EP4665382A2 - Compositions comprising an mtor inhibitor for use in treating diseases and disorders associated with leptin resistance and obesity - Google Patents

Abstract

The present invention provides compositions comprising mTOR inhibitors and their use for sensitizing cells to leptin and for treating or preventing diseases or disorders associated with leptin deficiency or leptin resistance.

Description

COMPOSITIONS COMPRISING AN MTOR INHIBITOR FOR USE IN TREATING DISEASES AND DISORDERS ASSOCIATED WITH LEPTIN RESISTANCE AND OBESITY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/484,756, filed February 14, 2023 which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Leptin, a hormone produced by adipose tissue, is the afferent signal in a negative feedback loop that maintains homeostatic control of food intake and body weight. Leptin modulates the activity of hypothalamic neurons including POMC and AGRP neurons that coexpress the leptin receptor. While leptin potently reduces food intake and body weight in lean animals, there is a disruption of leptin signaling in diet induced obese animals with hyperleptinemia and a resistance to leptin’s effect. To date, the underlying mechanisms leading to leptin resistance are unknown and there is no means for restoring leptin sensitivity.

[0003] There is thus a need in the art for compositions and methods for compositions to treat diseases and disorders associated with leptin resistance including obesity. The present invention addresses this unmet need in the art.

SUMMARY OF THE INVENTION

[0004] In one embodiment, the invention relates to a method of sensitizing cells to leptin, the method comprising contacting the cells with at least one mTOR inhibitor.

[0005] In one embodiment, the mTOR inhibitor is a small molecule, a small interfering RNA (siRNA), shRNA, a microRNA, a guide RNA, a microRNA, a gapmer, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, or a peptide.

[0006] In one embodiment, the mTOR inhibitor is rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, or a derivative thereof. [0007] In one embodiment, the cells are from a subject who has been diagnosed as having leptin resistance or a disease or disorder associated therewith, or who is at risk of developing a disease or disorder associated with leptin resistance.

[0008] In one embodiment, the disease or disorder is obesity or a co-morbidity thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity-related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes or type 2 diabetes.

[0009] In one embodiment, the method further comprises administering leptin, a leptin derivative, or a leptin receptor agonist in combination with the mTOR inhibitor.

[0010] In one embodiment, the leptin, leptin derivative, or leptin receptor agonist is administered concurrently with the mTOR inhibitor.

[0011] In one embodiment, the leptin is administered sequentially with the mTOR inhibitor.

[0012] In one embodiment, the invention relates to a method of treating or preventing a disease or disorder associated with leptin resistance or leptin deficiency in a subject in need thereof, the method comprising administering an effective amount of at least one mTOR inhibitor to a subject in need thereof.

[0013] In one embodiment, the mTOR inhibitor is a small molecule, a small interfering RNA (siRNA), shRNA, a microRNA, a guide RNA, a micro RNA, a gapmer, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, or a peptide.

[0014] In one embodiment, the mTOR inhibitor is rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, or a derivative thereof.

[0015] In one embodiment, the subject has been diagnosed as having leptin resistance or a disease or disorder associated therewith, or is at risk of developing a disease or disorder associated with leptin resistance.

[0016] In one embodiment, the disease or disorder is obesity or a co-morbidity thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity-related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes or type 2 diabetes.

[0017] In one embodiment, the method further comprises administering leptin, a leptin derivative, or a leptin receptor agonist, in combination with the mTOR inhibitor.

[0018] In one embodiment, the leptin, leptin derivative or leptin receptor agonist antibody is administered concurrently with the mTOR inhibitor.

[0019] In one embodiment, the leptin, leptin derivative or leptin receptor agonist is administered sequentially with the mTOR inhibitor.

[0020] In one embodiment, the invention relates to a pharmaceutical composition comprising a combination of at least one mTOR inhibitor and leptin, a leptin derivative, or a leptin receptor agonist.

[0021] In one embodiment, the mTOR inhibitor is rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, or a derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0023] Figures 1 A through IT depict exemplary experimental data demonstrating that rapamycin reduces food intake, fat mass, fat-to-lean ratio in DIO mice but not in WT-Chow or ob/ob. WT-D1O: DIO mice were treated with RAP or VEH daily for 14 days. (Figure 1A) Cumulative food intake, and (Figure IB) Weight, (Figure 1C) A Mass of weight, fat and lean tissue and (Figure ID) A Fat/Lean Ratio, (n = 11, 11 for each group; two-way ANOVA, with Sidak’s multiple comparisons for Figure 1 A - Figure 1C; Two-tailed Student’s t-tests for Figure ID). WT-DIO Prolonged treatment: DIO mice were treated with daily 2 mg/kg i.p. rapamycin (RAP) or vehicle (VEH) for 10 weeks. (Figure IE) Cumulative food intake, and (Figure IF) Weight, (Figure 1G) A Mass of weight, fat and lean tissue, (Figure 1H) A Fat/Lean Ratio (n = 8, 10 respectively; Two-way ANOVA, with Sidak’s multiple comparisons for Figure IE - Figure 1G; Two-tailed Student’s t-tests for Figure 1H. WT-Chow: Chow-fed lean mice were treated with daily RAP plus 600 ng/hr leptin (LEP), RAP plus VEH, LEP plus VEH, VEH plus VEH for 14 days. (Figure II) Cumulative food intake, and (Figure 1 J) Weight, (Figure IK) A Mass of weight, fat and lean tissue and (Figure IL) Fat/lean Ratio at day 14. (n = 12, 14, 14, 13 for RAP+LEP, RAP, LEP, VEH, respectively; Two-way ANOVA with Tukey’s multiple comparisons for Figure II - Figure IK; One-way ANOVA with Tukey’s multiple comparisons Figure IL). Aged-Chow: Aged, chow-fed mice (more than 16 months old) were treated with daily i.p. injections of RAP (2 mg/kg) plus LEP (600 ng/hr) vs. VEH plus LEP (600 ng/hr) for 14 days. (Figure IM) Cumulative food intake, and (Figure IN) Weight, (Figure 10) A Mass of weight, fat and lean tissue and (Figure IP) A Fat/Lean Ratio (a.u.) of (n = 6, 7 for RAP+LEP, LEP, respectively; Two-way ANOVA, with Sidak’s multiple comparisons for Figure IM - Figure. 10; Two-tailed Student’ s t tests for Figure IP). OB-Chow: Chow-fed, ob b mice were treated with daily i.p. injections of 2 mg/kg rapamycin (RAP) plus 300 ng/hr leptin (LEP), RAP plus vehicle (VEH), VEH plus LEP, or VEH plus VEH for 14 days. (Figure IQ) Cumulative food intake, (Figure 1R) Weight, (Figure IS) A Mass of weight, fat and lean tissue and (Figure IT) A Fat/Lean Ratio (a.u.), (n=5, 9, 7, 8 for RAP+LEP, RAP, LEP, VEH, respectively; Two- way ANOVA with Tukey’s multiple comparisons for Figure IQ - Figure IS; One-way ANOVA with Tukey’s multiple comparisons for Figure IT).

[0024] Figures 2A through 2N depict exemplary experimental data demonstrating that rapamycin increases the response to exogenous leptin in DIO. (Figure 2A) Schematic of DIO mice that were treated with daily i.p. injections of 2 mg/kg Rapamycin (RAP) vs. vehicle (VEH) followed by a leptin sensitivity test. Each group was treated with i.p. injections of 2 mg/kg leptin (LEP) twice a day for 3 days followed by i.p. injections of vehicle (VEH) twice a day for 3 days. (Figure 2B) Plasma leptin in DIO mice after 3-week RAP vs. VEH treatment prior to a leptin sensitivity test (n = 16, 14 for RAP, VEH, respectively; two-tailed Student’s t-tests). (Figure 2C) Cumulative food intake and (Figure 2D) weight during leptin sensitivity test (n = 8, 8 for each group; two-way ANOVA, with Sidak’s multiple comparisons). (Figure 2E) Schematic of DIO mice that were treated with daily i.p. injections of RAP vs. VEH. The group of mice treated with VEH were pairfed to the group of mice treated with RAP for 5 weeks. Leptin sensitivity test was conducted after 5-week treatment. For the leptin sensitivity test, each group of mice were treated with i.p. injections of 2 mg/kg LEP twice daily for 3 days followed by i.p. injections of VEH twice daily for 3 days. Both groups of mice had adlibitum access to food during the test. (Figure 2F) Plasma leptin in DIO mice after 3-week RAP vs. Pairfed- VEH treatment prior to the leptin sensitivity test (n = 6, 6 for each group; Two-tailed Student’s t-tests). (Figure 2G) Cumulative food intake and (Figure 2H) Weight of RAP vs. pairfed VEH-treated DIO mice were treated with LEP (n = 6, 6; Two-tailed Mann-Whitney tests). (Figure 21) Schematic of DIO mice that either remained on HFD with daily i.p. VEH-inj ections or transferred to chow-feed and given daily RAP vs. VEH injections. (Figure 2J) Weekly caloric food intake (Figure 2K) cumulative calorie food intake, (Figure 2L) Weight, (Figure 2M) A Mass of weight, fat and lean tissue, and (Figure 2N) A Fat/Lean Ratio (n=8, 7, 8 respectively; Two-way ANOVA, with Tukey’s multiple comparisons for Figure 2J - Figure 2M; One-way ANOVA, with Tukey’s multiple comparisons for Figure 2N).

[0025] Figures 3A through 3H depict exemplary experimental data demonstrating that exogenous leptin mitigates rapamycin-induced hyperglycemia. Adlibitum glucose levels in the WT-Chow (Figures 3A), WT-DIO (Figures 3B), OB-Chow (Figures 3C), DB-Chow (Figures 3D). (WT-Chow: n = 10, 8, 11, 10 for each group; WT-DIO: n = 17, 18, 17, 16 for each group; OB-Chow: n = 7, 6, 6, 5 for each group; One-way ANOVA, with Holm-Sidak’s multiple comparisons for Figures 3A - Figures 3C. DB-Chow: n = 8, 7 for each group; Two-tailed Student’s t tests for Figures 3D). (Figures 3E) Time-course of glucose levels from glucose tolerance test (GTT) in WT-Chow mice treated with RAP plus 600 ng/hr LEP, RAP plus VEH, LEP plus VEH or VEH plus VEH for 7 days and (Figures 3F) quantification of the area under curve (n = 10, 10, 10, 9, respectively; One-way ANOVA, with Holm-Sidak’s multiple comparisons). (Figures 3G) Time-course of glucose levels in GTT in DIO mice treated with RAP plus 600 ng/hr LEP, RAP plus VEH, LEP plus VEH and VEH plus VEH for 3 weeks and (Figures 3H) quantification of the area under curve (n = 5, 6, 4, 4, respectively; One-way ANOVA, with Holm-Sidak’s multiple comparisons). All error bars represent mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001.

DETAILED DESCRIPTION

[0026] The present invention relates generally to compositions comprising mechanistic target of rapamycin (mTOR) inhibitors and the use thereof for treating a disease or disorder associated with leptin resistance. The present invention further provides methods for use of the compositions comprising an mTOR inhibitor or a combination of an mTOR inhibitor and leptin for treating or preventing a disease or disorder associated with leptin resistance. In some embodiments, the mTOR inhibitor is rapamycin (also called sirolimus), everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, or a derivative thereof. Therefore, in some embodiments, the invention relates to the use of rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, or a derivative thereof, or in combination with leptin, a leptin derivative or a leptin receptor agonist for treating or preventing a disease or disorder associated with leptin resistance.

[0027] Diseases and disorders associated with leptin resistance that can be treated using the compositions of the invention include, but are not limited to, obesity and co-morbidities thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity-related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes and type 2 diabetes.

Definitions

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0029] As used herein, each of the following terms has the meaning associated with it in this section.

[0030] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0031] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations for example of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0032] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

[0033] “Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0034] “Antibody fragment” or “fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, singlechain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0035] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0036] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0037] A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0038] An “effective amount” or “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound. [0039] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

[0040] The term “microarray” refers broadly to both “DNA microarrays” and “DNA chip(s),” and encompasses all art-recognized solid supports, and all art-recognized methods for affixing nucleic acid molecules thereto or for synthesis of nucleic acids thereon.

[0041] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0042] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0043] “Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[0044] A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0045] “Promoter” as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.

[0046] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0047] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0048] As used herein, “treating a disease or disorder” means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient.

[0049] The phrase “therapeutically effective amount,” as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases.

[0050] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

[0051] In one embodiment, the invention provides a generic concept for inhibiting mTOR. In one embodiment, the inhibitor is selected from the group consisting of a small molecule, a small interfering RNA (siRNA), shRNA, a microRNA, a guide RNA, a micro RNA, a gapmer, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, and a peptide.

[0052] One skilled in the art will appreciate, based on the disclosure provided herein, that one way to decrease the mRNA and/or protein levels of mTOR in a cell is by reducing or inhibiting expression of the nucleic acid encoding mTOR. Thus, the protein level of mTOR in a cell can also be decreased using a molecule or compound that inhibits or reduces gene expression such as, for example, siRNA, an antisense molecule or a ribozyme. However, the invention should not be limited to these examples.

[0053] Inhibition of mTOR, can be assessed using a wide variety of methods, including those disclosed herein, as well as methods known in the art or to be developed in the future. That is, the person of skill in the art would appreciate, based upon the disclosure provided herein, that inhibiting the level or activity of a gene, or gene product, can be readily assessed using methods that assess the level of a nucleic acid encoding a gene product (e.g., mRNA), the level of polypeptide gene product present in a biological sample, the activity of polypeptide gene product present in a biological sample, or combinations thereof. Small molecule Inhibitors

[0054] One of skill in the art would readily appreciate, based on the disclosure provided herein, that an inhibitor of mTOR encompasses a chemical compound that modulates the level or activity of a gene, or gene product. Additionally, an inhibitor of mTOR encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts.

[0055] When the inhibitor of the invention is a small molecule or a derivative thereof, a small molecule inhibitor may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

[0056] Exemplary small molecule mTOR inhibitors include, but are not limited to rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, non-rapalog derived inhibitors, and derivatives thereof. Exemplary non-rapalog derived inhibitors that can be used in the compositions and methods of the invention include, but are not limited to, PI-103, BEZ235, XL765, PP242, AZD8055, OSI-027 and INK128 as described in Zheng et al. (Mol Cell Pharmacol. 2015; 7(2): 15-20).

[0057] Methods of identifying, or generating derivatives of, small molecule inhibitors are known in the art. Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

[0058] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.

Nucleic Acid Inhibitors

[0059] In one embodiment, the composition of the invention comprises one or more antisense nucleic acid molecules. For example, in one embodiment, the one or more antisense nucleic acid molecules are specific for targeting mTOR. Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of mTOR mRNA. When present in a cell, antisense oligonucleotides hybridize to an existing mRNA molecule and inhibit translation into a gene product or promote degradation of the RNA molecule. Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art, as are methods of expressing an antisense oligonucleotide in a cell. The methods of the invention include the use of antisense oligonucleotide to diminish the amount of mTOR activity or mTOR mRNA. Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art. As an example, an antisense oligonucleotide can be synthesized to be between about 10 and about 100, more preferably between about 15 and about 50 nucleotides long. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides.

[0060] Similarly, the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art.

[0061] Alternatively, inhibition of mTOR can be accomplished through the use of an siRNA, shRNA, antisense oligonucleotide or ribozyme. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.

[0062] In one embodiment, siRNA is used to decrease the level of mTOR is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe a chemical modification to siRNAs that aids in systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216.

Therefore, the present invention also includes methods of decreasing levels of mTOR at the mRNA or protein level using RNAi technology.

[0063] In certain embodiments, the modulators described herein comprise short hairpin RNA (shRNA) molecules. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.

[0064] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA, shRNA, gapmer or antisense molecule, inhibits mTOR, a regulator thereof, or an activator thereof.

Peptide inhibitors

[0065] In one embodiment, the composition of the present invention comprises an isolated peptide inhibitor of mTOR. [0066] The peptide of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0067] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0068] The peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0069] A peptide or protein of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of inhibiting mTOR.

[0070] A peptide or protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).

[0071] Cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, nonamino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0072] It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

[0073] The peptides and fusion proteins of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

Antibody Inhibitors

[0074] In some embodiments, the invention relates to compositions comprising at least one inhibitory antibody, or fragment thereof, specific for binding to mTOR. In one embodiment, the anti-mTOR antibody is a neutralizing antibody.

[0075] As used herein, the term “antibody” or “immunoglobulin” refers to proteins (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be tetrameric, comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length Ig molecule contains at least two binding sites for a specific target or antigen.

[0076] An anti-mTOR neutralizing antibody, or antigen-binding fragment thereof, includes, but is not limited to a polyclonal antibody, a monoclonal fusion proteins, antibodies or fragments thereof , chimerized or chimeric fusion proteins, antibodies or fragments thereof , humanized fusion proteins, antibodies or fragments thereof , deimmunized humfusion proteins, antibodies or fragments thereof , fully humfusion proteins, antibodies or fragments thereof , single chain antibody, single chain Fv fragment (scFv), Fv, Fd fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, diabody or antigen- binding fragment thereof, minibody or antigenbinding fragment thereof, triabody or antigen- binding fragment thereof, domain fusion proteins, antibodies or fragments thereof , camelid fusion proteins, antibodies or fragments thereof , dromedary fusion proteins, antibodies or fragments thereof , phage-displayed fusion proteins, antibodies or fragments thereof , or antibody, or antigen- binding fragment thereof, identified with a repetitive backbone array (e.g. repetitive antigen display).

[0077] As used throughout the present disclosure, the term “antibody” further refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that is generated by any one of a variety of methods that are known in the art and described herein. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a deimmunized human antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, nonhuman primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.

Genetic modification

[0078] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor of mTOR, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0079] The desired polynucleotide can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora of vectors which are readily available and/or well-known in the art. For example, a desired polynucleotide of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal viruse, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0080] In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0081] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.

[0082] For expression of the desired polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0083] Additional promoter elements, i.e., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0084] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0085] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001). The promoters employed may be constitutive, tissuespecific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous. [0086] A promoter sequence exemplified in the experimental examples presented herein is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue specific promoter, which promoter is active only in a desired tissue.

[0087] In order to assess the expression of the vector, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neo and the like.

[0088] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0089] Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of siRNA polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0090] In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. It is readily understood that the introduction of the expression vector comprising the polynucleotide of the invention yields a silenced cell with respect to a regulator.

[0091] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0092] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. [0093] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid nanoparticles. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0094] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0095] Any DNA vector or delivery vehicle can be utilized to transfer the desired polynucleotide to a cell in vitro or in vivo. In the case where a non-viral delivery system is utilized, a preferred delivery vehicle is a liposome. The above-mentioned delivery systems and protocols therefore can be found in Gene Targeting Protocols, 2ed., pp 1-35 (2002) and Gene Transfer and Expression Protocols, Vol. 7, Murray ed., pp 81-89 (1991).

[0096] “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. Methods

[0097] The invention provides methods and systems for administering the mTOR inhibitory compositions to sensitize cells to leptin. The invention also provides methods and systems for administering the mTOR inhibitory compositions to treat, ameliorate, inhibit, or prevent diseases or disorders associated with leptin deficiency or a disease or disorder associated with leptin resistance. Exemplary diseases or disorders that can be treated or prevented using the mTOR inhibitor compositions of the invention include, but are not limited to, obesity and comorbidities thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity- related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes and type 2 diabetes.

[0098] In one embodiment, the present invention provides a method comprising administering a mTOR inhibitor of the invention to a subject having leptin deficiency or a disease or disorder associated with leptin resistance.

[0099] In an embodiment, the term “administering” means that the compounds of the present invention are introduced into a subject using one or more known routes of administration. In some embodiments, the compositions of the invention are administrated by way of injection.

[0100] In one embodiment, one or more mTOR inhibitor of the invention is coadministered with one or more additional therapeutic agent or adjuvant. In one embodiment, one or more mTOR inhibitor of the invention is co-administered with leptin, a leptin derivative, or a leptin receptor agonist. “Co-administration” as used herein is understood as administration of one or more agents to a subject such that the agents are present and active in the subject at the same time. Co-administration does not require a preparation of an admixture of the agents or simultaneous administration of the agents.

[0101] One of skill in the art will appreciate that a mTOR inhibitor and leptin, a leptin derivative, or a leptin receptor agonist can be administered singly or in any combination thereof. Further, a mTOR inhibitor and leptin, a leptin derivative, or a leptin receptor agonist can be administered singly or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before, and/or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that a mTOR inhibitor and leptin, a leptin derivative, or a leptin receptor agonist can be used to prevent or treat diseases or disorders associated with leptin deficiency or a disease or disorder associated with leptin resistance.

[0102] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease associated with leptin deficiency or a disease or disorder associated with leptin resistance, encompasses administering to a subject a mTOR inhibitory composition as a preventative measure against the development of, or progression of, a disease associated with leptin deficiency or a disease or disorder associated with leptin resistance. As more fully discussed elsewhere herein, methods of modulating the level or activity of a gene, or gene product, encompass a wide plethora of techniques for modulating not only the level and activity of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both.

[0103] Additionally, as disclosed elsewhere herein, one skilled in the art would understand, once armed with the teaching provided herein, that the present invention encompasses methods of treating, or preventing, a wide variety of diseases associated with a disease associated with leptin deficiency or a disease or disorder associated with leptin resistance, where inhibiting the level or activity of a mTOR gene, or gene product treats or prevents the disease. Various methods for assessing whether a disease is associated with a disease associated with leptin deficiency or a disease or disorder associated with leptin resistance are known in the art. Further, the invention encompasses treatment or prevention of such diseases discovered in the future.

[0104] The invention encompasses administration of an inhibitor of a mTOR gene, or gene product. To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate inhibitor composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0105] The optimal effective amount of the compositions can be determined empirically and will depend on the type and severity of the disease, route of administration, disease progression and health, mass and body area of the individual. Such determinations are within the skill of one in the art. The effective amount can also be determined based on in vitro complement activation assays. Examples of dosages of molecules which can be used for methods described herein include, but are not limited to, an effective amount within the dosage range of any of about 0.01 mg/kg to about 300 mg/kg, or within about 0.1 mg/kg to about 40 mg/kg, or with about 1 mg/kg to about 20 mg/kg, or within about 1 mg/kg to about 10 mg/kg. In some embodiments, the amount of composition administered to an individual is about 10 mg to about 500 mg per dose, including for example any of about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 500 mg, about 500 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 200 mg, about 200 mg to about 300 mg, about 300 mg to about 400 mg, or about 400 mg to about 500 mg per dose.

[0106] The compositions may be administered in a single daily dose, or the total daily dose may be administered in divided dosages of two, three, or four times daily. The compositions can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The compositions may also be administered in a sustained release formulation, such as in an implant which gradually releases the composition for use over a period of time, and which allows for the composition to be administered less frequently, such as once a month, once every 2-6 months, once every year, or even a single administration. The sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, and the like) may be administered by injection or surgical implantation in various locations.

[0107] Dosage amounts and frequency will vary according the particular formulation, the dosage form, and individual patient characteristics. Generally speaking, determining the dosage amount and frequency for a particular formulation, dosage form, and individual patient characteristic can be accomplished using conventional dosing studies, coupled with appropriate diagnostics.

Pharmaceutical Compositions and Formulations

[0108] The invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention. Such a pharmaceutical composition may consist of at least one mTOR inhibitor composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one mTOR inhibitor composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. In some embodiments, the composition comprises a combination of at least one mTOR inhibitor composition and leptin, a leptin derivative, or a leptin receptor agonist. The compound or conjugate of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0109] In one embodiment, the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

[0110] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0111] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. A composition useful within the methods of the invention may be directly administered to the skin, vagina or any other tissue of a mammal. Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.

[0112] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0113] As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0114] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0115] In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0116] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.

[0117] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e g., other analgesic agents.

[0118] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0119] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid. [0120] The composition preferably includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0121] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n -propyl -para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0122] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0123] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0124] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0125] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0126] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0127] Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0128] The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0129] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention 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 subject, composition, and mode of administration, without being toxic to the subject.

[0130] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition 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.

[0131] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease in a subject.

[0132] In one embodiment, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.

[0133] Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0134] In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., a drug used for treating the same or another disease as that treated by the compositions of the invention) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. [0135] In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0136] The term “container” includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.

[0137] Routes of administration of any of the compositions of the invention include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intracerebral, epidural, intracerebroventricular, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0138] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Unit Dosages, Articles of Manufacture, and Kits

[0139] Also provided are unit dosage forms of compositions, each dosage containing from about 0.01 mg to about 50 mg, including for example any of about 0.1 mg to about 50 mg, about 1 mg to about 50 mg, about 5 mg to about 40 mg, about 10 mg to about 20 mg, or about 15 mg of the targeted molecule. In some embodiments, the unit dosage forms of targeted molecule composition comprise about any of 0.01 mg-0.1 mg, 0.1 mg-0.2 mg, 0.2 mg-0.25 mg, 0.25 mg- 0.3 mg, 0.3 mg-0.35 mg, 0.35 mg-0.4 mg, 0.4 mg-0.5 mg, 0.5 mg-1.0 mg, 10 mg-20 mg, 20 mg- 50 mg, 50 mg-80 mg, 80 mg-100 mg, 100 mg-150 mg, 150 mg-200 mg, 200 mg-250 mg, 250 mg-300 mg, 300 mg-400 mg, or 400 mg-500 mg targeted inhibitor molecule. In some embodiments, the unit dosage form comprises about 0.25 mg targeted molecule. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for an individual, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. These unit dosage forms can be stored in suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed.

[0140] The present invention also provides kits comprising compositions (or unit dosages forms and/or articles of manufacture) described herein and may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for performing any methods described herein.

[0141] In one embodiment, an article of manufacture containing mTOR inhibitor compositions useful for the treatment of the disorders described above is provided. In one embodiment, the article of manufacture contains an mTOR inhibitor composition and leptin, a leptin derivative, or a leptin receptor agonist. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for preventing or treating, for example, diseases or disorders associated with leptin deficiency or a disease or disorder associated with leptin resistance and may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

EXPERIMENTAL EXAMPLES

[0142] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0143] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, point out specific embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1:

[0144] In extensive preliminary studies, several potent mTOR ligands were found to be regulated by leptin in leptin-sensitive, but not in leptin-resistant animals. Thus, without being bound by theory, it was hypothesized that leptin resistance might be a result of increased mTOR signaling in leptin sensitive neurons in diet-induced-obese (DIO) mice. Indeed, it was found that rapamycin (RAP), an mTOR inhibitor, potently reduces food intake and fat mass in DIO mice, but not in leptin deficient ob/ob mice (Figure 1). This suggests that rapamycin restores leptin signaling. Furthermore, in electrophysiologic studies in neuronal slice preparations, reduced leptin signaling was found in POMC neurons with augmented mTOR signaling resulting from a knockout of Tscl, an endogenous Tor inhibitor. However, treatment of these cells with rapamycin restored leptin signaling. Consistent with this, mice with an ablation of POMC neurons become obese but do not lose weight on rapamycin, similar to ob/ob mice. Thus, without being bound by theory, it was hypothesized that leptin resistance is the result of increased mTOR activity in POMC neurons. It was further demonstrated that treating DIO animals with leptin together with rapamycin leads to a significant improvement in metabolic disease relative to the use of each agent alone; and that rapamycin reduced both food intake and body weight to a greater extent in DIO mice transferred to a standard chow diet compared to those treated with vehicle alone (Figure 2). Finally, it was demonstrated that exogenous leptin mitigates rapamycin- induced hyperglycemia (Figure 3).

The Materials and Methods used for the Experiments are now described.

Animals

[0145] Wild-type mice (#000664) and db/db mice (#000697) were acquired from Jackson Lab and ob/ob were Fl, bred in lab, from a cross between male ob/ob and female ob/+ (#000632). All crosses were bred in lab from the above animals. Animals were kept at ambient temperature and humidity-controlled housing with a 12hr light-dark cycle (lights on at 7am and off at 7pm) and on a standard-chow diet (PicoLab® Rodent Diet 205053) unless otherwise indicated. Diet-induced-obese (DIO) wild-type mice were fed on high-fat diet (HFD, Research Diets, Cat# DI 2492, Rodent Diet With 60 kcal% Fat) starting at ~6 weeks old and used for experiments starting at 24 weeks old (fed on HFD for at least 18 weeks). Aged wild-type mice (~15 months old, #000664) were acquired from Jackson Lab. All experiments were conducted according to AAALAC approved animal protocols #18050, #18051, #22012 and #21064. Males were used throughout. All experiments were internally sex and age matched.

Pharmacological administration [0146] Recombinant mouse leptin (R&D 498-OB-05M) was dissolved in PBS and injected intraperitoneally (i.p.) or delivered via a subcutaneous osmotic pump (Alzet Cat# 2002, 2004, or 2006). Osmotic pumps were filled and calibrated using the manufacturer’s instructions. They were inserted dorsally under the skin of an isoflurane-anesthetized mouse using sterile surgery techniques. Rapamycin (LC Laboratories, Cat# 53123-88-9) was first dissolved in DMSO at 200 mg/ml, then diluted in 5% PEG 400 and 5% Tween 80 (in PBS) to a final concentration of 0.5 mg/ml. I.p. injections were done at indicated concentrations using insulin syringes (Beckton Dickinson, Cat# 324911).

Magnetic resonance imaging (MRI)

[0147] Body fat mass was measured by MRI using Echo-MRI 100H (EchoMRI, LL). Body fat percentage was calculated by dividing fat mass over total body mass. Lean mass was calculated by subtracting fat mass from total body mass.

Leptin sensitivity test

[0148] Animals were administered with leptin (at indicated doses) or PBS either by i.p. injections every 12 hours or via osmotic pumps implanted 1 day prior to the start of the experiment. Food intake and body weight were measured through the course of treatment as indicated.

Glucose tolerance test

[0149] Animals fasted overnight were i.p. injected 5-20% glucose dissolved in PBS as indicated. Total amount of glucose injected was based on lean mass times the dosage. Dosage used for each experiment and cohort was indicated in the manuscript. Blood glucose in the GTT assay and ad libitum-fed conditions were measured by tail vein sampling using a Breeze2 glucometer (Bayer SKU: breeze2meter UPC: 301931440010). For the GTT in DIO mice, the groups of mice receiving 600 ng/hr leptin were i.p. injected 1 mg/kg leptin 1 hour prior to the glucose injection, while the other groups of mice were i.p. injected PBS 1 hour prior to the GTT started.

Leptin ELISA [0150] Blood was collected retro-orbitally using EDTA coated capillaries (Drummond Calibrated Micropipettes Glass Capillaries with EDTA lOOpl, Cat# 2-000- 100-D). Samples were spun for 20 minutes at 4°C and supernatant collected as plasma and frozen immediately in liquid nitrogen and stored at -80°C in screw cap tubes. Plasma leptin was measured by ELISA (Alpco, Cat# 22-LEPMS-E01) according to the manufacturer's protocol.

Pairfeeding

[0151] Pairfeeding was conducted by measuring the daily or weekly food intake of the group which shows lower food intake and feeding that same amount to the other group. Animals were single housed during experiments. Food intake and body weight were measured daily or weekly using an Ohaus Scale. For Figure 2e-3h, during weeks 19-23 (21 days), vehicle treated animals were fed, daily, what the rapamycin treated animals had eaten the preceeding 24hours.

Statistics and reproducibility

[0152] Statistical analyses were conducted using GraphPad Prism 9.0. Throughout the paper, values were reported as mean ± SEM (error bar). Each statistical test performed was denoted in the figure legends. P-values for pair-wise comparisons were obtained using two-tailed student’s t-test. P-values for two independent group comparisons were either obtained using two- tailed student’s t-test or nonparametric Mann-Whitney test based on data distributions. P-values for multiple group comparisons were conducted using one-way or two-way ANOVA (with repeated measures when possible) based on the number of factors and corrected for multiple comparisons using Fisher’s LSD test, Tukey’s test or Sidak’s test, indicated in the figure legends. The experiments were not randomized. Data for each experiment were repeated with at least two different cohorts. Representative trial data or pooled data from multiple cohorts were used for conducting statistics and plotting figures. The investigators were not blinded to allocation during experiments and outcome assessments.

[0153] A number of leptin deficiency syndromes treatable with leptin replacement therapy have been identified including leptin gene mutations, lipodystrophy and hypothalamic amenorrhea. In addition, this work has advanced our understanding of the pathogenesis of obesity. Most obese subjects are leptin resistant establishing that obesity is a hormone resistance syndrome. This research is being translated into potential new therapies for obesity and for treatment of Type 1 and Type 2 diabetes.

[0154] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No. 046531-5029-00WO CLAIMS

1. A method of sensitizing cells to leptin, the method comprising contacting the cells with at least one mTOR inhibitor.

2. The method of claim 1, wherein the mTOR inhibitor is selected from the group consisting of a small molecule, a small interfering RNA (siRNA), shRNA, a microRNA, a guide RNA, a micro RNA, a gapmer, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, and a peptide.

3. The method of claim 1, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, and a derivative thereof.

4. The method of claim 1, wherein the cells are from a subject who has been diagnosed as having leptin resistance or a disease or disorder associated therewith, or who is at risk of developing a disease or disorder associated with leptin resistance.

5. The method of claim 4, wherein the disease or disorder is selected from the group consisting of obesity and co-morbidities thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity-related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes and type 2 diabetes

6. The method of any one of claims 1-5 further comprising administering leptin, a leptin derivative, or a leptin receptor agonist in combination with the mTOR inhibitor.

7. The method of claim 6, wherein the leptin, leptin derivative, or leptin receptor agonist is administered concurrently with the mTOR inhibitor.

8. The method of claim 6, wherein the leptin is administered sequentially with the mTOR inhibitor.

9. A method of treating or preventing a disease or disorder associated with leptin resistance or leptin deficiency in a subject in need thereof, the method comprising administering an effective amount of at least one mTOR inhibitor to the subject.

10. The method of claim 9, wherein the mTOR inhibitor is selected from the group consisting of a small molecule, a small interfering RNA (siRNA), shRNA, a microRNA, a guide RNA, a micro RNA, a gapmer, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, and a peptide.

11. The method of claim 9, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, everolimus, temsirolimus, ridaforolimus, Torin-1, a non-rapalog derived inhibitor, and a derivative thereof.

12. The method of claim 9, wherein the subject has been diagnosed as having leptin resistance or a disease or disorder associated therewith, or is at risk of developing a disease or disorder associated with leptin resistance.

13. The method of claim 12, wherein the disease or disorder is selected from the group consisting of obesity and co-morbidities thereof, insulin resistance, high cholesterol, hypertension, atherothrombosis, obesity-related cardiovascular disease, myocardial injury, metabolic- and inflammatory-mediated injury, leptin gene mutations, lipodystrophy, hypothalamic amenorrhea, hyperglycemia, type 1 diabetes and type 2 diabetes.

14. The method of any one of claims 9-13 further comprising administering leptin, a leptin derivative, or a leptin receptor agonist, in combination with the mTOR inhibitor.

15. The method of claim 14, wherein the leptin, leptin derivative or leptin receptor agonist antibody is administered concurrently with the mTOR inhibitor.

16. The method of claim 14, wherein the leptin, leptin derivative or leptin receptor agonist is administered sequentially with the mTOR inhibitor.

17. A pharmaceutical composition comprising a combination of at least one mTOR inhibitor and leptin, a leptin derivative, or a leptin receptor agonist.

18. The pharmaceutical composition of claim 17, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, everolimus, temsirolimus, ridaforolimus, Torin- 1, a non-rapalog derived inhibitor, and a derivative thereof.