Patent Description:
Multiple sclerosis (MS), meaning "scar tissue in multiple areas", is an autoimmune disorder in which the immune system attacks the myelin sheath that surrounds and protects the nerve fibers of the central nervous system (CNS), causing inflammation. When myelin or nerve fibers are damaged or destroyed in MS, damage to areas of the CNS may produce a variety of neurological symptoms that vary among people with MS in type and severity. There are four types of MS: clinically isolated syndrome (CIS), relapse-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). The common symptoms include muscle weakness, numbness and tingling, Lhermitte's sign, bladder problems, bowel problems, fatigue, dizziness and vertigo, sexual dysfunction, spasticity and muscle spasms, tremor, vision problems, gait and mobility changes, emotional changes and depression, learning and memory problems, and pain.

The cause of MS is not known, but it is believed to involve genetic susceptibility, abnormalities in the immune system and environmental factors that combine to trigger the disease.

While available medications are helpful to slow progression by changing the way the immune system functions or relieve symptoms during a flare when a person experiences a worsening of symptoms, there is still an imperative need for new medications and methods for the treatment of MS.

<CIT> discloses a composition comprising gold clusters which is used to prepare drugs for preventing and treating Alzheimer's disease and/or Parkinson's disease (PD). It does not involve the pharmaceutical application of the gold clusters composition in other diseases, such as an autoimmune disease.

Bianco A, et al. involve the study of microglia activation in MS patients and its influence on the development of AD lesions. The disclosure fails to reveal any direct relation between MS syndrome and development of AD pathology. Aβ plaques was also investigated in the MS cases, where no difference in the density of Aβ plaques between the demyelinated and nondemyelinated area of the MS cortex was observed. The disclosure teaches away from the treatment of MS targeting to Aβ plaques.

David A, et al. disclose that Amyloid β, tau protein, and amyloid precursor protein associated with AD and PD (Parkinson's disease) are found in lesions and plaques of MS patients. But it does not confirm the role Amyloid β plays in MS pathogenesis, let alone in prevention or treatment of MS.

The previous report (<NPL>) discloses a clinical trial regarding to treatment of MS. But it does not involve pharmaceutical use of gold clusters or inhibition of Aβ plaques in MS.

<CIT> is a corresponding application of the present disclosure filed to CNIPA on the same application date.

The objective of the present disclosure is to provide a gold cluster (AuC) for use in the treatment of multiple sclerosis, the AuC comprises a gold core; and a ligand bonded the gold core;.

In certain embodiments of the AuC for use, the gold core has a diameter smaller than <NUM>.

In certain embodiments, the gold core has a diameter in the range of <NUM>-<NUM>.

The objectives and advantages of the disclosure will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.

Preferred embodiments according to the present disclosure will now be described with reference to the Figures, in which like reference numerals denote like elements.

The present disclosure may be understood more readily by reference to the following detailed description of certain embodiments of the disclosure. The following examples or embodiments which are not part of the scope of the claims are present for illustration purposes only.

Gold clusters (AuCs) are a special form of gold existing between gold atoms and gold nanoparticles. AuCs have a size smaller than <NUM>, and are composed of only several to a few hundreds of gold atoms, leading to the collapse of face-centered cubic stacking structure of gold nanoparticles. As a result, AuCs exhibit molecule-like discrete electronic structures with distinct HOMO-LUMO gap unlike the continuous or quasi-continuous energy levels of gold nanoparticles. This leads to the disappearance of surface plasmon resonance effect and the corresponding plasmon resonance absorption band (<NUM> ± <NUM>) at uv-vis spectrum that possessed by conventional gold nanoparticles.

The present disclosure provides a ligand-bonded AuC.

In certain embodiments, the ligand-bonded AuC comprises a ligand and a gold core, wherein the ligand is bonded to the gold core. In certain embodiments, the diameter of the gold core is in the range of <NUM> - <NUM>. In certain embodiments, the diameter of the gold core is in the range of <NUM> - <NUM>.

In certain embodiments, the ligand of the ligand-bonded AuC is a thiol-containing compound or oligopeptide. In certain embodiments, the ligand bonds to the gold core to form a ligand-bonded AuC via Au-S bond.

In certain embodiments, the ligand is one selected from the group consisting of L-cysteine, D-cysteine, and a cysteine derivative. In certain embodiments, the cysteine derivative is one selected from the group consisting of N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC).

In certain embodiments, the ligand is one selected from the group consisting of a cysteine-containing oligopeptide and its derivatives. In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In certain embodiments, the cysteine-containing dipeptide is one selected from the group consisting of L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-histidine-L-cysteine dipeptide (HC) and L-cysteine-L-histidine dipeptide (CH). In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing tripeptide. In certain embodiments, the cysteine-containing tripeptide is one selected from the group consisting of glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-lysine-L-cysteine-L-proline tripeptide (KCP), and L-glutathione (GSH). In certain embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In certain embodiments, the cysteine-containing tetrapeptide is one selected from the group consisting of glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) and glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

According to some embodiments not part of the scope of the claims, the ligand is a thiol-containing compound. According to some embodiments not part of the scope of the claims, thiol-containing compound is one selected from the group consisting of <NUM>-[(<NUM>)-<NUM>-methyl-<NUM>-thiol-<NUM>-oxopropyl]-L-proline, thioglycollic acid, mercaptoethanol, thiophenol, D-<NUM>-trolovol, N-(<NUM>-mercaptopropionyl)-glycine and dodecyl mercaptan.

The present disclosure provides a pharmaceutical composition for the treatment of multiple sclerosis in a subject. In certain embodiments, the subject is human. In certain embodiments, the subject is a pet animal such as a dog.

In certain embodiments, the pharmaceutical composition comprises a ligand-bonded AuC as disclosed above and a pharmaceutically acceptable excipient. In certain embodiments, the excipient is phosphate-buffered solution, or physiological saline.

The present disclosure provides a use of the above disclosed ligand-bonded AuCs for manufacturing a medication for the treatment of multiple sclerosis in a subject.

The present disclosure provides the above disclosed ligand-bonded AuCs for use in the treatment of multiple sclerosis in a subject using the above disclosed ligand-bonded AuCs. According to some embodiments not part of the scope of the claims, the treatment comprises administering a pharmaceutically effective amount of ligand-bonded AuCs to the subject. The pharmaceutically effective amount can be ascertained by routine in vivo studies.

The following examples are provided for the sole purpose of illustrating the principles of the present disclosure; they are by no means intended to limit the scope of the present disclosure.

As detected, the particle size of the powdery or flocculant substance obtained by the foregoing method is smaller than <NUM> (distributed in <NUM>-<NUM> in general). No obvious absorption peak at <NUM>. It is determined that the obtained powder or floc is ligand-bonded AuCs.

Taking ligand L-NIBC for example, the preparation and confirmation of AuCs bonded with ligand L-NIBC are detailed.

<NUM> After the reaction, the reaction solution is subjected to gradient centrifugation to obtain L-NIBC-AuCs powder with different particle sizes. Specific method: After the reaction is completed, the reaction solution is transferred to an ultrafiltration tube with MWCO of <NUM> and a volume of <NUM>, and centrifuged at 10000r/min for <NUM>, and the retentate in the inner tube is dissolved in ultrapure water to obtain powder with a particle size of about <NUM>. Then, the mixed solution in the outer tube is transferred to an ultrafiltration tube with a volume of <NUM> and MWCO of <NUM>, and centrifuged at <NUM>,<NUM> r/min for <NUM>. The retentate in the inner tube is dissolved in ultrapure water to obtain powder with a particle size of about <NUM>. Then the mixed solution in the outer tube is transferred to an ultrafiltration tube with a volume of <NUM> and MWCO of <NUM>, and centrifuged at <NUM>,500r/min for <NUM>. The retentate in the inner tube is dissolved in ultrapure water to obtain powder with a particle size of about <NUM>.

<NUM> Precipitate the powder in three different particle sizes obtained by gradient centrifugation, remove the solvent respectively, blow the crude product dry with N<NUM>, dissolve it in <NUM> of ultrapure water, put it in a dialysis bag (MWCO is 3KDa), put the dialysis bag in <NUM> of ultrapure water, change water every other day, dialyze it for <NUM> days, freeze-dry it and keep it for future use.

Characterization experiment was conducted for the powder obtained above (L-NIBC-AuCs). Meanwhile, ligand L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs) are used as control. The method for preparing gold nanoparticles with ligand being L-NIBC refers to the reference (<NPL>;<NPL>).

The test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water to <NUM>/L as samples, and then test samples were prepared by hanging drop method. More specifically, <NUM>µL of the samples were dripped on an ultrathin carbon film, volatized naturally till the water drop disappeared, and then observe the morphology of the samples by JEM-2100F STEM/EDS field emission high-resolution TEM.

The four TEM images of L-NIBC-AuNPs are shown in panels B, E, H, and K of <FIG>; the three TEM images of L-NIBC-AuCs are shown in panels B, E, and H of <FIG>.

The images in <FIG> indicate that each of L-NIBC-AuCs samples has a uniform particle size and good dispersibility, and the average diameter of L-NIBC-AuCs (refer to the diameter of gold core) is <NUM>, <NUM> and <NUM> respectively, in good accordance with the results in panels C, F and I of <FIG>. In comparison, L-NIBC-AuNPs samples have a larger particle size. Their average diameter (refer to the diameter of gold core) is <NUM>, <NUM>, <NUM> and <NUM> respectively, in good accordance with the results in panels C, F, I and L of <FIG>.

The test powders (L-NIBC-AuCs sample and L-NIBC-AuNPs sample) were dissolved in ultrapure water till the concentration was <NUM>·L-<NUM>, and the UV-vis absorption spectra were measured at room temperature. The scanning range was <NUM>-<NUM>, the sample cell was a standard quartz cuvette with an optical path of <NUM>, and the reference cell was filled with ultrapure water.

The UV-vis absorption spectra of the four L-NIBC-AuNPs samples with different sizes are shown in panels A, D, G and J of <FIG>, and the statistical distribution of particle size is shown in panels C, F, I and L of <FIG>; the UV-vis absorption spectra of three L-NIBC-AuCs samples with different sizes are shown in panels A, D and G of <FIG>, and the statistical distribution of particle size is shown in panels C, F and I of <FIG>.

<FIG> indicates that due to the surface plasmon effect, L-NIBC-AuNPs had an absorption peak at about <NUM>. The position of the absorption peak is relevant with particle size. When the particle size is <NUM>, the UV absorption peak appears at <NUM>; when the particle size is <NUM>, the UV absorption peak appears at <NUM>; when the particle size is <NUM>, the UV absorption peak appears at <NUM>, and when the particle size is <NUM>, the absorption peak appears at <NUM>. None of the four samples has any absorption peak above <NUM>.

<FIG> indicates that in the UV absorption spectra of three L-NIBC-AuCs samples with different particle sizes, the surface plasmon effect absorption peak at <NUM> disappeared, and two obvious absorption peaks appeared above <NUM> and the positions of the absorption peaks varied slightly with the particle sizes of AuCs. This is because AuCs exhibit molecule-like properties due to the collapse of the face-centered cubic structure, which leads to the discontinuity of the density of states of AuCs, the energy level splitting, the disappearance of plasmon resonance effect and the appearance of a new absorption peak in the long-wave direction. It could be concluded that the three powder samples in different particle sizes obtained above are all ligand-bonded AuCs.

Infrared spectra were measured on a VERTEX80V Fourier transform infrared spectrometer manufactured by Bruker in a solid powder high vacuum total reflection mode. The scanning range is <NUM>-<NUM>-<NUM> and the number of scans is <NUM>. Taking L-NIBC-AuCs samples for example, the test samples were L-NIBC-AuCs dry powder with three different particle sizes and the control sample was pure L-NIBC powder. The results are shown in <FIG>.

<FIG> shows the infrared spectrum of L-NIBC-AuCs with different particle sizes. Compared with pure L-NIBC (the curve at the bottom), the S-H stretching vibrations of L-NIBC-AuCs with different particle sizes all disappeared completely at <NUM>-<NUM>-<NUM>, while other characteristic peaks of L-NIBC were still observed, proving that L-NIBC molecules were successfully bonded to the surface of AuCs via Au-S bond. The figure also shows that the infrared spectrum of the ligand-bonded AuCs is irrelevant with its size.

AuCs bonded with other ligands were prepared by a method similar to the above method, except that the solvent of solution B, the feed ratio between HAuCl<NUM> and ligand, the reaction time and the amount of NaBH<NUM> added were slightly adjusted. For example: when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC) or N-isobutyryl-D-cysteine (D-NIBC) is used as the ligand, acetic acid is selected as the solvent; when dipeptide CR, dipeptide RC or <NUM>-[(<NUM>)-<NUM>-methyl-<NUM>-thiol-<NUM>-oxopropyl]-L-proline is used as the ligand, water is selected as the solvent, and so on and so forth; other steps are similar, so no further details are provided herein.

The present disclosure prepared and obtained a series of ligand-bonded AuCs by the foregoing method. The ligands and the parameters of the preparation process are shown in Table <NUM>.

The samples listed in Table <NUM> are confirmed by the foregoing methods. The characteristics of five different ligand-bonded AuCs are shown in <FIG> (CR-AuCs), in <FIG> (RC-AuCs), in <FIG> (Cap-AuCs) (Cap denotes <NUM>-[(<NUM>)-<NUM>-methyl-<NUM>-thiol-<NUM>-oxopropyl]-L-proline), in <FIG> (GSH-AuCs), and in <FIG> (D-NIBC-AuCs). <FIG> show UV spectra (panel A), infrared spectra (panel B), TEM images (panel C), and particle size distribution (panel D).

The results indicate that the diameters of AuCs bonded with different ligands obtained from Table <NUM> are all smaller than <NUM>. Ultraviolet spectra also show disappearance of peak at <NUM>±<NUM>, and appearance of absorption peak in other positions. The position of the absorption peak could vary with ligands and particle sizes as well as structures. In certain situations, there is no special absorption peak, mainly due to the formation of AuCs mixtures with different particles sizes and structures or certain special AuCs that moves the position of absorption peak beyond the range of UV-vis spectrum. Meanwhile, Fourier transform infrared spectra also show the disappearance of ligand thiol infrared absorption peak (between the dotted lines in panel B of <FIG>), while other infrared characteristic peaks are all retained, suggesting that all ligand molecules have been successfully bonded to gold atoms to form ligand-bonded AuCs, and the present disclosure has successfully obtained AuCs bonded with the ligands listed in Table <NUM>.

L-Cys-AuCs, the diameter of gold cores in the range of <NUM>-<NUM> (<NUM> ± <NUM>), were used as the testing sample; the preparation method followed the above described method with slight modification. <FIG> shows UV, infrared, TEM, and particle size distribution diagrams of ligand L-cysteine-bonded gold clusters (L-Cys-AuCs).

Prednisone (Shanghai Yuanye Biotechnology Co.

Prednisone was weighed required amount of prednisone and added proper volume of saline, gently vortexed the formulation to ensure proper mixing. L-Cys-AuCs was weighed, added with proper volume of saline, and vortexed to fully mix the suspension. All compounds were fresh prepared daily.

C57BL/6N female mice, <NUM>-<NUM> weeks old, were used. Animals were housed and maintained in compliance with the regulations.

On Day <NUM>, mice were grouped randomly by body weight, and then injected subcutaneously with <NUM>µg of myelin oligodendrocyte glycoprotein (MOG) peptide in complete Freund's adjuvant (CFA) into the right and left flank, <NUM>µl per site. Pertussis toxin (PTX) injected by i. p at <NUM> and <NUM> hours after MOG immunization. Compounds were given from day <NUM> to day <NUM> according to Table <NUM>. The animals (n=<NUM>) were scored daily for clinical signs of EAE. At the end of the experiments, spinal cords were used for HE staining (n=<NUM>), and analysis of TNF-a, IL-<NUM>, IFN-γ by Elisa kit.

The animals were scored daily for clinical signs of EAE according to the Clinical scoring rubric for EAE (Table <NUM>). As shown in <FIG>, L-Cys-AuCs at <NUM>/kg (50mpK), <NUM>/kg (20mpK) and <NUM>/kg (SmpK) prevented the paralysis of EAE mice from day 17th; prednisone is a positive control, it significantly reduced clinical signs of EAE from day 13th.

TNF-α, IL-<NUM> and IFN-γ analysis for spinal cord by ELISA kit. TNF-α, IL-<NUM> and IFN-γ are indicators for inflammation. L-Cys-AuCs at <NUM>/kg, <NUM>/kg and <NUM>/kg significantly reduced the production of these inflammatory factors in EAE mice (<FIG>).

HE staining for spinal cord was used to study the infiltration of immune cells into the spinal cord. During induction of EAE, mononuclear inflammatory cells infiltrate the spinal cord, contributing to the paralysis of EAE mice, HE staining reveal a significant increase in inflammatory cells in EAE mice, and L-Cys-AuCs at <NUM>/kg, <NUM>/kg and <NUM>/kg reduced immune cell infiltration (<FIG>) and showed significantly lower histological scales of inflammation (<FIG>).

Other sized L-Cys-AuCs and other ligand-bonded AuCs with different sizes also have the effects on inhibiting EAE, while their effects vary to certain extents. They would not be described in detail here.

Claim 1:
A gold cluster (AuC) for use in the treatment of multiple sclerosis, wherein said AuC comprises:
a gold core; and
a ligand bonded to the gold core;
wherein, the ligand is one selected from the group consisting of L-cysteine and its derivatives, D-cysteine and its derivatives, and cysteine-containing oligopeptides and their derivatives;
wherein the L-cysteine and its derivatives are selected from the group consisting of L-cysteine, N-isobutyryl-L-cysteine (L-NIBC), and N-acetyl-L-cysteine (L-NAC), and wherein the D-cysteine and its derivatives are selected from the group consisting of D-cysteine, N-isobutyryl-D-cysteine (D-NIBC), and N-acetyl-D-cysteine (D-NAC);
wherein the cysteine-containing oligopeptides and their derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides or cysteine-containing tetrapeptides;
wherein the cysteine-containing dipeptides are selected from the group consisting of L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-histidine-L-cysteine dipeptide (HC), and L-cysteine-L-histidine dipeptide (CH);
wherein the cysteine-containing tripeptides are selected from the group consisting of glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-lysine-L-cysteine-L-proline tripeptide (KCP), and L-glutathione (GSH);
wherein the cysteine-containing tetrapeptides are selected from the group consisting of glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR), and glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).