Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

• Aging of the skin is the most prominent feature of the aging process, being caused by multiple factors such as intrinsic aging process and UV light exposure.
• Age-related dermal disorders include for example dermal atrophy, actinic keratosis, pseudoscars, lentigines, focal areas of dermal thickening, and coarse wrinkles.
• Dermal atrophy also called skin atrophy or atrophy
• dermal atrophy is a disorder manifesting thinning or depression of skin due to reduction of underlying tissue.
• Dermal atrophy is a major clinical problem in the elderly population. Loss of dermal integrity leads to increased fragility of the skin and precludes the use of intravenous lines in many cases. Impairment in wound healing is an important clinical sequelae of reduced dermal integrity leading to an increase in the number of the infections and complications following injur ⁇ '.
• Pseudoscars are stellate lesions that occur spontaneously in elderly individuals which can occur as senile and presenile forms. These lesions can be found in 20% of patients over the age of 70.
• Lentigines are areas of hyperpigmentation occurring with age and may represent precursor lesions to lentigo maligna and melanoma. They may increase with age and become common in middle aged and elderly individuals. Seborrheic or actinic keratosis, which comprise focal areas of epidermal thickening, can occur, possibly representing a response to damage. Similarly, coarse wrinkles are thought to arise from a damage response.
• treatments for age-related dermal atrophy and related disorders include subdermal hyaluronic acid injection, injection of botulinum toxin or topical application of antioxidant such as vitamin C, green tea extract, and coenzyme Q, but these agents are not able to fully treat these conditions.
• Cellular senescence is a stress response activated by mammalian cells upon exposure to several insults, such as oxidative stress, genotoxic stress, telomere attrition, or dysregulated mitogenic signaling. These stresses activate the senescence response by triggering two pathways: the pathway, which are required to establish
• Senescence-inducing stimuli can cause DNA damage and trigger a sustained DNA damage response (DDR): in response to sustained, unresolved DNA damage, the Ataxia Telangiectasia Mutant (ATM) kinase activates p53 and its transcriptional target which arrests cellular proliferation by inhibiting cell-cycle- dependent kinases.
• DDR DNA damage response
• ATM Ataxia Telangiectasia Mutant
• the same senescence -inducing stimuli can trigger the activation of the Stress-Activated Protein Kinase independently of DNA damage.
• p38 MAPK then can promote the arrest of the cell-cycle and establish senescence by activating the transcription factor HBP!, which increases the expression of
• the DDR pathway usually mediate the initial arrest by increasing the levels of and only at later times senescence is reinforced by expression of Furthermore, the p53 and the p38 MAPK
• Mammalian/mechanistic target of rapamycin is an intracellular protein complex that is responsive to both growth factors and nutrient availability, and which also impacts mitochondrial function. It is comprised of the TOR kinase (originally identified in yeast, and known as mTOR in mammals), accessory proteins, and downstream mediators including the ribosomal S6 kinase (p70S6K) a key downstream target of TOR.
• the TOR signaling pathway is highly conserved in eukaryotes and is functionally defined as the target of the highly -specific antifungal, rapamycin.
• the proteins that comprise the core mTOR complex are the ser-thr kinase mTOR, also known as the FKBP-12-rapamycin associated protein (FRAPl), and mammalian lethal with SEC 13 protein 8 (mLST8). These core components have the capability of forming either of two complexes, mTORCl or mTORC2, which are distinguishable by their sensitivity to rapamycin.
• the rapamycin-sensitive mTORC l contains the scaffolding protein regulatory- associated protein of mTOR (Raptor), whereas the rapamycin-insensitive complex mTORC2 contains the scaffolding protein rapamycin-insensitive companion of mTOR (Rictor).
• scaffolding proteins function to direct mTORCl and mTORC2 to their respective targets. Additional components are unique to each complex.
• proline-rich Akt/PKB substrate 40 KDa PRAS40
• Rh stress-activated MAP kinase-interacting protein 1
• Rictor-1 protein observed with Rictor-1
• compositions and methods that can be used to treat or prevent certain age-related dermal conditions in a mammalian subject in need thereof, such as a human.
• the present invention fulfills this need.
• the invention provides a method of treating or preventing an age-related derrnal disorder in a mammalian subject in need thereof.
• the invention further provides a method of increasing the lifespan of a mammalian fibroblast.
• the invention further provides a method of preserving cell organization of a mammalian fibroblast.
• the invention further provides a method of pre venting or minimizing senescence of a mammalian fibroblast.
• the in vention further provides a kit for treating or preventing an age-related dermal disorder in a mammalian subject in need thereof.
• the method comprises topically administering to the subject a composition comprising a therapeutically effective amount of a mTORCl inhibitor or a salt, solvate, enantiomer or diastereoisomer thereof.
• the method comprises contacting the fibroblast with a composition comprising an effective amount of a mTORCl inhibitor or a salt, solvate, enantiomer or diastereoisomer thereof.
• the age-related dermal disorder is at least one selected form the group consisting of dermal atrophy, seborrheic or actinic keratosis, pseudoscars, ientigines, focal areas of dermal thickening, and coarse wrinkles.
• the mTORCl inhibitor is at least one selected from the group consisting of BEZ235, rapamycin, everolimus, AZD8055, Temsirolimus, KU-0063794, PT- 103, Torkinib, Tacrolimus, Ridaforolimus, INK- 128, Voxtalisib, Torin-1, Omipalisib, OSI- 027, PF-04691502, Aprtoiisib, GSK1059615, WYE-354, Gedatolisib, AZD-2014, Torm-2, WYE-125132, BGT226, Palomid-529, PP121 , WYE-687, CH5132799, Way-600, ETP- 46464, GDC-0349, XL388, and Zotarolimus, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the mTORC 1 inhibitor is at least one selected from the group consisting of rapamycin, Ridaforolimus, and Everolimus, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the mTORCl inhibitor is rapamycin, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the subject is a human. In other embodiments, the composition is applied topically to the skin of the subject.
• the composition comprises about 0.001-1% by weight of the mTORC l inhibitor, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the mTORCl inhibitor is rapamycin, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the composition further comprises a deployatologically acceptable carrier.
• the dermatologically acceptable carrier is at least one selected from the group consisting of a solvent, lubricant, emollient, emulsifier, moisturizer, thickening wax, softener, fragrance, preservative, and artificial color.
• the dermatologically acceptable carrier comprises petrolatum.
• the fibroblast is a dermal fibroblast.
• the dermal fibroblast is in vivo and part of a mammalian subject's skin.
• the kit comprises a composition comprising a therapeutically effective amount of a mTORC l inhibitor, or a salt, solvate, enantiomer or diastereoisomer thereof.
• the kit further comprises an applicator.
• the kit further comprises instructions for topically administering the composition to the subject.
• FIGs. 1A-1I are a set of graphs illustrating changes in mitochondrial parameters in response to NRTIs and rapamycin.
• Human cardiac fibroblasts were maintained with or without rapamycin (1 nM). After 7 days exposure to NRTIs, parameters of mitochondrial status and function were examined. Grey bars represent cells maintained under standard culture conditions and black bars represent cells maintained in the presence of rapamycin.
• FIG. 1 A is a bar graph illustrating mitochondrial membrane potential as assessed by tetramethylrhodamine ethyl ester, perchlorate (TMRE) staining.
• FIG. IB is a bar graph illustrating mitochondrial ROS levels.
• FIG. 1C is a bar graph illustrating mitochondrial mass.
• FIG. ID is a bar graph illustrating total cellular ROS.
• FIG. I E is a graph illustrating oxygen consumption/cell as a function of time following the addition of the mitochondrial inhibitors oligomycin, carbonyl cyanide p-triflouromethoxyphenylhydrazone (FCCP), or a combination of rotenone and antimycin A.
• FIG. IF is a bar graph illustrating the calculated rates of basal respiration.
• FIG. 1G is a bar graph illustrating the calculated rates of maximal respiration.
• FIG. 1H is a bar graph illustrating the calculated rates of ATP-linked respiration.
• FIG. II is a bar graph illustrating the calculated rates of proton leak. Each measurement represents a minimum of triplicate cultures and all measurements were repeated a minimum of 2 times with similar results. Bars marked with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05 and bars marked with an # represent values that are significantly different between rapamycm-treated and untreated cells within the same
• FIGs. 2A-2H illustrate steady state levels of electron transport chain subunits and mitochondrial proteins.
• FIG. 2A depicts steady state levels of the indicated electron transport chain (ETC) proteins, along with the outer mitochondrial membrane protein voltage-dependent anion channel (VDAC), assessed by immunoblot in cardiac fibroblasts exposed to NRTIs.
• FIG. 2B depicts the steady state levels of Pink 1, the Pink 1 cleavage product, and Parkin.
• FIG. 2C depicts the steady state levels p62 and actin.
• FIG. 2D depicts the results of a nanostring analysis of mRNA levels for the ETC subunits included in NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8 (NDUFB8) (complex 1).
• FIG. 1A depicts steady state levels of the indicated electron transport chain (ETC) proteins, along with the outer mitochondrial membrane protein voltage- dependent anion channel (VDAC), assessed by immunoblot in cardiac fibroblasts exposed to NRTIs.
• FIG. 2E depicts the results of a nanostring analysis of mRNA levels for ETC subunits included in ubiquinol-cytochrome c reductase core protein II (UQCRC2) (complex 3).
• FIG. 2F depicts the results of a nanostring analysis of mRNA levels for the ETC subunits included in succinate dehydrogenase
• FIG. 2G depicts the results of a nanostring analysis of mRNA levels for the ETC subunits included in cytochrome c oxidase subunit I (mt-Col) (complex 4).
• FIG. 2H depicts the results of a nanostring analysis of mRNA levels for the ETC subunits included in ATP5A1 (complex 5).
• Each immunoblot represents a minimum of two independent experiments with similar results. Gray bars represent data from control cultures while black bars represent data from rapamycin -treated cultures.
• Nanostring results are representative of 2 independent experiments.
• FIGs. 3A-3F illustrate the finding that NRTI exposure induces the senescence response, which is prevented by rapamycin.
• Human cardiac fibroblasts were maintained with or without rapamycin (1 nM) following exposure to NRTIs for 7 days. Markers of senescence were examined.
• FIG. 3A illustrates data from human cardiac fibroblasts showing steady state levels of pl6, p21, p53, lamin Bl, and 11. -6 known to be altered during the senescence response. Actin levels are presented as a loading control.
• FIG. 3B is a bar graph illustrating the percentage of ceils staining positive for SA- ⁇ gaiactosidase activity.
• 3C is a bar graph illustrating steady state mRNA levels for p21 determined by nanostring analysis in cells exposed to NRTIs.
• FIG. 3D is a bar graph illustrating steady state mRNA levels for lamin B l determined by nanostring analysis in cells exposed to NRTIs.
• FIG. 3E is a bar graph illustrating a comparative analysis of p21, comparing early passage ceils with cells that have undergone replicative senescence as well as cells that were maintained in rapamycin containing medium allowing lifespan extension.
• FIG. 3F is a bar graph illustrating a comparative analysis of lamin Bl, comparing early passage cells with cells that have undergone replicative senescence as well as cells that were maintained in rapamycin containing medium allowing lifespan extension.
• Grey bars represent cells maintained under standard culture conditions and black bars represent cells maintained in the presence of rapamycin. Bars marked with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05 and bars marked with an # represent values that are significantly different between rapamycin-treated and untreated cells within the same treatment group (e.g. control or exposed to NRTIs).
• FIGs. 4A-4E illustrate the finding that ROS scavengers prevent expression of senescence markers in cells exposed to NRTIs.
• Human cardiac fibroblasts were treated with the mitochondrial ROS scavenger mito-Q during exposure to NRTIs.
• FIG. 4A depicts the steady state levels of pi 6, p21, p53 and lamin B l known to be altered during the senescence response.
• FIG. 4B is a bar graph illustrating the percentage of cells staining positive for lysosomal SA- ⁇ gaiactosidase activity.
• FIG. 4C depicts the results of an immunoblot analysis showing steady state levels of pl6, p21 , p53, actin, and catalase known to be altered during the senescence response, after human cardiac fibroblasts were infected with an adenoviral vector expressing the mt-catalase protein or an empty vector during exposure to NRTIs. Immunoblot represents a minimum of two independent experiments with similar results.
• FIG. 4D is a bar graph illustrating the percentage of cells staining positive for lysosomal SA- ⁇ galactosidase activity, after human cardiac fibroblasts were infected with an adenoviral vector expressing the mt-catalase protein or an empty vector during exposure to NRTIs.
• FIG. 4D is a bar graph illustrating the percentage of cells staining positive for lysosomal SA- ⁇ galactosidase activity, after human cardiac fibroblasts were infected with an adenoviral vector expressing the
• 4E depicts steady state levels of pl6, p21, and lamin Bl associated with senescence.
• Grey bars represent cells maintained under standard culture conditions and black bars represent cells maintained in the presence of rapamycin .
• Bars marked with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05.
• Bars marked with an # represent values that are significantly different between rapamycin-treated and untreated cells within the same treatment group (control or exposed to NRTIs).
• FIGs. 5A-5D illustrate phosphorylation of ribosomal S6 protein and MDM2 in response to NRTI exposure.
• FIG. 5 A depicts the phosphorylation status of the ribosomal S6 protein and MDM2 in response to NRTI exposure as assessed by immunoblot analysis in human cardiac fibroblasts. Extracts from cultures maintained under standard culture conditions are shown in the left 3 lanes while cultures maintained in the presence of 1 nM rapamycin are shown in the 2 right hand lines. Cultures were exposed to 10 or 20 ⁇ , ⁇ NRTIs for 7 days in the case of control cultures while rapamycin treated cultures were exposed to 20 ⁇ NRTIs for 7 days.
• FIG. 5 A depicts the phosphorylation status of the ribosomal S6 protein and MDM2 in response to NRTI exposure as assessed by immunoblot analysis in human cardiac fibroblasts. Extracts from cultures maintained under standard culture conditions are shown in the left 3 lanes while cultures maintained in the presence of 1 nM rapamycin are shown
• FIG. 5B depicts the phosphorylation status of the ribosomal S6 protein and MDM2 after cells were exposed to NRTIs for 7 days followed by incubation with specific kinase inhibitors targeting either the p70 S6 kinase (PF-4708671 ) or MEK1 /2 (U0126) for the final 2 hours.
• FIG. 5C depicts the phosphorylation status of the ribosomal S6 protein and MDM2 after cells were exposed to NRTIs for 7 days followed by incubation with specific kinase inhibitors targeting MEK1/2 (U0126), Raf (GW5047), or p90RSK (BI- D1870), for the final 2 hours.
• FIG. 5D depicts the phosphorylation status of the ribosomal S6 protein and MDM2 after cells were treated with a specific p38 MAPK inhibitor (SB203580) during exposure to NRTIs.
• SB203580 a specific p38 MAPK inhibitor
• FIGs. 6A-6G illustrate effect of Mito-Q and mt-catalase on ribosomal S6 and MDM2 phosphorylation and mitochondrial activity in response to NRTIs.
• FIG. 6A depicts an immunoblot analysis of the phosphorylation status of the ribosomal S6 protein and MDM2 in cardiac fibroblasts cultured in the presence of mito-Q or the inactive carrier thiamine pyrophosphate (TPP) for the duration of NRTI exposure.
• TPP inactive carrier thiamine pyrophosphate
• FIG. 6B depicts an immunoblot analysis for phosphorylation of the ribosomal S6 protein and MDM2 in cardiac fibroblasts infected with adenoviral particles harboring a construct expressing the mt-catalase or an empty viral vector prior to NRTI exposure. Each immunoblot represents a minimum of two independent experiments with similar results and is shown with actin as a loading control.
• FIG. 6C is a Seahorse Bioanalyzer analysis illustrating calculated rates of basal respiration after cardiac fibroblasts treated with mito-Q or TPP during exposure to NRTIs.
• FIG. 6D is a Seahorse Bioanalyzer analysis illustrating calculated rates of maximal respirations after cardiac fibroblasts treated with mito-Q or TPP during exposure to NRTIs.
• FIG. 6E is a Seahorse Bioanalyzer analysis illustrating calculated rates of ATP-linked respiration after cardiac fibroblasts treated with mito-Q or TPP during exposure to NRTIs.
• FIG. 6F is a Seahorse Bioanalyzer analysis illustrating calculated rates of proton leak after cardiac fibroblasts treated with mito-Q or TPP during exposure to NRTIs.
• FIG. 6G is a bar graph illustrating the mean fluorescence intensity of mitochondrial ROS. Bars with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05 and bars marked with an # represent values that are significantly different between TPP and mito- Q-treated ceils within the same treatment group (control or exposed to NRTIs).
• FIGs. 7A-7B illustrate phosphorylation status of the ribosomal S6 protein and MDM2 in response to oxidative stress.
• FIG. 7 A depicts phosphorylation status of the ribosomal S6 protein and MDM2 after serum-deprived cardiac fibroblasts exposed to increasing concentrations of hydrogen peroxide for 2 hours. Serum-stimulated cultures are included in the right hand lane as a positive control for growth factor stimulation of ribosomal S6 phosphorylation.
• FIG. 7B depicts the phosphorylation status of the ribosomal S6 protein in ceils exposed to the indicated concentrations of rotenone for 16 hours. Serum deprived cardiac fibroblasts were exposed to rotenone at concentrations ranging from I to 50 nM. Steady state levels of actin are presented as a control for equal protein loading.
• FIGs. 8A-8C illustrates localization of Raptor to mitochondria in the presence of
• FIG. 8A is a set of images illustrating cells infected with an adenoviral construct harboring an expression construct that produces a GFP protein fused to a mitochondrial targeting sequence (green). These cells were fixed and stained with an antibody that recognizes Raptor (red) and counter stained with 4',6-diamidino-2-phenylindole (DAP1) to visualize DNA (blue).
• FIG. 8B is a representative confocal image of control fibroblasts (not exposed to NRTIs) illustrating a co-localization event in fibroblasts expressing the mitochondrial GFP following exposure to NRTIs for 7 days.
• FIG. 8A is a set of images illustrating cells infected with an adenoviral construct harboring an expression construct that produces a GFP protein fused to a mitochondrial targeting sequence (green). These cells were fixed and stained with an antibody that recognizes Raptor (red) and counter stained with 4',6-diamidino-2-phenylindole (DAP1) to
• 8C is a bar graph illustrating the quantification of co- localization events as determined by both confocal and deconvoiution microscopy. Bars marked with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05 and bars marked with an # represent values that are significantly different between rapamycm-treated and untreated cells within the same treatment group (control or exposed to NRTls).
• Co-localization experiments were performed by four independent evaluators over a series of experiments examining Raptor and mt-GFP co-localization by both confocai microscopy and deconvoiution microscopy. Quantitative data were generated from counts using deconvoiution microscopy.
• FIGs. 9A-9I illustrates the finding that fibroblasts display senescence elevated mitochondrial ROS and enhanced phosphorylation of ribosomal S6 protein and MDM2.
• FIG. 9A is a bar graph illustrating the levels of mitochondrial ROS in early passage and senescent cardiac fibroblasts.
• FIG. 9B is a bar graph illustrating the levels of total cellular ROS in early passage and senescent cardiac fibroblasts. Grey bars represent cells maintained under standard culture conditions and black bars represent cells maintained in the presence of 1 nM rapamycin.
• FIG. 9A-9I illustrates the finding that fibroblasts display senescence elevated mitochondrial ROS and enhanced phosphorylation of ribosomal S6 protein and MDM2.
• FIG. 9A is a bar graph illustrating the levels of mitochondrial ROS in early passage and senescent cardiac fibroblasts.
• FIG. 9B is a bar graph illustrating the levels of total cellular ROS in early passage and senescent cardiac fibro
• FIG. 9C is a graph illustrating oxygen consumption rate normalized to cell number, as a function of time following the addition of the mitochondrial inhibitors oligomycin, FCCP, or a combination of rotenone and antimycin A.
• FIG. 9D is a Seahorse Bioanalyzer analysis illustrating basal respiration of mitochondrial function in human cardiac fibroblasts.
• FIG. 9E is a Seahorse Bioanalyzer analysis illustrating maximal respiration of mitochondrial function in human cardiac fibroblasts.
• FIG. 9F is a Seahorse Bioanalyzer analysis illustrating ATP-linked respiration of mitochondrial function in human cardiac fibroblasts.
• FIG. 9G is a Seahorse Bioanalyzer analysis illustrating proton leak of mitochondrial function in human cardiac fibroblasts.
• FIG. 9H depicts protein lysates derived from cells at increasing population doublings probed for markers of senescence, p21 and pl6, as well as the phosphorylated forms of the ribosomal S6 and MDM2 proteins.
• FIG. 9H depicts protein lysates derived from cells at increasing population doublings probed for markers of senescence, p21 and pl6, as well as the phosphorylated forms of the ribosomal S6 and MDM2 proteins.
• 91 depicts protein lysates probed for the phosphorylation status of the ribosomal S6 protein and MDM2, after fully senescent cultures were treated with the ROS scavengers Trolox and N- acetylcysteine, the p90RSK inhibitor BI-D1870, or infected with adenoviral particles harboring a construct that expresses the mt-catalase protein or an empty vector control. Bars marked with an asterisk represent values that are significantly different from relative control values at P ⁇ 0.05, and bars marked with an # represent values that are same treatment group (control or exposed to NRTIs).
• FIG. 10 is a schematic illustration of a model for mTORCI integration of multiple signals to generate growth response or senescence arrest.
• additional inputs to mTORCl exist in the form of cellular redox status and mitochondrial function that may redirect mTORCl to support a senescent growth arrest through the p70 S6 kinase mediated modulation of MDM2 and p53 activity. These connections are shown in red (mTORC l ⁇ p7056K— j MDM2— j p53 -- ⁇ stabilization --> p53 --> senescence).
• FIGs. 11 A-l IF illustrate status of p53 in human cardiac fibroblast cultures exposed to
• FIG. 11A is a bar graph illustrating comet assay results for ceils exposed to NRTIs or hydrogen peroxide as a positive control.
• the white bar represents control data;
• grey bar represents data from cells exposed to 10 or 20 ug/ml NRTI for 7 days; and
• the black bar represents data from cells exposed to 200 ⁇ hydrogen peroxide for 2 hours.
• An asterisk represents values that are significantly different from relative control values at P ⁇ 0.05.
• FIG. 1 IB depicts the results of an imniunoprecipitation experiment using antibodies against p53.
• FIG. 1 iC depicts levels of p53, MDM2, and phosphoryiated MDM2 in the samples used for imniunoprecipitation in FIG. 1 1A.
• FIG. 1 ID depicts levels of p53 after control and rapamycin-treated cultures were exposed to NRTIs for 7 days followed by a 2-hour incubation with MG132 to inhibit proteasome activity. Modified p53 refers to higher molecular weight forms recognized by the anti-p53 antibody.
• FIG. 1 IE is an image illustrating cytosoiic level of MDM2 without exposure to NRTIs, and an image illustrating cytosoiic level of MDM2 with exposure to NRTIs. Representative photomicrographs of cells stained for MDM2 (red) and counter stained for DNA (blue).
• FIG. 1 IF is a bar graph illustrating relative intensity of cytosoiic MDM2 staining as determined by Image J Analysis.
• FIGs. 12A-12D are a set of images illustrating mitochondrial association of Raptor.
• FIGs. 12A-12B depict representative photomicrographs of cells expressing the mt-GFP protein (green) stained for Raptor (red) under control conditions.
• FIGs. 12C-12D depict representative photomicrographs of cells expressing the mt-GFP protein stained for Raptor following exposure to NRTIs. Co-localization events are indicated by arrows.
• FIG. 13 is a graph illustrating the finding that rapamycin treatment provides lifespan extension in human fibroblasts.
• Human fibroblast cells were growth in culture medium with or without rapamycin (1 nM). Cultures were split every 7 days and reseeded at identical cell number/cm 2 each week. The lifespan of normal human fibroblasts is counted by the number of times that the cells double. Rapamycin treated grow well beyond the normal lifespan for these cells.
• FIG. 14 illustrates the finding that rapamycin preserves cell organization during aging of human fibroblasts.
• Human fibroblasts maintained in the presence of Sow doses of rapamycin maintained an orderly growth pattern while untreated fibroblasts lost their ability to properly orient themselves with age.
• the ability to organize is a critical element of normal fibroblast function and contributes to tissue integrity in normal tissue. The disorganization which occurs with age contributes to functional decline.
• FIG. 15 are a set of images illustrating the finding that rapamycin preserves mitochondrial network in the face of damage.
• Panel A depicts that human fibroblasts expressing a fluorescent protein in the mitochondrial display a green mitochondrial network.
• Panel B depicts that rapamycin treated cells have a normal mitochondrial network.
• Panel C depicts that the mitochondrial network is destroyed by exposure to a mitochondrial toxin, ethidium bromide (EthBr).
• Panel D depicts that rapamycin treated cells are able to maintain their mitochondrial network following exposure to EthBr.
• FIG. 16 is a bar graph illustrating the finding that rapamycin increases the survival of human fibroblasts following mitochondrial stress. Human fibroblasts were exposed to ethidium bromide for 7 days and cell viability measured at that time.
• FIG. 17 is a bar graph illustrating the finding that rapamycin prevents the expression of genes that cause senescence.
• the p21 gene makes a protein that is critical for senescence of human fibroblasts. Cells grown in the presence of rapamycin do not produce p21 as they age while the control cells express p21 and enter senescence.
• FIG . 18 is a bar graph illustrating the finding that rapamycin prevents the expression of genes related to inflammation.
• the IL-6 gene makes a protein that activates the immune system, to recruit immune cells into an area of tissue damage. Cells grown in the presence of rapamycin do not produce IL-6 as they age while the control cells express IL-6 and enter senescence.
• FIG. 19 is a bar graph illustrating the finding that rapamycin increases dermal thickness in atrophic skin. Dermal thickness was measured using a Mitoutoyo digital caliper with certified accuracy to 0.001 mm following 14 days of rapamycin treatment in an emulsified gel preparation.
• FIG.20 is a photograph of a seborrheic keratosis lesion before and after 21 days of treatment with 10 ⁇ rapamycin in an emulsified gel preparation as in FIG. 19. A significant reduction in severity of the lesion was apparent upon visual inspection.
• the present invention relates, in one aspect, to the unexpected discovery that the compositions and methods of the invention can be used to treat or prevent age-related dermal disorders including, hut not limited to, dermal atrophy, seborrheic keratosis, actinic keratosis, pseudoscars, lentigines, focal areas of dermal thickening, and coarse wrinkles.
• compositions and methods of the invention are useful for treating or preventing dermal atrophy in a subject in need thereof.
• the compositions of the invention comprises therapeutically effectiv e amounts of at least one mTORCl inhibitor.
• the compositions of the invention comprise the mTORCl inhibitor as the only ingredient that is active against the age-related dermal condition.
• the mTORCl inhibitor is also a mTORC2 inhibitor.
• compositions and methods of the invention enhance lifespan of fibroblasts and improve the growth and stress resistance of normal fibroblasts. Without wishing to be limited by any theory, this may be associated with a decrease in inflammatory cytokine production.
• delivery of therapeutically effective amounts of a mTORCl inhibitor to the dermal layers induces mesenchymal responses that influence dermal homeostasis.
• delivery of therapeutically effective amounts of a mTORCl inhibitor to the dermal layers produces an increase in dermal thickness and improvement in skin function.
• mitochondrial ROS was identified as a novel input for mTORCl .
• the present results support the finding that mitochondrial -generated ROS serves to activate mTORCl (FIG. 10). This is reflected by increased phosphorylation of both the ribosomal S6 protein and MDM2 observed in the experimental setting. Inhibition of mTORCl by rapamycin prevented these responses, as did interventions aimed at reducing mitochondrial ROS, such as the expression of a mitochondrial-targeted catalase or treatment with ROS scavengers.
• This effect of rapamycin on p53 may underlay the lack of activation of p53 target genes, such as p21, in rapamycin-treated cells and contributed to the delayed senescence observed when cells were cultured in the presence of rapamycin at concentrations sufficient to influence mTORCl signaling but not sufficient to completely block proliferation.
• the present results show that mTORCl signaling through the p70S6K may be responsive to ROS generated by mitochondria.
• mTORCl/p70S6K occurred in settings of mitochondrial dysfunction, replicative senescence, and in aged tissue. Rapamycin ameliorated both the mitochondrial ROS production and blocks the mTORCl/p70S6K response. These effects of rapamycin supported the beneficial effects observed in terms of longevity and in age-related disorders like dermal atrophy following rapamycin treatment.
• the articles “a” and “ 'an” refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article.
• an element means one element or more than one element-
• the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used.
• the term “ 'about “ ' is meant to encompass variations of ⁇ 20% or ⁇ 10%, ⁇ 5%, ⁇ 1 %, or ⁇ 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.
• dermatologically acceptable carrier or “dermatologically acceptable excipient” refers to the compositions or components that are suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like.
• a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated, the animal's health continues to deteriorate.
• 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 uncreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
• the terms “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” of a compound are used interchangeably to refer to the amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered.
• the term to "treat,” as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the severity with which symptoms are experienced. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
• an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of a compound, composition, assay or method of the invention in a kit for suppressing or reducing systemic immune response in a subject.
• the instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composi tion , assay, or methods of the inventi on or be shipped together with a container that contains the identified compound, composition, assay, or method.
• the instructional material can be shipped separately from the container with the intention that the instructional material and the compound, composition, assay, or method be used
• module means, with respect to disease states or conditions associated with binding of a compound of the present invention to a receptor contemplated in the present invention, to produce, either directly or indirectly, an
• Modulation may occur by virtue of agonist activity, antagonist activity or mixed agonist/antagonist activity (depending on the receptor site).
• NRT1 refers to a nucleotide/nucleoside reverse transcriptase inhibitor.
• the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non- toxic, i. e. , the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
• composition refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
• the pharmaceutical composition facilitates
• administration of the compound to an organism.
• Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, intracranial and topical administration.
• the administration comprises topical administration.
• a "subject" refers to a human or non-human mammal.
• Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, fetine and murine mammals.
• the subject is human.
• TDF refers to tenofovir disoproxil fumarate, or a salt or solvate thereof.
• topical administration or “topical application” refers to a medication applied to body surfaces such as the skin or mucous membranes.
• treatment is defined as the application or administration of a therapeutic agent, i.e. , a composition useful within the invention (alone or in combination with another pharmaceutical agent), to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject ⁇ e.g. , for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder or the potential to develop the disease or disorder.
• Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
• 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.
• composition of the invention comprises a therapeutically effective amount of a mTORC 1 inhibitor, or salt, solvate, enantiomer or diastereoisomer thereof.
• the mTORC 1 inhibitor is at least one selected from the group consisting of BEZ235, rapamycin, everolimus, AZD8055, Temsirolimus, KU-0063794, PI- 103, Torkinib,
• the mTORCl inhibitor contemplated in the invention is rapamycin.
• the mTORC l inhibitor may be a modified form of rapamycin with improved delivery to specific intracellular compartments or organelles, such as the mitochondria, the nucleus, the lysosome, and/or the endoplasmic reticulum.
• the therapeutically effective amount of a mTORC l inhibitor in the composition ranges from about 0.001% to about I % by weight. In other embodiments, the therapeutically effective amount by weight of the mTORCl inhibitor in the composition ranges from about 0.002% to about 0.75%, about 0,005% to about 0.5%, about 0,008% to about 0.25%, about 0.01 % to about 0.2 %, about 0,02% to about 0.15%, or about 0.03% to about 0.1 %.
• the composition of the invention further comprises a dermatologically acceptable carrier.
• the compositions of the present invention may comprise from about 60% to about 99.9%, alternatively from about 70% to about 95%, and alternatively from about 80% to about 90%, of a dermatologically acceptable carrier.
• the dermatologically acceptable carrier is at least selected from the group consisting of solvent, lubricant, emollient, emulsifier, moisturizer, thickening wax, softener, fragrance, preservative, and artificial color(s ).
• the dermatologically acceptable carrier is at least one selected from the group consisting of water, fatty alcohols, and volatile organic alcohols.
• dermatologically acceptable carrier is petrolatum.
• the invention provides methods of increasing the lifespan of mammalian fibroblasts. In another aspect, the invention provides methods of preserving cell organization in mammalian fibroblasts. In yet another aspect, the invention provides methods of preventing or minimizing senescence in mammalian fibroblasts. In yet another aspect, the invention provides methods of treating or prev enting age-related dermal disorders including dermal atrophy, pseudoscars, seborrheic or actinic keratosis, ientigmes, focal areas of dermal thickening, and coarse wrinkles in a mammalian subject.
• the methods of the invention comprise topically
• compositions of the invention comprises a therapeutically effective amount of a mTORCl inhibitor.
• the composition further comprises a dermatologically acceptable carrier.
• the composition is applied topically to the affected skin area of the subject.
• topical formulations of the compositions contemplated within the invention are used for treating dermal atrophy.
• the invention provides a topical cream comprising a therapeutically effective amount of rapamycin for treating or preventing dermal atrophy.
• dermal atrophy is evaluated by measurement of the dermal layer utilizing a calibrated digital caliper measurement of the dermal layer.
• improvement in seborrheic keratosis, lentigines, pseudoscars, coarse wrinkles, and epidermal thickening is evaluated through an investigator evaluation rating scale of 1-4, in which 1 is normal with no sign of lesion; 2 represents minor lesions; 3 represents lesions that are distinct features relative to normal skin; and 4 represents lesions that are of high severity.
• lesions can be examined visually or with the aid of image analysis software such as ImageJ, an open source image analysis software available from the National Institutes of Health.
• lesions are evaluated by area measurement using manual measurement of the lesion or through analysis of images taken by investigators or research study staff.
• the relative amounts of the active ingredient, the dermatologically 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.
• the composition may comprise between about 0.001 % and about I % (w/w) of a mTORC I inhibitor.
• the therapeutically effective amount by weight of the mTORC 1 inhibitor in the composition ranges from about 0.002% to about 0.75%, about 0.005% to about 0.5%, about 0.008% to about 0.25%, about 0.01 % to about 0.2 %, about 0.02% to about 0.15%, or about 0.03% to about 0.1 %.
• compositions are generally suitable for administration to animals of all sorts.
• 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 can 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.
• composition of the invention can be administered to a mammal 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.
• Dosing regimens for administering the compositions of the invention may be once a day or twice a day.
• the frequency of the application and the concentration of the active agent is dependent on the skin condition and the response of the dermis.
• Application can be continued to achieve the desired effect on the dermis and the frequency of application can be reduced after a satisfactory result has been obtained.
• the administration lasts a minimum of 2 weeks to achieve results.
• Applications can continue beyond the initial 2 week period to obtain continued improvement and the frequency of application can be reduced once this result has been achieved.
• Applications may continue over the course of years with variable levels of application based upon the relative severity of lesions at any one time.
• the amount of the composition of the invention 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.
• 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, and so forth.
• compositions of the invention are formulated using one or more deployatologically acceptable excipients or carriers.
• the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a mTORCl inhibitor and a deployatologically acceptable carrier.
• Dennatologically acceptable carriers which are useful, include, but are not limited to, glycerol, water, saline, ethanol and other deployatologically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other deployatologically acceptable carriers are described in
• compositions of the present invention may comprise from about 60% to about 99.9%, alternatively from about 70% to about 95%, and alternatively from about 80% to about 90%, of a deployatologically acceptable carrier.
• the compositions of the present invention may comprise from about 60% to about 99.9%, alternatively from about 70% to about 95%, and alternatively from about 80% to about 90%, of a deployatologically acceptable carrier.
• the beatologically acceptable carrier is at least selected from the group consisting of solvent, lubricant, emollient, emulsifier, moisturizer, thickening wax, softener, fragrance, preservative, and artificial color(s ).
• the dermatologically acceptable carrier is at least one selected from the group consisting of water, fatty alcohols, and volatile organic alcohols.
• One non-limiting example of the dermatologically acceptable canier is petrolatum.
• 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.
• 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 which delays absorption, for example, aluminum monostearate or gelatin.
• Formulations may be employed in admixtures with conventional excipients, i.e. , pharmaceutically acceptable organic or inorganic earner substances suitable for topical 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.
• 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; 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 include antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
• Oilier ' " 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.
• 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.
• a particularly preferred preservative is a combination of about 0.5% to 2,0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
• the composition preferably includes an antioxidant and a chelating.
• Preferred antioxidants for some compounds are BUT, BHA, a-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.
• 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 ammopolycarboxyiic acid salts (e.g. disodium ethylenediaminetetraacetic acid) 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 rnetal ions in the composition which may be detrimental to the shelf life of the formulation.
• stratum corneum layer of the epidermis An obstacle for topical administration of pharmaceuticals is the stratum corneum layer of the epidermis.
• the stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells.
• One of the factors that limit the penetration rate (flux) of a compound through the stratum corneum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other tilings being equal.
• Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
• Such formulations may be applied to the skin directly or through the use of swabs, applicators, spatulas and the like, as well as in the form of transdermal patches.
• the patch minimizes loss of pharmaceuticals through washing, friction, scratching and/or rubbing of the skin.
• the patch increases absorption of the pharmaceutical through the skin, while minimizing the exposure of the skin to the pharmaceutical.
• Topically administrable formulations contemplated within the invention may, for example, comprise from about 0.001% to about 1% (w/w) a mTORCl inhibitor, although the concentration of the mTORCl inhibitor may be as high as the solubility limit of the active ingredient in the solvent.
• Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
• Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monoiaurate, PGML (polyethylene glycol monoiaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram,
• alkanecarboxylic acids dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.
• One acceptable vehicle for topical deliver ⁇ ' of some of the compositions of the invention may contain liposomes.
• the composition of the liposomes and their use are known in the art (for example, U.S. Patent No. 6,323,219).
• the topical formulation further comprises other ingredients such as adjuvants, antioxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like
• a permeation or penetration enhancer is included in the formulation and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum corneum with respect to a composition lacking the permeation enhancer.
• the topical formulation may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum corneum, and thus allows increased transport across the stratum corneum.
• hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.
• non-active ingredients in the topical formulation are well known in the art. These ingredients include, but are not limited to, humectants, emollients, pH stabilizing agents, chelating agents, gelling agents, thickening agents, emuisifiers, binders, buffers, carriers, antioxidants, etc. Additional examples of such ingredients are included in the U.S. Food & Drug Administration, Inactive Ingredients for Approved Drugs, available online.
• a gel formulation of the invention comprises about 0.001% to about 1% (w/w) of a mTORCl inhibitor, about 20-50% (w/w) dimethyl sulfoxide (DMSO), about 10-20% (w/w) polypropylene glycol , about 10-40% (w/w) polyethylene glycol (PEG) with a molecular weight from 100-800 (PEG 100-PEG800), about 1 -2% (w/w) gelling agents, and about 0-50% Water.
• DMSO dimethyl sulfoxide
• PEG polypropylene glycol
• PEG polyethylene glycol
• a gel formulation of the invention comprises about 0.001% to about 1% (w/w) of rapamycin, about 20-50% (w/w) dimethyl sulfoxide (DMSO), about 10- 20% (w/w) polypropylene glycol, about 10-40% (w/w) polyethylene glycol (PEG) with a molecular weight from 100-800 (PEGi00 ⁇ PEG800), about 1-2% (w/w) gelling agents, and about 0-50% Water.
• DMSO dimethyl sulfoxide
• PEG polypropylene glycol
• PEG polyethylene glycol
• a solution or spray formulation of the invention comprises about 0.001% to about 1% (w/w) of a mTORCl inhibitor in an aqueous solution having about 10-50% (w/w) of DMSO and about 10-50% (w/w) of PEG ,
• a solution or spray formulation of the invention comprises about 0.001% to about 1% (w/w) of rapamycin in an aqueous solution having about 10-50% (w/w) of DMSO and about 10-50% (w/w) of PEG ,
• a cream or lotion formulation of the invention comprises about 0.001% to about 1% (w/w) of a mTORCl inhibitor, mineral oil, any type of alcohol, a non-ionic detergent such as Triton X-100, emulsifying wax, glycerol monostearate (GMS), isopropyl myristate ( ⁇ ), and about 60-80% water.
• a non-ionic detergent such as Triton X-100, emulsifying wax, glycerol monostearate (GMS), isopropyl myristate ( ⁇ ), and about 60-80% water.
• a cream or lotion formulation of the invention comprises about 0.001% to about 1% (w/w) of rapamycin, mineral oil, any type of alcohol, a non-ionic detergent such as Triton X-100, emulsifying wax, glycerol monostearate (GMS), isopropyl myristate (IPM), and about 60-80% water.
• an ointment formulation of the invention comprises about 0.001% to about 1% (w/w) of a mTORCl inhibitor in an aqueous solution having about 10- 50% (w/w) of DMSO and about 10-50% (w/w) of PEG and about 1-60% (w/w) petrolatum.
• an ointment formulation of the invention comprises about 0.001% to about 1% (w/w) of rapamycin in an aqueous solution having about 10-50% (w/w) of DMSO and about 10-50% (w/w) of PEG and about 1-60% (w/w) petrolatum.
• Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
• the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticies, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions.
• Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention.
• single unit dosage forms suitable for topical administration such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, transdermal patches, and solutions or suspensions that are adapted for controlled-release are encompassed by the present invention.
• controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts.
• the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drag substance being employed to cure or control the condition in a minimum amount of time.
• Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance.
• controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drag, and thus can affect the occurrence of side effects.
• controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time.
• the drug In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drag being metabolized and excreted from the body.
• Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
• controlled-release component in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient.
• the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
• sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drag over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
• the period of time may be as long as a month or more and should be a release that is longer that the same amount of agent administered in bolus form.
• the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
• the compositions of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
• delayed release is used herein in its conventional sense to refer to a drag formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
• pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drag administration.
• immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drag administration.
• short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drag administration.
• rapid-offset refers to any period of tim e up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drag administration.
• reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g. , nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
• Ceil culture experiments utilizing NRTls were of the following design. Cultures of either human lung or cardiac fibroblasts were cultivated according to standard culture protocols for these cells (Cristofalo, et ai, Journal of Tissue Culture Methods 1980, 6: 117- 121). Parallel sets of cultures were maintained in normal growth media or in normal growth media with the addition of 1 riM rapamycin. Cultures were maintained with 1 nM rapamycin (Enzo Biologicals) for two weeks before exposure to NRTIs. Cell cultures were exposed to NRTls at indicated concentrations in individual experiments (generally 10-20 ⁇ g/mi) for 7 day s, with a change of media and fresh NRTls at day 4. Mitochondrial measurements, bioanalyzer measurements, immunoblotting for protein expression and phosphorylation status, and assays for senescence were performed at the end of this 7-day period.
• Cell culture reagents were from Celigro, unless indicated. WI-38 fetal-lung primary human fibroblasts or human cardiac fibroblasts were grown in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine, MEM vitamins, and MEM non-essential amino acids. Cells were maintained in a 37°C in 5% CO?, incubator. For treatment studies, cells were treated with 1 nM rapamycin (Enzo Biologicals) for two weeks before treatment with NRTIs. Cells were maintained by trypsmization and reseeding at a cell density of 1 x 10 4 /cm 2 every 7 to 10 days.
• Enzo Biologicals Enzo Biologicals
• Emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) were kindly provided by the NIH AIDS Research & Reference Reagent program .
• the pharmacologic inhibitors PD98059 (10 ⁇ , Santa Cruz
• Vector and mt-cataiase adenoviruses were purchased from Gene Transfer Vector Core from the Uni versity of Iowa. Western blotting and co -unmunoprecipitation
• Cell protein extracts were prepared by extracting with radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Sigma- Aldrich) and phosphatase inhibitors, NaF and sodium orthovanadate. Protein concentration was quantified using a bicinchoninic acid (BCA) assay (Pierce Biotechnology). Western blot analysis was performed using 15 to 30 ug of protein extracts that were ran on SDS-PAGE and transferred onto nitrocellulose (Biorad) membranes.
• RIPA radioimmunoprecipitation assay
• BCA bicinchoninic acid
• Western blot analysis was performed using 15 to 30 ug of protein extracts that were ran on SDS-PAGE and transferred onto nitrocellulose (Biorad) membranes.
• Blots were incubated with antibodies specific for: beta-actin (Sigma- Aldrich), TFAm, parkin, pi 6, phospho(S82)HSP27, HSP27 (Santa Cruz Biotechnologies), p53, 21, catalase, MDM2 (HMD Millipore), VDAC, phospho(S235/236)- ribosomal protein S6, ribosomal protein S6, phospho(S166)-MDM2, phospho(S473)-AKT, AKT, beta-tubulin (Cell Signaling), p62 (Enzo Biologicals), IL-6 (NeoBiolab), lamin B l, ⁇ 1, and Mitoprofile Total OXPHOS Cocktail (complex FNDUFB8 subumt, CIFSDHB subunit, CIIIUQCRC2 subunit, and CIV -mitochondrial COX1 subunit) (Abcarn) according to manufacturers' instructions.
• beta-actin Sigma- Aldrich
• TFAm parkin
• pi 6 phospho
• Immunocomplexes were precipitated using 100 ul of protein A/G beads overnight at 4 °C. Immunocomplexes bound to the beads were centrifuged at 2000 rpm for 5 minutes at 4 °C and washed three times using HNTG buffer. Following the addition of 2X sample buffer and boiling at 100 °C, protein samples were used for western blotting.
• Mitochondrial membrane potential was detected by incubating cells with 25 nM TMRE (Molecular Probes). Mitochondrial mass was evaluated by incubating the ceils with 100 nM Mitotracker Green FM (Molecular Probes). Mitochondrial superoxide anion levels were detected by incubating the cells with 5 ⁇ MitoSox Red (Molecular Probes). Total cellular levels of ROS were detected by incubating the cells with 10 ⁇ 2',7' ⁇
• DCF-DA dichlorofluoroscein diacetate
• Mitochondrial function was measured using the XF cell mito stress test kit on a
• Oxygen consumption was measured in triplicate prior to and following sequential addition of oligomycin, FCCP, and rotenone/antimycin A, Respiration rates and proton leak were assessed as outlined in published methods (Hill, et al, Biological chemistry 2012, 393: 1485- 1512). Mitochondrial respiration was calculated based upon oxygen consumption rate measurements in triplicates from cells seeded in at least quadruplicates. Basal respiration represents the initial oxygen consumption rate
• ATP-linked respiration is represented as the oligomycin- sensitive oxygen change to basal oxygen consumption rate.
• Proton leak represents the oligmycin insensitive oxygen consumption rate.
• Non-mitochondrial sources of oxygen consumption were subtracted by normalizing to the rotenone/antimycin A-insensitive oxygen consumption rate measurements. All data was normalized to cell number by counting cells in each well at the completion of the mitochondrial assessments.
• SA-p-gal activity was performed by plating cells following respective treatments at low density (0.5 x 10 4 /cm 2 ) to prevent false positive staining known to occur in high density cultures. Seeding was performed for SA-fi-gal staining following NRTI treatment for one week and staining was performed 24 hours following seeding.
• Ceils were washed with PBS, fixed with 2% formaldehyde-0.2% glutaraldehyde for 5 minutes at room, temperature, washed with PBS once more, and incubated overnight at 37°C in a staining solution containing 50 mg/ml X-gal, 100 mM potassium, ferricyanide, 100 mM potassium ferrocyanide, 5 M NaCl, 1 M MgCl 2 , and 0.2 M citric acid/phosphate buffer (pH 6.0).
• Cells were seeded onto acid-washed coverslips at standard density following the designated treatment. Following 24 hours, cells were fixed using 4% paraformaldehyde, permeabilized using 0.1% Triton-PBS, and blocked with applicable animal serum.
• Results are representative of at least three independent experiments and statistical significance was determined using an unpaired two-tailed Student's t-test. Data sets were subjected to normality tests to verify normal distribution of data. One-way Anova with Bonferroni post-hoc analysis was performed on multiple comparison groups involving control, NRTI, rapamycin, and rapamycin NRTI where appropriate.
• An exemplary non-limiting emulsion formulation of the invention (referred to as "Formulation R" hereinafter) comprises about 0.001% to about 1% (w/w) of rapamycin, palmitate at about 4-6%, glycerin at about 6-8%, and the balance consisting of water.
• Example 1 Mitochondrial effects of nucleoside/nucleotide analogs are relieved by rapamycin.
• NRTIs The effects of combination treatment with TDF and FTC (referred to as NRTIs for simplicity) were examined at concentrations relevant to serum levels in patients receiving anti-retro viral therapy, on mitochondria in both human cardiac and lung fibroblasts.
• Parallel cultures were grown in the additional presence of 1 nM rapamycin. This concentration of rapamycin was found to extend replicative lifespan and improve the mitochondria] profile of human fibroblasts.
• Exposure to NRTIs for 7 days produced a significant increase in mitochondrial membrane potential, mitochondrial ROS production, and mitochondrial mass in the human cardiac fibroblasts (FIGs. 1A-1C). Similar results were observed in the human lung fibroblasts. Additionally, total cellular ROS increased significantly following exposure to NRTIs in both fibroblast populations (FIG. ID). Cultures grown in the presence of rapamycin did not exhibit the same level of increase in mitochondrial membrane potential, mitochondrial ROS, or total cellular ROS following exposure to NRTIs.
• rapamycm-treated cells did not exhibit an increase in proton leak following exposure to NRTIs (FIG. II).
• Human lung fibroblasts gave similar results when subjected to the same analyses (i.e., basal and maximal respiration increased significantly, as did proton leak in cells treated with NRTIs). Similar to the cardiac fibroblasts, rapamycin-treated lung fibroblasts exhibited no increase in these parameters following exposure to NRTIs.
• Example 2 Alterations in electron transport chain components in response to nucleoside/nucleotide analogs and rapamycin
• NRTIs (FIGs. 3A-3F).
• Levels of p53, p21, and p!6 increased in fibroblasts exposed to NRTIs, while in rapamycin treated cells the levels of these senescence associated proteins did not increase (FIG. 3A).
• levels of lamin Bl which is known to decrease during senescence, decreased in cells exposed to NRTIs. Rapamycin prevented this decrease (FIG. 3A).
• intracellular levels of IL-6 which is a component of the senescence associated secretory program, increased following exposure to NRTIs.
• rapamycin treated cells showed no increase in the levels of IL-6 following exposure to NRTIs (FIG. 3A).
• the percentage of the ceil population expressing the senescence marker, senescence associated ⁇ -galactosidase (SA-p-gal) following exposure to NRTIs was examined.
• a dose dependent increase in the percent of cardiac fibroblasts expressing SA-fi-gal was observed (9% in control cells compared to 28% and 48% in cells exposed to 10 or 20 ug/ml of NRITs respectively, FIG, 3B).
• rapamycin treated cultures showed no increase in SA ⁇ -gal (FIG. 3B).
• Lung fibroblasts showed identical changes in response to NRTIs in terms of senescence markers and the protection afforded by rapamycin treatment. There was no reduction in viability of cells exposed to NRTIs as judged by vital dye exclusion assay under any of the conditions tested and no apparent markers of apoptosis, such as caspase cleavage. Alkaline comet assay showed no evidence of increased DNA damage, indicating that the response to NRTIs was primarily a growth inhibition and not due to DNA damage.
• MDM2 key regulator
• mitochondrial ROS The role of mitochondrial ROS in the NRTI-induced senescence response was examined by treating control cultures (grown without rapamycin) with mito-Q, a ROS scavenger that targets mitochondrial ROS or by introducing a mitochondrial targeted catalase (mt-catalase) into cardiac fibroblasts.
• Mito-Q ameliorated both mitochondrial ROS production following exposure to NRTIs and the increase in senescence-associated proteins p53, p21, and pl6 (FIG. 4A).
• the percentage of cells positive for SA-p-gal staining was reduced in cells treated with mito-Q (FIG. 4B). Similar results were obtained when cells were infected with the mt-catalase construct.
• MDM2 phosphorylation mediated by p70S6K
• p70S6K phosphorylation receptor 1
• phosphorylation status of MDM2 Phosphorylation of both the ribosomal S6 protein and MDM2 increased in cardiac fibroblasts following exposure to NRTIs.
• rapamycin-treated cultures showed a complete lack of ribosomal S6 phosphorylation and no increase in MDM2 phosphorylation (FIG. 5A).
• NRTI-induced p70S6K activity was examined by treating ceils exposed to NRTIs with troiox/N -acetylcysteine and examining ribosomal S6 phosphorylation. Both ribosomal S6 phosphorylation and MDM2 phosphorylation were decreased when cells exposed to NRTIs were treated with troiox/N -acetylcysteine (FIG. 5C). Involvement of the NADPH oxidase system was examined using apocyanin, an inhibitor of NADPH oxidases. However, treatment of NRTI-exposed cells with apocyanin did not prevent the increase in ribosomal S6 phosphorylation. Similar results in terms of p7-S6 kinase and MDM phosphorylation were obtained when human lung fibroblasts were exposed to NRTIs.
• FIGs. 6A-6B phosphorylation following exposure to NRTIs
• mito-Q treated cells were examined by Seahorse Bioanalyzer to assess mitochondrial activity. This analysis revealed that rnito-Q treatment partially alleviated the increase in basal respiration, ATP- linked respiration, and proton leak, while maximal respiration was less affected (FIGs. 6C- 6F). In addition, mito-Q treated cells exhibited a lower level of mitochondrial ROS when exposed to NRTIs (FIG. 6G).
• cardiac fibroblasts were placed in serum-free medium for 48 hours to abrogate growth factor signaling that might influence mTORCl activity. The cells were then exposed to hydrogen peroxide at concentrations ranging from 1 to 400 ⁇ for 2 hours. Both ribosomal S6 phosphorylation and MDM2 phosphorylation were increased at the lower concentrations of hydrogen peroxide, with maximal activation at 50 uM and inhibition of both ribosomal S6 phosphorylation and MDM2 phosphorylation occurred at concentrations above 100 uM (FIG. 7A).
• the role of mitochondrial ROS in senescence was examined by treating senescent cardiac fibroblasts with the mitochondrial ROS scavenger mito-Q or by introducing a mitochondrial targeted catalase. Initially, levels of mitochondrial and total cellular ROS were examined in late passage cells. This assessment revealed elevated levels of mitochondrial ROS and total cellular ROS in late passage cells (FIGs. 9A-9B). Assessment of
• a single site open label study was performed. A patient presenting with an area of dermal atrophy and actinic keratosis on the hand was evaluated for application of
• Formulation R the contralateral hand with similar dermal thickness but no actinic keratosis was used as a control.
• the patient was provided with Formulation R with instructions for twice daily application.

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