavenging effect of the clusters reduced the final concentration of ROS in the sample by 10-fold.

[0070] Example 4: In vitro evaluation of the proliferation and ROS production of mesenchymal stem cells derived from the bone marrow of FRDA patients

[0071] Mesenchymal stem cells (MSCs) were isolated from the bone marrow derived from FRDA patients.

[0072] After collecting the informed consent forms, samples were obtained from 3 FRDA subjects. 6 ml of bone marrow was aseptically aspirated from the left posterior iliac crest under local anesthesia in an aseptic manner. The collected bone marrow was filtered through a cell strainer (100 μm) to remove any bone spicules or clots present. MSC extraction was performed using the method of lysing red blood cells. The collected sample was transferred to a 50 ml conical centrifuge tube, and red blood cell lysis buffer, namely ACK solution (NH4CI 150 mM, KHCO3 10 mM and Na2EDTA 0.1 mM), was added at a ratio of 1:5 (v / v). The tube was manually stirred for 1 minute and then centrifuged at 480 g for 5 minutes. Then the precipitated bone marrow was diluted at a ratio of 1:1 with the corresponding medium. Lymphoprep TM (1.077 g / ml) was used for gradient density centrifugation to isolate the mononuclear cell fraction (MNC) from the bone marrow. 2.5 ml of Lymphoprep TM was collected in a 15 ml sterile centrifuge tube and layered with 5 ml of diluted bone marrow (ratio 1:2) without mixing with the Lymphoprep layer. Then the sample was centrifuged at 1800 rpm for 20 minutes at room temperature (RT). The MNCs accumulating at the plasma preparation interface and buffy coat in the lymphatic fluid were carefully separated by aspiration, transferred to a new 15 ml centrifuge tube and suspended in the medium. The entire volume of the resuspended pellet was transferred to a vented culture flask of 175 cm 2 and cultured in DMEM medium containing 10% FBS in an incubator under standard conditions of 5% CO2, 37 °C for 24 hours. After 24 hours, the medium was removed and the cells were washed with phosphate-buffered saline (PBS) to remove non-adherent cells. MSC basal medium (DMEM / F12, 1:1) (Thermo Fisher Scientific - US) containing 10% FBS (Thermo Fisher Scientific - US) was used for the subsequent culture of MSCs. The medium was completely changed every 3 - 4 days. When the adherent cells reached confluence, the MSCs were treated with trypsin-EDTA (Invitrogen, UK), washed twice with PBS, counted and seeded at a density of 2 × 10 6 cells per flask in new 175 cm 2 flasks and incubated in an incubator under standard conditions (5% CO2, 37 °C).

[0073] Image analysis

[0074] Bone marrow mesenchymal stem cells (BM-MSCs) derived from healthy donors (ctr) or patients (ftx) were seeded at 75000 cells / cm 2Cells were seeded in 24-well plates at a concentration such that there were three experimental replicates for each test condition. Twenty-four hours after seeding, when the cells reached 70% confluence, three wells containing BM-MSC ctr and three wells containing BM-MSC ftx were treated with 5 and 10 μM of Au8-pX. Images were acquired using an IncuCyte Vive cell analysis system (Sartorius). The experiment lasted for 24 hours, and four pictures were taken for each well with a 10× objective lens every 4 hours. The results were analyzed using IncuByte software (Sartorius), setting the instrument to create a cell mask that best fit all the different test conditions and correlating the cell area of each well with time.

[0075] As Figure 10 shown in panel A of, the results obtained showed that, compared to the proliferation in the absence of treatment, MSCs derived from FRDA bone marrow (MSC ftx) exhibited significantly higher proliferation in the presence of 5 or 10 μM of Au8-pX. The degree of proliferation achieved was comparable to that observed in MSC ctr.

[0076] ROS assay

[0077] To evaluate the reactive oxygen species (ROS) that might be generated in the culture, cells were seeded as for the image analysis test. The analysis was performed 24 hours after adding Au8-pX to the culture medium. Using the ROS-Glo TM H2O2 (Promega) assay according to the manufacturer's protocol. A non-lithic assay was performed, and the relative light units were measured using a plate reader (GloMax discover, Promega).

[0078] As Figure 10 shown in panel B of, the results obtained showed that MSCs ftx exposed to 5 or 10 μM Au8-pX produced lower levels of ROS than those observed in untreated MSCs ftx.

[0079] The results indicate that Au8-pX is able to reduce ROS levels, thereby limiting the cytotoxicity of ROS without having their inherent cytotoxicity.

[0080] Example 5: Evaluation of the role of Au8-pX in the FRDA mouse model

[0081] Animal model

[0082] The Ataxin YG8R mouse (Jackson Laboratory, stock number 024113) is a recognized murine model of Friedreich's ataxia (Anjomani Virmouni et al., 2015 Mol Neurodegener. 10:22). In particular, Ataxin YG8sR mice start to show signs of cardiomyopathy and motor deficits at 9 months of age, also showing defects in glucose tolerance and insulin resistance, as well as histological signs of cell damage in the brain, muscle, and DRG. The animals generally appear normal, they can eat on their own, but they have difficulty reproducing. The animals were maintained on a mixed genetic background C57BL6 / J.

[0083] Preparation of Au8-pX and injection

[0084] For each experimental group, 10 12-month-old Ataxin YG8R mice (5 males and 5 females) were evaluated. Using a syringe with a fine needle (to avoid deposition of clusters), all animals received intravenous (IV) injection of Au8-pX in the tail vein. Based on in vitro evidence, the therapeutic dose of Au8-pX was estimated to be 10 μM, equivalent to 300 μg of clusters for a 20 g mouse. To avoid in vivo cluster aggregation and lung injury during intravenous injection, the animals received a dose of 100 μg of Au8-pX suspended in 100 μl of saline per week for three weeks.

[0085] Motor testing

[0086] Footprint: To obtain paw prints, the mouse legs were dipped into non-toxic water-based edible dye. The mice were allowed to walk along a 40 cm long and 9.5 cm wide (with 7 cm high side walls) channel with white paper covering the bottom plate. All mice had one running training session and then underwent three tests. Three steps in the central part of each run were measured, a total of nine steps per mouse, for the length of the posterior left pitch and anterior left pitch, the length of the posterior right pitch and anterior right pitch, the width of the anterior base (width between the right and left forelimbs), and the width of the posterior base (width between the right and left hindlimbs).

[0087] Treadmill: A treadmill was used to test the resistance to exercise. Mice were placed on a transparent running belt (CleverySys Inc) with a constant slope of 10% and the rotational speed was gradually increased. The following program was used: speed 18 cm / sec for 0 to 10 minutes; speed 28 cm / sec for 10 to 20 minutes, speed 38 cm / sec for 20 to 25 minutes, speed 42 cm / sec for 25 to 30 minutes. Mice were trained for three weeks before enrollment. Data collection began at a speed of 38 cm / sec. At each time point, the cumulative number of errors was recorded; if obvious physical exhaustion occurred before the end of the test, the animal was removed from the device and an arbitrary value was assigned based on the total distance traveled. The test was repeated every other week.

[0088] Echocardiogram

[0089] Transthoracic echocardiography was performed on the animals using a small high-resolution imaging system (VeVo2100, VisualSonics, Inc, Toronto, Canada) equipped with a 22 - 55 MHz transducer (MicroScan transducer, MS500D). Mice were anesthetized by inhalation of iso-uranus (2%) and maintained by mask ventilation (iso-uranus 1%). The mice were placed in the left lateral decubitus position and strict temperature regulation (37 ± 1 °C) was performed to optimize physiological conditions and reduce hemodynamic changes. The fur was removed from the chest by applying cosmetic cream to obtain clear images. Echocardiographic parameters (M-mode) were measured at the papillary muscle in the parasternal short-axis view. The calculation method of the LV fractional shortening rate was as follows: FS = ((LVEDD - LVESD) / LVEDD) × 100, where LVFS represents the LV fractional shortening rate; LVEDD, LV end-diastolic diameter; and LVESD, LV end-systolic diameter. The LV ejection fraction was automatically calculated by the echocardiography system. All measurements were averaged over 5 consecutive cardiac cycles for each experiment and cardiac function was evaluated at a heart rate of 450 to 500 bpm.

[0090] Real-time qPCR

[0091] For the quantitative analysis of mRNA expression, tissue fragments isolated and sectioned from injected and control (non-injected) animals at the time of sacrifice were immediately immersed in Trizol reagent (Roche) and extracted according to the manufacturer's instructions. RNA quality, primer efficiency, and correct product size were verified by RT-PCR and agarose gel electrophoresis. A LightCycler (Roche) was used, and real-time qPCR was performed using FastStart DNAMasterPLUS SYBR-Green I (Roche). 2 μl of cDNA was used in each reaction. All samples were tested in triplicate. Primer specificity and dimer absence were controlled by the melting curve; only one melting peak was observed for each mRNA detected. Normalization was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and calculations were performed using LightCycler software 3.5.3.

[0092] Biochemical analysis of cell metabolism

[0093] Tissues were sectioned and immediately frozen by immersion in liquid nitrogen, then pulverized and stored at -70 °C. To isolate the mitochondrial fraction, tissue powder was washed twice in 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 minutes at +4 °C: the supernatant was collected and centrifuged at 13,000 g for 5 minutes at +4 °C. The pellet containing mitochondria was washed once with lysis buffer and resuspended in 0.25 ml of resuspension buffer consisting of 250 mmol / L sucrose, 15 mmol / L K2HPO4, 2 mmol / L MgCl2, and 0.5 mmol / L EDTA. 50 μl aliquots were sonicated and used for protein content measurement 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, United Kingdom).

[0094] The amount of ROS in whole cell or mitochondrial extracts was measured by labeling the samples with a ROS 5-(E-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester (DCFDA-AM) sensitive fluorescent probe. The results were expressed as nmol / mg cellular protein or mitochondrial protein.

[0095] To measure the electron flow from complex I to complex III (as an indicator of mitochondrial respiratory activity), 50 μg of non-sonicated mitochondrial samples were resuspended in 0.2 ml of buffer A (5 mmol / L KH2PO4, 5 mmol / L MgCI2, 5% w / v BSA) and transferred to a quartz spectrophotometer cuvette. Then 0.1 ml of buffer B (25% w / v saponin, 50 mmol / L KH2PO4, 5 mmol / L MgCI2, 5% w / v BSA, 0.12 mmol / L oxidized form of cytochrome C, 0.2 mmol / L NaN3) was added at room temperature and incubated for 5 minutes. The reaction was initiated with 0.15 mmol / L NADH and followed for 5 minutes, and the absorbance at 550 nm was read using a Packard EL340 microplate reader (Bio-Tek Instruments, Winoski, VT, USA). The results were expressed as nmol / min / mg of reduced cytochrome C mitochondrial protein. The amount of ATP in mitochondrial extracts was measured using a bioluminescent ATP assay kit (Sigma-Aldrich). The results were expressed as nmol / mg mitochondrial protein.

[0096] The amount of ATP generated by oxidative phosphorylation was measured for 20 μg of mitochondrial protein using an ATP bioluminescent assay kit (FL-AA; Sigma Chemical Co.). The data were converted to nmol / mg mitochondrial protein using a pre-set calibration curve. The amount of oxidative damage in total tissue extracts and mitochondrial extracts was measured by two independent assays: 1) ELISA quantitative measurement of lipid peroxidation (OH-nonenal) (Abcam, Cancridge, United Kingdom), and the results were expressed as nmol / mg cellular protein or nmol / mg mitochondrial protein; 2) ELISA (Abcam Cambridge, United Kingdom) quantitative measurement of 8-oxo-deoxy-guanine (DNA damage), and the results were expressed as nmol / μg of DNA.

[0097] To measure the activities of SOD1 and SOD2, mitochondria were isolated as previously reported (Riganti et al., 2013). Using 10 μg of each extract, the activities of cytosolic SOD1 and mitochondrial SOD2 were measured by incubating with 50 μmol / L xanthine, 5 U / mL xanthine oxidase, and 1 μg / mL oxidized cytochrome C.

[0098] The reduction rate of cytochrome C inhibited by the presence of SOD was monitored for 5 minutes by reading the absorbance at 550 nm using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT). The results were expressed as μmol / min / mg cytosolic or mitochondrial protein of reduced cytochrome C.

[0099] As described in detail (Riganti et al., 2006), the contents of total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured colorimetrically using a Packard EL340 microplate reader (Bio-Tek Instruments). The results were expressed as pmol of glutathione / mg cellular protein. For each sample, GSH was obtained by subtracting GSSG from total glutathione. According to the manufacturer's instructions, a glutathione S-transferase (GST) assay kit (Sigma Chemicals. Co) was used to measure GST activity. The results were expressed as μmol / min / mg protein of CDNB-GSH adduct.

[0100] Lipid peroxidation: A lipid peroxidation kit (4-HNE) was used to assay 100 μg of whole tissue homogenate protein and 50 μg of isolated mitochondrial protein (Riganti et al., 2013) to evaluate the amount of 4-hydroxynonenal (4-HNE), an index of protein oxidation. The results were expressed as nmol / mg total protein or mitochondrial protein.

[0101] DNA damage: 50 ng of DNA extracted from whole tissue homogenate and 10 ng of mitochondrial DNA extracted from isolated mitochondria (Riganti et al., 2013) were evaluated using an 8-hydroxy-2'-deoxyguanosine ELISA kit (Abcam, Cambridge, United Kingdom) to detect oxidative damage to DNA. The results were expressed as nmol / μg mitochondrial DNA or total DNA, respectively.

[0102] Results:

[0103] Treatment with Au8-pX improves neuromotor and cardiac function in aged YG8R mice.

[0104] Overall, locomotor ability was assessed in untreated aged YG8R mice (n = 10; 5 females and 5 males) and treated aged YG8R mice (n = 10; 5 females and 5 males) by using the footprint test for motor coordination and the exhaustion time and resistance time obtained from treadmill performance. When the YG8R mice (n = 10) treated with Au8-pX developed clinical symptoms of coordination and motor deficits (12 months old), they were injected and tested from 2 months old (asymptomatic) to 6 months after injection until sacrifice (18 months old). As previously described, YG8R mice showed a progressive decrease in locomotor activity and coordination deficits (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, there was a significant improvement in the footprint test of motor coordination in treated YG8R mice ( Figure 5 A, 5B). In particular, the resistance time measured at time points 1, 2, and 3 was improved in aged YG8R mice treated with Au8-pX, increasing by approximately 40% compared with the resistance time of untreated YG8R animals ( Figure 5 C). Echocardiographic analysis of 18-month-old YG8R mice showed that, as Figure 6 shown, after treatment with Au8-pX (n = 10), there was a decrease in LV end-systolic / diastolic volume (n = 10), accompanied by a significant improvement in left ventricular (LV) ejection fraction. (LV end-systolic volume: p = 0.0123; LV end-diastolic volume: p = 0.0362; ejection fraction: p = 0.0130). In addition, a significant decrease was observed in the stroke volume (SV) scale (volume of the scalestroke) (p = 0.0049), left ventricular internal diameter in diastole (LVID; p = 0.0148), and the diameter lengths in systole (diameter; s) and diastole (diameter; d) (p = 0.0309 and p = 0.0394, respectively).

[0105] Au8-pX affects the redox pathway in YG8R mice.

[0106] 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. Sci Transl Med. 2017 9:413). Significant changes in the expression of peroxiredoxin, glutaredoxin, glutathione-S-transferase, and Nrf2 were found in the brain, spinal cord, DRG ( Figure 7 ), pancreas, and muscle tissues of YG8R mice treated with Au8-pX. No differences in ROS levels were found between untreated and treated YG8R mice in total tissue extracts and isolated mitochondria (data not shown). In nerve tissue samples (cortex, cerebellum, basal ganglia) and skeletal muscle (TA, VM, and soleus) of mice injected with Au8-pX, lipid peroxidation (OH-nonenal, Figure 8 D) and ROS-dependent DNA damage (deoxy-guanine backbone, Figure 8 E) in the cytosol and mitochondria were reduced, while increased SOD2 and GSH levels and decreased GSSG levels were observed, along with increased GST activity ( Figure 9 ). In all of these tissues and the heart, the mitochondrial electron transport chain ( Figure 8 C) and mitochondrial ATP levels ( Figure 8 A) were significantly increased in YG8R mice treated with Au8-pX compared to untreated YG8R mice. Collectively, these data suggest that injection of Au8-pX reduces oxidative damage and improves mitochondrial function in YG8R mice, which is consistent with the improvement of neuromotor and cardiac function.

Claims

1. Use of the superstructured gold cluster Au-pX in the preparation of a medicament for the treatment of Friedreich's ataxia (FRDA), said superstructured gold cluster Au-pX being composed of gold atoms and the ligand 11-mercaptoundecanoic acid, wherein the number of said gold atoms in the cluster is 8, whereby said superstructured gold cluster is designated Au8-pX.

2. Use according to claim 1, wherein said treatment is by the intravenous route.

3. Use according to any one of claims 1 to 2, wherein said treatment consists of a single intravenous administration.

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