US 2012019687OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0196870 A1 Arbiser (43) Pub. Date: Aug. 2, 2012

(54) COMBINATION THERAPY AND CANCER A6IP3L/2 (2006.01) A63L/439 (2006.01) (75) Inventor: Jack L. Arbiser, Atlanta, GA (US) A6IP35/00 (2006.01) (73) Assignee: EMORY UNIVERSITY, Atlanta, (52) U.S. Cl...... 514/252.18; 514/291 GA (US) (21) Appl. No.: 13/357,920 (57) ABSTRACT (22) Filed: Jan. 25, 2012 The disclosure relates to methods of treating tuberous scle rosis in a Subject, comprising administering a composition Related U.S. Application Data comprising an mTOR inhibitor and a tyrosine kinase inhibitor to a Subject that is diagnosed with, Suspected of, or exhibiting (60) Provisional application No. 61/436,760, filed on Jan. symptoms of cancer. In some embodiments, the cancer is 27, 2011. tuberous sclerosis. In some embodiments, the mTOR inhibi O O tor is and the tyrosine kinase inhibitor is imatinib. In Publication Classification Some embodiments, the disclosure relates to a composition (51) Int. Cl. comprising an mTOR inhibitor and a tyrosine kinase inhibi A 6LX 3L/2197 (2006.01) tor. In some embodiments, the disclosure relates to a compo A6IP3I/00 (2006.01) sition comprising sirolimus and imatinib. Patent Application Publication Aug. 2, 2012 Sheet 1 of 7 US 2012/0196870 A1

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COMBINATION THERAPY AND CANCER position comprising an mTOR inhibitor and a tyrosine kinase inhibitor to a subject that is diagnosed with, Suspected of, or CROSS REFERENCE TO RELATED exhibiting symptoms of cancer. In some embodiments, the APPLICATIONS cancer is tuberous Sclerosis. In some embodiments, the 0001. This application claims priority to U.S. Provisional mTOR inhibitor in the method or composition is Sirolimus, Application No. 61/436,760 filed Jan. 27, 2011, which appli , Deforolimus, AZD8055, BEZ235, cation is hereby incorporated by this reference in its entirety. GSK105.9615, KU-0063794, WYE-354, PI-103, Temsiroli mus, , , and (also ACKNOWLEDGEMENTS known as Biolimus or Biolimus A9). In some embodiments, the tyrosine kinase inhibitor in the method or composition is 0002 This invention was made with government support imatinib, linifanib, ponatinib, regorafenib, Vargatef, or Suni under Grants RO1-AR47901, P30-AR42687, CA87986, tinib, or CP 673451. In some embodiments, the disclosure CA116552 and CA99163 awarded by the National Institutes relates to a composition comprising an mTOR inhibitor and a of Health. The government has certain rights in the invention. tyrosine kinase inhibitor. In some embodiments, the disclo Sure relates to a composition comprising sirolimus and ima FIELD tinib. 0003. The disclosure relates to methods of treating tuber 0007. In some embodiments, the administration of the ous Sclerosis in a subject, comprising administering a com composition is performed under conditions such that the position comprising an mTOR inhibitor and a tyrosine kinase tuberous Sclerosis is no longer detected. In some embodi inhibitor to a subject that is diagnosed with, Suspected of, or ments, the disclosure relates to treating or preventing tuber exhibiting symptoms of cancer. In some embodiments, the ous sclerosis by administering an mTOR inhibitor and a cancer is tuberous Sclerosis. In some embodiments, the tyrosine kinase inhibitor to a Subject also diagnosed with mTOR inhibitor is sirolimus and the tyrosine kinase inhibitor another type of cancer. In some embodiments, the Subject is is imatinib. In some embodiments, the disclosure relates to a diagnosed with a mutation inhamartin (tSc1) or tuberin (tsc2). composition comprising an mTOR inhibitor and a tyrosine 0008. In some embodiments, the subject is diagnosed with kinase inhibitor. In some embodiments, the disclosure relates tuberous sclerosis, skin cancer, breast cancer, prostate cancer, to a composition comprising sirolimus and imatinib. lung cancer, bladder cancer, melanoma, colon and rectal can cer, non-hodgkin’s lymphoma, endometrial cancer, pancre BACKGROUND atic cancer, renal cell cancer, leukemia, and/or thyroid cancer. 0004 Tuberous Sclerosis (TS) is an autosomal dominant 0009. In some embodiments, the subject is a mammal, disorder that is characterized by the development of benign typically a human. and malignant tumors of the brain, kidney, lung, and skin. TS 0010. In some embodiments, the tyrosine kinase inhibitor is characterized by mutations or deletions in one of two genes, is a platelet-derived growth factor receptor (PDGFR) inhibi either hamartin (tsc 1) or tuberin (tsc2). Furthermore, TS is tor. In some embodiments, the tyrosine kinase inhibitor is an associated with neuro-developmental complications, includ Abelson (Abl) protein inhibitor. In some embodiments, the ing autism and seizures, leading to significant morbidity. tyrosine kinase inhibitor is a CD117 (Mast/stem cell growth Although there is no known strict genotype-phenotype rela factor receptor) inhibitor. In some embodiments, the tyrosine tionship with TS, the disease is more severe in patients with kinase inhibitor is imatinib, linifanib, ponatinib, regorafenib, tSc2 mutations. Loss of heterozygosity, in which the unaf Vargatef, or Sunitinib, or CP 673451. In some embodiments, fected allele is deleted, is necessary for the development of the disclosure may include therapeutic methods and compo certain neoplastic features of TS, including renal angiomyo sitions targeting the active site of the TK domain in abl (the lipomas, lymphangiomyomatosis, and skin lesions. Abelson proto-oncogene), c-kit, and PDGF-R (platelet-de 0005 TS is associated with a number of complex signal rived growth factor receptor). ing pathways. Tsc2 mutations often involve regions impli 0011. In some embodiments, the subject undergoes immu cated in controlling rheb, although other signaling pathways nological monitoring. In some embodiments, the disclosure have been implicated in TS-related neoplasia, including relates to methods of treating a subject diagnosed with tuber mTORC1, notch, p42/44. MAP kinase, NFkB, and Akt. The ous sclerosis and either treating or preventing a viral infection use of sirolimus as a therapy for TS has been based on the by administration of an mTOR inhibitor, a tyrosine kinase upregulation of mTORC1 in the neoplasms of TS patients. inhibitor, and one or more anti-viral agents, wherein the Sub Although sirolimus resulted in a partial regression of kidney, ject is immunocompromised. In typical embodiments, the lung and skin lesions, there was not a complete disappearance immunocompromised Subject is an organ transplant recipi of tumors. See, e.g., Franz, D. N. et al. Rapamycin causes ent, undergoing hemodialysis, diagnosed with cancer, receiv regression of astrocytomas in tuberous Sclerosis complex. ing an , and/or diagnosed with an Ann Neurol. 2006 March; 59(3):490-8. Furthermore, tumor HIV-infection. In some embodiments, the subject is at risk of regrowth was observed upon cessation of Sirolimus therapy. an infection because the sexual partner of the Subject is diag Bissler, J.J. et al. Sirolimus for angiomyolipoma in tuberous nosed with a virus. Sclerosis complex or lymphangioleiomyomatosis. N Engl J 0012. In some embodiments, the subject is diagnosed with Med. 2008 Jan. 10; 358(2):140-51. These clinical observa influenza A virus including subtype H1N1, influenza B virus, tions indicate that other signaling pathways may be active in influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavi TS-related tumors. rus D, rotavirus E, SARS coronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types SUMMARY 16, 18, 31, 33,35, 39, 45, 51, 52, 56,58, 59, parvovirus B19, 0006. The disclosure relates to methods of treating tuber molluscum contagiosum virus, JC virus (JCV), BK virus, ous Sclerosis in a subject, comprising administering a com Merkel cell polyomavirus, coxsackie A virus, norovirus, US 2012/0196.870 A1 Aug. 2, 2012

Rubella virus, lymphocytic choriomeningitis virus (LCMV), Cefepime), Moxolactam, Carbapenems (Imipenem, Ertap yellow fever virus, measles virus, mumps virus, respiratory enem, Meropenem) Monobactams (AZtreonam). Oxytetracy syncytial virus, rinderpest virus, California encephalitis cline, Chlortetracycline, Clomocycline, Demeclocycline.Tet virus, hantavirus, rabies virus, ebola virus, marburg virus, racycline, Doxycycline, Lymecycline, Meclocycline, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 Methacycline, Minocycline, Rolitetracycline, Chloram (HSV-2), varicella Zoster virus (VZV), Epstein-Barr virus phenicol, Amikacin, Gentamicin, Framycetin, Kanamycin, (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Neomicin, Neomycin, Netilmicin, Streptomycin, Tobramy roseolovirus, Kaposi's sarcoma-associated herpesvirus, cin, Azithromycin, Clarithromycin, Dirithromycin, Erythro hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), mycin, Roxithromycin, Telithromycin, Polymyxin-B, Colis hepatitis D (HDV), hepatitis E (HEV), human immunodefi tin, Bacitracin, Tyrothricin Notrifurantoin, Furazolidone, ciency virus (HIV), The Human T-lymphotropic virus Type I Metronidazole, Tinidazole, Isoniazid, Pyrazinamide, (HTLV-1), Friend spleen focus-forming virus (SFFV) or Ethionamide, Nystatin, Amphotericin-B, Hamycin, Micona Xenotropic MuDV-Related Virus (XMRV). Zole, Clotrimazole, Ketoconazole, Fluconazole, Rifampacin, 0013. In some embodiments, the subject is diagnosed with Lincomycin, Clindamycin, Spectinomycin, Chlorampheni a virus, as described above, and is administered a pharmaceu col, Clindamycin, Colistin, Fosfomycin, Loracarbef, Metron tical composition comprising sirolimus, imatinib, and an anti idazole, Nitrofurantoin, Polymyxin B, Polymyxin B Sulfate, viral agent. The antiviral agent may be abacavir, acyclovir, Procain, Spectinomycin, Tinidazole, Trimethoprim, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbi Ramoplanin, Teicoplanin, Vancomycin, Trimethoprim, Sul dol, atazanavir, atripla, boceprevir, cidofovir, combivir, famethoxazole, and/or Nitrofurantoin. darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, BRIEF DESCRIPTION OF THE FIGURES fomivirsen, foSamprenavir, foScarnet, foSfonet, ganciclovir, 0015 FIG.1. Effect of sirolimus, imatinib or combination ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, of sirolimus and imatinib on tsc2angl cell line proliferation. inosine, type III, interferon type II, interferon type 0016 FIG. 2. Phospho-PDGFR-B is strongly downregu I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, lated in combinational treatment. methisaZone, nelfinavir, nevirapine, nexavir, oseltamivir 0017 FIG. 3. Combination therapy significantly down (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, ple regulates Angpt2, Nrarp, and VEGF. conaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, 0018 FIG. 4. A. List of genes with greatest change in ritonavir, pyramidine, saquinavir, stavudine, tenofovir, teno sirolimus-treated vs. control cells. B. List of genes with great fovir disoproxil, tipranavir, trifluridine, trizivir, tromanta est change in sirolimus-treated VS. combination therapy dine, truvada, Valaciclovir (Valtrex), Valganciclovir, Vicrivi treated cells. roc, Vidarabine, Viramidine Zalcitabine, Zanamivir (Relenza), (0019 FIG. 5. Relative expression levels of A. Aurora and/or zidovudine. kinase A, B. Aurora kinase B, C. Centromere protein A in 0014. In some embodiments, the subject is diagnosed or tSc2angl cells. D. Western blot analysis comparing therapies. Susceptible to a bacterial infection. In some embodiments, the 0020 FIG. 6. Substrate gel electrophoresis using gelatin Subject is administered one or more antibiotics including, but as the Substrate. not limited to, Sulfonamides, Diaminopyrimidines, Quinolo 0021 FIG. 7. Western blot analysis using FoxM1 mono nes, Beta-lactam antibiotics, Cephalosporins, Tetracyclines, clonal . Notribenzene derivatives, Aminoglycosides, Macrollide anti 0022 FIG.8. Effect of sirolimus and imatinib on in vivo biotics, Polypeptide antibiotics, Nitrofuran derivatives, growth oftsc2angl Xenografts in nude mice. Nitroimidazoles, Nicotinin acid derivatives, Polyene antibi otics, Imidazole derivatives or Glycopeptide, Cyclic lipopep DETAILED DESCRIPTION tides, Glycylcyclines and Oxazolidinones. In further embodi ments, these antibiotics include but are not limited to 0023 mTOR Inhibitor Sulphadiazine, Sulfones—Dapsone (DDS) and Paraami (0024. As used herein, “mTOR’ inhibitor refers to any nosalicyclic (PAS), Sulfanilamide, Sulfamethizole, Sul therapeutic capable of inhibiting the mammalian target of famethoxazole, Sulfapyridine, Trimethoprim, rapamycin. Typically, the mTOR inhibitor is sirolimus. As Pyrimethamine, Nalidixic acids, Norfloxacin, Ciproflaxin, used herein, “sirolimus' may refer to sirolimus (also known Cinoxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Grepa as rapamycin) or any formulation (e.g. Rapamune), func floxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, tional equivalent, or derivative thereof Functional equivalents Ofloxacin, Pefloxacin, Sparfloxacin, Trovafloxacin, Penicil and derivatives may include other mammalian target of rapa lins (Amoxicillin, Ampicillin, AZlocillin, Carbenicillin, mycin (mTOR) inhibitors including, but not limited to, Cloxacillin, Dicloxacillin, Flucloxacillin, Hetacillin, Oxacil Everolimus, Deforolimus, AZD8055, BEZ235, lin, Mezlocillin, Penicillin G, Penicillin V. Piperacillin), GSK105.9615, KU-0063794, WYE-354, PI-103, Temsiroli Cephalosporins (Cefacetrile, Cefadroxil, Cefalexin, Cefa mus, Ridaforolimus, Zotarolimus, and Umirolimus (also loglycin, Cefalonium, Cefaloridin, Cefalotin, Cefapirin, known as Biolimus or Biolimus A9). Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefradine, 0025 Sirolimus is a type of macrollide (i.e. possesses a Cefroxadine, Ceftezole, Cefaclor, Cefonicid, Ceforanide, ringed chemical structure) first isolated from bacteria found Cefprozil, Cefuroxime, CefuZonam, Cefnetazole, Cefoteta, on Rapa Nui (Easter Island). Sirolimus is currently used as an Cefoxitin, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, anti-proliferative agent in cancer as well as an immunosup Cefetamet, Cefixime, Cefnmenoxime, Cefodizime, Cefopera pressant and may act by inhibiting cell cycle progression. In Zone, Cefotaxime, Cefotiam, Ce?pimizole, Ce?piramide, Cef particular, sirolimus has shown promise in the treatment of podoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolen, Cefti TS, but remains problematic as a monotherapy due to the Zoxime, Ceftriaxone, CefoperaZone, Ceftazidime, return of TS following cessation of sirolimus therapy. See, US 2012/0196.870 A1 Aug. 2, 2012

e.g., Tsai, P. Mechanisms of neurocognitive dysfunction and FRB-mediated pathway, especially since mTORC1 activation therapeutic considerations in tuberous Sclerosis complex. and PDGFRB signaling have been shown to have an inverse Curr Opin Neurol. 2011 April; 24(2):106-13. relationship. 0026. As disclosed herein, sirolimus monotherapy may induce PDGFR signaling, as well as upregulate Aurora kinase Combination Therapy signaling, thus providing a rationale for the failure of Siroli 0030. In some embodiments, the disclosure relates to mus monotherapy in tuberous sclerosis. Platelet-derived treating Tuberous Sclerosis with combination therapy com growth factor B receptor (PDGFR f) is both present and prising a composition comprised of an mTOR inhibitor, typi active in human and murine TS lesions. There is an inverse cally sirolimus, and a tyrosine kinase inhibitor, typically ima relationship between mTOR activation and the PDGFR B tinib. The combination of sirolimus and imatinib, two FDA levels in TS-derived cells. Therefore, mTOR blockade might approved drugs, is well tolerated in vivo. Given that sirolimus be compensated for by a PDGF activator, such as imatinib, monotherapy does not cause long lasting remissions in tuber both in vitro and in vivo. ous Sclerosis, as disclosed herein, a rationale exists for the combination of these two drugs in humans. Tyrosine Kinase Inhibitor 0031 One previous study tested sirolimus and imatinib when using SK-LMS-1 cells as a model for human leiomyo 0027. As used herein, the term “tyrosine kinase inhibitor sarcoma. That study, however, explicitly stated that there was may refer to any tyrosine kinase inhibitor. The tyrosine kinase no advantage to adding imatinib to the sirolimus mono inhibitor may be a platelet-derived growth factor receptor therapy. Merimsky, O. etal. Molecular impacts of rapamycin (PDGFR) inhibitor, an Abelson (Abl) protein inhibitor, or a based drug combinations: Combining rapamycin with gem CD117 inhibitor. Tyrosine kinase inhibitors include, but are citabine or imatinib mesylate (Gleevec) in a human not limited to, imatinib, linifanib, ponatinib, regorafenib, leiomyosarcoma model. Int J Oncol. 2007 July; 31 (1):225 Vargatef, or Sunitinib, or CP 673451. Typically, the tyrosine 32. Sirolimus and imatinib in a nanoparticle formulation with kinase inhibitor is imatinib. As used herein, the term “ima an albumin carrier protein has been suggested as part of a list tinib' may refer to imatinib or any formulation (e.g. Gleevec of numerous potential combinations, for the treatment of or Glivec, a mesylate salt formulation), functional equivalent, cancer. No demonstration of its utility in TS, which was listed or derivative thereof Imatinib is a tyrosine kinase (TK) among more than 100 other forms of cancer, was presented. enzyme inhibitor, acting by occupying the TK active site Published International Patent Application WO 2008/109163 thereby diminishing TK activity. Functional equivalents may A1. include therapeutics targeting the active site of the TK domain in abl (the Abelson proto-oncogene), c-kit, and PDGF-R Formulations (platelet-derived growth factor receptor). 0032 Generally, for pharmaceutical use, the compositions Tuberous Sclerosis may be formulated as a pharmaceutical preparation compris ing an mTOR inhibitor, typically sirolimus, in combination 0028. As used herein, “tuberous sclerosis” (TS) refers to a with a tyrosine kinase inhibitor, typically imatinib multisystem disorder characterized by benign and malignant 0033. The pharmaceutical preparations of the disclosure neoplasia, as well as autism and seizures. No effective therapy are preferably in a unit dosage form, and may be suitably currently exists for TS. Renal lesions such as angiomyolipo packaged, for example in a box, blister, Vial, bottle, Sachet, mas may cause massive bleeding and compromise renal func ampoule or in any other Suitable single-dose or multi-dose tion, often requiring a kidney transplant. Lymphangiomyo holder or container (which may be properly labeled); option matosis, a neoplastic complication most commonly seen in ally with one or more leaflets containing product information young women, can only be cured by lung transplantation. and/or instructions for use. Generally, such unit dosages will Such transplantation is itself problematic, requiring immuno contain between 1 and 40 mg, and usually between 2 and 6 Suppression and typically failing within approximately five mg, of the mTOR inhibitor, typically sirolimus, e.g. about 2 years. Deforming skin lesions cause significant psychologi mg per unit dosage, and 1 and 1000 mg. A one-time bolus, of cal distress. Brain tumors, such as subependymal giant cell sirolimus for example, may be given initially followed by astrocytomas, can cause morbidity and mortality, and mul lower Subsequent doses, e.g. 6 mg on day one followed by 2 tiple tubers result in refractory seizures and exacerbation of mg per day thereafter. The unit dosage is usually between 100 mental retardation. These complications demonstrate that and 800 mg of the tyrosine kinase inhibitor, typically ima effective therapy for TS is urgently needed. TS may also refer tinib, e.g. about 10, 25, 50, 100, 200, 300, 400, or 800 mg per to a Subject diagnosed with other forms of cancer in addition unit dosage. to TS 0034. The compositions can be administered by a variety 0029 Multiple signaling pathways are impacted by muta of routes including the oral, ocular, rectal, transdermal, Sub tions in tsc1 and tsc2. One of the most well studied pathways cutaneous, intravenous, intramuscular or intranasal routes, is rheb, a ras like protein which is inhibited by the heterodimer depending mainly on the specific formulation used. Typically, oftsclftsc2. If rheb is activated, then, mTORC1 and notch are the mTOR inhibitor is administered orally, either in pill or activated downstream, but notch activation is independent of liquid formulations. Typically, the tyrosine kinase inhibitor is mTORC1 because notch activation is not blocked by siroli administered orally in pill form. The composition will gen mus. The lack of total blockade by sirolimus is consistent erally be administered in an “effective amount', by which is with the incomplete clinical response of tumors to systemic meant any amount of a composition that, upon Suitable Sirolimus, implying that either additional pathways are administration, is sufficient to achieve the desired therapeutic already present in these tumors or are induced by Sirolimus or prophylactic effect in the subject to which it is adminis monotherapy. High among the candidate pathways is a PDG tered. Depending on the condition to be prevented or treated US 2012/0196.870 A1 Aug. 2, 2012

and the route of administration, an effective amount of either 0038. When rectally administered in the form of supposi the mTOR inhibitor or tyrosine kinase inhibitor may be deter tories, the formulations may be prepared by mixing the com mined by the subject's body weight. The amount(s) to be positions with a suitable non-irritating excipient, such as administered, the route of administration and the further treat cocoa butter, synthetic glyceride esters or polyethylene gly ment regimen may be determined by the treating clinician, cols, which are solid at ordinary temperatures, but liquefy depending on factors such as the age, gender and general and/or dissolve in the rectal cavity to release the drug. condition of the patient and the nature and severity of the 0039. In some embodiments, it is contemplated that these disease/symptoms to be treated. Reference is made to U.S. compositions can be extended release formulations. Typical extended release formations utilize an enteric coating. Typi Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. cally, a barrier is applied to oral medication that controls the 6,369,087 and U.S. Pat. No. 6,372,733 as well as to standard location in the digestive system where it is absorbed. Enteric handbooks, such as the latest edition of Remington's Phar coatings prevent release of medication before it reaches the maceutical Sciences. Small intestine. Enteric coatings may contain polymers of 0035. For an oral administration form, the composition polysaccharides, such as maltodextrin, Xanthan, Scleroglucan can be mixed with Suitable additives, such as excipients, dextran, starch, alginates, pullulan, hyaloronic acid, chitin, stabilizers or inert diluents, and brought by means of the chitosan and the like; other natural polymers, such as proteins customary methods into the Suitable administration forms, (albumin, gelatin etc.), poly-L-lysine; sodium poly(acrylic Such as tablets, coated tablets, hard capsules, aqueous, alco acid); poly(hydroxyalkylmethacrylates) (for example poly holic, or oily solutions. Examples of suitable inert carriers are (hydroxyethylmethacrylate)); carboxypolymethylene (for gum arabic, magnesia, magnesium carbonate, potassium example CarbopolTM); carbomer; polyvinylpyrrolidone: phosphate, lactose, glucose, or starch, in particular, corn gums, such as guar gum, gum arabic, gum karaya, gum ghatti, starch. Oily excipients or solvents are vegetable or animal locust bean gum, tamarindgum, gellangum, gum tragacanth, oils, such as sunflower oil or cod liver oil. Solvents for aque agar, pectin, glutenand the like; poly(vinyl alcohol); ethylene ous or alcoholic Solutions are water, ethanol, Sugar Solutions, vinyl alcohol; polyethylene glycol (PEG); and cellulose or mixtures thereof Polyethylene glycols and polypropylene ethers, such as hydroxymethylcellulose (HMC), hydroxyeth glycols are also useful as further auxiliaries for other admin ylcellulose (HEC), hydroxypropylcellulose (HPC), methyl istration forms. As immediate release tablets, these composi cellulose (MC), ethylcellulose (EC), carboxyethylcellulose tions may contain microcrystalline cellulose, dicalcium phos (CEC), ethylhydroxyethylcellulose (EHEC), carboxymeth phate, starch, magnesium Stearate and lactose and/or other ylhydroxyethylcellulose (CMHEC), hydroxypropylmethyl excipients, binders, extenders, disintegrants, diluents and cellulose (HPMC), hydroxypropylethylcellulose (HPEC) lubricants known in the art. and sodium carboxymethylcellulose (Na CMC); as well as 0036 When administered by nasal aerosol or inhalation, copolymers and/or (simple) mixtures of any of the above the compositions may be prepared according to techniques polymers. Certain of the above-mentioned polymers may well-known in the art of pharmaceutical formulation and may further be crosslinked by way of standard techniques. be prepared as Solutions in Saline, employing benzyl alcohol 0040. The choice of polymer will be determined by the or other Suitable preservatives, absorption promoters to nature of the active ingredient/drug that is employed in the enhance bioavailability, fluorocarbons, and/or other solubi composition of the disclosure as well as the desired rate of lizing or dispersing agents known in the art. Suitable phar release. In particular, it will be appreciated by the skilled maceutical formulations for administration in the form of person, for example in the case of HPMC, that a higher aerosols or sprays are, for example, Solutions, Suspensions or molecular weight will, in general, provide a slower rate of emulsions of the compositions of the disclosure or their release of drug from the composition. Furthermore, in the physiologically tolerable salts in a pharmaceutically accept case of HPMC, different degrees of substitution of methoxy able solvent, Such as ethanol or water, or a mixture of Such groups and hydroxypropoxyl groups will give rise to changes Solvents. If required, the formulation can also additionally in the rate of release of drug from the composition. In this contain other pharmaceutical auxiliaries Such as Surfactants, respect, and as stated above, it may be desirable to provide compositions of the disclosure in the form of coatings in emulsifiers and Stabilizers as well as a propellant. which the polymer carrier is provided by way of a blend of 0037 For subcutaneous or intravenous administration, the two or more polymers of for example, different molecular compositions, if desired with the Substances customary there weights in order to produce a particular required or desired fore such as solubilizers, emulsifiers or further auxiliaries are brought into Solution, Suspension, or emulsion. The compo release profile. sitions can also be lyophilized and the lyophilizates obtained 0041 Administration of the therapies described herein used, for example, for the production of injection or infusion may include a method of sustained release. Sustained release preparations. Suitable solvents are, for example, water, physi may be achieved in multiple ways known to one ordinarily ological saline solution or alcohols, e.g. ethanol, propanol, skilled in the art. These methods include, but are not limited glycerol, Sugar Solutions such as glucose or mannitol Solu to, implantable osmotic pumps and formulations of sirolimus tions, or mixtures of the various solvents mentioned. The and/or imatinib that are not soluble at physiological pH, injectable solutions or Suspensions may be formulated resulting in the gradual dissolving after, for example, injec according to known art, using Suitable non-toxic, parenter tion. ally-acceptable diluents or solvents, such as mannitol, 1.3- butanediol, water, Ringer's solution or isotonic sodium chlo Aurora Kinases ride solution, or Suitable dispersing or wetting and 0042 Aurora kinases are a family of serine/threonine Suspending agents. Such as Sterile, bland, fixed oils, including kinases that are necessary for mitosis and meiosis. Aurora synthetic mono- or diglycerides, and fatty acids, including kinases A and B are the most prominent. Although there are oleic acid. structural similarities between Aurora kinases A and B, each US 2012/0196.870 A1 Aug. 2, 2012 has a different role in both cytokinesis and tumorigenesis. The pendently selected from nitrogen, oxygen and Sulfur which activation of Aurora kinases is required for the optimal may be saturated or unsaturated (but not aromatic), monocy growth of tumor cells. Furthermore, different tumors appear clic or polycyclic, and wherein the nitrogen and Sulfur het to have differential sensitivity to Aurora kinase inhibitors. eroatoms may be optionally oxidized, and the nitrogen het The amplification of Aurora kinase A is associated with eroatom may be optionally quaternized. Heterocarbocycles highly aneuploid tumors as well as p53 dysfunction, whereas include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidi Aurora kinase B is highly associated with tumors with wild nyl, hydantoinyl, Valerolactamyl, oxiranyl, oxetanyl, tetrahy type p53. drofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahy droprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, Terms tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothi 0043. As used herein, the term “immunocompromised' or opyranyl, and the like. “immunosuppressed’ refers to a subject with an impaired or 0048. The term “aryl” refers to aromatic homocyclic (i.e., ablated immune response. The impairment or ablation may be hydrocarbon) mono-, bi- or tricyclic ring-containing groups complete or partial. Partial may or may preferably having 6 to 12 members such as phenyl, naphthyl not result in the inability to mount an appropriate immune and biphenyl. Phenyl is a preferred aryl group. The term response. Typically, immunocompromised refers to the “substituted aryl” refers to aryl groups substituted with one or depletion of a particular cell type, such as lymphocytes. A more groups, preferably selected from alkyl, Substituted Subject may be immunocompromised, for example, by taking alkyl, alkenyl (optionally substituted), aryl (optionally Sub immunosuppressive drugs in order to prevent allograft rejec stituted), heterocyclo (optionally Substituted), halo, hydroxy, tion Immunosuppressive drugs include, but are not limited to, alkoxy (optionally Substituted), aryloxy (optionally Substi antithymocyte globulin (ATG). ATG-fresenius, anti-CD3 tuted), alkanoyl (optionally Substituted), aroyl, (optionally (mAb), anti-CD20 mAb, anti-TNF substituted), alkylester (optionally substituted), arylester (op mAb, sirolimus, anti-CD25 mAb, and inhibitors tionally Substituted), cyano, nitro, amino, Substituted amino, (e.g. cyclosporine). amido, lactam, urea, urethane, Sulfonyl, and, the like, where 0044 As used herein, the term "derivative' refers to a optionally one or more pair of substituents together with the structurally similar compound that retains Sufficient func atoms to which they are bonded form a 3 to 7 member ring. tional attributes of the identified analogue. The derivative 0049. As used herein, "heteroaryl” or "heteroaromatic” may be structurally similar because it is lacking one or more refers an aromatic heterocarbocycle having 1 to 4 heteroat atoms, substituted, a salt, in different hydration/oxidation oms selected from nitrogen, oxygen and sulfur, and contain states, or because one or more atoms within the molecule are ing at least 1 carbon atom, including both mono- and poly Switched, such as, but not limited to, replacing a oxygenatom cyclic ring systems. Polycyclic ring systems may, but are not with a Sulfur atom or replacinga amino group with a hydroxyl required to, contain one or more non-aromatic rings, as long group. The derivative may be a prodrug. Derivatives may be as one of the rings is aromatic. Representative heteroaryls are prepare by any variety of synthetic methods or appropriate furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, adaptations presented in synthetic or organic chemistry text indolyl, isoindolyl azaindolyl pyridyl, quinolinyl, isoquino books, such as those provide in March's Advanced Organic linyl, oxazolyl, isooxazolyl, benzoxazolyl pyrazolyl, imida Chemistry: Reactions, Mechanisms, and Structure, Wiley, Zolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiaz 6th Edition (2007) Michael B. Smith or Domino Reactions in olyl pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incor cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated porated by reference. that the use of the term "heteroaryl' includes N-alkylated 0045. As used herein, “alkyl means a noncyclic straight derivatives such as a 1-methylimidazol-5-yl substituent. chain or branched, unsaturated or Saturated hydrocarbon Such as those containing from 1 to 10 carbonatoms. Representative 0050. As used herein, "heterocycle” or "heterocyclyl saturated Straight chain alkyls include methyl, ethyl, n-pro refers to mono- and polycyclic ring systems having 1 to 4 pyl. n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and heteroatoms selected from nitrogen, oxygen and Sulfur, and the like; while saturated branched alkyls include isopropyl. containing at least 1 carbon atom. The mono- and polycyclic sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsat ring systems may be aromatic, non-aromatic or mixtures of urated alkyls contain at least one double or triple bond aromatic and non-aromatic rings. Heterocycle includes het between adjacent carbonatoms (referred to as an “alkenyl' or erocarbocycles, heteroaryls, and the like. “alkynyl', respectively). Representative straight chain and 0051. “Alkylthio’ refers to an alkyl group as defined above branched alkenyls include ethylenyl, propylenyl, 1-butenyl, with the indicated number of carbon atoms attached through 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1- a Sulfurbridge. An example of an alkylthio is methylthio. (i.e., butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the —S-CH3). like; while representative straight chain and branched alky 0.052 Alkoxy' refers to an alkyl group as defined above nyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, with the indicated number of carbon atoms attached through 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like. an oxygen bridge. Examples of alkoxy include, but are not 0046 Non-aromatic mono or polycyclic alkyls are limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, referred to herein as “carbocycles' or “carbocyclyl groups. S-butoxy, t-butoxy, n-pentoxy, and S-pentoxy. Preferred Representative Saturated carbocycles include cyclopropyl. alkoxy groups are methoxy, ethoxy, n-propoxy, i-propoxy, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while n-butoxy, S-butoxy, t-butoxy. unsaturated carbocycles include cyclopentenyl and cyclohex 0053 Alkylamino” refers an alkyl group as defined above enyl, and the like. with the indicated number of carbon atoms attached through 0047. “Heterocarbocycles” or heterocarbocyclyl groups an amino bridge. An example of an alkylamino is methy are carbocycles which contain from 1 to 4 heteroatoms inde lamino, (i.e., —NH-CH3). US 2012/0196.870 A1 Aug. 2, 2012

0054 Alkanoyl refers to an alkyl as defined above with Sclerosis, and may include even minimal reductions in one or the indicated number of carbon atoms attached through a more measurable markers of the disease or condition. “Treat carbonyl bride (i.e., —(C=O)alkyl). ment” does not necessarily indicate complete eradication or 0055 “Alkylsulfonyl refers to an alkyl as defined above cure of tuberous Sclerosis, or associated symptoms thereof. with the indicated number of carbonatoms attached through The Subject receiving this treatment is any animal in need, a Sulfonyl bridge (i.e., —S(=O)2alkyl) such as mesyl and the including primates, typically humans, and other mammals like, and "Arylsulfonyl refers to an aryl attached through a Such as equines, cattle, Swine and sheep as well as poultry and sulfonyl bridge (i.e., —S(=O)2aryl). other pets. 0056 “Alkylsulfamoyl refers to an alkyl as defined above 0063. In some embodiments, the subject has a compro with the indicated number of carbonatoms attached through mised or Suppressed immune system. Immunosuppression a sulfamoyl bridge (i.e., NHS(=O)2alkyl), and an 'Aryl reduces the activation or efficacy of the immune system. sulfamoyl refers to an alkyl attached through a sulfamoyl Deliberately induced immunosuppression is typically done to bridge (i.e., (i.e., NHS(=O)2aryl). prevent the body from rejecting an organ transplant (e.g., 0057. “Alkylsulfinyl refers to an alkyl as defined above bone marrow, heart, kidney, liver), treating graft-Versus-host with the indicated number of carbonatoms attached through disease after a bone marrow transplant, or for the treatment of a sulfinyl bridge (i.e. —S(=O)alkyl). auto-immune diseases (e.g., , multiple 0058. The term “substituted refers to a molecule wherein Sclerosis, myasthenia gravis, Systemic lupus erythematosus, at least one hydrogen atom is replaced with a Substituent. Crohn's disease, pemphigus, and ulcerative colitis). Depend When substituted, one or more of the groups are “substitu ing on the dose used, the mTOR described herein may result ents.” The molecule may be multiply substituted. In the case in detectable immunosuppression, via the induction of regu of an oxo substituent ("—O”), two hydrogen atoms are latory T lymphocytes by Sirolimus for example Immunosup replaced. Example Substituents within this context may pression may be accomplished by certain agents (immuno include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, OXo, Suppressants) Such as, but not limited to, dactinomycin, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocar , , , terifluno bocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, mide, , , , , NRaRb, NRaC(=O)Rb, NRaC(=O)NRaNRb, , gusperimus, , , Sirolimus, - NRaC(=O)CRb, NRaSO2Rb, C(=O)Ra, C(=O) deforolimus, everolimus, , Zotarolimusm, bioli ORa, C(=O)NRaRb, OC(=O)NRaRb, ORa, mus A9, anti-thymocyte globulin (ATG). T-cell receptor —SRa, -SORa, S(=O)2Ra, OS(=O)2Ra and directed (e.g., muromonab), and IL-2 receptor —S(=O)2ORa. Ra and Rb in this context may be the same or directed antibodies (e.g., and ). Sur different and independently hydrogen, halogen hydroxyl, gery (splenectomy), plasmapharesis, or radiation may also alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, car cause a Suppressed immune system. Since certain immuno bocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocar Suppressants act non-selectively, the immune system may be bocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl. less able to combat tumorogenesis. A person who is under 0059. The term “optionally substituted,” as used herein, going immunosuppressant therapy, or whose immune system means that substitution is optional and therefore it is possible is weak for other reasons (for example, chemotherapy, HIV. for the designated atom to be unsubstituted. and Lupus) is said to be immunocompromised. Neonates are 0060. The terms “cycloalkyl and “cycloalkenyl refer to considered to immunocompromised. Certain viruses Such as mono-, bi-, or trihomocyclic ring groups of 3 to 15 carbon HIV will comprise the host immune system. In some embodi atoms which are, respectively, fully saturated and partially ments, the disclosure relates to treating or preventing Tuber unsaturated. The term “cycloalkenyl' includes bi- and tricy ous Sclerosis by administering sirolimus and imatinib com clic ring systems that are not aromatic as a whole, but contain bination therapy to an immunocompromised subject. aromatic portions (e.g., fluorene, tetrahydronapthalene, dihy 0064. As used herein, the term “combination with or droindene, and the like). The rings of multi-ring cycloalkyl “combination therapy” when used to describe administration groups may be either fused, bridged and/or joined through of an mTOR inhibitor and a tyrosine kinase inhibitor means one or more spiro unions. The terms “substituted cycloalkyl that either agent may be administered prior to, together with, and “substituted cycloalkenyl refer, respectively, to or after the other agent, or a combination thereof. cycloalkyl and cycloalkenyl groups substituted with one or 0065. As used herein, the term “subject” refers to any more groups, preferably selected from aryl, Substituted aryl, animal, preferably a human patient, livestock, or domestic heterocyclo, substituted heterocyclo, carbocyclo, substituted pet. carbocyclo, halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally Substituted), alkylester (optionally Sub EXPERIMENTAL stituted), arylester (optionally Substituted), alkanoyl (option Example 1 ally Substituted), aryol (optionally Substituted), cyano, nitro, amino, Substituted amino, amido, lactam, urea, urethane, Sul Sirolimus and Imatinib Combination Therapy Inhib fonyl, and the like. its TSC2 Cell Proliferation 0061. The terms “halogen' and “halo” refer to fluorine, 0066. A proliferation assay with Tsc2 cells indicated a chlorine, bromine, and iodine. An unspecified “R” group is a decrease in the viable cell numbers when treated indepen hydrogen, lower alkyl, or aryl all of which may be optionally dently with either rapamycin (5 nM) or imatinib (10 uM) for substituted with one or more substituents. Throughout the 24 hours. 10tsc2ang1 cells per well were plated in 24-well specification, groups and Substituents thereof may be chosen dishes in triplicate. The next day, fresh medium containing to provide stable moieties and compounds. the compounds or vehicle controls was added. Cells were 0062. The terms “treatment” or “treating include any incubated at 37°C. Tsc2angl (ATCC CRL 2620) is a murine desirable effect on the symptoms or pathology of tuberous cell line derived from a cutaneous sarcoma that arose in the US 2012/0196.870 A1 Aug. 2, 2012 extremity of a mouse heterozygous for tsc2; these mice tially expressed genes in each comparison group was ana develop cutaneous sarcomas at a frequency of about 10 to lyzed for statistically enriched or depleted biological 15%. The cells were cultured in complete DMEM medium classifications using the GoMiner database engine. supplemented with 10% FBS (Sigma Aldrich, St Louis, Mo.). 0069. Equal numbers of Tsc2ang1 cells were seeded in Combination treatment of rapamycin (5 nM) and imatinib (10 three T-75 flasks and treated with Vehicle control, rapamycin, uM) showed a significant decrease in the viable cell number imatinib or imatinib and rapamycin for 24 hours. RNA was compared with DMSO treated control cells (FIG. 1). Cells extracted and purified using QIAGEN RNeasy Mini Kit (no. were treated with vehicle control (DMSO), rapamycin (5 74104) and quantified spectrophotometrically. RNA (1 lug) nM), imatinib (10 uM) or imatinib (10 uM)+rapamycin (5 was used after DNase treatment (no. 18068-015; Invitrogen) nM) (shown along X-axis) for 24 h and counted using a followed by first-strand cDNA synthesis and RT-PCR (no. Coulter counter. The Y-axis represents cell number with error 12371-019; SuperScript, Invitrogen) using a 96-well Optical bars representing the SEM. The effect of imatinib and rapa Reaction Plate (no. 128; ABI PRISM, Applied Biosystems) mycin in combination was significantly different than either for the RT-PCR reaction. A measure of 2.5 ul of template, rapamycin (p<0.05) or imatinib (p<0.05) monotherapy. which had been diluted 1:10 in molecular grade water (Cell gro; Mediatech Inc.) was used and each data point was set in Example 2 in triplicate. Angpt2, Nrarp, Vegf. Aurora kinase A, Aurora kinase B, Centromere protein A, Cyclin B1 and 18S TaqMan Sirolimus and Imatinib Combination Therapy Inhib Gene Expression primer assay from Applied Biosystems its Activation of PDGFRB Tsc2 angl Cells were used along with master mix (TaqMan Fast Universal 0067. The impact of imatinib (10 um), rapamycin (10 PCR Master Mix2x. Applied Biosystems). The reaction nM), and a combination of rapamycin and imatinib on the was run on the 7500 Applied Biosystems Reader for absolute phosphorylated PDGFR-3 signaling pathway was tested in quantification for 96-well plates. Gene expression data were Tsc2angl cells. Treatment oftsc2angl cells with rapamycin automatically calculated by Sequence Detection Software, monotherapy led to upregulation of PDGFRB and increased version 1.3.1 (Applied Biosystems) as mean+/-SEM. phosphorylation at Tyr-1009 compared with DMSO control Imatinib treatment, however, led to a downregulation of Example 4 PDGFRB phosphorylation; this decrease was maintained in the presence of rapamycin (FIG. 2). The upregulation of Transcriptional Profiling Analysis PDGFR? by rapamycin monotherapy suggests that the acti 0070 RNA was extracted fromtsc2ang1 cells treated with vation of PDGFRB is a compensatory mechanism to the rapamycin, imatinib and a combination of the two for 24 mTOR blockade. hours. A coordinate downregulation of Aurora kinase signal ing pathway was seen in cells treated with rapamycin and Example 3 imatinib combination therapy (FIG. 4a and FIG. 4b, showing change in gene expression) compared to vehicle control treat Sirolimus and Imatinib Combination Therapy’s ment. Effect on Angiogenic Mediators 0071. To verify the effect of imatinib and rapamycin on the 0068 Tsc2ang1 cells were treated with control (DMSO), levels of Aurora kinases A and B, imatinib (10 uM), rapamy rapamycin (5 nM), imatinib (10 uM), and imatinib (10 uM)+ cin (10 nM), and a combination of both were tested in vitro rapamycin (5 nM) for 24 hours. Total RNA was then extracted with Tsc2ang1 cells. The combination therapy inhibited the and the levels of expression of the angiogenic genes relative expression levels of both Aurora kinase A (FIG. 5a) Angiopoietin-2 (Angpt2) (FIG. 3a), NOTCH-regulated and Aurora kinase B (FIG.5b) by 70% compared to DMSO ankyrin repeat protein (Nrarp) (FIG.3c), and Vascular endot treated control. The cells were also treated with the combi helial growth factor (VEGF) (FIG. 3b) were measured by nation of imatinib (10 uM) and rapamycin (10 nM) for 24 real-time PCR. Significant downregulation of Angpt2 (p<0. hours before being lysed and analyzed by using antibodies 001), Nrarp (p<0.001), and VEGF (p<0.05) levels were specific for the Aurora kinase A and Aurora kinase B. BActin observed in the imatinib (10 uM)+rapamycin (5 nM) combi was used as the loading control by using monoclonal anti-B- nation therapy group. Total RNA was extracted from cell Actin antibody. Western blot analysis (FIG. 5d) revealed that pellets pooled from two identical and independent experi the combination therapy modestly increased the level of ments using the RNeasy mini column (Qiagen). 10 ug total Aurora kinase A protein and caused a steep decrease in the RNA pools from different experimental conditions were level of Aurora kinase B protein. independently and randomly labeled with either Cy3 or Cy5 0072 Lysates of Tsc2ang1 cells treated with vehicle or fluorophores using the Agilent Fluorescent Direct Label kit indicated drugs were prepared in NP-40 lysis buffer (1% (Agilent Technologies). Labeled RNA pools were then com NP-40, 150 mmol/L NaCl, 10% glycerol, 20 mmol/L petitively hybridized to Agilent murine cDNA microarray HEPES, 1 mmol/L phenylmethylsulfonyl fluoride, 2.5 slides, which contain 2 identical array areas. The above pro mmol/L EDTA, 100 umol/L Na3VO4, and 1% aprotinin) cedures were repeated 3 times on RNA isolated from a dif Protein concentration in cleared lysates was determined using ferent batch of cells, giving 6 arrays in an in 3 (biological an Eppendorf BioPhotometer. Samples were resolved using replicates), n=2 (technical replicates) arrangement for each SDS-PAGE (National Diagnostics) and transferred to nitro comparison group. Fluorescence intensities of each hybrid cellulose membranes. The membranes were blocked with 5% ized spot were determined by using the Agilent Array Scanner nonfat dry milk in 10 mmol/L Tris/0.1% Tween 20/100 and the Agilent Feature Extraction Software. Two different mmol/L NaCl and incubated with primary antibodies fol statistical methods were used to determine differentially lowed by horseradish peroxidase-conjugated secondary anti expressed genes: mixed ANOVA (MXANOVA) and signifi body. The immunoreactive bands were visualized by cance analysis of microarrays (SAM). The list of differen enhanced chemiluminescence (Amersham Biosciences). The US 2012/0196.870 A1 Aug. 2, 2012 antibodies used were: Phospho-PDGFR-B antibody (Tyr cells were injected Subcutaneously into nude mice (n=4) in 1021) (Cell signaling Laboratories); Phospho-PDGFR-3 each treatment group. Two days later, intraperitoneal treat antibody (Tyr 1009) (Cell Signaling Laboratories); Aurora ment with control, imatinib, rapamycin, and combination kinase A antibody (4718) (Cell signaling Laboratories), therapy of imatinib and rapamycin was conducted for 30 Aurorakinase B (3094) antibody (Cell signaling Laborato days. The treatment resulted in a 97% decreased tumor vol ries) monoclonal anti-B-tubulin antibody (Sigma) was used ume in the imatinib and rapamycin combination treatment as a loading control. group compared with control (FIG. 8). 0077. The compounds were suspended in 0.1 ml of ethanol Example 5 and 0.9 ml of Intralipid solution (Fresenius Kabi, Uppsala, Sweden). No local or systemic toxicity was observed in any of Matrix Metalloproteinase Activity the animals. Injections were given over a period of 4 weeks, 0073. The matrix metalloproteinase (MMP) activity of after which the mice were sacrificed due to overwhelming tSc2angl cells treated with either imatinib or rapamycin tumor burden in the control group. Date are presented as monotherapy showed a decrease in the level of MMP-2 com average tumor Volume (mm3) in each of the two groups vs. pared with control. A greater reduction in the MMP bioactiv controls with bars representing SEM. A representative ity, however, was found when the cells were treated with vehicle-treated mouse is shown on the left (FIG. 8a), and a imatinib and rapamycin in combination (FIG. 6). Specifi representative treated mouse is shown on the right (FIG. 8b). cally, Substrate gel electrophoresis using gelatin as the Sub The y-axis represents tumor Volume and the X-axis treatment. strate indicated that the treatment oftsc2angl cells with rapa Column 1 represents vehicle-treated mice (n=3), and column mycin, imatinib, and a combination of rapamycin and 2, 3 and 4 represent imatinib, rapamycin, and combination imatinib for 24 hours resulted in a decrease in the gelatinase therapy of imatinib and rapamycin (n=4), respectively. P=0. activity of MMP-2 (-65 kDa) in comparison to control 076 using the two-tailed ttest. (DMSO-treated) treatment. 0074 The presence and activity of specific MMP species Example 8 were detected using gelatin Zymography. An aliquot of each conditioned media sample was centrifuged at 4,000 rpm for Sirolimus and Imatinib combination Therapy for 1-5 min at 4°C. and supernatant was used. A buffer of 4% Tuberous Sclerosis SDS, 0.15 mol/L Tris (pH 6.8), 20% glycerol and 0.5% (w/v) 0078. A subject diagnosed with tuberous sclerosis will be bromophenol blue was added to the conditioned media treated with a combination therapy comprising sirolimus and sample. Condition media samples mixed with buffer were imatinib. Specifically, the subject will be treated with Rapa directly added to 10% SDS-acrylamide gel containing 0.1% mune and Gleevec. The treatment will comprise an initial (w/v) gelatin (BioRad) and separated by running on a minigel bolus of 6 mg Rapamune followed by 2 mg daily as well as apparatus at 15 mA/gel, and then gels gently rocked in a 2.5% 400 mg Gleevec daily. Potential toxicity resulting from the Triton X-100 solution for 30 min at room temperature. Gels treatment will be monitored during said treatment. This com were then incubated over night at 37° C. in substrate buffer bination therapy will resultina reduction of the tumor volume containing 50 mmol/L Tris-HCL (pH 8), 5 mmol/L CaCl2 associated with tuberous sclerosis in the Subject. and 0.02% NaN3. After incubation, gels were stained for 30 min in 0.5% Coomassie Blue R-250 dissolved in acetic acid, What is claimed: isopropyl alcohol and water (1:3:6); destained in acetic acid, 1. A method of treating or preventing cancer comprising ethanol and water (1:3:6). The finalized gel was scored for the administering an mTOR inhibitor in combination with a presence/absence MMP activity by a blinded evaluator and tyrosine kinase inhibitor to a subject in need thereof. photographed. 2. The method of claim 1 wherein the tyrosine kinase inhibitor is a platelet-derived growth factor receptor Example 6 (PDGFR) inhibitor, an Abelson (Abl) protein inhibitor, or a CD117 inhibitor. Effect of Therapy on FoxM1 3. The method of claim 1, wherein the tyrosine kinase 0075. In order to understand the differential effect of com inhibitor is imatinib, linifanib, ponatinib, regorafenib, bination therapy on Aurora kinase B over Aurora kinase A, Vargatef, or Sunitinib, or CP 673451. FoxM1 expression was examined. FoxM1 is a transcription 4. The method of claim 1 wherein the mTOR inhibitor is factor that is required for the expression of Aurora kinase B Sirolimus, Everolimus, Deforolimus, AZD8055, BEZ235, but not Aurora kinase A. Combination therapy of tSc2angl GSK105.9615, KU-0063794, WYE-354, PI-103, Temsiroli cells with imatinib (10 uM) and rapamycin (10 nM) for 24 mus, Ridaforolimus, Zotarolimus, and Umirolimus (also hours resulted in the downregulation of FoxM1 protein com known as Biolimus or Biolimus A9). pared with DMSO control (FIG. 7). This was determined by 5. The method of claim 1, wherein the subject is diagnosed western blot analysis using a FoxM1 monoclonal antibody with tuberous sclerosis, skin cancer, breast cancer, prostate following cell lysis. BActin was used as the loading control by cancer, lung cancer, bladder cancer, melanoma, colon and using a monoclonal anti-B-Actin antibody. rectal cancer, non-hodgkin's lymphoma, endometrial cancer, pancreatic cancer, renal cell cancer, leukemia, and/or thyroid Example 7 CaCC. In Vivo Tumorigenesis 6. The method of claim 1, wherein the subject is diagnosed with a mutation in hamartin (tSc1) or tuberin (tsc2). 0076. To determine whether compounds that inhibit 7. The method of claim3, whereinimatinib is administered VEGF, phosphorylated forms of PDGFR-3, and angiopoietin to a human subject orally at about 400 mg. 600 mg. or 800 mg 2 in vitro would affect tumor formation in vivo, 10° Tsc2ang1 per day. US 2012/0196.870 A1 Aug. 2, 2012

8. The method of claim 4, wherein rapamycin is adminis 12. A pharmaceutical composition comprising an mTOR tered to a human subject orally at a dose of about 0.5 mg, 1 inhibitor and a tyrosine kinase inhibitor. mg, or 2 mg per day. 13. The pharmaceutical composition of claim 12, wherein 9. The method of claim 8, wherein a one-time bolus of the tyrosine kinase inhibitor is a platelet-derived growth fac about 6 mg of rapamycin is administered to a subject at the tor receptor (PDGFR) inhibitor, an Abelson (Abl) protein initiation of treatment. inhibitor, or a CD117 inhibitor. 10. The method of claim 1, wherein the subject is immu 14. The pharmaceutical composition of claim 12, wherein nocompromised. the mTOR inhibitor is rapamycin and the tyrosine kinase 11. The method of claim 10, wherein the subject is treated inhibitor is imatinib. with one or more anti-viral therapies and/or one or more antibiotics.