Metformin nanoformulations and methods of use thereof

Abstract

The present invention provides metformin nanoparticles and methods of using the same.

Description

[0001] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 322,270, filed on March 22, 2022. The foregoing application is incorporated herein by reference. Technical Field

[0002] The present invention relates generally to the delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for delivering metformin to a patient. Background Art

[0003] Metformin is a commonly prescribed drug for the treatment of diabetes, which reduces the glucose produced by the liver and regulates many metabolic pathways. Due to the effects of metformin on AMP-activated protein kinase (AMPK) dependent and independent mechanisms, the inhibitory effects on mitochondrial respiration and the inhibitory effects on mitochondrial glycerolphosphate dehydrogenase, metformin has been identified as a potential therapeutic candidate for the treatment of cancer. It is known that metformin also affects the differentiation of monocytes into macrophages, as well as the secretion of proinflammatory cytokines. Metformin can be used to regulate the abnormal macrophage activity associated with many immune disorders and diseases. However, a problem with using free metformin is that metformin will not accumulate in tumors and other target tissues at physiologically relevant concentrations when administered at a tolerated dose. It is necessary to improve the administration of metformin. Summary of the invention

[0004] According to the present invention, nanoparticles comprising a dendrimer covalently linked or attached to metformin are provided. In certain embodiments, the dendrimer is a poly(amidoamine) (PAMAM) dendrimer. In certain embodiments, the PAMAM dendrimer is a 3rd generation PAMAM dendrimer, a 4th generation PAMAM dendrimer, or a 5th generation dendrimer. In certain embodiments, the PAMAM dendrimer is a 4th generation PAMAM dendrimer. In certain embodiments, the PAMAM dendrimer is conjugated to metformin via a linker. In certain embodiments, the linker comprises polyethylene glycol and / or a carbamate bond. In certain embodiments, the nanoparticle is P4-MET. In certain embodiments, the nanoparticle is PAMAM-PEG-MET. Compositions comprising at least one nanoparticle of the present invention and at least one pharmaceutically acceptable carrier are also provided.

[0005] According to another aspect of the present invention, there is provided a method for treating, suppressing and / or preventing a disease or condition in a subject in need. In certain embodiments, the method comprises administering to the subject at least one nanoparticle of the present invention or a composition comprising the same. In certain embodiments, the disease is cancer. In certain embodiments, the cancer is brain cancer, such as glioblastoma. In certain embodiments, the disease is a neurodegenerative disease, such as dementia, including but not limited to Alzheimer's disease, neurocognitive deficits induced by traumatic brain injury, radiation and / or chemotherapy. In certain embodiments, the method further comprises administering at least one additional therapy, such as radiotherapy, chemotherapy, immunotherapy and / or targeted therapy. In certain embodiments, the method further comprises administering radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 A graph of the release of metformin from P4-MET dendrimers over time (hours) at the indicated pH values ​​is provided.

[0007] Figures 2A-2D Metformin ( Figure 2A )、PAMAM-OH( Figure 2B ), PAMAM-OH and metformin ( Figure 2C ) and P4-MET( Figure 2D ) is a graph of U87MG growth under.

[0008] Figures 3A-3D Metformin ( Figure 3A )、PAMAM-OH( Figure 3B ), PAMAM-OH and metformin ( Figure 3C ) and P4-MET( Figure 3D ) diagram of Ln229 growth.

[0009] Figures 4A-4D Metformin ( Figure 4A )、PAMAM-OH( Figure 4B ), PAMAM-OH and metformin ( Figure 4C ) and P4-MET( Figure 4D ) is a graph of MGPP3 growth under .

[0010] Figures 5A-5D Metformin ( Figure 5A )、PAMAM-OH( Figure 5B ), PAMAM-OH and metformin ( Figure 5C ) and P4-MET( Figure 5D ) is a graph of the growth of bone marrow-derived macrophages (BMDM).

[0011] Fig. 6A A graph of survival of glioblastoma tumor-bearing mice treated with vehicle (Ctrl) or with metformin (Met) or P4-MET is presented. ns = not significant. *p<0.05. Figure 6B Provided are graphs of body weights of glioblastoma tumor-bearing mice that were untreated (Ctrl) or treated with metformin (Met) or P4-MET at the indicated times after tumor inoculation. Figure 6C A graph is provided of the survival of mice bearing glioblastoma tumors treated with vehicle (Ctrl) or with metformin (Met) or P4-MET, optionally with brain radiation therapy (RT). Asterisks indicate the degree of significance.

[0012] Fig. 7A The tissue (brain) / background ratio (TBR) of P4-MET-IR800 after one injection is provided. P4-MET-IR800 or free dye (IR800) was injected into control mice (no tumor) or mice with glioblastoma tumors. Figure 7B The tissue (brain) / background ratio (TBR) of P4-MET-IR800 combined with brain radiation therapy (RT) is provided. P4-MET-IR800 or free dye (IR800) was injected into control mice (no tumor) or mice with glioblastoma tumors. Brain RT was administered 24 hours after the first injection of P4-MET-IR800, and a second injection of P4-MET-IR800 was given.

[0013] Figure 8 The structure of PAMAM-OH G4 is provided. DETAILED DESCRIPTION

[0014] The present invention describes nanoformulations of metformin that allow rapid distribution of metformin into target tissues, increased cellular uptake, enhanced permeability across the blood-brain barrier, enhanced anti-tumor activity, and improved modulation of macrophage activity compared to free metformin. The compositions of the present invention can be used to treat a variety of diseases, such as cancer, diabetes, and immune disorders and diseases.

[0015] According to the present invention, nanoparticles (also referred to as nanoformulations) comprising metformin are provided. In certain embodiments, the nanoparticles comprise poly(amidoamine) (PAMAM) dendrimers conjugated and / or linked to metformin. Metformin can be conjugated and / or linked to the PAMAM dendrimers directly or through a linker (e.g., through a functional group at the end group). Dendrimers include a molecular structure having an inner core, layers / generations of repeating units connected to and extending from the inner core, and an outer surface of the end groups (e.g., connected to the outermost layer / generation). PAMAM dendrimers may comprise different cores and / or end groups having amidoamines.

[0016] In certain embodiments, nanoparticles of the invention have a diameter (e.g., z-average diameter) or longest dimension of less than about 500 nm, particularly less than about 100 nm, less than about 50 nm, or less than about 25 nm. In certain embodiments, nanoparticles of the invention have a diameter of about 0.1 nm to about 50 nm, about 1 nm to about 25 nm, about 1 nm to about 20 nm, or about 1 nm to about 15 μm.

[0017] PAMAM dendrimers and methods of synthesis are well known in the art (e.g., de Araujo et al., Molecules (2018) 23(11):2849; Maiti et al., Macromolecules (2004) 37:6236-6254; Abbasi et al., Nanoscale Res. Lett. (2014) 9:247; Santos et al., Molecules (2016) 21:686; all incorporated herein by reference). For example, layers / generations of PAMAM dendrimers can be added by reacting an amino-terminated core or layer with methyl acrylate to produce an ester-terminated outer layer and reacting with ethylenediamine to obtain a new amino-terminated surface. The PAMAM dendrimers of the present invention can be of any size / generation. In certain embodiments, the PAMAM dendrimer is of generation 1 to 10, particularly generation 1 to 6, generation 1 to 5, generation 2 to 6, generation 2 to 5, generation 3 to 6, or generation 3 to 5. In certain embodiments, the PAMAM dendrimer is of generation 3, 4, or 5; generation 3 or 4; or generation 4 or 5. In certain embodiments, the PAMAM dendrimer is of generation 4.

[0018] The PAMAM dendritic polymers of the present invention may include one or more different end groups (surface groups). In certain embodiments, the PAMAM dendritic polymers include one type of end groups. Examples of PAMAM dendritic polymer end groups include, but are not limited to, amide ethanol end groups, amide ethyl ethanolamine end groups, amino end groups, hydroxyl end groups, hexylamide end groups, mixed (bifunctional) end groups, sodium carboxylate end groups, succinamic acid end groups, trimethoxysilyl end groups, tris (hydroxymethyl) amidomethane end groups, and 3-methoxypyrrolidone end groups. In certain embodiments, the end groups include hydroxyl groups. In certain embodiments, the end groups include -NH2 groups.

[0019] Typically, the number of end groups is determined by the generation of the PAMAM dendrimer. In certain embodiments, the PAMAM nanoparticles comprise 1 to 5,000 end groups. In certain embodiments, the PAMAM nanoparticles comprise 1 to 1,000 end groups. In certain embodiments, the PAMAM nanoparticles comprise 1 to 100 end groups.

[0020] The PAMAM dendrimer core comprises a primary amine, particularly a linear chain molecule comprising a primary amine. Examples of PAMAM core types include, but are not limited to: ammonia, ethylenediamine (2-carbon core), 1,4-diaminobutane (4-carbon core), 1,6-diaminohexane (6-carbon core), 1,12-diaminododecane (12-carbon core) and cystamine core (cleavable core). In certain embodiments, the PAMAM core is ethylenediamine.

[0021] As described above, the PAMAM dendrimer is conjugated and / or connected to metformin directly or through a linker. In certain embodiments, metformin is conjugated and / or connected to the surface of the PAMAM dendrimer. In certain embodiments, metformin is conjugated and / or connected through a functional group (e.g., -OH or -NH2) at the end group of the PAMAM dendrimer. In certain embodiments, the hydroxyl or amino surface groups of the PAMAM dendrimer are activated with 1,1'-carbonyldiimidazole to conjugate and / or connect metformin. In certain embodiments, the PAMAM dendrimer is conjugated and / or connected to metformin through a cross-linking agent. In certain embodiments, the linker comprises at least one carbamate bond (-O(CO)NH-). In certain embodiments, the conjugation and / or connection of metformin to the PAMAM dendrimer results in the formation of at least one carbamate bond (-O(CO)NH-).

[0022] In certain embodiments, the linker comprises polyethylene glycol (PEG). The PEG used can have various ethylene glycol units. In certain embodiments, the PEG has 1 to 100 ethylene glycol units. In certain embodiments, the PEG has 1 to 10 ethylene glycol units.

[0023] The PAMAM dendrimers of the present invention are conjugated and / or linked to one or more metformin molecules. Metformin can be conjugated and / or linked to one, all, or any number of end groups or surface functional groups of the PAMAM dendrimers. For example, for the 4th generation (G4) PAMAM dendrimers, there may be 64 end groups for connecting metformin. Metformin can be conjugated and / or linked to 1 to 64 (i.e., all) end groups of G4 PAMAM. In certain embodiments, 1% to 100% of the end groups of the PAMAM dendrimers are conjugated and / or linked to metformin (e.g., through a linker (e.g., PEG)). In certain embodiments, 35% to 100% of the end groups or surface functional groups of the PAMAM dendrimers are conjugated and / or linked to metformin (e.g., through a linker (e.g., PEG)). In certain embodiments, 1% to 35% of the terminal groups or surface functional groups of the PAMAM dendrimer are conjugated and / or linked to metformin (e.g., via a linker (e.g., PEG)). In certain embodiments, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or about 100% of the terminal groups or surface functional groups of the PAMAM dendrimer are conjugated and / or linked to metformin (e.g., via a linker (e.g., PEG)).

[0024] In certain embodiments, the nanoparticles of the invention comprise a PAMAM dendrimer (e.g., a G4PAMAM dendrimer) comprising an amino terminal group linked and / or conjugated to a linker (e.g., PEG) that is linked and / or conjugated to metformin (e.g., PAMAM-PEG-MET). In certain embodiments, the nanoparticles of the invention comprise the formula:

[0025]

[0026] wherein A is a PAMAM dendrimer (e.g., a G4 PAMAM dendrimer), y is the number of end groups or surface functional groups of the PAMAM dendrimer, x is 1 to y, and n is 1 to 10. In certain embodiments, the nanoparticles of the invention comprise the formula:

[0027]

[0028] wherein A is a G4 PAMAM dendrimer, x is 1-64, and n is 1-10.

[0029] In certain embodiments, the nanoparticles of the invention comprise a PAMAM dendrimer (e.g., a G4 PAMAM dendrimer) comprising a hydroxyl (e.g., aminoethanol) terminal group linked and / or conjugated (e.g., via 1,1'-carbonyldiimidazole) to metformin (e.g., P4-MET). In certain embodiments, the nanoparticles of the invention comprise the formula:

[0030]

[0031] wherein A is a PAMAM dendrimer (eg, a G4 PAMAM dendrimer), y is the number of end groups or surface functional groups of the PAMAM dendrimer, and n is 1 to y. In certain embodiments, the nanoparticles of the invention comprise the formula:

[0032]

[0033] wherein A is a G4 PAMAM dendrimer and n is 1-64.

[0034] The present invention encompasses compositions (e.g., pharmaceutical compositions) comprising at least one nanoparticle of the present invention and at least one carrier (e.g., a pharmaceutically acceptable carrier). In certain embodiments, the composition comprises at least one nanoparticle of the present invention and at least one pharmaceutically acceptable carrier. The composition of the present invention (e.g., pharmaceutical composition) may further comprise other compounds or therapeutic agents.

[0035] As explained herein, the nanoparticles (such as PAMAM-PEG-MET and P4-MET) comprising metformin of the present invention rapidly distribute the drug to tissues compared with free metformin, and enhance cellular uptake and penetration across the blood-brain barrier. Compared with free metformin, these nanoparticles can significantly increase drug delivery to irradiated tissues (such as the brain), and significantly increase the overall survival rate of animals suffering from glioblastoma. The metformin nanoparticles of the present invention can be used in medicine. More specifically, the metformin nanoparticles can be used to enhance the efficacy of other cancer therapies such as radiotherapy, chemotherapy, immunotherapy and targeted therapy. Compared with free metformin, the metformin nanoparticles of the present invention can also be used to significantly improve the regulation of macrophage activity.

[0036] The present invention also includes methods for preventing, inhibiting and / or treating diseases or conditions. The methods include administering the nanoparticles of the present invention (e.g., in a composition) to a subject in need thereof. The diseases and conditions include, but are not limited to, cancer, immune diseases and / or conditions, neurodegenerative conditions, and diabetes.

[0037] In certain embodiments, the method prevents, inhibits and / or treats cancer in a subject in need. Cancers to be treated include, but are not limited to, brain cancer (e.g., glioma, glioblastoma), CNS cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer, prostate cancer, leukemia, lymphoma, lung cancer, liver cancer, gastric cancer, and skin cancer. In certain embodiments, the cancer is CNS cancer. In certain embodiments, the cancer is brain cancer. In certain embodiments, the cancer is a glioma. In certain embodiments, the cancer is a glioblastoma. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the method includes administering the nanoparticles of the present invention (e.g., in a composition) to a subject in need. In certain embodiments, the method also includes administering at least one other therapeutic agent (e.g., anticancer agent, chemotherapeutic agent). In certain embodiments, the method also includes administering at least one additional cancer therapy to the subject. Cancer therapy includes, but is not limited to, radiotherapy, chemotherapy, immunotherapy, and targeted therapy. When additional therapy is administered, the additional therapy can be administered sequentially (e.g., before and / or after) and / or synchronously (e.g., simultaneously) with the nanoparticles or compositions comprising the nanoparticles. In certain embodiments, the additional therapy is administered at least after the administration of the nanoparticles or compositions comprising the nanoparticles (e.g., at least after the first dose of the regimen of the nanoparticles or compositions comprising the nanoparticles). In certain embodiments, the additional therapy is administered at least within the same time frame as the administration of the nanoparticles or compositions comprising the nanoparticles (e.g., at least during the regimen of the nanoparticles or compositions comprising the nanoparticles).

[0038] In certain embodiments, the cancer treatment method includes administering a nanoparticle of the present invention (e.g., in a composition) and radiotherapy. In certain embodiments, the radiotherapy is CNS radiotherapy, and the cancer is CNS cancer. In certain embodiments, the radiotherapy is brain radiotherapy, and the cancer is brain cancer (e.g., glioma, glioblastoma). In certain embodiments, the radiotherapy is administered at least after administering nanoparticles or compositions comprising nanoparticles (e.g., at least after the first dose of a scheme of a nanoparticle or a composition comprising nanoparticles). In certain embodiments, the radiotherapy is administered at least within the same time frame of administering nanoparticles or compositions comprising nanoparticles (e.g., at least during a scheme of a nanoparticle or a composition comprising nanoparticles).

[0039] In certain embodiments, the method prevents, inhibits and / or treats immune diseases and / or conditions in a subject in need thereof. Immune diseases and / or conditions include, but are not limited to, Crohn's disease, ulcerative colitis, rheumatoid arthritis, osteoarthritis, psoriasis, radiation pneumonitis, radiation necrosis, spinal cord injury, and traumatic brain injury. In certain embodiments, the disease is a neurodegenerative disease. Neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, and amyotrophic lateral sclerosis. In certain embodiments, the method further comprises administering at least one additional therapy to the subject.

[0040] Nanoparticles of the present invention (e.g., in compositions) can be administered to animals, particularly mammals, more particularly humans, to treat / suppress / prevent diseases or conditions. Pharmaceutical compositions of the present invention can also include at least one other compound or therapeutic agent. Additional compounds can also be administered in pharmaceutical compositions separate from the compositions of the present invention. The pharmaceutical compositions can be administered at the same time or at different times (e.g., sequentially).

[0041] The dosage range of the nanoparticles and / or compositions of the present invention is a dosage range that is large enough to produce the desired effect (e.g., cure, alleviate, treat and / or prevent a disease or condition, its symptoms or its predisposition to disease). The dosage should not be too large to avoid causing significant adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Typically, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by those skilled in the art. In the event of any contraindications, the dosage can be adjusted by an individual physician.

[0042] The nanoparticles described herein will generally be administered to a patient as a pharmaceutical composition. The term "patient" as used herein refers to a human or animal subject. These nanoparticles can be used therapeutically under the guidance of a physician.

[0043] The pharmaceutical composition comprising the nanoparticle of the present invention can be conveniently formulated for use with any pharmaceutically acceptable carrier. For example, the complex can be formulated with an acceptable medium, such as water, buffered saline, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oil, detergent, suspending agent or their suitable mixture, particularly aqueous solution. The concentration of the nanoparticle in the selected medium can vary, and the medium can be selected based on the desired route of administration of the pharmaceutical composition. Unless any conventional medium or medicament is incompatible with the nanoparticle to be used, it is considered to be used in the pharmaceutical composition.

[0044] The dosage and administration regimen of the nanoparticles according to the present invention suitable for use in a specific patient can be determined by a doctor taking into account the patient's age, sex, weight, general medical condition, and the specific condition and severity of the nanoparticles used. The doctor can also consider the route of administration, the drug carrier, and the biological activity of the nanoparticles.

[0045] The selection of suitable pharmaceutical composition will also depend on the selected mode of administration. For example, the nanoparticles of the present invention can be administered by direct injection or injection into the bloodstream (e.g., intravenously). In this case, the pharmaceutical composition comprises nanoparticles dispersed in a medium compatible with the injection site.

[0046] Nanoparticles of the present invention can be used by any method. For example, nanoparticles of the present invention can be used by, but not limited to, parenteral, subcutaneous, oral, topical, pulmonary, rectal, vaginal, intravenous, intraperitoneal, intrathecal, intracerebral, epidural, intramuscular, intradermal or intracarotid. In a specific embodiment, the nanoparticles are parenteral. In a specific embodiment, the nanoparticles are oral, intramuscular, subcutaneous or applied to the bloodstream (e.g., intravenous). In a specific embodiment, the nanoparticles are intramuscular or subcutaneous. Pharmaceutical compositions for injection are known in the art. If injection is selected as a method for applying the nanoparticles, measures should be taken to ensure that a sufficient amount of molecules or cells reach their target cells to exert a biological effect. Dosage forms for oral administration include, but are not limited to, tablets (e.g., coated and uncoated, chewable), gelatin capsules (e.g., soft or hard), lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders / granules (e.g., reconstituted or dispersible), gums, and effervescent tablets. Dosage forms for parenteral administration include, but are not limited to, solutions, emulsions, suspensions, dispersants, and powders / granules for reconstitution. Dosage forms for topical administration include, but are not limited to, creams, gels, ointments, salves, patches, and transdermal delivery systems.

[0047] Pharmaceutical compositions containing nanoparticles of the present invention as active ingredients intimately mixed with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical formulation techniques. The carrier can take a variety of forms, depending on the form of the pharmaceutical composition desired to be administered, such as intravenous, oral, direct injection, intracranial and intravitreal administration.

[0048] The pharmaceutical composition of the present invention can be formulated into a dosage unit form for ease of administration and uniformity of dosage. The dosage unit form used herein refers to a physical discrete unit of the pharmaceutical composition suitable for patients receiving treatment. Each dose should contain a certain amount of active ingredient, which produces the desired effect together with the selected pharmaceutical carrier. The procedure for determining a suitable dosage unit is well known to those skilled in the art. In a specific embodiment, the nanoparticles of the present invention only need to be administered once to a subject due to their long-acting therapeutic effect.

[0049] The dosage unit may be increased or decreased proportionally according to the patient's body weight. As known in the art, the appropriate concentration for alleviating a particular pathological condition may be determined by calculation of a dose concentration curve.

[0050] The appropriate dosage unit for nanoparticle administration can be determined by evaluating their toxicity in animal models. Nanoparticles of various concentrations in the pharmaceutical composition can be administered to mice, and the minimum and maximum doses can be determined based on the beneficial results and side effects observed as a result of the treatment. Suitable dosage units can also be determined by evaluating the efficacy of nanoparticle therapy in combination with other standard drugs. The dosage unit of the nanoparticle can be determined individually or in combination with each treatment based on the detected effect.

[0051] The pharmaceutical composition comprising nanoparticles can be administered at appropriate intervals until the pathological symptoms are alleviated or relieved, after which the dosage can be reduced to a maintenance level. The appropriate interval in a particular case generally depends on the patient's condition.

[0052] definition

[0053] The following definitions are provided to facilitate the understanding of the present invention.

[0054] The singular forms ("a," "an," and "the") include plural referents unless the context clearly dictates otherwise.

[0055] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0056] "Carrier" refers to diluents, adjuvants, preservatives (e.g., thimerosal (Thimersol), benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), solubilizers (e.g., polysorbate 80), emulsifiers, buffers (e.g., Tris HCl, acetates, phosphates), antimicrobial agents, fillers (e.g., lactose, mannitol), excipients, adjuvants, or carriers that are administered with the active drug of the invention. Pharmaceutically acceptable carriers can be sterile liquids such as water and oils (including water and oils of petroleum, animal, plant, or synthetic origin). Water or aqueous saline solutions and aqueous glucose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin (Mack Publishing Co., Easton, PA); Gennaro, AR, Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0057] As used herein, the term "treatment" refers to any type of treatment that benefits a patient with a disease, including improving the patient's condition (eg, one or more symptoms), slowing the progression of the condition, and the like.

[0058] As used herein, the term "prevention" refers to prophylactic treatment of a subject at risk for a condition (eg, cancer) thereby reducing the likelihood that the subject will develop the condition.

[0059] A "therapeutically effective amount" of a compound or pharmaceutical composition is an amount effective to prevent, inhibit, treat or alleviate the symptoms of a particular disease or condition. Cancer treatment herein may refer to curing, alleviating and / or preventing cancer, its symptoms or its predisposition to the disease.

[0060] As used herein, the term "therapeutic agent" refers to a chemical compound or biomolecule, including but not limited to nucleic acids, peptides, proteins, and antibodies, which is useful for treating a condition, disease, or disorder or alleviating the symptoms of a condition, disease, or disorder.

[0061] "Linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently connects at least two compounds. The linker can be connected to any synthetically feasible position of the compound, but is preferably connected in a manner to avoid blocking the desired activity of the compound. Linkers are well known in the art. In a specific embodiment, the linker can contain 0 (i.e., bond) to about 100 atoms, 0 to about 50 atoms, 0 to about 10 atoms, or about 1 to about 5 atoms.

[0062] The term "cross-linker" refers to a molecule capable of forming covalent bonds between compounds.

[0063] The following examples provide illustrative methods of practicing the invention and are not intended to limit the scope of the invention in any way.

[0064] Example 1

[0065] Metformin (1,1-dimethylbiguanide) is a synthetic biguanide that is an FDA-approved drug for the treatment of type 2 diabetes. Metformin is considered a safe drug, with the most notable side effects being gastrointestinal-related (e.g., abdominal pain, bloating, diarrhea, nausea, and / or vomiting), which occur in approximately 20-30% of patients.

[0066] Mitochondria are the main target of metformin (Vial et al., Front. Endocrinol. (2019) 10: 294). It is worth noting that metformin has anti-tumor properties at least in part through this mitochondrial effect. In fact, both in cell lines and in retrospective clinical studies, metformin has shown strong and sustained antiproliferative effects on many types of cancer (including breast cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer and prostate cancer). For example, metformin selectively affects glioblastoma tumor initiation cells (cancer stem cells) and exerts antiproliferative activity on glioblastoma cells (Wurth et al., Cell Cycle (2013) 12 (1): 145-56). In addition, metformin reduces the risk of cardiovascular disease (Lamanna et al., Diabetes Obes. Metab. (2011) 13 (3): 221-8), provides anti-aging properties, and can treat central nervous system (CNS) diseases.

[0067] Despite these positive findings, metformin has not shown significant clinical benefit in many prospective clinical trials. It is worth noting that the typical effective concentration of metformin in cell culture is in the mM range, while the drug concentration in patients taking metformin orally is usually less than 50 μM (Badrick et al., Eur. J. Cancer (2014) 50(12): 2119-25).

[0068] To improve the delivery of metformin, polyamidoamine (PAMAM) dendrimers were used. PAMAM dendrimers are hyperbranched polymers with molecular uniformity and narrow molecular weight distribution. Dendrimer PAMAM-OHG4 (Mw = 14277.19 g / mole) was used. Figure 8 The structure of PAMAM-OH G4 is provided.

[0069] To conjugate metformin to PAMAM-OH G4, conjugation through the 64 terminal -OH groups was utilized. Briefly, PAMAM-OH G4 (P4-OH) was first reacted with 1,1'-carbonyldiimidazole (CDI) and then with metformin to give PAMAM G4-conjugated metformin (P4-MET). A schematic diagram of P4-MET showing metformin attachment is provided:

[0070]

[0071] The circles represent PAMAM G4, and n ranges from 1 to 64.

[0072] Dynamic light scattering (DLS) was used to determine the size of the dendrimer. eff ) was 7 nm ± 1 nm and the polydispersity index (PDI) was 0.17. P4-MET was determined to have a particle size (D eff ), PDI is 0.19.

[0073] In addition, the zeta potential of the dendrimer was measured. The zeta potential of P4-OH was measured to be 0 mV, and the zeta potential of P4-MET was measured to be 10 mv±2 mv.

[0074] Using an LH20 column, PAMAM-OH or P4-MET conjugate was passed through the column and eluted with methanol. Appropriate fractions were collected and the solvent removed. PAMAM-OH and P4-MET were dried in vacuo and then passed through 1 H-NMR (D2O) analysis and confirmation.

[0075] The ability of P4-MET to release metformin was then determined. Figure 1 As shown, metformin is released faster from P4-MET at acidic pH. In fact, P4-MET retains more than 50% of conjugated metformin for more than 150 hours at pH 7.4 and pH 6.8. However, metformin is rapidly released from P4-MET at pH 4.5. The presence of lower pH in tumors and tumor environments can lead to more drug release at the targeted site.

[0076] The ability of dendrimers to deliver metformin to U87MG cells, a human glioma cell line that is sensitive to metformin, was tested. Specifically, U87MG cells were exposed to metformin, PAMAM-OH, PAMAM-OH and metformin, or P4-MET. Figures 2A-2D As shown, P4-MET greatly increased the delivery of metformin to U87MG cells, as evidenced by the decrease in optical density observed in the cultures (eg, less cell growth and / or more cell death). 50 The values ​​are listed in Table 1.

[0077]

[0078]

[0079] Table 1: IC of U87MG cells 50 value.

[0080] As shown in Table 1, P4-MET significantly reduced the concentration of metformin required to inhibit the growth of U87MG (approximately 70-fold).

[0081] The ability of the dendrimers to deliver metformin to Ln229 cells, a human glioblastoma cell line that is sensitive to metformin, was also tested. Specifically, Ln229 cells were exposed to metformin, PAMAM-OH, PAMAM-OH and metformin, or P4-MET. Figures 3A-3D As shown, P4-MET greatly increased the delivery of metformin to Ln229 cells, as evidenced by the decrease in optical density observed in the cultures. 50 The values ​​are listed in Table 2.

[0082] Ln229 <![CDATA[IC 50 (mM)]]> Metformin 0.99 PAMAM-OH - PAMAM-OH+Metformin 1.46 P4-MET 0.13

[0083] Table 2: IC of Ln229 cells 50 value.

[0084] As shown in Table 2, P4-MET significantly reduced the concentration of metformin required to inhibit the growth of Ln229 (approximately 7-fold).

[0085] The ability of the dendrimers to deliver metformin to MGPP3 cells, a mouse glioblastoma cell line that is sensitive to metformin, was also tested. Specifically, MGPP3 cells were exposed to metformin, PAMAM-OH, PAMAM-OH and metformin, or P4-MET. Figures 4A-4D As shown, P4-MET greatly increased the delivery of metformin to MGPP3 cells, as evidenced by the decrease in optical density observed in the cultures.50 The values ​​are listed in Table 3.

[0086]

[0087]

[0088] Table 3: IC of MGPP3 cells 50 value.

[0089] As shown in Table 3, P4-MET significantly reduced the concentration of metformin required to inhibit the growth of MGPP3 (approximately 250-fold).

[0090] The ability of dendrimers to deliver metformin to bone marrow-derived macrophages (BMDM) was also tested. Specifically, BMDM cells were exposed to metformin, PAMAM-OH, PAMAM-OH and metformin, or P4-MET for 72 hours. Figures 5A-5D As shown, P4-MET greatly increased the delivery of metformin to BMDM, as evidenced by the decrease in optical density observed in the cultures. 50 Values ​​are listed in

[0091] Table 4.

[0092] BMDM <![CDATA[IC 50 (mM)]]> Metformin 3.53 PAMAM-OH - PAMAM-OH+Metformin 4.89 P4-MET 0.061

[0093] Table 4: IC of BMDM cells 50 value.

[0094] As shown in Table 4, P4-MET significantly reduced the concentration of metformin required to inhibit BMDM growth (approximately 60-fold).

[0095] Furthermore, the in vitro cellular uptake of P4-MET was confirmed by confocal microscopy. 488P4-MET for 24 hours. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Mitochondria were stained with MitoTracker TM Deep Red staining. Alexa Fluor 488P4-MET staining and MitoTracker TM Deep Red staining overlaps.

[0096] The ability of the dendrimers to deliver metformin to UNKPC cells, a pancreatic cancer cell line, was also tested. Specifically, MGPP3 cells were exposed to metformin, PAMAM-OH, or P4-MET. P4-MET greatly increased the delivery of metformin to UNKPC, as evidenced by the decrease in optical density observed in the cultures. 50 The values ​​are listed in Table 5.

[0097] UNKPC <![CDATA[IC 50 (mM)]]> Metformin 1.638 PAMAM-OH - P4-MET 0.1702

[0098] Table 5: IC of UNKPC cells 50 value.

[0099] As shown in Table 5, P4-MET significantly reduced the concentration of metformin required to inhibit the growth of UNKPC (approximately 10-fold).

[0100] The ability of the dendrimers to deliver metformin to Panc-1 cells, a human pancreatic cancer cell line, was also tested. Specifically, Panc-1 cells were exposed to metformin, PAMAM-OH, and P4-MET. P4-MET greatly increased the delivery of metformin to Panc-1, as evidenced by the decrease in optical density observed in the cultures. 50 The values ​​are listed in Table 6.

[0101] Panc-1 <![CDATA[IC 50 (mM)]]> Metformin 5.244 PAMAM-OH - P4-MET 0.4024

[0102] Table 6: IC50 values ​​for UNKPC cells.

[0103] As shown in Table 6, P4-MET significantly reduced the concentration of metformin required to inhibit the growth of Panc-1 (approximately 13-fold).

[0104] The ability of P4-MET to treat glioblastoma in vivo was also tested. Glioblastomas were implanted in mice on day 0. On day 8, the mice were intraperitoneally administered vehicle (control) or 200 mg / kg metformin equivalents of metformin or P4-MET. The mice were treated daily until day 28. Fig. 6A As shown, mice treated with P4-MET had a statistically significant higher survival rate than mice treated with metformin. Figure 6B As shown, mice treated with P4-MET maintained their body weight through 7 weeks of observation.

[0105] These mice also received brain radiation therapy (RT). On day 11 after tumor inoculation, mice were treated with brain RT (8 Gy). Figure 6C As shown, P4-MET sensitized brain RT and significantly improved the overall survival rate of mice.

[0106] We also determined whether brain radiation therapy increased the penetration of P4-MET. (IR800) labels P4-MET. Fig. 7A The tissue (brain) / background ratio (TBR) of P4-MET-IR800 after one injection is presented. Figure 7BThe tissue (brain) / background ratio (TBR) of P4-MET-IR800 after the first injection at 0 hours and the second injection at 24 hours and brain radiation therapy is provided. Figure 7B As shown, brain RT increased the presence of P4-MET in the brain.

[0107] Finally, P4-MET was determined to be well tolerated by mice. Mice treated with P4-MET showed no statistically significant differences in body weight, blood cell counts, or comprehensive metabolic panel (CMP) analysis.

[0108] In summary, P4-MET releases free metformin under acidic conditions. P4-MET can penetrate into cancer and benign cells (e.g., BMDM). Although BMDM is very sensitive to P4-MET, no significant adverse effects of P4-MET were observed in mice. P4-MET significantly increased the efficacy compared to free metformin at the same concentration. In addition, the biological effects of increased P4-MET were significantly greater than the additive effects of PAMAM and metformin when mixed together.

[0109] Example 2

[0110] Another PAMAM conjugated to metformin was synthesized, in which metformin was attached to the PAMAM dendrimer via a polyethylene glycol (PEG) linker. Typically, a short PEG linker (e.g., where n=1-10) was used. Briefly, PEG was first reacted with 1,1'-carbonyldiimidazole (CDI) to generate CDI-PEG-CDI.

[0111]

[0112] Then PAMAM G4 with -NH2 group as the terminal group reacted with CDI-PEG-CDI to connect PEG-CDI to PAMAM-NH2 through a carbamate bond.

[0113]

[0114] The circles represent PAMAM G4, and x ranges from 1 to 64. The product was then reacted with metformin (DMSO, RT, 48 h, N2) to give PAMAM-PEG-metformin.

[0115]

[0116] The circles represent PAMAM G4, and x ranges from 1 to 64. 1 The product was analyzed and confirmed by H-NMR.

[0117] Throughout the foregoing specification, numerous publications and patent documents are cited to describe the state of the art to which the invention pertains. The entire disclosure of each of these citations is incorporated herein by reference.

[0118] Although certain preferred embodiments of the present invention have been described and specifically illustrated above, it is not intended that the present invention be limited to these embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as described in the following claims.

Claims

1. A nanoparticle comprising a poly(amidoamine) (PAMAM) dendrimer conjugated to metformin.

2. The nanoparticle of claim 1, wherein the PAMAM dendrimer is a 3rd generation PAMAM dendrimer, a 4th generation PAMAM dendrimer, or a 5th generation PAMAM dendrimer.

3. The nanoparticle of claim 1, wherein the PAMAM dendrimer is a 4th generation PAMAM dendrimer.

4. The nanoparticle of claim 1, wherein the PAMAM dendrimer is conjugated to the metformin via a linker.

5. The nanoparticle of claim 4, wherein the linker comprises polyethylene glycol. The nanoparticle of claim 4 , wherein the linker comprises a carbamate bond.

7. The nanoparticle of claim 1, wherein the nanoparticle comprises the formula: wherein A is a PAMAM dendrimer, y is the number of end groups of the PAMAM dendrimer, x is 1 to y, and n is 1 to 10.

8. The nanoparticle of claim 1, wherein the nanoparticle comprises the formula: wherein A is a 4th generation PAMAM dendrimer, x is 1-64, and n is 1-10.

9. The nanoparticle of claim 1, wherein the nanoparticle comprises the formula: wherein A is a PAMAM dendrimer, y is the number of end groups of the PAMAM dendrimer, and n is 1 to y.

10. The nanoparticle of claim 1, wherein the nanoparticle comprises the formula: wherein A is a 4th generation PAMAM dendrimer and n is 1-64.

11. A composition comprising at least the nanoparticle according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A method of treating, inhibiting and / or preventing a disease in a subject in need thereof, the method comprising administering to the subject a nanoparticle according to any one of claims 1 to 10.

13. The method of claim 12, wherein the disease is cancer.

14. The method of claim 13, wherein the cancer is brain cancer.

15. The method of claim 13, wherein the cancer is glioblastoma or glioma.

16. The method of claim 13, further comprising administering at least one of radiation therapy, chemotherapy, immunotherapy, and targeted therapy.

17. The method of claim 13, further comprising administering radiation therapy.

18. The method of claim 13, further comprising administering brain radiation therapy, wherein the cancer is brain cancer.

19. The method of claim 18, wherein the brain cancer is glioblastoma or glioma.

20. The method of claim 12, wherein the disease is an immune disorder.

21. The method of claim 20, wherein the immune disorder is an autoimmune disease.

22. The method of claim 20, wherein the immune disorder is rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, multiple sclerosis, radiation pneumonitis, or radiation necrosis.

23. The method of claim 12, wherein the disease is a neurodegenerative disorder.

24. The method according to claim 23, wherein the neurodegenerative disorder is dementia, neurocognitive deficits induced by radiation, chemotherapy or traumatic brain injury.

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