METHODS OF TREATING DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/043,986, filed June 25, 2020, the contents of which are hereby incorporated herein by reference in their entirety.

BACKGROUND

Metformin is the most widely prescribed diabetes medication in the world and top four of all medications in the USA. Metformin is also used for treatment of polycystic ovarian syndrome (PCOS) and is in clinical trials for cancer and other age-related illnesses. It was developed in 1922 as a semi-synthetic version of an herb found in the French Lily. Despite being used all over the world for decades, the direct protein target of metformin has never been described. In consequence, the drug has never been subjected to target-based medicinal chemistry. It is therefore highly likely that a rational approach with target in hand could lead to improved potency and efficacy.

SUMMARY OF THU INVENTION

In certain aspects, the present disclosure provides methods of treating disease, comprising inhibiting one or more mitochondrial proteins in a subject in need thereof. In certain embodiments, the disease is diabetes, polycystic ovarian syndrome, a cancer, or an age-related illness.

In certain embodiments, the one or more mitochondrial proteins are selected from UQCRC2, UQCRB, CYC1, UQCRC1, BCKDHB, UQCRFS1, UQCRQ, and BCKDHA, such as UQCRC1, UQCRC2, or UQCRB, such as UQCRC1.

In certain embodiments, inhibiting one or more mitochondrial proteins comprises administering a therapeutically effective amount of an inhibitor of the one or more mitochondrial proteins to a subject in need thereof.

In certain such embodiments, the inhibitor is hydrogen peroxide, epigallocatechin gallate, A1938, A1893, terpestacin, HDNT, 3-(l-(phenylamino)ethylidene)-chroman-2,4- dione, or a derivative of any of the foregoing.

In other such embodiments, the inhibitor is a small molecule characterized by: at least 3 nitrogen atoms; at least 2 hydrogen bond donors; 2 to 12 carbon atoms; a molecular weight of about 100 to about 500 Da; and a ClogP value of about -1 to about -1.5. In certain such embodiments, the inhibitor is not metformin or phenformin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB show mitochondrial respiration by the Seahorse assay in primary murine hepatocytes. In Figl A cells were treated for 3hs with metformin and 1 mM but not 100 uM inhibits respiration, while in FiglB cells are treated for 24hs at the lower 100 uM concentration does inhibit mitochondrial respiration.

Figure 2 shows a comparison of the structures of biguanide, metformin, phenformin, and a thiol-metformin.

Figure 3 shows affinity enrichment of specific mitochondrial proteins on a metformin column. Several parallel columns were prepared with or without the attachment of the modified metformin, and both columns were eluted with PBS containing 1% SDS. Quantitative protein mass spectrometry using isobaric labeling was done on SDS eluates from duplicate columns in both cases, and the top fold-enriched proteins in the metformin column vs the control column are shown in the top box. Components of Complex I are shown in lower box. GPD2 is shown in lowest box.

Figure 4 shows elution of mitochondrial proteins from metformin affinity column with control buffer (PBS), metformin or phenformin (100 uM) or rosiglitazone (100 uM). SDHA and COX4 are shown as control subunits from Complex II and CIV respectively.

Figure 5 shows direct ³ H-metformin binding to holo-Complex III.

Figure 6 shows NMR analyses of UQCRC1 (Cl) and separately, UQCRC2 (C2), using 5uM of ¹³ C labeled metformin. Cold is the same preparation with lOOuM of non- ¹³ C labeled metformin. The binding of the labeled metformin is indicated by the loss of NMR signal with the Cl subunit but not the C2 subunit. Binding is further indicated by the fact that the addition of non-labeled metformin brings this signal back as the binding of ¹³ C metformin is lost.

PET ATT, ED DESCRIPTION OF TUI W E M l ON

In certain embodiments, the invention relates to the discovery that certain proteins of Complex III of the electron transport chain (ETC) bind metformin. Two of these proteins, UQCRC1 and UQCRC2, have been found to be direct binding partners of metformin. These two proteins form a heterodimeric unit within Complex III. In certain aspects, the present disclosure provides methods of treating disease, comprising inhibiting one or more mitochondrial proteins in a subject in need thereof. In certain embodiments, the disease is diabetes, polycystic ovarian syndrome, a cancer, or an age-related illness. Cancers that can be treated according to the methods of the present disclosure include, but are not limited to, breast cancer (including metastatic breast cancer, estrogen receptor positive metastatic breast cancer, HER2 -positive breast cancer, and localized breast cancer), head and neck cancer (such as head and neck squamous cell cancer), endometrial hyperplasia (such as atypical endometrial hyperplasia), endometrial cancer, prostate cancer (such as adenocarcinoma of the prostate), multiple myeloma (such as relapsed and/or refractory multiple myeloma), uterine cancer, cervical cancer, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, leukemia (such as acute lymphocytic leukemia or chronic lymphocytic leukemia (which may be relapsed or untreated)), thyroid cancer (such as differentiated thyroid cancer or medullary thyroid cancer), malignant solid tumors, melanoma, colon cancer, pancreatic cancer (such as metastatic pancreatic adenocarcinoma, acinar cell adenocarcinoma of the pancreas, duct cell adenocarcinoma of the pancreas, or recurrent pancreatic cancer), lung cancer (such as lung neoplasms or non-small cell lung carcinoma), genograph, colorectal cancer (including colorectal adenoma or polyps), glioma, liver cancer, gastric cancer (also called stomach cancer), gastric carcinoma, non metastatic cancers, cancers with a low degree of malignity, lymphoma, esophageal cancer, rectal cancer, and kidney cancer. Based on recent analyses and studies, metformin reduces the proliferation of cancer cells and the possibility of malignancies in different types of cancer, including gastric carcinoma, pancreatic cancer, uterine cancer, medullary thyroid cancer and a number of other cancers, such as prostate, colon, pancreas, and breast. (Saraei P, Asadi I, Kakar MA, Moradi-Kor N. : The beneficial effects of metformin on cancer prevention and therapy: a comprehensive review of recent advances. Cancer Manag Res. 2019 Apr 17; 11:3295-3313.) Breast cancer patients to be treated may have atypical hyperplasia or in situ breast cancer. Cancers to be treated may be metastatic or localized and may be refractory to other treatments. Cancer patients suitable for treatment with the methods of the present disclosure include those being treated concomitantly with other therapies, such as endocrine therapy. Age-related illnesses that can be treated according to the methods of the present disclosure include, but are not limited to, diabetes, NAFLD, hypertension, obesity,

Afzheimers disease, Parkinson’s disease and AL8. In certain embodiments, the one or more mitochondrial proteins are selected from UQCRC2, UQCRB, CYC1, UQCRC1, BCKDHB, UQCRFS1, UQCRQ, and BCKDHA, such as UQCRC1, UQCRC2, or UQCRB, such as UQCRC1 or UQCRC2, such as UQCRC1.

In certain embodiments, inhibiting one or more mitochondrial proteins comprises administering a therapeutically effective amount of an inhibitor of the one or more mitochondrial proteins to a subject in need thereof.

In certain such embodiments, the inhibitor is hydrogen peroxide, epigallocatechin gallate, A1938, A1893, terpestacin, HDNT, 3-(l-(phenylamino)ethylidene)-chroman-2,4- dione, or a derivative of any of the foregoing, or a salt thereof. Epigallocatechin gallate has the following structure: . A1938 has the following structure: (Scientific Reports (2018) 8:12407). A1893 has the following structure: Oncology (2018)

52(1):241-251). Terpestacin has the following structure: . HDNT has the following structure: (Bioorg. Med. Chem. Lett.

(2011) 21(3): 1052-6). Derivatives of HDNT include, but are not limited to, those disclosed in J. Med. Chem. (2014) 57(19):7990-7998. 3-(l-(phenylamino)ethylidene)-chroman-2,4-dione has the following structure:

In other such embodiments, the inhibitor is a small molecule characterized by at least three of the following features, for example, at least four of the following features, preferably all five of the following features: at least 3 nitrogen atoms; at least 2 hydrogen bond donors; 2 to 12 carbon atoms; a molecular weight of about 100 to about 500 Da; and a ClogP value of about -1 to about -1.5. In certain such embodiments, the inhibitor is not metformin or phenformin. In certain embodiments, the small molecule contains at least 5 nitrogen atoms. In certain embodiments, the small molecule contains 2, 4, or 10 carbon atoms. In certain embodiments, the small molecule is characterized by a molecular weight of about 100 to about 500 Da, such as about 100 to about 400, about 100 to about 300, or about 100 to about 200 Da. In certain embodiments, the small molecule is characterized by a ClogP value of about -1 to about -1.5, such as about -1.0, about -1.1, about -1.2, about -1.3, about -1.4, or about -1.5. In certain embodiments, the inhibitor is not metformin or phenformin. In certain embodiments, the inhibitor is not a biguanide. In certain embodiments, the inhibitor is administered at a dose less than 100 mg/day, for example, less than 85 mg/day, preferably less than 50 mg/day.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference). In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1 -(2-hydroxy ethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1, 5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1- pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid acid salts.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et ah, “Molecular Cell Biology, 4th ed ”, W. H. Freeman & Co., New York (2000); Griffiths et ah, “Introduction to Genetic Analysis, 7th ed ”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et ah, “Developmental Biology, 6th ed ”, Sinauer Associates, Inc., Sunderland, MA (2000).

Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

“Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(0)0-, preferably alkylC(0)0-.

The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.

The term “Cx- _y ” or “Cx-C _y ”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci- ₆ alkyl group, for example, contains from one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term “amide”, as used herein, refers to a group wherein R ⁹ and R ¹⁰ each independently represent a hydrogen or hydrocarbyl group, or R ⁹ and R ¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R ⁹ , R ¹⁰ , and R ¹⁰ ’ each independently represent a hydrogen or a hydrocarbyl group, or R ⁹ and R ¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The term “carbamate” is art-recognized and refers to a group wherein R ⁹ and R ¹⁰ independently represent hydrogen or a hydrocarbyl group.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as used herein, refers to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably a carbocycle ring contains from 3 to 10 atoms, more preferably from 5 to 7 atoms. The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group -OCO2-.

The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

The term “ester”, as used herein, refers to a group -C(0)0R ⁹ wherein R ⁹ represents a hydrocarbyl group.

The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the poly cycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group -OSChH, or a pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae wherein R ⁹ and R ¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SCbH, or a pharmaceutically acceptable salt thereof.

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

The term “thioester”, as used herein, refers to a group -C(0)SR ⁹ or -SC(0)R ⁹ , wherein R ⁹ represents a hydrocarbyl.

The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity. The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers).

Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

“Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

The term “logP” means the logarithm of the octanol/water partition coefficient for a compound; it is a measure of the lipophilicity or hydrophobicity of the compound. The term “ClogP,” calculated log P, of a compound is the log P calculated based on the Pomona College Medicinal Chemistry program based on the sum of the compound’s non-overlapping molecular fragments.

EXAMPLES

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Decreased mitochondrial respiration by Seahorse analyses at uM doses of metformin.

Primary murine hepatocytes were isolated and 24 hrs later were treated with metformin (1 mM or 100 uM) or PBS for 3 or 24 hrs and oxygen consumption was measured using the Seahorse apparatus with the Cell Mito Stress Test Kit (Agilent). In Fig 1 A, it can be seen that 1 mM but not 100 uM of metformin inhibits respiration within 3 hours. However, by 24 hrs, as shown in Fig. IB, 100 uM metformin does inhibit mitochondrial respiration. This effect at 24 hrs is half-maximal at ~50 uM (not shown). This data shows that metformin does have an effect on mitochondrial respiration of hepatocytes at uM levels, but it is not immediate, requiring up to 24 hours to be effective.

Example 2: Affinity Enrichment of Mitochondrial Proteins on a Metformin Column.

Because metformin and phenformin are both clinically active in diabetes and place very different chemical groups on the same nitrogen atom of biguanide (Fig. 2), this site was used to attach a thiol group, which was subsequently used to couple to reactive iodoacetamide beads for metformin affinity chromatography. Hepatocyte extracts were prepared and eluted with either an SDS-containing buffer (Fig. 3) or with metformin, phenformin or a non-related drug, rosiglitazone (Fig. 4).

Interestingly, this resulted in the enrichment of several components of Complex III of the electron transport chain (ETC). Furthermore, several of these subunits (UQCRCR1, UQCRC2 and UQCRB) are in close contact with each other in the crystal structure of CIII (Iwata, S., Lee J.W., Okada K., Lee J.K., Iwata M., Rasmussen B., Link T.A., Ramaswamy S., Jap B.K.: Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome bcl Complex. Science. 1998 Jul 3;281(5373):64-71), so these proteins being enriched as a group is plausible. Importantly, no subunits of Complex I were enriched (middle box), nor was mGPD (GPD2, lowest box).

The specificity of binding of these same proteins to the metformin columns was examined by ligand-selective elution using metformin, phenformin, and a structurally unrelated drug, rosiglitazone. Mitochondrial holo-complexes were partially purified from hepatocyte mitochondria by standard chromatographic procedures (Ljungdahl PO, Pennoyer JD, Robertson DE, Trumpower BL.: Purification of highly active cytochrome bcl complexes from phylogenetically diverse species by a single chromatographic procedure. Biochim Biophys Acta. 1987 May 6;891(3):227-41). ³ H-metformin was purchased from Movarek, Inc. Analysis by Mass Spectrometry and HPLC indicated that this metformin was radiochemically pure (no other ³ H compounds in vial) but contained large amounts of cold metformin. Fractions that were >80% pure Complex I or Complex III were incubated with ³ H-metformin for 2 hrs, then the void (macromolecules) were separated from the inclusion volume (small molecules) on an acrylamide P6 gel filtration column. Complex III bound to ³ H-metformin but Complex 1 had minimal binding. As shown in Fig 5, UQCRC1, UQCRC2 and UQCRB are all eluted with 100 uM of metformin or phenformin but not eluted with PBS plus appropriate levels of DMSO or with rosiglitazone. This selective elution with the biguanide drugs supports the specificity of interactions between the Complex III proteins and metformin.

Example 3: Direct 3H-metformin binding to purified UQCRC1.

To approach the key target proteins within Complex III, in silico docking of metformin and phenformin was performed within Complex III. The analyses strongly suggested UQCRC1 as the most likely target of metformin and phenformin binding (not shown). Recombinant murine UQCRC1 were prepared in bacteria and purified, as well as a control subunit of COX7A2L. These were incubated for 2 hours with ³ H-metformin and then chromatographed on a P6 gel filtration column exactly as in Fig. 5.

Example 4; Measuring Kd of metformin and phenformin to Complex III and subcomplexes thereof.

Metformin and phenformin will be characterized by 'H NMR. Each will then be combined at various concentrations with a fixed, saturating amount of Complex III, thereby allowing definition of a binding event from the proton shifts in metformin (or phenformin). The concentration dependence of the characteristic shifts will allow derivation of a Kd for each of metformin and phenformin. This analysis will be repeated for subcomplexes of Complex III.

Example 5: Identification of a minimal subcomplex of Complex III that binds metformin.

A His-tagged version of UQCRC1 (e.g., a C-terminal His-tagged version) will be expressed in bacteria. The protein will be purified from the bacterial extracts using Ni ⁺⁺ affinity column chromatography and analyzed by SDS-PAGE for purity.

Gel -filtration chromatography and subsequently NMR will be used to study binding of UQCRC1 to metformin. If any binding is observed, NMR will be used to derive a Kd value for this interaction, as described in Example 4 above.

If binding is not observed or is weaker than for the whole Complex III, then the His- tagged UQCRC1 construct will be expressed along with an untagged version of UQCRC2; these two proteins interact tightly in the known crystal structure of Complex III (Iwata, S., Lee J.W., Okada K., Lee J.K., Iwata M., Rasmussen B., Link T.A., Ramaswamy S., Jap B.K.: Complete Structure of the 11-Subunit Bovine Mitochondrial Cytochrome bcl Complex. Science. 1998 Jul 3;281(5373):64-71). The UQCRC1 will be purified as described above. If UQCRC2 co-purifies with UQCRC1, as ascertained by SDS-PAGE, that will suggest that UQCRC1 and UQCRC2 are in a heterodimeric complex. If instead the data do not indicate metformin binding and/or protein complex formation, more subunits of Complex III will be expressed, up to and including the four other subunits observed in the affinity chromatography shown in Fig. 3.

Example 6: Hydrogen-Deuterium Exchange (HDX) Experiments.

Peptide coverage maps are generated by Mascot analysis of MS/MS experiments on intact Complex III or on individual recombinant proteins from Complex III under various HDX quench conditions. HDX experiments will be carried out as described previously [Feng, L., V. et ah, The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS Chem Biol, 2016, 11, 1313-21] using a fully automated system. Both Complex III or recombinant proteins (fully assembled Complex III purified from mouse liver or single subunits from this Complex expressed and purified from bacteria) will be tested and will be premixed with 50-fold excess of metformin allowed to form a complex on for several hours on ice prior to HDX analysis, to allow binding. For HDX analysis, 5 mΐ aliquots of 10 mM protein and protein-metformin complex will be mixed with 20 mΐ of D20-containing HDX buffer and incubated for a range of on exchange times from 10 sec to 1 hr (or longer if necessary) before quenching exchange with an acidic quench solution that will be optimized for Complex III or individual recombinant proteins from Complex III. Protease digestion will be performed in line with chromatography using an immobilized pepsin column. Mass spectra will be acquired on either a Q Exactive or Fusion Lumos Tribrid ETD Orbitrap mass spectrometer (Thermo Scientific) and HDX experiments for each pairwise comparison will be run separately under the same conditions. Differential exchange values are attained for each peptide at each time point using an intensity weighted centroid m/z method within HDX Workbench [Pascal, B.D., et al., HDX workbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom, 2012, 23, 1512-21]

Example 7: Identification of amino acids involved in binding of metformin to Complex III.

Preference for mutational analyses will be given to amino acids where hydrogen bonding is predicted to occur via R-group side chains, as opposed to the peptide backbone (all amino acid residues present the same amide hydrogens in the backbone). All chosen “candidate” amino acids will be mutated to an alanine in the bacterial protein(s). After protein production, binding of the different proteins will be done by gel filtration chromatography and NMR to determine specific binding affinities for metformin. After obtaining this binding data with mutants in these bacterial proteins, mammalian cellular systems will be studied.

Both primary murine hepatocytes and AML 12 immortalized murine hepatocytes will be used for these experiments. In both cases, both gain and loss of function experiments will be completed. The same mutants that decrease metformin binding in adenoviral expression vectors will be created. Viral vectors expressing both the wild-type and mutant alleles for a subunit such as UQCRC1, and others as the data suggest, will be injected into the tail vein of mice. Since the mitochondrial complexes contain subunit proteins in a fixed stoichiometry, expression of elevated levels of a mutant protein should displace the wild-type protein in the full complex. With each mutation, it will be important to determine whether the mutant proteins are expressed at the same levels as the wild-type protein, and that it is incorporated into mitochondria. Proper insertion into Complex III can be determined by analyses of this complex on Blue Native Gels (Wittig, T, Braun, H. & Schagger, H. Blue native PAGE. Nat Protoc 1, 418-428 (2006). https://doi.org/10.1038/nprot.2006.62). Example S: Effects of mutations on metformin activity in hepatocvtes.

This experiment will evaluate the effects of the mutations on metformin activity in hepatocytes at the doses studied above (20 uM to 100 uM). The metformin activities to be studied include respiration, activation of AMPK signaling (J Clin Invest. 2001;108(8):1167- 1174), secretion of GDF15 (Nature. 2020 Feb;578(7795):444-448. doi: 10.1038/s41586-019- 1911-y. Epub 2019 Dec 25. Erratum in: Nature. 2020 Feb 13) and secretion of glucose under the influence of glucagon (Yu Bl, Pugazhenthi S, Khandelwal RL.: Effects of metformin on glucose and glucagon regulated gluconeogenesis in cultured normal and diabetic hepatocytes. Biochem Pharmacol. 1994 Aug 30;48(5):949-54). These experiments will also explore elucidation of effects that are on-target and off-target. This need is acute since many different (and high) concentrations have been used in various experimental studies in diabetes, cancer and aging. Hence, the effects of treating hepatocytes for 24 hrs with 100 uM of metformin will be studied, and gene expression by RNA-seq will be analyzed. Signal transduction will be examined by analyzing the phospho-proteome in these cells by protein Mass Spectrometry, to assess on- and off-target effects at least with regard to this target.

Example 9: Genetic and physiological analyses of mice with altered metformin binding.

The loss of function mutation(s) which are insensitive to metformin will be installed into mice, using CRISPR technology. This will be a whole body change.

Mice that are unable to bind and respond to metformin via the mutation in the site mapped in Complex III, as described in Examples 7 and 8, will be rendered obese with a typical high fat diet (HFD). Obesity with insulin resistance usually develops by 10-12 weeks. The mice will then be treated with metformin added to the drinking water, according to well- established protocols (Madiraju AK et al: Metformin inhibits gluconeogenesis via a redox- dependent mechanism in vivo. Nat Med. 2018 Sep;24(9): 1384-1394. doi: 10.1038/s41591- 018-0125-4. Epub 2018 Jul 23). Diabetes in these mice will be determined by glucose and insulin tolerance tests. Further analysis, if needed, will be done by hyperinsulinemic- euglycemic clamps, the gold standard for examining insulin sensitivity. This method will be combined with a radiotracer on glucose to determine which tissues show altered insulin- sensitivity. Since phenformin is thought to utilize a very similar anti-diabetic mechanism as metformin, and also competes with metformin binding to CIII proteins (Fig 4), these physiological experiments will be repeated with phenformin. References:

Feng, L., V. Dharmarajan, E. Serrao, A. Hoyte, R.C. Larue, A. Slaughter, A. Sharma, M.R. Plumb, J.J. Kessl, J.R. Fuchs, F.D. Bushman, A.N. Engelman, P.R. Griffin and M. Kvaratskhelia, The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS Chem Biol, 2016, 11, 1313-21.

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INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EOUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.