Method for treating and / or preventing lymphangiogenesis-related diseases using gold nanoclusters

Abstract

To provide a method for treating and / or preventing lymphangiogenesis-related diseases in a subject.SOLUTION: The present invention provides a method that includes the step for administering to the subject an effective amount of gold nanoclusters coated with dihydrolipoic acid (DHLA), wherein the gold nanoclusters have a particle size ranging from 1 to 10 nm. The DHLA-coated gold nanoclusters are each composed of a gold nanocluster formed from a plurality of gold nanoparticles and a plurality of DHLA molecules covering the gold nanocluster.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present disclosure relates generally to the field of treating lymphangiogenesis-associated diseases and promoting lymphangiogenesis in a subject. More specifically, the present disclosure relates to a method of treating lymphangiogenesis-associated diseases and promoting lymphangiogenesis by using gold nanoclusters coated with dihydrolipoic acid (DHLA). [Background technology]

[0002] Lymphangiogenesis-associated diseases refer to a group of disorders characterized by abnormal lymphatic vessel growth that leads to poor lymphatic drainage and subsequent tissue dysfunction. The pathogenesis of these diseases involves dysregulation of factors involved in lymphatic vessel development and remodeling, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoietin. These factors promote lymphatic endothelial cell proliferation, migration and tube formation. Dysfunctional lymphangiogenesis can result from genetic mutations, inflammation, tumor growth or chronic conditions such as lymphedema.

[0003] Epidemiologically, lymphangiogenesis-associated diseases vary according to the specific condition. For example, lymphedema affects approximately 140-250 million people worldwide, with primary lymphedema often occurring due to genetic abnormalities, and secondary lymphedema resulting from surgery, radiation, or infection. Other conditions such as lymphangioma, lymphangioleiomyomatosis (LAM), and lymphatic malformations have different prevalence and clinical presentations. Treatment approaches currently used for lymphangiogenesis-associated diseases are primarily aimed at managing symptoms and improving quality of life. Conservative management options may include compression therapy, physical therapy, and skin care measures to reduce tissue swelling and prevent complications in conditions such as lymphedema. Surgical interventions such as lymphovenous anastomosis or lymph node transfer may be performed in some selected cases. In some cases, drug therapy targeting lymphangiogenesis-associated factors is utilized. For example, the use of VEGF-C / VEGF-D or certain diuretics has shown promise in suppressing lymphatic vessel growth in lymphedema. However, these therapies are not designed to specifically modulate lymphatic vessel growth and function. Current treatments focus on managing symptoms and improving quality of life rather than directly targeting the underlying pathogenic mechanisms. Furthermore, the heterogeneity and complexity of lymphangiogenesis-related diseases pose challenges in developing universal treatment strategies.

[0004] In view of the above, there is a need in the related art for improved methods of specifically and effectively treating lymphangiogenesis-associated diseases, thereby effectively addressing the needs of patients with lymphangiogenesis-associated diseases. Summary of the Invention

[0005] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an exhaustive overview of the disclosure, nor does it identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that follows.

[0006] As realized and broadly described herein, one aspect of the disclosure is directed to a method of treating and / or preventing lymphangiogenesis-associated disease in a subject. The method comprises administering to the subject an effective amount of gold nanoclusters coated with dihydrolipoic acid (DHLA) having a particle size in the range of 1-10 nm, preferably in the range of 1-5 nm, and more preferably 2 nm. The DHLA-coated gold nanoclusters comprise a gold nanocluster formed by a plurality of gold nanoparticles and a plurality of DHLA coated on the gold nanoclusters.

[0007] According to embodiments of the present disclosure, an effective amount of DHLA coated gold nanoclusters is about 0.001-10 mg / kg body weight per day, preferably about 0.01-1 mg / kg body weight per day. According to one particular embodiment, an effective amount of DHLA coated gold nanoclusters is about 0.057 mg / kg body weight per day.

[0008] According to an embodiment of the present disclosure, the lymphangiogenesis-related diseases described herein are lymphedema and systolic heart failure. Thus, in one advanced embodiment, the method may further comprise administering an additional pharmaceutical agent in combination with the DHLA-coated gold nanoclusters to treat the above diseases. The additional pharmaceutical agent may be administered to the subject prior to, in conjunction with, or after administration of the DHLA-coated gold nanoclusters.

[0009] For example, if the lymphangiogenesis-related disorder is lymphedema, the additional medications may be VEGF-C, VEGF-D, acetazolamide, furosemide, azosemide, bumetanide, ethacrynic acid, etozolin, indacrinone, muzolimine, ozolinone, piretanide, thienilic acid, torasemide, altizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, epithizide, hydrochlorothiazide, hydroflumethiazide, mebutizide, and methyclothiazide. The at least one agent may be selected from the group consisting of thiazides, polythiazides, trichlormethiazide, chlorthalidone, clofenamide, clopamide, chlorexolone, fenquizone indapamide, mefruside, meticrane, metolazone, quinethazone, xipamide, amiloride, benzamyl, triamterene, spironolactone, canrenone, eplerenone, potassium canrenoate, finerenone, conivaptan, mozavaptan, satavaptan, and tolvaptan.

[0010] Alternatively, when the lymphangiogenesis-associated disease is systolic heart failure, the additional pharmaceutical agent can be any one agent selected from the group consisting of carvedilol, metoprolol, bisoprolol, losartan, valsartan, candesartan, sacubitril, lisinopril, enalapril, ramipril, spironolactone, eplerenone, digoxin, hydralazine, isosorbide dinitrate, ivabradine, atorvastatin, simvastatin, aspirin, clopidogrel, warfarin, apixaban, rivaroxaban, metolazone, nebivolol, and milrinone.

[0011] Preferably, the subject is a human.

[0012] Another aspect of the present disclosure relates to a method of promoting lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a DHLA-coated gold nanocluster, the DHLA-coated gold nanocluster consisting of a gold nanocluster formed by a plurality of gold nanoparticles and a plurality of DHLA coated on the gold nanocluster.

[0013] According to certain embodiments of the present disclosure, the DHLA coated gold nanoclusters are about 1-10 nm in diameter, preferably about 1-5 nm in diameter, and more preferably about 2 nm in diameter.

[0014] According to embodiments of the present disclosure, an effective amount of DHLA coated gold nanoclusters is about 0.001-10 mg / kg body weight per day, preferably about 0.01-1 mg / kg body weight per day. According to one particular embodiment, an effective amount of DHLA coated gold nanoclusters is about 0.057 mg / kg body weight per day.

[0015] Preferably, the subject is a human.

[0016] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0017] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings. [Brief description of the drawings]

[0018] [Figure 1A] FIG. 1A shows the effect of DHLA-coated gold nanoclusters (shown as fluorescent gold nanoclusters (FANC) in the figure) on cell proliferation in human lymphatic endothelial cells (LEC). Data are presented as the mean ± SEM of at least five independent experiments. *P<0.05 compared to the control group. [Figure 1B] FIG. 1B shows the effect of DHLA-coated gold nanoclusters (shown as fluorescent gold nanoclusters (FANC) in the figure) on migration (i.e., wound healing) in human lymphatic endothelial cells (LEC). Data are presented as the mean ± SEM of at least five independent experiments. *P<0.05 compared to the control group. [Diagram 2]Figure 2 shows the effect of FANC on tube formation in human LEC. Data are presented as the mean ± SEM of at least five independent experiments. *P<0.05 compared with the control group. [Diagram 3] Figure 3 shows the expression of lymphatic endothelium-specific marker, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), in mice treated with isoproterenol (ISO) and / or FANC. Data are expressed as mean ± SEM. N=4 for control, N=3 for ISO, N=6 for FANC+ISO. *P<0.05 compared to ISO group. [Figure 4A] FIG. 4A shows echocardiographic results in mice treated with ISO, FANC and / or MAZ-51, where MAZ-51 is (3E)-3-[[4-(dimethylamino)naphthalen-1-yl]methylidene]-1H-inden-2-one. FIG. 4B shows fractional shortening (FS) results. Data are expressed as mean±SEM. N=3 for control, N=3 for ISO, N=3 for FANC+ISO, N=4 for FANC+ISO+MAZ-51. *P<0.05 compared to control group. †P<0.05 compared to ISO group. ‡P<0.05 compared to ISO+FANC group. [Figure 4B] FIG. 4B shows echocardiographic results in mice treated with ISO, FANC and / or MAZ-51, where MAZ-51 is (3E)-3-[[4-(dimethylamino)naphthalen-1-yl]methylidene]-1H-inden-2-one. FIG. 4B shows tissue Doppler (TDI-s´) results. Data are expressed as mean ± SEM. N=3 for control, N=3 for ISO, N=3 for FANC+ISO, N=4 for FANC+ISO+MAZ-51. *P<0.05 compared to control group. †P<0.05 compared to ISO group. ‡P<0.05 compared to ISO+FANC group. [Diagram 5]Figure 5 shows in vivo lymphangiogenesis in zebrafish treated with FANC, showing the percentage of embryos with 7 or more somites containing intersomitic lymphatic vessels (ISLVs) in somites 5-12. Each experiment was obtained from 20 juvenile fish. Data are expressed as the mean ± SEM from at least three independent experiments. *P<0.05 compared to the control group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The detailed description provided below in conjunction with the accompanying drawings is intended as a description of the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or utilized. The description sets forth the functions of the present embodiment and the sequence of steps for constructing and operating the embodiment, although the same or equivalent functions and sequences may be accomplished by different embodiments.

[0020] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are summarized here. Unless otherwise specified herein, scientific and technical terms employed in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art. Furthermore, singular terms shall include their plurals and plural terms shall include the singular, unless otherwise required by context. Specifically, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Furthermore, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more. The practice of the present invention employs, unless otherwise indicated, conventional techniques of biochemistry, general biology, animal testing, and the like, which are within the skill of those of ordinary skill in the art. Such techniques are fully described in the literature.

[0021] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within the acceptable standard error of the mean as considered by one of ordinary skill in the art. All numerical ranges, amounts, values ​​and percentages, such as those for amounts of materials, durations, temperatures, operating conditions, ratios of amounts, and the like, disclosed herein, other than in the operating / examples, or unless expressly stated otherwise, should be understood in each instance as being modified by the term "about." Thus, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0022] The terms "treatment" and "treating" as used herein may refer to interventions intended to prevent the progression of lymphangiogenesis-related disorders or to alter their pathology. The concept of treatment or treating is used in the broadest sense and specifically includes prevention (prophylaxis), reduction, reduction, cure and palliation of lymphangiogenesis-related disorders at any stage. Thus, "treatment" refers to both therapeutic treatment and preventative or prophylactic measures, or also to palliative measures, the goal being to prevent, slow down (alleviate) or improve lymphangiogenesis-related disorders such as lymphedema and systolic heart failure. Subjects in need of treatment include those already suffering from the disorder, as well as those susceptible to the disorder or those in whom the disorder is to be prevented. The disorder may result from any cause, including idiopathic or psychogenic causes, or those secondary to these causes or other conditions such as surgery, radiation or infection.

[0023] The term "subject" or "patient" refers to an animal, including the human species, treatable by the methods of the present disclosure. The term "subject" or "patient" is intended to refer to both genders, male and female, unless one gender is specifically indicated. Thus, the term "subject" or "patient" includes any mammal that may benefit from administration of DHLA-coated gold nanoclusters. Examples of "subject" or "patient" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. According to a preferred embodiment, the subject is a human.

[0024] The terms "administered," "administering," or "administration" are used interchangeably herein to refer to either administering the present DHLA-coated gold nanoclusters directly or administering a pharmaceutical composition containing the DHLA-coated gold nanoclusters.

[0025] As used herein, the term "effective amount" refers to an amount of gold nanoclusters effective at the dosage and for the period of time necessary to achieve the desired result in terms of treating lymphangiogenesis-related diseases or promoting lymphangiogenesis in a subject. For therapeutic purposes, an effective amount is also one in which any toxic or detrimental effects of the components are outweighed by the therapeutically beneficial effects. The specific effective or sufficient amount will vary depending on factors such as the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of the concurrent therapy (if any), and the specific formulation and structure of the compound or its derivative employed. An effective amount may be expressed, for example, in grams, milligrams, or micrograms, or as milligrams per kilogram of body weight (mg / kg). Alternatively, an effective amount may be expressed in terms of the concentration of the active component (e.g., the present DHLA-coated gold nanoclusters), such as molar concentration, mass concentration, volume concentration, molar concentration, mole fraction, mass fraction, and mixture ratio. One skilled in the art would be able to calculate the human equivalent dose (HED) for a pharmaceutical (such as the present DHLA-coated gold nanoclusters) based on the dose determined from an animal model. For example, when estimating the maximum safe dose for use in human subjects, industry guidance issued by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" can be followed.

[0026] As used herein, the term "lymphangiogenesis-associated disorder" refers to a disease, condition, or disorder resulting from or exacerbated by impaired lymphangiogenesis in a subject; a lymphangiogenesis-associated disorder can be, for example, lymphedema or systolic heart failure.

[0027] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is to be administered. Generally, the amount of active compound (i.e., the present DHLA-coated gold nanoclusters) is present in the pharmaceutical composition at a level of about 0.01% to 99% by weight, preferably at least 0.1% by weight, more preferably at least 1% by weight, even more preferably at least 5% by weight, even more preferably at least 10% by weight, and even more preferably at least 25% by weight, based on the total weight of the pharmaceutical composition. For clinical use of the present invention, the pharmaceutical composition is formulated to be a formulation suitable for the intended route of administration.

[0028] The term "pharmaceutical acceptable excipient" as used herein means a pharmaceutical acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting a subject drug from one organ or part of the body to another. Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The pharmaceutical formulation contains the present DHLA coated gold nanoclusters in combination with one or more pharmaceutical acceptable ingredients. The excipient may be a solid, semi-solid or liquid diluent, cream or capsule. These pharmaceutical preparations are further subjects of the present invention.

[0029] II. Description of the Invention The present disclosure is based, at least in part, on the discovery that DHLA-coated gold nanoclusters enhance characteristics of lymphangiogenesis both in vitro (e.g., increasing cell proliferation, migration and tube formation) and in vivo (e.g., improving cardiac function in cardiac-injured mice and promoting lymphangiogenesis in zebrafish), suggesting that DHLA-coated gold nanoclusters are promising candidates for the development of pharmaceuticals for the treatment and / or prevention of lymphangiogenesis-associated diseases.

[0030] The present DHLA-coated gold nanoclusters are structurally composed of gold nanoclusters formed by a plurality of gold nanoparticles and a plurality of DHLA coated on the gold nanoclusters. The skilled artisan will be familiar with the DHLA-coated gold nanoclusters utilized in the present disclosure and their method of production (Lin et al., ACS Nano 2009, 3(2), pp395-401), so further elaboration of the details of their preparation is unnecessary at this point. The DHLA-coated gold nanoclusters have a fluorescence emission at 650 nm under an excitation wavelength of about 420 nm, so that the emitted fluorescence is in the red to near infrared spectrum range. Each of the DHLA-coated gold nanoclusters has a particle size in the range of 0.1-20 nm, preferably in the range of 1-10 nm, more preferably in the range of 1-5 nm, and even more preferably in the range of 2 nm. The above dimensions for the gold nanoclusters of the present disclosure are in the dry state, but it is advantageous if the gold nanoclusters used in the present disclosure are water-soluble or at least dispersible in aqueous media and / or water. The hydrodynamic size of the gold nanoclusters of the present disclosure can be significantly larger than that in the dry state due to the binding of surrounding solvent molecules such as water. In one specific example embodiment, the gold nanoclusters have a hydrodynamic size equivalent to a 1-30 kDa polyethylene glycol (PEG) molecule (Lin et al., ACS Nano 2009, 3(2), pp395-401).

[0031] According to one aspect of the present disclosure, the gold nanoclusters coated with DHLA are used to prepare a composition for treating and / or preventing lymphangiogenesis-related diseases or promoting lymphangiogenesis in a subject. The composition may be utilized in the form of a pharmaceutical composition or medicine or a health functional food (including nutritional supplements) depending on the purpose. The composition comprises an effective amount of the gold nanoclusters coated with DHLA together with an optional pharma- ceutically acceptable excipient.

[0032] Methods for preparing pharmaceutical compositions are well known in the art of pharmacology.For application of the present invention, the DHLA-coated gold nanoclusters of the present disclosure can be manufactured into desired formulations, such as tablets, sugar-coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions, solutions, ointments, creams or gels or any, by using pharma-ceutical suitable excipients, particularly inert and essentially non-toxic.

[0033] According to some embodiments of the present disclosure, the composition is used as a preparation, in which case the pharmaceutical composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.The pharmaceutical composition may be formulated and used in the form of oral administration such as powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injections by conventional methods.The carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.For formulation, it is prepared using excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate or talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as simple diluents, various excipients such as wetting agents, sweeteners, flavorings, and preservatives may be included in the preparations. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, food oils such as olive oil, and injectable esters such as ethyl oleate. Witepsol, macrogol, Tween-61, cocoa butter, lauric butter, and glycerogelatin may be used as bases for suppositories.

[0034] According to an embodiment of the present disclosure, the composition is used as a health functional food, which can be any type of liquid and solid / semi-solid material used to provide nutrition to humans and animals, to benefit the treatment of lymphangiogenesis-related diseases, and / or to improve or alleviate symptoms associated with lymphangiogenesis-related diseases. Ingredients commonly used in health functional foods may be included in addition to the active ingredient, and when the composition is manufactured as a health functional drink, for example, citric acid, oligosaccharides, taurine, concentrated fruit juice, etc. The health functional food can be a food (e.g., tea-based drinks, juices, soft drinks, coffee, milk, jellies, cookies, cereals, chocolates, snack bars, herbal extracts, dairy products (e.g., ice cream and yogurt)), a food / nutraceutical, or a nutraceutical formulation.

[0035] The DHLA-coated gold nanoclusters provided herein are usually formulated in dosage unit form for ease of administration and uniformity of dosage. However, it should be understood that the total daily use of the compositions described herein will be determined by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organ will depend on a variety of factors, including the severity of the disease and disorder being treated; the activity of the specific active ingredient employed; the specific composition employed; the subject's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific active ingredient employed; the duration of treatment; drugs used in combination with or simultaneously with the specific active ingredient employed; and similar factors well known in the medical arts of that kind.

[0036] In another aspect of the present disclosure, the present disclosure provides a method of treating a lymphangiogenesis-associated disease and promoting lymphangiogenesis in a subject, the method comprising administering to the subject an effective amount of DHLA-coated gold nanoclusters or a composition comprising same as described herein.

[0037] In certain embodiments, the DHLA-coated gold nanoclusters described herein are provided in an effective amount of a pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., an amount effective to treat lymphangiogenesis-associated disease). In certain embodiments, the effective amount is a prophylactically effective amount (e.g., an amount effective to prevent lymphangiogenesis-associated disease in a subject in need thereof).

[0038] The exact amount of DHLA-coated gold nanoclusters required to achieve an effective dose will vary between subjects, depending, for example, on the species, age and general condition of the subject, the severity of side effects or disorders, the mode of administration, etc. An effective dose can be contained in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject, any two of the multiple doses contain different or substantially the same amount of DHLA-coated gold nanoclusters as described herein. In certain embodiments, when multiple doses are administered to a subject, the frequency with which the multiple doses are administered to the subject is three doses per day, two doses per day, one dose per day, one dose per every other day, one dose per three days, one dose per week, one dose per every other week, one dose per month, or one dose per every other month. In certain embodiments, the frequency with which the multiple doses are administered to a subject is one dose per day. In certain embodiments, the frequency of administration of multiple doses to the subject is two doses per day. According to certain examples of the present disclosure, the subject is a mouse. To elicit a therapeutic effect on the mouse, the present DHLA-coated gold nanoclusters are administered to the subject in an amount of about 0.01-150 mg / kg body weight per day, preferably about 0.1-15 mg / kg body weight per day, more preferably about 0.1-1.5 mg / kg body weight per day. According to one embodiment, the present DHLA-coated gold nanoclusters are administered to the subject in an amount of 0.57 mg / kg body weight per day.

[0039] One of skill in the art can readily determine the human equivalent dose (HED) of the present DHLA-coated gold nanoclusters based on the doses determined from the animal studies provided in the Examples of this application. Thus, amounts of the present DHLA-coated gold nanoclusters suitable for use in human subjects range from 0.001 to 10 mg / kg of body weight per day, e.g., 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.0 2, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051, 0.052, 0 .053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.06, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.07, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.08, 0.081, 0.082, 0.083, 0.084, 0.0 The amount of DHLA coated gold nanoclusters for treating a human subject may be about 0.01-1 mg / kg of body weight per day. More preferably, the amount of the present DHLA coated gold nanoclusters for treating a human subject is about 0.01-0.1 mg / kg of body weight per day.According to one embodiment, the amount of the present DHLA coated gold nanoclusters for treating a human subject is about 0.046 mg / kg of body weight per day. According to one embodiment, the amount of the present DHLA coated gold nanoclusters for treating a human subject is about 0.057 mg / kg of body weight per day.

[0040] The dosage ranges as described herein provide guidance for administration of the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a physician or person skilled in the art and may be less than or the same as the amount administered to an adult.

[0041] The DHLA-coated gold nanoclusters and compositions comprising same provided herein can be administered by any suitable route, such as enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, interdermal, subcutaneous, intradermal, rectal, intracavity, intraperitoneal, topical (such as by powder, ointment, cream and / or infusion). Specifically, possible routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via the blood and / or lymphatic supply, and / or direct administration to the affected area. In general, the optimal route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the intestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0042] As will be appreciated, the method can be applied to a subject alone or in combination with an additional therapy that has some beneficial effect on the treatment of lymphangiogenesis-related disease. Depending on the intended purpose, the method can be applied to the subject before, during, or after the additional therapy. The specific type of additional therapeutic agent will depend on the particular condition being treated (i.e., lymphedema or systolic heart failure).

[0043] If the lymphangiogenesis-related disorder is lymphedema, the additional medications include VEGF-C, VEGF-D, acetazolamide, furosemide, azosemide, bumetanide, ethacrynic acid, etozolin, indacrinone, muzolimine, ozolinone, piretanide, thienilic acid, torasemide, altizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, epitizide, hydrochlorothiazide, hydroflumethiazide, mebutizide, The agent may be any one of the following: methyclothiazide, polythiazide, trichlormethiazide, chlorthalidone, clofenamide, clopamide, chlorexolone, fenquizone indapamide, mefruside, meticrane, metolazone, quinethazone, xipamide, amiloride, benzamyl, triamterene, spironolactone, canrenone, eplerenone, potassium canrenoate, finerenone, conivaptan, mozavaptan, satavaptan, or tolvaptan.

[0044] When the lymphangiogenesis-associated disorder is systolic heart failure, the additional pharmaceutical agent can be at least one of the following agents: carvedilol, metoprolol, bisoprolol, losartan, valsartan, candesartan, sacubitril, lisinopril, enalapril, ramipril, spironolactone, eplerenone, digoxin, hydralazine, isosorbide dinitrate, ivabradine, atorvastatin, simvastatin, aspirin, clopidogrel, warfarin, apixaban, rivaroxaban, metolazone, nebivolol, or milrinone.

[0045] In certain embodiments, the subject is a mammal, preferably a human.

[0046] The following examples are provided to illustrate certain aspects of the present invention and to assist those skilled in the art in practicing the invention. These examples are not to be construed as limiting the scope of the present invention in any manner. Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. EXAMPLES

[0047] 1. Preparation of DHLA-coated gold nanoclusters (FANCs) The fluorescent DHLA-coated gold nanoclusters used in this study were prepared based on precursor-induced gold (Au) nanoparticle (NP) etching (Lin et al., ACS Nano 2009, 3(2), pp395-401). Briefly, 6 nm gold nanoparticles stabilized by didodecyldimethylammonium bromide (DDAB) were first synthesized in toluene by adding 0.8 ml of gold precursor (7.5 mg / ml AuCl3 prepared in 100 mmol / L DDAB solution) to a reducing agent containing 1 ml of freshly prepared tetrabutylammonium borohydride (TBAB) (100 mmol / L in DDAB solution) and 0.675 ml of decanoic acid (100 mmol / L in toluene). Additional gold precursor was added dropwise until the color changed to a transparent yellow, resulting in non-plasmonic gold nanoclusters, which could be further delivered to the aqueous phase by ligand exchange with reduced α-lipoic acid. α-Lipoic acid (0.206 g, Sigma-Aldrich, St. Louis, MO, USA) was reduced to dihydrolipoic acid (DHLA) by adding 5 ml of TBAB (50 mmol / L) in DDAB solution. DHLA-coated gold nanoclusters were formed by mixing an equal amount of gold precursor into DHLA and additional UV light (302 nm) annealing for 30 min, and stable fluorescent signals were obtained in live cells. For simplicity, DHLA-coated gold nanoclusters are abbreviated as "FANC". After removing the supernatant by centrifugation, FANC were further purified by a subsequent methanol / chloroform washing step, reprocessed in borate buffer (pH 9) for overnight incubation at 55 °C, and collected and buffer exchanged by 30-100 kDa centrifugal filters (EMD Millipore).

[0048] 2.Cell culture Human lymphatic endothelial cells (LECs) were obtained from a commercial source (Lonza; Walkersville, MD). LECs were grown in endothelial cell growth medium (EGM-2MV medium) containing EBM-2 basal medium plus endothelial cell growth supplement (Lonza). Cells were seeded on plasticware coated with 1% gelatin and cultured at 37°C with 5% CO2 for subsequent processing.

[0049] 3. Cell Proliferation Assay 5 × 10 LECs per well in a 96-well plate 3 Cells were seeded at a density of 100x100 / ml. After 24 h of incubation, the culture medium was removed and the cells were treated with EGM-2MV medium with or without FANC for another 24 h. The reaction was then terminated by fixing the cells with 50% trichloroacetic acid (TCA) and treating with 0.4% sulforhodamine B (SRB) (Sigma; St. Louis, MO, USA) in 1% acetic acid. After 15 min of incubation, the plate containing the cells was washed and treated with 10 mM Tris buffer. The absorbance of the plate at 515 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0050] 4. Cell Migration Assay LECs were seeded in 6-well plates and grown until they reached 90-100% confluence. Wounds were generated in the cells by scratching a gap using a 1 mm size scratcher tip (SPL Life Sciences, Pocheon, Korea), and the cells were washed twice with Hank's Balanced Salt Solution (HBSS). Cells were incubated in fresh growth medium with or without FANC at the indicated concentrations. Images were taken by microscope at 0, 16, and 24 h after treatment, followed by analysis. Migration areas were calculated using ImageJ with MRI Wound Healing Tool.

[0051] 5. Capillary Tube Formation Assay Matrigel (BD Biosciences; Bedford, MA) was dissolved overnight at 4° C. and 96-well plates were coated with 50 μl of Matrigel per well, followed by incubation for 30 min at 37° C. LECs were cultured at 2 × 10 per well in EGM-2MV medium at the indicated FANC concentrations. 4 Cells were seeded at a density of 100 / 200 μl. After 24 h of incubation at 37° C., tube formation of LECs was assessed by microscopy and each well was photographed. The number of tube branches and total tube length were calculated using MacBiophotonics ImageJ software.

[0052] 6. Isoproterenol (ISO)-induced heart failure model Male 7-week-old C57BL / 6 mice were purchased from BioLasco Inc. (Yilan, Taiwan). On day 0, the mice were treated with FANC (20 μM; approximately 0.57 mg / kg body weight) by abdominal implantation using an osmotic pump system. Then, on day 14, the mice were subcutaneously treated with ISO (150 mg / kg). 14 days after ISO injection, on day 28, cardiac ultrasound was performed to measure the cardiac function of the mice. The animals were then sacrificed and their hearts were collected for histological and pathological examination.

[0053] 7. Immunohistochemistry (IHC) staining Mouse left ventricular tissue sections were deparaffinized with xylene and rehydrated by adding ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval was performed for all sections in 0.01 M sodium citrate buffer, pH 6, at 95°C for 25 min. Antibody against mouse LYVE-1 was applied at a dilution of 1:200 and incubated overnight at 4°C. Antibody binding signals were detected using the NovoLink Polymer Detection System (Leica Microsystems) and visualized using diaminobenzidine reaction. Sections were counterstained with hematoxylin and eosin (H&E). Quantification of LYVE-1 expression was analyzed using MacBiophotonics Image-J software.

[0054] 8. Cardiac ultrasound examination Experimental echocardiography was performed on mice after they were anesthetized with isoflurane (Abbott Laboratories Ltd., England; delivered to achieve a concentration of 1–2% in a combination of oxygen and nitrous oxide mixed in a 50:50 ratio with air). All mice underwent cardiac structural and functional assessment using a 10 MHz sector array transducer connected to a Vivid i echo system (GE Vingmed Ultrasound, Horten, Norway). Mice were positioned in a left lateral decubitus position with ultrasound gel placed on the anterior chest wall. The depth of the M-mode linear scan was set to 1.0–1.5 cm at the maximum achievable frame rate. Systolic myocardial performance was assessed using ECG-gated offline analysis on an EchoPac Workstation with parameters including tissue Doppler (TDI-S´), used to identify contractile velocity, deformation-based circumferential systolic strain measurements, and a quantification index of tissue structure integrated with backscatter intensity due to dynamic myocardial motion.

[0055] 9. In vivo lymphangiogenesis model The DNA construct used to establish the transgenic zebrafish line Tg(lyve1:TRFP) was generated as follows. A 6.2 kb promoter sequence of lymphatic endothelial hyaluronan receptor 1b (lyve1b) gene was linked to red fluorescent protein (TRFP) for expression to enable marking of developing lymphatic cells in transgenic zebrafish. Accordingly, the transgenic zebrafish line Tg(lyve1:TRFP) was established using the aforementioned DNA construct. Red fluorescent signals were detected in the majority of proliferating lymphatic cells in the head and trunk regions of zebrafish embryos at 144 hours post fertilization (hpf). FANC at a concentration of 5 nM was injected into 72 hpf embryos of the zebrafish line Tg(lyve1:TRFP), and lymphatic vessel growth in the trunk region was observed at 144 hpf. Finally, we assessed in vivo lymphangiogenic function by determining the percentage of embryos with ≥7 somites that contained intersomitic lymphatic vessels (ISLVs) in somites 5–12.

[0056] 10. Data Analysis Experimental results, expressed as mean±SEM, were analyzed using one-way analysis of variance or Student's t-test. Data were considered statistically significant when P value<0.05.

[0057] Example 1 In vitro effect of FANC on lymphangiogenesis The effects of FANC on lymphangiogenesis, including promoting cell proliferation, enhancing cell migration, and promoting the formation of capillary-like structures in lymphatic endothelial cells (LECs), were investigated in this example. The results are given in Figures 1 and 2. Specifically, FANC was found to increase cell proliferation of LECs in a concentration-dependent manner (Figure 1A). With regard to the effect of FANC on the migration and tube-forming abilities that are important for endothelial cells during lymphangiogenesis, FANC was found to significantly increase the wound-healing area of ​​LECs in a concentration- and time-dependent manner (Figure 1B) and significantly induce the formation of capillary-like structures in LECs (Figure 2).

[0058] Taken together, the results collectively suggested that FANC has the ability to promote in vitro lymphangiogenesis in human LECs.

[0059] Example 2 In vivo effects of FANC on lymphangiogenesis and cardiac function in mice with isoproterenol (ISO)-induced cardiac injury The in vivo effect of FANC on lymphangiogenesis was investigated by using an isoproterenol (ISO)-induced cardiac injury mouse model, which is a standard pathogenesis of post-myocardial infarction (MI) and heart failure models. First, to verify the role of lymphangiogenesis in the therapeutic effects brought about by FANC, we performed immunohistological analysis to examine LYVE-1, a major specific marker for lymphatic vessels. FANC significantly increased the expression of LYVE-1 compared with cardiac tissue derived from the ISO-treated group, as shown in Figure 3. The results confirmed that FANC can enhance lymphangiogenesis in cardiac injury mice.

[0060] Since FANC improved lymphangiogenesis in ISO-induced cardiac injury mice, the cardiac function of these mice was further investigated. As shown in Figure 4A-4B, treatment with FANC improved the left ventricular fractional shortening (FS) and tissue Doppler imaging (TDI-s´) in ISO-treated mice. FS acts as an indicator for ventricular systolic function, while TDI-s´ acts as an indicator for myocardial contraction velocity. The results clearly demonstrated that improved cardiac function is ensured if there is improved lymphangiogenesis in cardiac injury tissue after treatment with FANC. Furthermore, the improved cardiac function by FANC was abolished by co-treatment with lymphangiogenesis inhibitor MAZ-51, further confirming that the enhanced cardiac function by FANC is due to its ability to promote lymphangiogenesis. Taken together, these results support that FANC has the ability to promote lymphangiogenesis in the heart and improves cardiac function in cardiac injury mice as well.

[0061] Example 3 In vivo effects of FANC on lymphangiogenesis in zebrafish In this example, the in vivo effect of FANC on lymphangiogenesis was investigated in zebrafish. For this purpose, a genetically modified zebrafish line, Tg(lyve1:TRFP), was generated. In particular, a 6.2 kb promoter sequence of the lymphatic endothelial hyaluronan receptor 1b (lyve1b) gene was used to express red fluorescent protein (TRFP) to allow visualization of developing lymphatic cells when observing the head and trunk regions of zebrafish embryos at 144 hours post fertilization (hpf). FANC was administered intraperitoneally to Tg(lyve1:TRFP) zebrafish embryos at 72 hpf, and lymphatic vessel growth in the trunk region was observed at 144 hpf. The percentage of embryos with lymphatic vessel (ISLV) growth in 7 or more of the 8 somites between the 5th and 12th somites was calculated. The untreated control group showed a percentage of 8.3±2.4%, whereas the FANC-treated group showed a percentage of 45.0±10.8% (Figure 5). Based on these findings, the promotion of lymphatic vessel growth by FANC was again demonstrated in a zebrafish model.

[0062] In conclusion, the data in the Examples demonstrate that DHLA-coated gold nanoclusters can promote lymphangiogenesis in vitro and in vivo. Thus, administration of DHLA-coated gold nanoclusters to individuals in need may potentially ameliorate symptoms resulting from abnormal lymphangiogenesis, such as lymphedema and systolic heart failure. The present method comprising administering DHLA-coated gold nanoclusters and / or pharmaceutical compositions comprising same may provide a promising means of effectively treating or preventing subjects from suffering from lymphangiogenesis-related diseases.

[0063] It should be understood that the above description of the embodiments is given by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of the exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, one skilled in the art could make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. 1. A method of treating and / or preventing lymphangiogenesis-associated diseases in a subject, comprising administering to the subject an effective amount of dihydrolipoic acid (DHLA) coated gold nanoclusters having a particle size in the range of 1-10 nm, the DHLA coated gold nanoclusters consisting of gold nanoclusters formed by a plurality of gold nanoparticles and a plurality of DHLA coated on the gold nanoclusters.

2. The method of claim 1, wherein the DHLA-coated gold nanoclusters have a particle size in the range of 1-5 nm.

3. The method of claim 2 , wherein the DHLA-coated gold nanoclusters have a particle size of 2 nm.

4. The method of claim 1, wherein the gold nanoclusters are administered in an amount of about 0.001 to 10 mg / kg of body weight per day.

5. The method of claim 4, wherein the gold nanoclusters are administered in an amount of about 0.01 to 1 mg / kg of body weight per day.

6. 2. The method of claim 1, wherein the lymphangiogenesis-associated disease is lymphedema and systolic heart failure.

7. When the lymphangiogenesis-related disease is lymphedema, VEGF-C, VEGF-D, acetazolamide, furosemide, azosemide, bumetanide, ethacrynic acid, etozolin, indacrinone, muzolimine, ozolinone, piretanide, thienilic acid, torasemide, altizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, epitizide, hydrochlorothiazide, hydroflumethiazide, mebutizide, methyclothiazide, polythiazide, trichlormethiazide, 7. The method of claim 6, further comprising administering to the subject at least one agent selected from the group consisting of thiazides, chlorthalidone, clofenamide, clopamide, chlorexolone, fenquizone indapamide, mefruside, meticrane, metolazone, quinethazone, xipamide, amiloride, benzamyl, triamterene, spironolactone, canrenone, eplerenone, potassium canrenoate, finerenone, conivaptan, mozavaptan, satavaptan, and tolvaptan.

8. 7. The method of claim 6, further comprising administering to the subject a drug selected from the group consisting of carvedilol, metoprolol, bisoprolol, losartan, valsartan, candesartan, sacubitril, lisinopril, enalapril, ramipril, spironolactone, eplerenone, digoxin, hydralazine, isosorbide dinitrate, ivabradine, atorvastatin, simvastatin, aspirin, clopidogrel, warfarin, apixaban, rivaroxaban, metolazone, nebivolol, and milrinone, when the lymphangiogenesis-associated disorder is systolic heart failure.

9. The method of claim 1 , wherein the subject is a human.

10. A method of promoting lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a DHLA-coated gold nanocluster having a particle size in the range of 1-10 nm, the DHLA-coated gold nanocluster consisting of a gold nanocluster formed by a plurality of gold nanoparticles and a plurality of DHLA coated on the gold nanocluster.

11. The method of claim 10, wherein the DHLA-coated gold nanoclusters have a particle size in the range of 1-5 nm.

12. The method of claim 11 , wherein the DHLA-coated gold nanoclusters have a particle size of 2 nm.

13. The method of claim 10, wherein the gold nanoclusters are administered in an amount of about 0.001 to 10 mg / kg of body weight per day.

14. The method of claim 13, wherein the gold nanoclusters are administered in an amount of about 0.01 to 1 mg / kg of body weight per day.

15. The method of claim 10 , wherein the subject is a human.

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