US20150157580A1 - Methods of regulating cannabinoid receptor activity-related disorders and diseases - Google Patents

Methods of regulating cannabinoid receptor activity-related disorders and diseases Download PDF

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US20150157580A1
US20150157580A1 US14/403,516 US201314403516A US2015157580A1 US 20150157580 A1 US20150157580 A1 US 20150157580A1 US 201314403516 A US201314403516 A US 201314403516A US 2015157580 A1 US2015157580 A1 US 2015157580A1
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Irving William Wainer
Michel Bernier
Rajib Kumar Paul
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US Department of Health and Human Services
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    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present disclosure relates to the field of cannabinoid receptors and, in particular, to methods of regulating cannabinoid (CB) receptor activity-related disorders and diseases, such as activating CB receptors, including treating a disorder or disease, such as a glioblastoma, hepatocellular carcinoma, liver cancer, colon cancer, and/or lung cancer, which is associated with altered cannabinoid receptor activity by administration of specific fenoterol analogues.
  • CB cannabinoid
  • Cancer is the second leading cause of human death next to coronary disease in the United States. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. Cancer is soon predicted to become the leading cause of death. Many types of cancers, including brain and liver cancers, have no effective clinical treatments.
  • This disclosure concerns the discovery that specific fenoterol analogues are cannabinoid (CB) receptor modulators and can be used to treat a disorder or disease such as a tumor, including, but not limited to, a glioblastoma or hepatocellular carcinoma that is associated with altered CB receptor activity or expression (or both), such as altered expression or activity (or both) of the GPR55 cannabinoid receptor.
  • CB cannabinoid
  • the inventors have discovered that administration of specific fenoterol analogues inhibits one or more signs or symptoms (such as tumor growth) associated with a tumor that expresses a CB receptor.
  • the inventors developed the disclosed methods of treating a CB receptor-modulated disorder or disease, including treatment of a tumor expressing a CB receptor; for example, a glioblastoma or hepatocellular carcinoma expressing a CB receptor.
  • the method includes administering to a subject having or at risk of developing a disorder or disease regulated by CB receptor activity an effective amount of a compound to reduce one or more symptoms associated with the disorder or disease regulated by CB receptor activity, wherein the compound has the formula
  • Y 1 , Y 2 and Y 3 independently are hydrogen, halogen, sulphur-containing moiety including SH, sulfoxides, sulphones, sulphanamides and related alkyl and aromatic substituted moieties, lower —OR 6 and —NR 7 R 8 ;
  • R 6 is H or lower alkyl;
  • R 7 and R 8 independently are hydrogen, lower alkyl, alkoxy carbonyl, acyl or amino carbonyl and wherein the compound is optically active, thereby reducing the one or more symptoms associated with the disorder or disease in the subject regulated by CB receptor activity.
  • administering comprises administering a therapeutically effective amount of a compound, wherein R 4 within the compound is selected from methyl, ethyl, n-propyl, and isopropyl.
  • administering comprises administering a therapeutically effective amount of a compound, wherein R 4 within the compound is methyl.
  • administering comprises administering a therapeutically effective amount of a compound, wherein R 6 within the compound is methyl.
  • administering comprises administering a therapeutically effective amount of a compound, wherein R 5 within the compound is one of
  • administering comprises administering a therapeutically effective amount of a compound, wherein R 1 -R 3 within the compound are hydrogen.
  • administering comprises administering a therapeutically effective amount of (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF), or a combination thereof.
  • MNF methoxy-1-napthylfenoterol
  • administering comprises administering a therapeutically effective amount of MNF, NF or a combination thereof.
  • administering comprises administering a therapeutically effective amount of MNF.
  • the method includes administering a therapeutically effective amount of a pharmaceutical composition containing any of the disclosed fenoterol analogues capable of regulating a CB receptor-associated disorder or disease and a pharmaceutically acceptable carrier to treat the disorder or disease regulated by a CB receptor, such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • a pharmaceutical composition containing any of the disclosed fenoterol analogues capable of regulating a CB receptor-associated disorder or disease and a pharmaceutically acceptable carrier to treat the disorder or disease regulated by a CB receptor, such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • the disclosed (R,R′)-MNF, (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)—NF, (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-aminoNF, (R,R′)-hydroxyNF, or a combination thereof are effective at treating a glioblastoma or hepatocellular carcinoma expressing a CB receptor, such as a GPR55 expressing glioblastoma or hepatocellular carcinoma.
  • the method further includes selecting a subject having or at risk of developing a disorder or disease regulated by a CB receptor.
  • a subject is selected for treatment by determining that the disorder or tumor is associated with CB receptor expression, such as GPR55 expression.
  • the method further includes selecting a subject with a disorder and/or disease, which is not associated with altered ⁇ 2-AR function.
  • the disorder or disease does not respond to a treatment targeting ⁇ 2-AR activity.
  • the method includes administering one or more therapeutic agents in addition to the fenoterol analogue or combination thereof.
  • the methods can include administration of the one or more therapeutic agents separately, sequentially or concurrently, for example in a combined composition with a fenoterol analogue or combinations thereof.
  • the method is for use in treating a tumor expressing a CB-receptor.
  • the disorder or disease is selected from the group consisting of a primary brain tumor expressing a CB-receptor, a glioblastoma expressing a CB-receptor, a hepatocellular carcinoma expressing a CB-receptor, colon cancer, liver cancer, and lung cancer.
  • inhibiting one or more signs or symptoms associated with the disease or disorder comprises inhibiting cellular growth, such as tumor and/or cancer cell growth, tumor volume or a combination thereof.
  • the method is used to treat a disorder or disease regulated by a CB receptor, which is GPR55, such as diabetes.
  • FIGS. 1A-1C illustrate responses of HepG2 cells exposed to ⁇ -agonist stimulation.
  • FIG. 1A is a digital image of soluble extracts which were prepared from HepG2 (lane 1) and 1321N1 (lane 2) cells maintained in complete medium, and subjected to Western blot analysis. Cellular content in ⁇ 2-adrenergic receptor ( ⁇ 2-AR) and ⁇ -actin was measured using specific primary antibodies.
  • FIG. 1B is a bar graph illustrating increases in cAMP accumulation in HepG2 cells with forskolin, but not with (R)-isoproterenol (Iso) or (R,R′)-fenoterol (Fen). Data shown are from a single study conducted in quadruplicate.
  • FIG. 1C is a digital image of an immunoblot. Serum-starved HepG2 cells were incubated in the presence of (R)-isoproterenol (Iso; 1 ⁇ M) or (R,R′)-Fen (1 ⁇ M) for 5, 10 and 30 minutes. Cell lysates were immunoblotted with antibodies against phosphorylated (Ser473) and total Akt, as well as phosphorylated ERK1/2 and total ERK2. The studies shown in FIGS. 1B and 1C were repeated twice with comparable results. The migration of molecular mass markers (values in kilodaltons) is shown on the left of immunoblots.
  • FIGS. 2A-D illustrate the effects of (R)-isoproterenol, (R,R′)-Fen and derivatives on cell growth are cell-type specific. Serum-starved HepG2 cells were incubated with vehicle or the indicated concentrations of (R)-isoproterenol (Iso), (R,R′)-Fen, (R,R′)-aminoFen (NH 2 -fen) or (R,R′)-MNF for 24 hours, and levels of [ 3 H]-thymidine incorporation was measured. Representative dose-response curves are shown in FIGS. 2A and 2B .
  • FIG. 2D illustrates the findings when HepG2 and 1321N1 cells were incubated without (SFM) or with serum (CM) in the presence of the indicated concentrations of Iso or (R,R′)-Fen (fen). Quantification of percent change in [ 3 H]-thymidine incorporation versus control are expressed as means ⁇ SE and represent results from two to six independent studies, each performed in triplicate dishes. In most instances, error bars are smaller than the symbols.
  • FIGS. 3A-3D demonstrate that a ⁇ 2-AR antagonist does not inhibit the anti-proliferative action of (R,R′)-MNF in HepG2 cells.
  • Serum-depleted HepG2 cells were incubated with the indicated concentrations of the ⁇ 2-AR antagonist, ICI-118,551 (ICI), for 1 hour followed by the addition of vehicle ( FIG. 3A ), (R,R′)-Fen ( FIG. 3B , left panel), or (R,R′)-MNF ( FIG. 3B , right panel) for 24 hours, and levels of [ 3 H]-thymidine incorporation were measured.
  • Representative dose-response curves for (R,R′)-Fen and (R,R′)-MNF are shown FIG. 3B .
  • FIGS. 3C and 3D are bar graphs illustrating quantification of percent change in [ 3 H]-thymidine incorporation versus control expressed as means ⁇ SE and represent results from three independent studies, each performed in triplicate
  • FIG. 4 illustrates (R,R′)-MNF increases the number of sub-G1 events in HepG2 cells.
  • Serum-depleted HepG2 cells were harvested after 6-hour, 12-hour or 24-hour treatment with vehicle, (R,R′)-Fen (1 ⁇ M) or (R,R′)-MNF (1 ⁇ M). Cells were fixed, stained, and analyzed for DNA content using flow cytometry, Representative DNA content analysis in various phases of the cell cycle after 24-hour treatment with vehicle, (R,R′)-Fen, or (R,R′)-MNF are shown.
  • FIG. 5 illustrates the results of flow cytometry studies in which (R,R′)-MNF induced apoptosis in HepG2 cells.
  • Serum-depleted HepG2 cells were treated with vehicle, (R,R′)-Fen (1 ⁇ M), or (R,R′)-MNF (1 ⁇ M) for 24 hours; stained with Annexin V and propidium iodide (PI); and then analyzed by flow cytometry. Representative profiles are shown.
  • FIGS. 6A-6C illustrate the role of cannabinoid receptor activation in the anti-proliferative action of (R,R′)-MNF in HepG2 cells.
  • FIG. 6A Total RNA was extracted from HepG2, 1321N1 and U87MG cells, and then analyzed semi-quantitatively by PCR. A non-template control (NTC) has been included (lane 1).
  • FIGS. 6B and 6C Serum-depleted HepG2 cells were incubated with the cannabinoid receptor agonist, WIN 55,212-2 (Win; 1 ⁇ M), ( FIG.
  • AM251 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide; 1 ⁇ M) or AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone, 0.5 ⁇ M), ( FIG. 6C ) for 1 hour followed by the addition of vehicle, (R,R′)-Fen (0.5 ⁇ M), or (R,R′)-MNF (0.25 ⁇ M) for 24 hours. Quantification of percent change in [ 3 H]-thymidine incorporation versus control are expressed as means ⁇ SD and represent results from three independent studies, each performed in triplicate dishes.
  • FIG. 7 is a series of graphs illustrating selective inhibition of (R,R′)-Fen-mediated cell proliferation control by the ⁇ 2-AR antagonist, ICI-118,551.
  • HepG2, 1321N1 and U87MG cells were incubated with the ⁇ 2-AR antagonist, ICI-118,551 (ICI, 1 ⁇ M), for 1 hour followed by the addition of vehicle, (R,R′)-Fen (0.5 ⁇ M), or (R,R′)-MNF (0.25 ⁇ M) for 24 hours, and levels of [ 3 H]-thymidine incorporation was measured. Quantification of percent change in [ 3 H]-thymidine incorporation versus control are expressed as means ⁇ SD and represent the results from three independent studies, each performed in triplicate dishes.
  • FIGS. 8A-8D are bar graphs illustrating cannabinoid receptors play no role in cell proliferation control by (R,R′)-Fen. 1321N1 ( FIG. 8A ) and U87MG ( FIG. 8C ) cells were incubated with the cannabinoid receptor agonist, WIN 55,212-2 (Win; 0.5-1 ⁇ M) or the cannabinoid receptor antagonists, AM251 (0.5-1 ⁇ M) or AM630 (0.25-0.5 ⁇ M) ( FIGS. 8B , 8 D) for 1 hour followed by the addition of vehicle, (R,R′)-Fen (0.5 ⁇ M), or (R,R′)-MNF (0.25 ⁇ M) for 24 hours. Quantification of percent change in [ 3 H]-thymidine incorporation versus control is expressed as means ⁇ SD and represents the results from three independent studies, each performed in triplicate.
  • FIG. 9 illustrates cellular uptake of TocriFluor 1117 (T1117), a fluorescent AM251 analog, in HepG2 cells.
  • T1117 TocriFluor 1117
  • FIG. 9 illustrates cellular uptake of TocriFluor 1117 (T1117), a fluorescent AM251 analog, in HepG2 cells.
  • Cells were treated with (R,R′)-Fen (1 ⁇ M), (R,R′)-MNF (1 ⁇ M), or AM251 (10 ⁇ M) for 1 hour followed by addition of T1117 (0.1 ⁇ M).
  • Cells were mounted on confocal microscope and maintained at 37° C. with CO 2 . Images were captured every 30 seconds for up to one hour.
  • FIG. 10 is a graph illustrating metabolic stability of (R,R′)-MNF on human and rat liver microsomes.
  • FIG. 11 is a bar graph illustrating cytochrome p450 (CYP) inhibition by (R,R′)-MNF.
  • Human liver microsomes were incubated with 8 different CYP substrates and 1 or 10 ⁇ M MNF.
  • MNF at 10 ⁇ M was determined to inhibit CYP2D6 and CYP3A4.
  • the primary metabolite was determined to be O-demethylated-MNF.
  • FIGS. 12 and 13 are tracings illustrating plasma and brain tissue concentrations of (R,R′)-MNF.
  • FIG. 12 illustrates the analysis of a plasma sample obtained 30 minutes post IV administration of 10 mg/kg MNF. MNF and Gluc-MNF, in the insert of FIG. 12 , are shown with no interfering peaks being present in the control plasma matrix.
  • FIG. 13 illustrates the analysis of brain tissue obtained 30 minutes post IV administration of 10 mg/kg MNF. The peak at 6.39 minutes is an unidentified compound present in control brain matrix (see insert of FIG. 13 ).
  • the MNF concentration in brain tissue was 200 ng/mg tissue at 10 minutes after administration and peaked at 30 minutes at 800 ng/mg tissue.
  • the relative distribution between the concentration of MNF in blood (measured as ng/ml) and brain tissue (measured as ng/mg tissue) was 0.2 at 10 minutes and 1.0 at 30 minutes and 60 minutes reflecting an equivalent distribution between both the central and peripheral body compartments.
  • FIG. 15 is a series of bar graphs illustrating that MNF does not produce significant negative effects on the central nervous system relative to the effects produced by tetrahydrocannabinol (presented in FIG. 16 ).
  • FIG. 16 is a series of bar graphs illustrating the effects of tetrahydrocannabinol on central nervous system function.
  • FIG. 17 is a series of graphs illustrating [3H]-Thymidine incorporation in 96-well culture plates including Hep3B cells, PC3 cells or LN229 cells.
  • FIGS. 18A , 18 B and 18 C illustrate MNF reduces proliferation of rat C6 glioma cell line.
  • FIG. 18A Cell proliferation assay was performed in rat C6 glioma cells treated with increasing concentrations of MNF for 24 hours followed by the addition of [ 3 H]-thymidine for 16 hours.
  • FIG. 18B Cells were preincubated without or with the selective ⁇ 2-AR blocker, ICI-118,551 (3 nM) for 30 minutes followed by the addition of vehicle or 20 nM of MNF, (R,R′)-Fen or isoproterenol (ISO) for 24 hours.
  • FIG. 18A Cell proliferation assay was performed in rat C6 glioma cells treated with increasing concentrations of MNF for 24 hours followed by the addition of [ 3 H]-thymidine for 16 hours.
  • FIG. 18B Cells were preincubated without or with the selective ⁇ 2-AR blocker, ICI-118,551 (3 nM) for 30
  • FIGS. 18B and 18C C6 glioma cells were pretreated in the presence or absence of cannabinoid receptor inverse agonists, AM251 (0.5 and 1 ⁇ M) and AM630 (0.5 ⁇ M), for 30 minutes followed by the addition of vehicle or 20 nM MNF for 24 hours.
  • FIGS. 18B and 18C [ 3 H]-thymidine was determined after a 16 hour-incubation. Bars represent the average ⁇ SD of a single experiment performed in triplicate wells. Similar results were obtained in 2-3 independent experiments.
  • FIG. 18D Changes in cell morphology were observed for C6 cells incubated with 20 nM MNF for 48 hours.
  • FIGS. 19A and 19B each include a graph illustrating MNF reduces tumor growth in vivo in a rat C6 glioma xenograft model.
  • C6 tumor-bearing female nude mice were assigned randomly to either the vehicle or the MNF group. Treatment was given by injecting either 2 mg/kg MNF or citrate in PBS five days a week for 19 days. Tumor volume was monitored daily and mice were sacrificed on the day after the last treatment.
  • FIGS. 20A-20D illustrate gene expression profiling in MNF-treated C6 tumor-bearing mice.
  • FIG. 20A Gene clustering in the rat C6 xenografts: Principal component analysis (PCA) of rat C6 glioma xenograft treated with MNF vs. vehicle control. PCA was applied to the six independent samples (3 MNF, 3 controls), and numbers refer to individual sample labels. Analysis reveals clustering of samples into treatment groups.
  • FIG. 20B Cluster analysis of 100 gene sets altered by MNF treatment, as compared to the control group, in cohort #1, cohort #2, and combined cohorts (1+2).
  • FIG. 20A Gene clustering in the rat C6 xenografts: Principal component analysis (PCA) of rat C6 glioma xenograft treated with MNF vs. vehicle control. PCA was applied to the six independent samples (3 MNF, 3 controls), and numbers refer to individual sample labels. Analysis reveals clustering of samples into treatment groups.
  • FIG. 20C Zratios of selected genes of interest is depicted, showing either up- or down-regulated expression after pairwise comparison between MNF and the vehicle-treated group.
  • FIG. 20E demonstrates the negative impact of MNF on cyclin expression in C6 tumor xenografts. Lysates from tumor samples were separated by SDS-PAGE and Western blotting was carried out primary antibodies raised against cyclin A and cyclin D. Membranes were reprobed for Hsp90, which served as a loading control. Upper panel, representative immunoblots; lower panels, scatter plot of data showing significant differences in cyclin A and cyclin D1 expression between xenograft tumors from vehicle and MNF-treated mice. *, p ⁇ 0.05; **, p ⁇ 0.01 using two-tailed Student's t-test. FIG.
  • C6 glioma cells were incubated with 20 nM MNF for 6 and 24 hours after which lysates were prepared and immunoblotted for cyclin A and cyclin D1.
  • Membranes were reprobed for ⁇ -actin, which served as a loading control.
  • Upper panel representative blots; lower panel, Bars represent densitometric quantification of each blot with the values in vehicle-treated cells set at 1.0. Bars represent means ⁇ SEM from three independent studies. a, b: significant difference between groups at P ⁇ 0.01.
  • FIGS. 21A-21D illustrate rapid and saturable incorporation of T1117 in HepG2 cells.
  • Cellular entry of T1117 was measured on a Zeiss 710 confocal microscope with thermoregulated chamber system for live cell imaging.
  • FIG. 21A Serum-depleted HepG2 cells were incubated in the presence of increasing concentrations of T1117 (2.5-100 nM). Plots of signal intensity versus time were generated from defined regions of interest (ROIs). Results are from 2-3 independent studies.
  • FIG. 21B Relative AUC data versus T1117 concentrations is shown, and the T1117-100 nM values were set at 1.
  • FIGS. 22A-22D illustrate a role for GPR55 in cellular incorporation of T1117.
  • FIG. 22A HepG2 cells were transfected with siRNA oligos either against CB 1 R, CB 2 R or GPR55 or the non-silencing siRNA control for 48 hours. Cells were maintained in serum-free medium for 3 hours, followed by the addition of T1117. Plots of signal intensity vs. time were generated from defined ROIs. Error bars indicate mean ⁇ S.D. of two independent studies, each performed with 3-4 ROIs.
  • FIG. 22B Relative AUC data, and the control siRNA values were set at 1.
  • FIG. 22A Relative AUC data, and the control siRNA values were set at 1.
  • FIGS. 23A-25D illustrate the effect of MNF on cellular uptake of T1117.
  • Serum-depleted HepG2 ( FIG. 23A , FIG. 23B ) and PANC-1 ( FIG. 18C , FIG. 23D ) cells were pretreated or not with MNF (1 ⁇ M) or AM251 (10 ⁇ M) for 30 minutes followed by the addition of vehicle (0.1% DMSO), AM251 or MNF for an additional 30 minutes. Cells were then incubated with 10 nM T1117.
  • FIGS. 23A , C Plots of signal intensity vs. time were generated from defined ROIs. Error bars indicate mean ⁇ S.D. of two independent studies, each performed with 3-4 ROIs.
  • FIGS. 23B , D Relative AUC data, and the DMSO values were set at 1.
  • FIGS. 24A and 24B show MNF impairs ligand-induced internalization of GPR55.
  • FIGS. 25A-25D illustrates impairment in GPR55 downstream signaling by MNF.
  • Serum-depleted HepG2 ( FIG. 25A , FIG. 25B ) and PANC-1 ( FIG. 25C , FIG. 25D ) cells were pretreated or not in the presence of MNF (1 ⁇ M) for 10 minutes followed by the addition of vehicle, O-1602 (2.5 and 10 ⁇ M), or 10% FBS for an additional 10 minutes.
  • Cell lysates were prepared, separated by reducing SDS-PAGE gel electrophoresis and immunoblotted for total and phosphoactive forms of ERK.
  • FIGS. 25A , C Representative immunoblots; FIGS.
  • D phospho-ERK1/2 bands were normalized to total ERK2, and the O-1602-10 ⁇ M values were set at 1. Data are means of two independent dishes ⁇ range. The migration of molecular-mass markers (values in kilodaltons) is shown on the left of immunoblots.
  • FIGS. 26A-26C show MNF interferes with inducible changes in cell morphology and expression of EGFR.
  • Serum-starved HepG2 ( FIG. 26A ) and PANC-1 ( FIG. 26B ) cells were pre-incubated in the presence of DMSO (0.1%) or MNF (1 ⁇ M) for 30 minutes followed by the addition of AM251 (5 ⁇ M) or O-1602 (5 ⁇ M) for 16 hours.
  • Unstimulated PANC-1 cells displayed cuboidal morphology with and without MNF.
  • White arrows show individual cells with filopodia.
  • FIG. 26C Cell lysates were prepared from similar studies and immunoblotted for EGFR. The membranes were reprobed for Hsp90, which served as a loading control.
  • FIGS. 27A-27D show MNF inhibits ligand-induced motility of HepG2 and PANC-1 cells in a wound-healing assay.
  • Confluent HepG2 ( FIG. 27A , FIG. 27B ) and PANC-1 ( FIG. 27C , FIG. 27D ) cells were subjected to scratch wound. Cells were incubated in the presence of DMSO (0.1%) or the GPR55 agonist AM251 (1 ⁇ M) for 30 minutes, followed by the addition of MNF (1 ⁇ M) where indicated. Images were captured at various time-points.
  • FIGS. 27A , C The relative wound surface area was measured over time and plotted, and values at time 0 were set at 1.
  • FIGS. 27B , D The relative wound surface area of four independent observations at the 24-hour time point is plotted. *, ***P ⁇ 0.05 and 0.001.
  • FIG. 28A provides the structure of 5′-TAMRA-3-phenylpropan-1-amine (TAMRA-PPA) and T1117.
  • FIG. 28B provides the mass spectrum of TAMPRA-PPA ion, m/z equals 548.0.
  • FIG. 28C provides a comparison of the cellular accumulation of T1117 (10 nM) vs. TAMRA-PPA (20 nM) in serum-depleted HepG2 cells. Note the absence of TAMRA-PPA incorporation in cells.
  • FIGS. 29A-29C are captured images of a representative wound-healing assay.
  • Confluent HepG2 ( FIG. 29A ) and PANC-1 ( FIG. 29B ) cells were subjected to scratch wound and treated as described above for FIGS. 27A-27D . Similar profiles were obtained in four independent assays.
  • FIG. 29C Confluent HepG2 and PANC-1 cells were subjected to scratch wound. Cells were incubated in the presence of the atypical cannabinoid O-1602 (1 ⁇ M) for 30 minutes, followed by the addition of vehicle (DMSO, 0.1%) or MNF (1 ⁇ M). Images were captured at various time-points. The relative wound surface area at the 24-hour time point is plotted. **P ⁇ 0.01.
  • fenoterol analogues such as MNF
  • MNF fenoterol analogues
  • the inventors performed a series of studies to characterize fenoterol analogues and determine their possible therapeutic activities.
  • MNF was observed to inhibit the growth of human-derived hepatocellular carcinoma cells (HepG2) and human- (U87MG) and rat- (C6) derived glioblastoma cells using in vitro incubation and in vivo in flank implanted C6 xenograft in nude mice.
  • MNF is a ⁇ 2-AR agonist and this class of compounds had been shown to increase cellular growth in HepG2 cells. Binding and functional studies were performed which revealed that MNF acts as an inhibitor of the GPR55 cannabinoid receptor and, as such, represents one of the first potential drugs directed at this target. Initial pK studies demonstrated that the compound crosses the blood brain barrier and initial toxicity studies indicated that the drug had little off-target effects. The ⁇ 2-AR agonist properties were a positive indication and suggest that MNF may have cardio-protective effects. MNF was also found to be capable of significantly inhibiting additional types of tumor cell growth, including, but not limited to, colon cancer cells, lung cancer cells and liver cancer cells.
  • fenoterol compounds such as (R,R′)-1-naphthylfenoterol (NF) were found to inhibit hepatocellular carcinoma cell growth.
  • CB receptor related disorders and diseases such as (R,R′)-1-naphthylfenoterol (NF)
  • NF neurotrophic factor-1-naphthylfenoterol
  • AKAP A-kinase anchoring protein
  • AM251 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide
  • AM630 1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole
  • AR adrenergic receptor
  • BBB blood brain barrier
  • ⁇ 2-AR ⁇ 2-adrenergic receptor
  • ERK extracellular regulated kinase
  • GPR55 G protein-coupled receptor 55
  • GPCR G protein-coupled receptor
  • IAM-PC immobilized artificial membrane chromatographic support
  • ICI 118,551 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol
  • IP intraperitoneal
  • IV intravenous
  • UV ultraviolet
  • any quantitative values are approximate whether the word “about” or “approximately” or the like are stated or not.
  • the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Any molecular weight or molecular mass values are approximate and are provided only for description. Except as otherwise noted, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Loudon, Organic Chemistry , Fourth Edition, New York: Oxford University Press, 2002, pp.
  • Acyl A group of the formula RC(O)— wherein R is an organic group.
  • Acyloxy A group having the structure —OC(O)R, where R may be an optionally substituted alkyl or optionally substituted aryl. “Lower acyloxy” groups are those where R contains from 1 to 10 (such as from 1 to 6) carbon atoms.
  • compositions such as a pharmaceutical composition including one or more fenoterol analogues by any effective route.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal (IP), and intravenous (IV)), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Alkoxy A radical (or substituent) having the structure —O—R, where R is a substituted or unsubstituted alkyl. Methoxy (—OCH 3 ) is an exemplary alkoxy group. In a substituted alkoxy, R is alkyl substituted with a non-interfering substituent. “Thioalkoxy” refers to —S—R, where R is substituted or unsubstituted alkyl. “Haloalkyloxy” means a radical —OR where R is a haloalkyl.
  • Alkoxy carbonyl A group of the formula —C(O)OR, where R may be an optionally substituted alkyl or optionally substituted aryl. “Lower alkoxy carbonyl” groups are those where R contains from 1 to 10 (such as from 1 to 6) carbon atoms.
  • Alkyl An acyclic, saturated, branched- or straight-chain hydrocarbon radical, which, unless expressly stated otherwise, contains from one to fifteen carbon atoms; for example, from one to ten, from one to six, or from one to four carbon atoms. This term includes, for example, groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl, heptyl, octyl, nonyl, decyl, or dodecyl.
  • the term “lower alkyl” refers to an alkyl group containing from one to ten carbon atoms.
  • alkyl groups can either be unsubstituted or substituted.
  • An alkyl group can be substituted with one or more substituents (for example, up to two substituents for each methylene carbon in an alkyl chain).
  • substituents include, for instance, amino groups, amide, sulfonamide, halogen, cyano, carboxy, hydroxy, mercapto, trifluoromethyl, alkyl, alkoxy (such as methoxy), alkylthio, thioalkoxy, arylalkyl, heteroaryl, alkylamino, dialkylamino, alkylsulfano, keto, or other functionality.
  • Amino carbonyl (carbamoyl): A group of the formula C(O)N(R)R′, wherein R and R′ are independently of each other hydrogen or a lower alkyl group.
  • Anti-diabetic agent A chemical or pharmaceutical anti-hyperglycemic agent or drug capable of treating diabetes, including, but not limited to agents for alleviating the symptoms associated with type 2 diabetes or slowing the progression or onset of type 2 diabetes.
  • Anti-diabetic agents are generally categorized into six classes: biguanides; thiazolidinediones; sulfonylureas; inhibitors of carbohydrate absorption; fatty acid oxidase inhibitors and anti-lipolytic drugs; and weight-loss agents.
  • the anti-diabetic agents include those agents disclosed in Diabetes Care , 22(4):623-634 (1999), herein incorporated by reference.
  • an anti-diabetic agent is a disclosed fenoterol analogue capable of modulating a CB receptor activity, such as GPR55 activity.
  • Astrocytoma A tumor of the brain that originates in astrocytes.
  • An astrocytoma is an example of a primary tumor.
  • Astrocytomas are the most common glioma, and can occur in most parts of the brain and occasionally in the spinal cord. However, astrocytomas are most commonly found in the cerebrum.
  • an astrocytoma is inhibited by administering to a subject a therapeutic effective amount of fenoterol, a fenoterol analogue or a combination thereof, thereby inhibiting astrocytoma growth.
  • ⁇ 2-adrenergic receptor A subtype of adrenergic receptors that are members of the G-protein coupled receptor family. ⁇ 2-AR subtype is involved in respiratory diseases, cardiovascular diseases, premature labor and, as disclosed herein, tumor development. Increased expression of ⁇ 2-ARs can serve as therapeutic targets.
  • drugs e.g., albuterol, formoterol, isoproterenol, or salmeterol have ⁇ 2-AR agonist activities.
  • fenoterol and fenoterol analogues are ⁇ 2-AR agonists.
  • Blood-brain barrier The barrier formed by epithelial cells in the capillaries that supply the brain and central nervous system. This barrier selectively allows entry of substances such as water, oxygen, carbon dioxide, and nonionic solutes such as glucose, alcohol, and general anesthetics, while blocking entry of other substances. Some small molecules, such as amino acids, are taken across the barrier by specific transport mechanisms. In one example, fenoterol or disclosed fenoterol analogues are capable of passing through the barrier.
  • Body Mass Index A mathematical formula for measuring body mass in humans, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height 2 (in meters 2 ). The current standards for both men and women accepted as “normal” are a BMI of 20-24.9 kg/m 2 . In one embodiment, a BMI of greater than 25 kg/m 2 can be used to identify an obese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m 2 . Grade II obesity corresponds to a BMI of 30-40 kg/m 2 ; and Grade III obesity corresponds to a BMI greater than 40 kg/m 2 (Jequier, Am. J Clin. Nutr ., 45:1035-47, 1987). Ideal body weight will vary among individuals based on height, body build, bone structure, and sex.
  • Cannabinoid Receptors A class of cell membrane receptors under the G protein-coupled receptor superfamily. The cannabinoid receptors contain seven transmembrane spanning domains. Cannabinoid receptors are activated by three major groups of ligands, endocannabinoids (produced by the mammalian body), plant cannabinoids (such as THC, produced by the cannabis plant) and synthetic cannabinoids (such as HU-210). All of the endocannabinoids and plant cannabinoids are lipophilic, i.e. fat soluble, compounds. Two subtypes of cannabinoid receptors are CB 1 (see GenBank Accession No.
  • the CB 1 receptor is expressed mainly in the brain (central nervous system, CNS), but also in the lungs, liver and kidneys.
  • the CB 2 receptor is expressed mainly in the immune system and in hematopoietic cells. Additional non-CB 1 and non-CB 2 include GPR55 (GenBank Accession No.
  • NM — 005683.3 or NP — 005674.2 protein each of which is hereby incorporated by reference as of May 23, 2012
  • GPR119 GenBank Accession No. NM — 178471.2 or NP — 848566.1 protein, each of which is hereby incorporated by reference as of May 23, 2012
  • GPR18 also known as N-arachidonyl glycine receptor and involved in microglial migration, GenBank Accession No. NM — 001098200 mRNA, NP — 001091670.1, each of which is hereby incorporated by reference as of May 23, 2012).
  • CB 1 and CB 2 receptors are about 44% similar. When only the transmembrane regions of the receptors are considered, amino acid similarity between the two receptor subtypes is approximately 68%. In addition, minor variations in each receptor have been identified. Cannabinoids bind reversibly and stereo-selectively to the cannabinoid receptors. The affinity of an individual cannabinoid to each receptor determines the effect of that cannabinoid. Cannabinoids that bind more selectively to certain receptors are more desirable for medical usage. GPR55 is coupled to the G-protein G 13 and/or G 11 and activation of the receptor leads to stimulation of rhoA, cdc42 and rac1.
  • GPR55 is activated by the plant cannabinoids ⁇ 9 -THC and cannabidiol, and the endocannabinoids anandamide, 2-AG, noladin ether in the low nanomolar range.
  • CB 1 and CB 2 receptors are coupled to inhibitory G proteins. This indicates that both types of receptors will have different readouts. For example, activation of CB1 causes apoptosis whereas increase in GPR55 activity is oncogenic.
  • the CB1 receptor antagonist (also termed ‘inverse agonist’) compound, AM251 is, in fact, an agonist for GPR55. It binds GPR55 and is readily internalized. This illustrates the opposite behavior of these two GPCRs.
  • MNF is shown herein to be a CB1 receptor agonist (similar to WIN55,212-2) but acts as an inhibitor of GPR55, hence the pro-apoptotic behavior of MNF in select cancer cells.
  • fenoterol analogues such as MNF and NF
  • cannabinoid receptor regulators such as regulators of GPR55.
  • a fenoterol analogue either alone or in combination with other agents is administered to a subject to reduce or inhibit one or more symptoms or signs associated with a disorder (such as a metabolic, inflammatory, pain or the like disorder) or disease (such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease) modulated by cannabinoid receptors (such as GPR55).
  • a disorder such as a metabolic, inflammatory, pain or the like disorder
  • disease such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease modulated by cannabinoid receptors (such as GPR55).
  • Carbamate A group of the formula —OC(O)N(R)—, wherein R is H, or an aliphatic group, such as a lower alkyl group or an aralkyl group.
  • Chemotherapy; chemotherapeutic agents any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth.
  • a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors, including a tumor associated with CB receptor activity and/or expression.
  • a chemotherapeutic agent is radioactive molecule.
  • a CB receptor regulator such as one or more fenoterol analogues or a combination thereof is a chemotherapeutic agent.
  • a chemotherapeutic agent is carmustine, lomustine, procarbazine, streptozocin, or a combination thereof.
  • a chemotherapeutic agent of use e.g., see Slapak and Kufe, Principles of Cancer Therapy , Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy , Ch. 17 in Abeloff, Clinical Oncology 2 nd ed., ⁇ 2000 Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).
  • control refers to a sample or standard used for comparison with a test sample.
  • the control is a sample obtained from a healthy subject or a tissue sample obtained from a patient diagnosed with a disorder or disease, such as a tumor, that did not respond to treatment with a ⁇ 2-agonist.
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects which do not have a tumor expressing CB receptors or group of samples that represent baseline or normal values, such as the level of CB receptors in tumor tissue that does not respond to treatment with fenoterol, a fenoterol analogue or a combination thereof).
  • Diabetes mellitus A disease caused by a relative or absolute lack of insulin leading to uncontrolled carbohydrate metabolism, commonly simplified to “diabetes,” though diabetes mellitus should not be confused with diabetes insipidus. As used herein, “diabetes” refers to diabetes mellitus, unless otherwise indicated. A “diabetic condition” includes pre-diabetes and diabetes. Type 1 diabetes (sometimes referred to as “insulin-dependent diabetes” or “juvenile-onset diabetes”) is an autoimmune disease characterized by destruction of the pancreatic 0 cells that leads to a total or near total lack of insulin. In diabetes type 2 (sometimes referred to as “non-insulin-dependent diabetes” or “adult-onset diabetes”), the body does not respond to insulin, though it is present.
  • Symptoms of diabetes include: excessive thirst (polydipsia); frequent urination (polyuria); extreme hunger or constant eating (polyphagia); unexplained weight loss; presence of glucose in the urine (glycosuria); tiredness or fatigue; changes in vision; numbness or tingling in the extremities (hands, feet); slow-healing wounds or sores; and abnormally high frequency of infection.
  • Diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load.
  • FPG fasting plasma glucose
  • OGTT oral glucose tolerance test
  • a subject exhibiting one or more of the following risk factors is considered to have a heightened or substantial risk of developing diabetes type 2:
  • Obesity such as a BMI greater than or equal to about 30 kg/m 2 ; 2. Elevated fasting blood glucose (FPG) levels; 3. Impaired glucose tolerance (IGT); 4. Non-Caucasian ethnicity;
  • non-diabetic or “normal” subject does not have any form of diabetes, such as type 1 diabetes, type 2 diabetes, or pre-diabetes.
  • a derivative such as a fenoterol analogue
  • a biological activity CB receptor activation
  • a fenoterol analogue capable of regulating a CB receptor, such as GPR55
  • Effective amount An amount of agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and/or underlying causes of any of a disorder or disease. In some examples, an “effective amount” is a “therapeutically effective amount” in which the agent alone with an additional therapeutic agent(s) (for example a chemotherapeutic agent) induces the desired response such as treatment of a tumor. In one example, a desired response is to decrease tumor size or metastasis in a subject to whom the therapy is administered.
  • Tumor metastasis does not need to be completely eliminated for the composition to be effective.
  • a composition can decrease metastasis by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the tumor), as compared to metastasis in the absence of the composition.
  • a composition can decrease the number of cancer cells by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable cancer cells), as compared to the number of cancer cells in the absence of the composition.
  • an effective amount is the amount of (R,R′)- or (R,S′)-fenoterol analogue(s) useful in reducing, inhibiting, and/or treating a disorder or disease associated with CB receptor, such as GPR55, expression and/or activity.
  • a therapeutically effective amount of an agent is an amount sufficient to reduce, inhibit, and/or treat the disorder in a subject without causing a substantial cytotoxic effect in the subject.
  • the effective amount of a composition useful for reducing, inhibiting, and/or treating a disorder in a subject will be dependent on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
  • Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for a reduction in tumor size or improvement of physiological condition of a subject having a tumor, such as a brain tumor. Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
  • Glioblastoma A common and malignant form of a primary brain tumor.
  • a glioblastoma is a grade IV astrocytoma and usually spreads rapidly in the brain.
  • a glioblastoma is inhibited by administering a therapeutic effective amount of a fenoterol analogue with other agents capable of regulating a CB receptor, such as GPR55, to a subject, thereby inhibiting one or more symptoms associated with the glioblastoma.
  • Inflammation When damage to tissue occurs, the body's response to the damage is usually inflammation.
  • the damage may be due to trauma, lack of blood supply, hemorrhage, autoimmune attack, transplanted exogenous tissue or infection.
  • This generalized response by the body includes the release of many components of the immune system (for instance, IL-1 and TNF), attraction of cells to the site of the damage, swelling of tissue due to the release of fluid and other processes.
  • a disclosed fenoterol analogue capable of regulating CB receptor activity is used to treat, such as reduce or inhibit, one or more signs or symptoms associated with inflammation.
  • Isomers Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that contain two or more chiral centers and are not mirror images of one another are termed “diastereomers.” Steroisomers that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or ( ⁇ ) isomers, respectively).
  • a chiral compound can exist as either an individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”
  • the compounds described herein may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R), (S), (R,R′), (R,S′)-stereoisomers or as mixtures thereof.
  • R reactive oxygen species
  • S secondary amine
  • R,R′ asymmetric center
  • R,S′ stereoisomers
  • mixtures thereof mixtures thereof.
  • the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof.
  • the methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (see, e.g., March, Advanced Organic Chemistry , 4th edition, New York: John Wiley and Sons, 1992, Chapter 4).
  • Obesity A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res . 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure.
  • the Body Mass Index (BMI) is used to assess obesity.
  • BMI Body Mass Index
  • a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 is overweight, while a BMI of 30 kg/m 2 is obese.
  • waist circumference is used to assess obesity.
  • a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese.
  • Strong evidence shows that obesity affects both the morbidity and mortality of individuals.
  • an obese individual is at increased risk for heart disease, non-insulin-dependent (type 2) diabetes, hypertension, stroke, cancer (e.g., endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys . 63:2185, 2001).
  • Oral glucose tolerance test A diagnostic test for diabetes. After fasting overnight, a subject is provided a concentrated sugar solution to drink, usually containing 50 to 100 grams of glucose. The subject's blood is sampled periodically over the next few to several hours to test blood glucose levels over time. In a non-diabetic subject, blood glucose concentration shows a slight upward shift and returns to normal within 2-3 hours. In a diabetic subject, blood glucose concentration is generally higher than normal after fasting, rises more after the subject drinks the glucose solution, and may take several hours to return to normal. An OGTT of greater than or equal to 140 mg/dl and less than 200 mg/dl indicates that a subject has pre-diabetes.
  • An OGTT of greater than or equal to 200 mg/dl indicates that a subject has frank diabetes, and an OGTT of less than 140 mg/dl indicates that a subject is normal (healthy) and does not have pre-diabetes or diabetes.
  • Overweight An individual who weighs more than their ideal body weight.
  • An overweight individual can be obese, but is not necessarily obese.
  • an overweight human individual is any individual who desires to decrease their weight.
  • an overweight human individual is an individual with a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 .
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Phenyl groups may be unsubstituted or substituted with one, two or three substituents, with substituent(s) independently selected from alkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, halo, haloalkyl (such as —CF 3 ), nitro, cyano, —OR (where R is hydrogen or alkyl), —N(R)R′ (where R and R′ are independently of each other hydrogen or alkyl), —COOR (where R is hydrogen or alkyl) or —C(O)N(R′)R′′ (where R′ and R′′ are independently selected from hydrogen or alkyl).
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified preparation is one in which a desired component such as an (R,R′)-enantiomer of fenoterol is more enriched than it was in a preceding environment such as in a ( ⁇ )-fenoterol mixture.
  • a desired component such as (R,R′)-enantiomer of fenoterol is considered to be purified, for example, when at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight is composed of the desired component.
  • Purity of a compound may be determined, for example, by high performance liquid chromatography (HPLC) or other conventional methods.
  • HPLC high performance liquid chromatography
  • the specific fenoterol analogue enantiomers are purified to represent greater than 90%, often greater than 95% of the other enantiomers present in a purified preparation.
  • the purified preparation may be essentially homogeneous, wherein other stereoisomers are less than 1%.
  • a compound described herein may be obtained in a purified form or purified by any of the means known in the art, including silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography , 2nd Edition, ed. by Snyder and Kirkland, New York: John Wiley and Sons, 1979; and Thin Layer Chromatography , ed. by Stahl, New York: Springer Verlag, 1969.
  • a compound includes purified fenoterol or fenoterol analogue with a purity of at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight relative to other contaminants.
  • a compound includes at least two purified stereoisomers each with a purity of at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% of a sample by weight relative to other contaminants.
  • a compound can include a substantially purified (R,R′)-fenoterol analogue and a substantially purified (R,S′)-fenoterol analogue.
  • subject includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, rats, mice, and cows.
  • mammal includes both human and non-human mammals.
  • Tissue A plurality of functionally related cells.
  • a tissue can be a suspension, a semi-solid, or solid.
  • Tissue includes cells collected from a subject such as the brain or a portion thereof.
  • a primary tumor is tumor growing at the anatomical site where tumor progression began and proceeded to yield this mass.
  • a primary brain tumor (also referred to as a glioma) is a tumor that originates in the brain. Exemplary primary brain tumors include astrocytomas, glioblastomas, ependymoma, oligodendroglomas, and mixed gliomas.
  • a primary brain tumor expresses CB receptors, such as a glioblastoma associated with CB receptor expression.
  • under conditions sufficient for includes administering one or more fenoterol analogues, fenoterol or a combination thereof to a subject to at a concentration sufficient to allow the desired activity.
  • the desired activity is reducing or inhibiting a sign or symptom associated with a disorder or disease, such as a primary brain tumor, hepatocellular carcinoma, liver cancer, colon cancer, or lung cancer
  • a disorder or disease such as a primary brain tumor, hepatocellular carcinoma, liver cancer, colon cancer, or lung cancer
  • the desired activity is preventing or inhibiting tumor growth, such as astrocytoma, glioblastoma, or hepatocellular carcinoma growth.
  • Tumor growth does not need to be completely inhibited for the treatment to be considered effective.
  • a partial reduction or slowing of growth such as at least about a 10% reduction, such as at least 20%, at least about 30%, at least about 40%, at least about 50% or greater is considered to be effective.
  • R 1 -R 3 independently are hydrogen, acyl, alkoxy carbonyl, amino carbonyl or a combination thereof;
  • R 4 is H or lower alkyl
  • R 5 is lower alkyl
  • X and Y independently are selected from hydrogen, lower —OR 6 and —NR 7 R 8 ;
  • R 6 is lower alkyl or acyl
  • R 7 and R 8 independently are hydrogen, lower alkyl, alkoxy carbonyl, acyl or amino carbonyl.
  • Y may be —OH.
  • R 5 is a 1- or 2-napthyl derivative optionally having 1, 2 or 3 substituents. Examples of such R 5 groups are represented by the formula
  • Y 1 , Y 2 and Y 3 independently are hydrogen, halogen, sulphur-containing moiety including SH, sulfoxides, sulphones, sulphanamides and related alkyl and aromatic substituted moieties, lower —OR 6 and —NR 7 R 8 ;
  • R 6 is independently for each occurrence selected from lower alkyl and acyl; and
  • R 7 and R 8 independently are hydrogen, lower alkyl, alkoxy carbonyl, acyl or amino carbonyl (carbamoyl).
  • compounds at least one of Y 1 , Y 2 and Y 3 is —OCH 3 .
  • Particular R 5 groups include those represented by the formulas
  • R 6 is lower alkyl, such as methyl, ethyl, propyl or isopropyl or acyl, such as acetyl.
  • R 5 groups include
  • R 4 is lower alkyl and R 5 is
  • X and Y independently are selected from H, lower alkyl —OR 6 and —NR 7 R 8 ; R 6 is lower alkyl; and R 7 and R 8 independently are hydrogen or lower alkyl.
  • R 4 is selected from ethyl, n-propyl, and isopropyl and R 5 has the formula
  • X is H, —OR 6 or —NR 7 R 8 .
  • R 6 may be methyl or R 7 and R 8 are hydrogen.
  • R 5 has the formula
  • R 4 is selected from methyl, ethyl, n-propyl and isopropyl and R 5 represents
  • R 1 -R 3 independently are hydrogen; R 4 is a lower alkyl (such as, CH 3 or CH 2 CH 3 ); R 5 is lower alkyl,
  • X, Y 1 , Y 2 and Y 3 independently are hydrogen, —OR 6 and —NR 7 R 8 ;
  • R 6 is independently hydrogen, lower alkyl, acyl, alkoxy carbonyl or amino carbonyl;
  • R 7 and R 8 independently are hydrogen, lower alkyl, alkoxy carbonyl, acyl or amino carbonyl and wherein the compound is optically active.
  • R 1 -R 3 independently are hydrogen; R 4 is a methyl or an ethyl; R 5 is
  • X is —OH or —OCH 3 .
  • R 1 -R 3 independently are hydrogen; R 4 is a methyl or an ethyl; R 5 is
  • Exemplary compounds include, but are not limited to, (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), or (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF).
  • R 1 -R 3 examples include, without limitation, acyl, acyloxy and alkoxy carbonyl groups.
  • Compounds having such cleavable groups are referred to as “prodrugs.”
  • the term “prodrug,” as used herein, means a compound that includes a substituent that is convertible in vivo (e.g., by hydrolysis) to a hydroxyl group.
  • Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs , Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology , Vol.
  • administering comprises administering a therapeutically effective amount of MNF, NF or a combination thereof. In some embodiments, administering comprises administering a therapeutically effective amount of MNF.
  • the method includes administering a therapeutically effective amount of a pharmaceutical composition containing any of the disclosed fenoterol analogues capable of regulating a CB receptor disorder or disease and a pharmaceutically acceptable carrier to treat the disorder or disease regulated by a CB receptor, such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • a CB receptor such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • a CB receptor such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • a CB receptor such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • a CB receptor such as a glioblastoma or hepatocellular carcinoma expressing GPR55.
  • An exemplary (R,R′)-compound has the chemical structure of:
  • solvates such as hydrates
  • pharmaceutically acceptable salts and/or different physical forms of (R,R′)-fenoterol or any of the fenoterol analogues herein described.
  • Solvate means a physical association of a compound with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including by way of example covalent adducts and hydrogen bonded solvates. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include ethanol-associated compound, methanol-associated compounds, and the like. “Hydrate” is a solvate wherein the solvent molecule(s) is/are H 2 O.
  • the disclosed compounds also encompass salts including, if several salt-forming groups are present, mixed salts and/or internal salts.
  • the salts are generally pharmaceutically acceptable salts that are non-toxic. Salts may be of any type (both organic and inorganic), such as fumarates, hydrobromides, hydrochlorides, sulfates and phosphates. In an example, salts include non-metals (e.g., halogens) that form group VII in the periodic table of elements. For example, compounds may be provided as a hydrobromide salt.
  • salt-forming groups include, but are not limited to, a carboxyl group, a phosphonic acid group or a boronic acid group, that can form salts with suitable bases.
  • These salts can include, for example, nontoxic metal cations, which are derived from metals of groups IA, IB, IIA and IIB of the periodic table of the elements.
  • alkali metal cations such as lithium, sodium or potassium ions, or alkaline earth metal cations such as magnesium or calcium ions can be used.
  • the salt can also be a zinc or an ammonium cation.
  • the salt can also be formed with suitable organic amines, such as unsubstituted or hydroxyl-substituted mono-, di- or tri-alkylamines, in particular mono-, di- or tri-alkylamines, or with quaternary ammonium compounds, for example with N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower alkyl)amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine, or N-methyl-D-glucamine, or quaternary ammonium compounds such as tetrabutylam
  • Exemplary compounds disclosed herein possess at least one basic group that can form acid-base salts with inorganic acids.
  • basic groups include, but are not limited to, an amino group or imino group.
  • inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid.
  • Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and, in addition, with amino acids, for example with ⁇ -amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonic acid, ethane-1,2-disulfonic acid
  • a pharmaceutically acceptable salt may also serve to adjust the osmotic pressure of a composition.
  • the compounds used in the method are provided are polymorphous.
  • the compounds can be provided in two or more physical forms, such as different crystal forms, crystalline, liquid crystalline or non-crystalline (amorphous) forms.
  • any of the above described compounds e.g., (R,R′) and/or (R,S′) fenoterol analogues or a hydrate or pharmaceutically acceptable salt thereof
  • a CB receptor such as GPR55
  • a CB receptor-regulated disorder such as a metabolic, inflammatory, pain or the like disorder
  • disease such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease
  • cannabinoid receptors such as GPR55
  • the disclosed fenoterol analogues can be synthesized by any method known in the art including those described in U.S. patent application Ser. No. 12/376,945 filed Feb. 9, 2009, U.S. patent application Ser. No. 13/333,866 filed Dec. 21, 2011 and WO 2011/112867 filed Mar. 10, 2011, each of which is hereby incorporated by reference in its entirety.
  • Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure , Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis , Fourth Edition, New York: Longman, 1978).
  • Compounds as described herein may be purified by any of the means known in the art, including chromatographic means, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. Most typically the disclosed compounds are purified via open column chromatography or prep chromatography.
  • Scheme I An exemplary synthesis of 4 stereoisomers of 1-6 including the coupling of the epoxide formed from either (R)- or (S)-3′,5′-dibenzyloxyphenylbromohydrin with the (R)- or (S)-enantiomer of the appropriate benzyl-protected 2-amino-3-benzylpropane (1-5) or the (R) or (S)-enantiomer of N-benzyl-2-aminoheptane (6).
  • Scheme III describes an exemplary synthesis for the chiral building blocks used in Scheme II.
  • the (R)- and (S)-3′,5′-dibenzyloxyphenyl-bromohydrin enantiomers were obtained by the enantiospecific reduction of 3,5-dibenzyloxy- ⁇ -bromoacetophenone using boron-methyl sulfide complex (BH 3 SCH 3 ) and either (1R,2S)- or (1S,2R)-cis-1-amino-2-indanol.
  • BH 3 SCH 3 boron-methyl sulfide complex
  • the disclosed fenoterol analogues can be useful, at least, for reducing or inhibiting one or more symptoms or signs associated with a disorder (such as a metabolic, inflammatory, pain or the like disorder) or disease (such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease) modulated by cannabinoid receptors (such as GPR55).
  • a disorder such as a metabolic, inflammatory, pain or the like disorder
  • disease such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease
  • cannabinoid receptors such as GPR55
  • Formulations for pharmaceutical compositions are well known in the art. For example, Remington's Pharmaceutical Sciences , by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of (R,R′)-fenoterol and disclosed fenoterol analogues. Pharmaceutical compositions comprising at least one of these compounds can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration (e.g., oral or parenteral) and/or on the disorder to be treated (e.g., a tumor associated with CB receptor, such as GPR55 receptor, activity or expression). In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a fenoterol compound.
  • a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a fenoterol compound.
  • compositions useful for the disclosed methods and compositions are conventional in the art.
  • the nature of a pharmaceutical carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • solid compositions such as powder, pill, tablet, or capsule forms conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can optionally contain minor amounts of non-toxic auxiliary substances or excipients, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate.
  • auxiliary substances or excipients such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate.
  • excipients include, nonionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations.
  • compositions may be formulated as a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydriodic acid, and phosphoric acid.
  • Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like.
  • suitable pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences , 19th Edition, Mack Publishing Company, Easton, Pa., 1995.
  • a pharmaceutically acceptable salt may also serve to adjust
  • the dosage form of a disclosed pharmaceutical composition will be determined by the mode of administration chosen.
  • oral dosage forms may be employed.
  • Oral formulations may be liquid such as syrups, solutions or suspensions or solid such as powders, pills, tablets, or capsules. Methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • compositions comprising a disclosed compound may be formulated in unit dosage form suitable for individual administration of precise dosages.
  • amount of active ingredient such as (R,R′)-MNF or NF administered will depend on the subject being treated, the severity of the disorder, and the manner of administration, and is known to those skilled in the art.
  • the formulation to be administered will contain a quantity of the extracts or compounds disclosed herein in an amount effective to achieve the desired effect in the subject being treated.
  • compositions are provided in the form of a tablet containing from about 1.0 to about 50 mg of the active ingredient, particularly about 2.0 mg, about 2.5 mg, 5 mg, about 10 mg, or about 50 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated.
  • a tablet containing from about 1 mg to about 50 mg (such as about 2 mg to about 10 mg) active ingredient is administered two to four times a day, such as two times, three times or four times.
  • a suitable dose for parental administration is about 1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose of about 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • mg/kg milligram per kilogram
  • a dose of about 10 mg/kg to about 80 mg/kg such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • other higher or lower dosages also could be used, such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 to about 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.
  • Single or multiple administrations of the composition comprising one or more of the disclosed compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • multiple doses are administered.
  • the composition is administered parenterally once per day.
  • the composition can be administered twice per day, three times per day, four times per day, six times per day, every other day, twice a week, weekly, or monthly.
  • Treatment will typically continue for at least a month, more often for two or three months, sometimes for six months or a year, and may even continue indefinitely, i.e., chronically. Repeat courses of treatment are also possible.
  • the pharmaceutical composition is administered without concurrent administration of a second agent for the treatment of a tumor that expresses a CB receptor, such as GPR55.
  • a CB receptor such as GPR55
  • one or more of the disclosed compositions is administered without concurrent administration of other agents, such as without concurrent administration of an additional agent also known to target the tumor.
  • a therapeutically effective amount of a disclosed pharmaceutical composition is administered concurrently with an additional agent, including an additional therapy (such as, but not limited to, a chemotherapeutic agent, an additional CB receptor regulator (such as regulator of GPR55), an anti-inflammatory agent, an anti-oxidant, or other agents known to those of skill in the art).
  • the disclosed compounds are administered in combination with a chemotherapeutic agent, anti-oxidants, anti-inflammatory drugs or combinations thereof.
  • a disclosed pharmaceutical composition is administered an adjuvant therapy.
  • a pharmaceutical composition containing one or more of the disclosed compounds is administered orally daily to a subject in order to prevent or retard tumor growth.
  • a composition containing equal portions of two or more disclosed compounds is provided to a subject.
  • a composition containing unequal portions of two or more disclosed compounds is provided to the subject.
  • a composition contains unequal portions of a (R,R′)-fenoterol derivative and a (S,R′)-fenoterol derivative and/or a (R,S′)-derivative.
  • the composition includes a greater amount of the (S,R′)- or (R,S′)-fenoterol derivative.
  • Such therapy can be given to a subject for an indefinite period of time to inhibit, prevent, or reduce tumor reoccurrence.
  • the present disclosure includes methods of treating disorders including reducing or inhibiting one or more signs or symptoms associated with a disorder (such as a metabolic, inflammatory, pain or the like disorder) or disease (such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease) modulated by cannabinoid receptors (such as GPR55).
  • a disorder such as a metabolic, inflammatory, pain or the like disorder
  • disease such as hepatocellular carcinoma, glioblastoma, liver cancer, lung cancer, colon cancer, brain cancer, diabetes, or an inflammatory disease
  • cannabinoid receptors such as GPR55.
  • the tumor is a primary tumor, such as a primary brain tumor expressing or regulated by CB receptors, such as GPR55.
  • the tumor is a glioblastoma or hepatocellular carcinoma expressing CB receptors, such as GPR55.
  • the tumor is a glioblastoma or hepatocellular carcinoma expressing CB receptors, such as GPR55, but not expressing ⁇ 2-AR.
  • the tumor is a glioblastoma or hepatocellular carcinoma expressing both CB receptors, such as GPR55, and ⁇ 2-AR.
  • the fenoterol analogue and/or fenoterol, such as (R,R′) fenoterol, itself is administered depending upon the tumor receptor population.
  • a tumor expressing or regulated by a CB receptors, such as GPR55 is treated by administering one or more disclosed fenoterol analogues possessing CB receptor modulatory activity, such as (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF), or a combination thereof.
  • MNF methoxy-1
  • a tumor expressing or regulated by a CB receptor such as GPR55, and ⁇ 2-AR is treated by administering one or more disclosed fenoterol analogues possessing CB receptor regulatory activity, such as (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF), and one or more fenoterol analogues or fenoterol itself having ⁇ 2-AR stimulatory activity in combination.
  • MNF methoxy-1-napthylfenoterol
  • R,S′
  • Disclosed methods include administering fenoterol, such as (R,R′)-fenoterol, a disclosed fenoterol analogue or a combination thereof (and, optionally, one or more other pharmaceutical agents) depending upon the receptor population of the tumor, to a subject in a pharmaceutically acceptable carrier and in an amount effective to treat the tumor expressing a ⁇ 2-AR, a CB receptor or combination thereof, such as a primary tumor.
  • Treatment of a tumor includes preventing or reducing signs or symptoms associated with the presence of such tumor (for example, by reducing the size or volume of the tumor or a metastasis thereof).
  • Such reduced growth can in some examples decrease or slow metastasis of the tumor, or reduce the size or volume of the tumor by at least 10%, at least 20%, at least 50%, or at least 75%, such as between 10%-90%, 20%-80%, 30%-70%, 40%-60%, including a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% reduction.
  • treatment includes reducing the invasive activity of the tumor in the subject, for example by reducing the ability of the tumor to metastasize.
  • treatment using the methods disclosed herein prolongs the time of survival of the subject.
  • Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (IV), intraperitoneal (IP), rectal, topical, ophthalmic, nasal, and transdermal as described in detail above.
  • IV intravenous
  • IP intraperitoneal
  • rectal topical
  • ophthalmic nasal
  • transdermal transdermal
  • an effective amount of fenoterol such as (R,R′)-fenoterol, or a disclosed fenoterol analogue or combination thereof will depend, at least, on the particular method of use, the subject being treated, the severity of the tumor, and the manner of administration of the therapeutic composition.
  • a “therapeutically effective amount” of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject being treated. For example, this may be the amount of (R,R′)-fenoterol, a disclosed fenoterol analogue or a combination thereof necessary to prevent or inhibit tumor growth and/or one or more symptoms associated with the tumor in a subject.
  • a therapeutically effective amount of (R, ′R)-fenoterol or a disclosed fenoterol analogue is an amount sufficient to prevent or inhibit a tumor, such as a brain or liver tumor growth and/or one or more symptoms associated with the tumor in a subject without causing a substantial cytotoxic effect on host cells.
  • Therapeutically effective doses of a disclosed fenoterol compound or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving concentrations that are at least as high as the IC 50 of the applicable compound disclosed in the examples herein.
  • An example of a dosage range is from about 0.001 to about 10 mg/kg body weight orally in single or divided doses.
  • a dosage range is from about 0.005 to about 5 mg/kg body weight orally in single or divided doses (assuming an average body weight of approximately 70 kg; values adjusted accordingly for persons weighing more or less than average).
  • compositions are, for example, provided in the form of a tablet containing from about 1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg, about 5 mg, about 10 mg, or about 50 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated.
  • a tablet containing from about 1 mg to about 50 mg active ingredient is administered two to four times a day, such as two times, three times or four times.
  • a suitable dose for parental administration is about 1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose of about 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • mg/kg milligram per kilogram
  • a dose of about 10 mg/kg to about 80 mg/kg such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • other higher or lower dosages also could be used, such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 to about 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.
  • Single or multiple administrations of the composition comprising one or more of the disclosed compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • multiple doses are administered.
  • the composition is administered parenterally once per day.
  • the composition can be administered twice per day, three times per day, four times per day, six times per day, every other day, twice a week, weekly, or monthly.
  • Treatment will typically continue for at least a month, more often for two or three months, sometimes for six months or a year, and may even continue indefinitely, i.e., chronically. Repeat courses of treatment are also possible.
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy.
  • Subjects can be screened prior to initiating the disclosed therapies, for example to select a subject in need of or at risk of developing a disorder or disease regulated by CB receptor activity or expression.
  • the method can include screening subjects to determine if they have or are at risk of developing a GPR55-regulated disorder or disease, such as if the subject is in need of tumor inhibition.
  • Subjects having a tumor that expresses a CB receptor, such as GPR55, or is regulated by CB receptor activity, such as a primary tumor, including a primary brain tumor, such as a glioblastoma, hepatocellular carcinoma, liver cancer, lung cancer, or colon cancer or at risk of developing such a tumor are selected.
  • subjects are diagnosed with the tumor by clinical signs, laboratory tests, or both.
  • a tumor such as a primary brain tumor
  • characteristic clinical signs such as headaches, vomiting, seizures, dizziness, weight loss and various associated complaints.
  • Diagnosis is generally by imaging analysis such as by magnetic resonance imaging (MRI) and confirmed by histology.
  • MRI magnetic resonance imaging
  • a subject is selected that does not have a bleeding disorder, such as an intracerebral hemorrhage.
  • a subject in need of the disclosed therapies is selected by detecting a tumor expressing a CB receptor (e.g., GPR55) or regulated by its activity, such as by detecting CB receptor activity or expression in a sample obtained from a subject identified as having, suspected of having or at risk of acquiring such a tumor.
  • a CB receptor e.g., GPR55
  • a subject is selected by detecting a primary brain tumor such as an astrocytoma or glioblastoma by MRI or positron emission tomography (PET) in a subject.
  • a primary brain tumor such as an astrocytoma or glioblastoma by MRI or positron emission tomography (PET)
  • a subject is selected by determining the subject has or is at risk of developing a disorder or disease, such as a tumor and/or cancer, which does not respond to ⁇ 2-AR stimulation.
  • Pre-screening is not required prior to administration of the therapeutic agents disclosed herein (such as those including fenoterol, a fenoterol analogue or a combination thereof).
  • Exemplary tumors include tumors that express a CB receptor, such as GPR55, or regulated by such, including primary tumors, such as a primary brain tumor.
  • a primary brain tumor includes astrocytomas, glioblastomas, ependymoma, oligodendroglomas, and mixed gliomas.
  • tumors associated with CB receptor activity or expression include hematological tumors, such as leukemias, including acute leukemias (such as 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
  • acute leukemias such as
  • Examples of possible solid tumors which may express a CB receptor or be regulated by CB receptor activity include sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, liver cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, me
  • a tumor is a brain cancer, liver cancer, or lung cancer that expresses a CB receptor, such as GPR55.
  • Tumors expressing a CB receptor, such as GPR55 can be identified by routine methods known to those of skill in the art including Western blot and histological studies with antibodies capable of detecting a CB receptor, such as GPR55
  • subjects having a disorder or disease regulated by CB receptor activity can be monitored for decreases in tumor growth, tumor volume or in one or more clinical symptoms associated with the tumor.
  • subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art including those described herein including imaging analysis.
  • a partial response is a reduction, such as at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, or at least a 70% reduction in one or more signs or symptoms associated with the disorder or disease, such as a tumor regulated by CB receptor activity, including tumor size or volume.
  • the method further includes administering a therapeutic effective amount of fenoterol, a fenoterol analogue or a combination thereof with additional therapeutic treatments.
  • the subject prior to, during, or following administration of a therapeutic amount of an agent that prevents or inhibits a tumor regulated by CB receptor activity, the subject can receive one or more other therapies.
  • the subject receives one or more treatments to remove or reduce the tumor prior to administration of a therapeutic amount of a composition including fenoterol, a fenoterol analogue or combination thereof.
  • Such therapies include, but are not limited to, surgical treatment for removal or reduction of the tumor (such as surgical resection, cryotherapy, or chemoembolization), as well as anti-tumor pharmaceutical treatments which can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents.
  • additional therapeutic agents include microtubule-binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.
  • Microtubule-binding agent refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division.
  • microtubule-binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No.
  • Taxoids such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.
  • DNA and/or RNA transcription regulators including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies.
  • DNA intercalators and cross-linking agents that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.
  • DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5′-deoxyuridine, 5-fluorouracil and analogs thereof.
  • suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.
  • alkylating agents include carmustine or lomustine.
  • Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.
  • Kinase inhibitors include Gleevac, Iressa, and Tarceva that prevent phosphorylation and activation of growth factors.
  • anti-tumor agents for example anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the disclosed therapies.
  • agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogues thereof.
  • the tumor such as the primary brain tumor
  • the disclosed therapies such as administration of fenoterol, a fenoterol analogue or a combination thereof.
  • a subject having a primary brain tumor associated with CB receptor activity can have at least a portion of the tumor surgically excised prior to administration of the disclosed therapies.
  • one or more chemotherapeutic agents are administered following treatment with a composition including fenoterol, a fenoterol analogue or a combination thereof.
  • the subject has a primary brain tumor and is administered radiation therapy, chemoembolization therapy, or both concurrently with the administration of the disclosed therapies.
  • the disclosed fenoterol analogues possessing CB receptor modulatory activity can be used to treat other conditions associated with CB receptor regulation, such as metabolic disorders and disease (e.g., obesity and diabetes), or inflammatory and neuropathic pain disorders, diseases associated with aging such as Alzheimer's, bone loss, muscle wasting (sarcopenia), osteoarthritis and loss of appetite, central nervous system conditions such as depression and anxiety and other diseases and disorders associated with CB receptor regulation.
  • metabolic disorders and disease e.g., obesity and diabetes
  • inflammatory and neuropathic pain disorders e.g., diseases associated with aging such as Alzheimer's, bone loss, muscle wasting (sarcopenia), osteoarthritis and loss of appetite
  • central nervous system conditions such as depression and anxiety and other diseases and disorders associated with CB receptor regulation.
  • metabolic disorders/diseases such as obesity, loss of appetite, and/or diabetes
  • bone and/or muscle disorders or diseases such as bone loss, muscle wasting, osteoarthritis
  • central nervous system conditions such as depression and anxiety
  • inflammatory disorders/diseases such as inflammation, neuropathic pain disorders and diseases associated with inflammation and/or aging (such as Alzheimer's disease, osteoarthritis)
  • methods of preventing and treating obesity, diabetes, and related disorders are disclosed.
  • a method of treating obesity or a condition associated with obesity, such as diabetes, in a subject comprising administering to a subject an effective amount of at least one fenoterol analogue with CB receptor modulatory activity, such as GPR55 activity (e.g., a disclosed naphthylfenoterol analogue, such as (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF), or a combination thereof), to reduce or inhibit one or more signs or symptoms associated with obesity or
  • methods of treating one or more signs or symptoms associated with an inflammatory disorder and/or disease comprising administering to a subject an effective amount of at least one fenoterol analogue with CB receptor modulatory activity, such as GPR55 activity (e.g., a disclosed naphthylfenoterol analogue, such as (R,R′)-4′-methoxy-1-napthylfenoterol (MNF), (R,S′)-MNF, (R,R′)-ethylMNF, (R,R′)-napthylfenoterol (NF), (R,R′)-ethylNF, (R,S′)—NF and (R,R′)-4′-amino-1-napthylfenoterol (aminoNF), (R,R′)-4′-hydroxy-1-napthylfenoterol (hydroxyNF), or a combination thereof), to reduce or inhibit one or more signs or symptoms associated with the inflammatory disorder/
  • Disclosed methods include administering a disclosed fenoterol analogue with CB receptor modulatory activity (and, optionally, one or more other pharmaceutical agents) to a subject in a pharmaceutically acceptable carrier and in an amount effective to treat the disorder or disease regulated by CB receptor activity, such as GPR55 activity.
  • Treatment of the disorder or disease includes preventing or reducing signs or symptoms associated with the particular disorder or disease.
  • the signs and symptoms associated with the particular disorder or disease are known to one of ordinary skill in the art and can be measured by assays disclosed herein as well as those known to those skilled in the art.
  • treatment using the methods disclosed herein prolongs the time of survival of the subject.
  • Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (IV), intraperitoneal (IP), rectal, topical, ophthalmic, nasal, and transdermal as described in detail above.
  • IV intravenous
  • IP intraperitoneal
  • rectal topical
  • ophthalmic nasal
  • transdermal transdermal
  • an effective amount of a disclosed fenoterol analogue or combination thereof will depend, at least, on the particular method of use, the subject being treated, the severity of the disorder/disease, and the manner of administration of the therapeutic composition.
  • a “therapeutically effective amount” of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject being treated.
  • a therapeutically effective amount of a disclosed fenoterol analogue is an amount sufficient to prevent or inhibit one or more symptoms associated with the particular disorder/disease in a subject without causing a substantial cytotoxic effect on host cells.
  • Therapeutically effective doses of a disclosed fenoterol compound or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving concentrations that are at least as high as the IC 50 of the applicable compound disclosed in the examples herein.
  • An example of a dosage range is from about 0.001 to about 10 mg/kg body weight orally in single or divided doses.
  • a dosage range is from about 0.005 to about 5 mg/kg body weight orally in single or divided doses (assuming an average body weight of approximately 70 kg; values adjusted accordingly for persons weighing more or less than average).
  • compositions are, for example, provided in the form of a tablet containing from about 1.0 to about 50 mg of the active ingredient, particularly about 2.5 mg, about 5 mg, about 10 mg, or about 50 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated.
  • a tablet containing from about 1 mg to about 50 mg active ingredient is administered once to four times a day, such as one time, two times, three times or four times.
  • a suitable dose for parental administration is about 1 milligram per kilogram (mg/kg) to about 100 mg/kg, such as a dose of about 10 mg/kg to about 80 mg/kg, such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • mg/kg milligram per kilogram
  • a dose of about 10 mg/kg to about 80 mg/kg such including about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg or about 100 mg/kg administered parenterally.
  • other higher or lower dosages also could be used, such as from about 0.001 mg/kg to about 1 g/kg, such as about 0.1 to about 500 mg/kg, including about 0.5 mg/kg to about 200 mg/kg.
  • compositions comprising one or more of the disclosed compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • multiple doses are administered.
  • the composition is parenterally administered once per day.
  • the composition can be administered twice per day, three times per day, four times per day, six times per day, every other day, twice a week, weekly, or monthly. Treatment will typically continue for at least one month, more often for two or three months, sometimes for six months or a year, and may even continue indefinitely, that is, chronically. Repeat courses of treatment are also possible.
  • one or more disclosed fenoterol analogues with CB receptor activity is orally administered to a subject daily to treat one or more symptoms associated with an aging disorder or disease (such as Alzheimer's, sarcopenia, bone loss, or combinations thereof) or a central nervous system disorder or disease (such as anxiety or depression).
  • an aging disorder or disease such as Alzheimer's, sarcopenia, bone loss, or combinations thereof
  • a central nervous system disorder or disease such as anxiety or depression
  • Subjects can be screened prior to initiating the disclosed therapies, for example to select a subject in need of or at risk of developing a disorder or disease regulated by CB receptor activity or expression.
  • the method can include screening subjects to determine if they have or are at risk of developing a GPR55-regulated disorder or disease.
  • Subjects having a disorder or disease that expresses a CB receptor, such as GPR55, or is regulated by CB receptor activity are selected.
  • subjects are diagnosed by clinical signs, laboratory tests, or both known to those of ordinary skill in the art or disclosed herein (or both).
  • Pre-screening is not required prior to administration of the therapeutic agents disclosed herein (such as those including fenoterol, a fenoterol analogue or a combination thereof).
  • a partial response is a reduction, such as at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, or at least a 70% reduction in one or more signs or symptoms associated with the disorder or disease.
  • the method further includes administering a therapeutic effective amount of one or more fenoterol analogues with additional therapeutic treatments.
  • the subject prior to, during, or following administration of a therapeutic amount of an agent that prevents or inhibits a tumor regulated by CB receptor activity, the subject can receive one or more other therapies.
  • the subject receives one or more treatments to remove or reduce one or more signs or symptoms associated with the CB receptor regulated disorder/disease prior to administration of a therapeutic amount of a composition including one or more fenoterol analogues.
  • the subject prior to, during, or following administration of a therapeutic amount of a disclosed fenoterol analogue composition that reduces or inhibits one or more signs or symptoms of obesity or a condition associated with obesity, the subject can receive one or more other therapies.
  • the subject receives one or more treatments to remove or reduce one or more conditions associated with obesity, such as diabetes.
  • Examples of such therapies include, but are not limited to, anti-diabetic agents, insulin sensitizers, insulin secretagogues, agents that preserve and/or increase ⁇ -cell mass, agents that enhance glucose-stimulated insulin secretion and glucose uptake in peripheral organs of insulin action (skeletal muscle, liver, adipose tissue), agents that suppress endogenous glucose production, and anti-obesity agents.
  • the disclosed therapies can be administered with anti-diabetic agents such as biguanides.
  • the biguanide antidiabetic agent is metformin.
  • Metformin is manufactured by Lyonnaise Industrielle Pharmaceutique SA (Lyons, France), also known by its acronym LIPHA SA, and commercially distributed in the United States as a hydrochloride salt by the Bristol-Myers Squibb Company (Princeton, N.J.) as GLUCOPHAGE® XR. Additionally, Bristol-Myers Squibb distributes a pharmaceutical having a combination of metformin and glyburide as GLUCOVANCE®.
  • Anti-diabetic agents other than biguanides can also be administered to the identified subject.
  • the anti-diabetic agent is a thiazolidinedione, such as troglitazone.
  • the anti-diabetic agent is an incretin or dipeptidyl peptidase-4 inhibitor, but the anti-diabetic agent can be any agent of interest.
  • a therapeutically effective amount of an anti-diabetic agent may be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the course of treatment may last for any length of time, such as a day or several days, a week or several weeks, a month or several months, or a year or several years, so long as the therapeutic effect is observed, such as inhibiting the onset of type II diabetes in a subject diagnosed with pre-diabetes, or inducing a subject diagnosed with type 2 diabetes or pre-diabetes to a normal glucose tolerance.
  • the subject can be monitored while undergoing treatment using the methods described herein in order to assess the efficacy of the treatment protocol. In this manner, the length of time or the amount given to the subject can be modified based on the results obtained using the methods disclosed herein.
  • the therapeutically effective amount will depend on the anti-diabetic agent being used, the characteristics of the subject being treated (such as age, BMI, physiological condition, etc.), the severity and type of the affliction, and the manner of administration of the agent.
  • the therapeutically effective dose can be determined by various methods, including generating an empirical dose-response curve, predicting potency and efficacy by using quantitative structure-activity relationships (QSAR) methods or molecular modeling, and other methods used in the pharmaceutical sciences.
  • the therapeutically effective amount of metformin (or a related biguanide analog or homolog) is at least about 1000 mg per day, such as at least about 1500 mg per day, or even at least about 1700 mg per day.
  • the total amount of metformin is divided into smaller doses, such as two or three doses per day, for example 850 mg twice a day (b.i.d.) or 500 mg three times a day (t.i.d.). In alternative, non-limiting examples, the total amount of metformin is about 500 mg or less per day.
  • the subject can be monitored at different doses of an agent using the assays described herein, in order to determine a therapeutically effective amount for the subject of interest.
  • purified therapeutically active agents are generally combined with a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations may contain only one type of anti-diabetic agent or may be composed of a combination of several types of anti-diabetic agents, such as a combination of two or more anti-diabetic agents.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Anti-diabetic agents may be administered by any means that achieve their intended purpose.
  • the anti-diabetic agents may be administered to a subject through systemic administration, such as intravenous, intraperitoneal, intralesional, suppository, or oral administration.
  • the anti-diabetic agent can be administered alone or in combination with another anti-diabetic agent. In certain embodiments, the anti-diabetic agent is administered in the absence of administering any other anti-diabetic agent.
  • a subject may be instructed, trained, or induced to adopt anti-diabetic lifestyle modifications.
  • the subject can be counseled to reduce caloric intake or to exercise.
  • the methods disclosed herein can be used to monitor the effectiveness of these alternative measures to determine if pharmaceutical intervention is warranted for a subject of interest.
  • [ 3 H]-Thymidine (70-90 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, Mass.). Eagle's Minimum Essential Medium (E-MEM), trypsin solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), 100 ⁇ solutions of sodium pyruvate (100 mM), L-glutamine (200 mM), and penicillin/streptomycin (a mixture of 10,000 units/ml penicillin and 10,000 ⁇ g/ml streptomycin) were obtained from Quality Biological (Gaithersburg, Md.). WIN 55,212-2, AM251, and AM630 were purchased from Cayman Chemical (Ann Arbor, Mich.). ICI 118,551 hydrochloride and (R)-isoproterenol were obtained from Sigma-Aldrich (St. Louis, Mo.).
  • Human HepG2 hepatocarcinoma cells and human U87MG glioma cells were maintained in EMEM medium supplemented with 1% L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin, and 10% FBS (Hyclone, Logan, Utah).
  • the human 1321N1 astrocytoma cells European Collection of Cell Cultures, Sigma-Aldrich) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and penicillin/streptomycin. All cell lines were cultured at 37° C. in 5% CO 2 , and the medium was replaced every 2-3 days.
  • cells at 70-80% confluence were depleted of serum for 3 hours, followed by the addition of ICI 118551, AM251, AM630 or WIN 55-212.2 for 1 hour before treatment with vehicle, fenoterol or fenoterol derivatives at the indicated concentrations.
  • the lysate was then mixed with 3 mL of liquid scintillation cocktail (Beckman Coulter, Inc., Brea, Calif.), and radioactivity was measured by liquid scintillation counting using Beckman Coulter LS6000IC Scintillation Counter. Data are shown as CPM incorporated compared to the control cells.
  • liquid scintillation cocktail Beckman Coulter, Inc., Brea, Calif.
  • HepG2 cells were seeded in 96-well plates and grown to confluency. Cells were rinsed in Krebs-HEPES buffer, pH 7.4, pre-incubated for 10 minutes with the buffer, and then 10 ⁇ M (R)-isoproterenol or (R,R′)-fenoterol was added followed by incubation for an additional 10 minutes. The levels of cAMP accumulated in cells were determined and normalized to the amount of protein per well.
  • Cell cycle distributions were performed by flow cytometry on propidium iodide-stained nuclei prepared by the NIM technique.
  • DNA histograms of at least 10,000 cells acquired on a Becton-Dickinson FACScanto II (BD Biosciences, San Jose, Calif.) were deconvoluted using the Multicycle program (Phoenix Flow Systems) for estimates of the percentage of cells in the G0/1, S, and G2+M phases of the cell cycle. Debris and doublets were removed from the analysis by software algorithms.
  • the degree of apoptosis induced by drug treatment was assayed by flow cytometry using the Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) following the standard manufacturer's protocol. Briefly, HepG2 cells (5 ⁇ 10 5 ) were grown on 100-mm dishes for 24 hours followed by treatment with vehicle, (R,R′)-fenoterol, or (R,R′)-MNF, all in serum-free medium.
  • Cells were subsequently harvested after a 24-hour incubation, washed in cold PBS, and resuspended in 100 ⁇ L of 1 ⁇ annexin-binding buffer to maintain a density ⁇ 1 ⁇ 10 6 cells/mL, after which 5 ⁇ L Alexa Fluor® 488 annexin V and 1 ⁇ L 100 ⁇ g/mL propidium iodide were added to the cell suspensions. Cells were then incubated at room temperature for 15 minutes and 400 ⁇ L 1 ⁇ annexin-binding buffer was added followed by gentle mixing. Stained cells were analyzed on a BDFACSCanto II flow cytometer.
  • the visualization of immunoreactive bands was performed using the ECL Plus Western Blotting Detection System (GE Healthcare, NJ) and their quantification was done by volume densitometry using Image J software and normalization to ⁇ -actin.
  • the primary antibody for ⁇ 2-AR was obtained from Enzo Life Sciences, Inc. (Cat. # ADI-905-742-100, Farmingdale, N.Y.); rabbit anti-phospho-Akt (Ser-473), phospho-ERK1/2, total Akt and total ERK2 were from Cell Signaling Technology (Beverly, Mass.), and anti- ⁇ -actin was from Abcam (Cambridge, Mass.).
  • the antibodies were used at a dilution recommended by the manufacturer.
  • Results were expressed as relative to the control value. Studies were performed in at least two to three different culture preparations, and two to three dishes for each test condition were plated in each preparation. Results are expressed as means ⁇ S.E. Student's t-test was used to make statistical comparisons between groups. Analyses were performed using the SigmaPlot Software (Systat Software, Inc. San Jose, Calif.), Graphpad Prism 4 (GraphPad Software, Inc., La Jolla, Calif.) and Microsoft® Office Excel, 2003 (Microsoft Corp., Redmond, Wash.), with p values ⁇ 0.05 considered significant.
  • This example describes a series of studies performed to characterize the ability of fenoterol and disclosed fenoterol analogs to modulate cannabinoid receptor activity.
  • (R,R′)-Fenoterol, (R,R′)-Fen is a potent and selective agonist of the ⁇ 2 -adrenergic receptor ( ⁇ 2 -AR), which has a 43-fold higher affinity for the ⁇ 2 -AR relative to the ⁇ 1 -AR and an EC 50cAMP value of 0.3 nM for the stimulation of cAMP accumulation in HEK cells expressing human ⁇ 2 -AR.
  • the inventors have disclosed the synthesis and characterization of a number of (R,R′)-Fen analogs and stereoisomers with a range of ⁇ 2 -AR selectivity and potency (see, for example International Patent Publication No.
  • ⁇ 2 -ARs associate with heterotrimeric G proteins (e.g., G S , G i ), ion channels and cytosolic scaffold proteins, including ⁇ -arrestin, to initiate various signaling pathways and modulate the activity of intracellular effectors such as adenyl cyclase and mitogen-activated protein kinase (MAPK).
  • G S G S , G i
  • ion channels e.g., G S , G i
  • cytosolic scaffold proteins including ⁇ -arrestin
  • the difference in the G protein and ⁇ -arrestin signaling by ⁇ 2 -AR agonists has been attributed to interaction with ligand-specific GPCR conformations and functional selectivity, which is based upon the assumption that the ⁇ 2 -AR exists in an inactive (R) state and one or more ligand-specific active conformations (R* n ).
  • the basis for the ligand-specific differences in pharmacological outcome lies in the interplay between the molecular structure of the agonist and the cellular environment of the receptor.
  • the inventors have shown that the G S /G i selectivity of Fen is a function of molecular structure and stereochemistry as (R,R′)-Fen preferentially activated G S signaling in a cardiomyocyte contractility model while (S,R′)-Fen and (R,R′)-MNF activated both G S and G i proteins.
  • ⁇ 2 -AR agonists such as (R,R′)-Fen and isoproterenol exert anti-proliferative effects in the human-derived 1321N1 astrocytoma cell line specifically through the cAMP-dependent pathway while Yuan and colleagues ( Oncol Rep 23:151-157, 2010) reported that isoproterenol dose-dependently induced the growth of the human-derived HepG2 hepatocellular carcinoma cell line.
  • the current example describes the effect of the molecular structure and stereochemistry of ⁇ 2 -AR agonists on [ 3 H]-thymidine incorporation in HepG2 cells and to compare these results to similar studies conducted using the 1321N1 and human-derived U87MG glioblastoma cells lines.
  • the initial data demonstrated that (R,R′)-Fen and isoproterenol induced [ 3 H]-thymidine incorporation in HepG2 cells, reduced proliferation in 1321N1 cells and had no effect on U87MG cells and that the effects in the HepG2 and 1321N1 cells could be attenuated by the ⁇ 2 -AR antagonist ICI 118551.
  • the protein levels of the ⁇ 2 -AR were determined by Western blot analysis in total extracts of HepG2 hepatocarcinoma cells, 1321N1 astrocytoma cells, and U87MG glioma cells ( FIG. 1A ).
  • ⁇ 2 -AR protein level was the highest in 1321N1 cells when compared to HepG2 cells.
  • U87MG cells were previously found by the inventors to be devoid of ⁇ 2 -AR at the cell surface.
  • the inventors determined that the incorporation of a naphthyl moiety into the Fen molecule reduced the potency of the resulting compound, but not the inhibitory effect on mitogenesis in 1321N1 cells. Further, the opposite effect was observed as (R,R′)-MNF and 1-naphthylFen inhibited [ 3 H]-thymidine incorporation with IC 50 values of 0.39 ⁇ 0.09 ⁇ M and 0.21 ⁇ 0.07 ⁇ M, respectively (Table 1; FIG. 2B ).
  • ⁇ 2 -AR Antagonism does not Inhibit the Anti-Proliferative Action of (R,R′)-MNF while Preventing (R,R′)-Fen's Growth Promoting Effects in HepG2 Cells.
  • Sub-G 1 events occur when cells have proceeded to the late stage of apoptosis or are already dead.
  • flow cytometry analysis with Annexin V/PI staining was carried out in HepG2 cells.
  • the percentage of apoptotic cells induced by a 24-hour treatment with (R,R′)-MNF (1 ⁇ M) increased 5.7-fold compared to the control (P ⁇ 0.01).
  • (R,R′)-Fen treatment markedly reduced apoptosis when compared to control untreated cells ( FIG. 5 ).
  • AM251 and AM630 are synthetic inverse agonists for CB1R and CB2R, respectively.
  • Cell pretreatment with AM251 or AM630 had no impact on the mitogenic responses of (R,R′)-Fen ( FIG. 6C ), indicating that basal-level activity of these two cannabinoid receptors does not play a major role in the proliferative action of (R,R′)-Fen.
  • NF naphthylfenoterol
  • This example describes a series of studies performed to further characterize the ability of fenoterol and disclosed fenoterol analogs to modulate cannabinoid receptor activity.
  • T1117 is a fluorescent form of the cannabinoid CB1 receptor inverse agonist AM251, which binds GPR55 with high affinity, but has modest binding with CB1 receptors and no interaction with CB2 receptors.
  • HepG2 cells were incubated with fenoterol (1 ⁇ M), MNF (1 ⁇ M) or AM251 (10 ⁇ M) for 1 hour followed by addition of T1117 (0.1 ⁇ M).
  • fenoterol 1 ⁇ M
  • MNF 1 ⁇ M
  • AM251 10 ⁇ M
  • T1117 uptake was dramatically reduced in cells treated with MNF or AM251 prior to T1117 as compared to either vehicle or fenoterol.
  • This example describes the metabolic stability of MNF on human and rat liver microsomes.
  • FIG. 10 presents the data from the in vitro metabolic stability study in which MNF was incubated (in duplicate) with active and heat-inactivated human (HLM) and rat (RLM) liver microsomes and cofactors at 37° C. Aliquots were removed at 0, 15, 30 and 60 minutes and the amount of test compound (MNF) remaining at each time point was determined by LC-MS/MS. Although the results indicated that MNF is somewhat unstable under the incubation conditions, based on the decrease in concentration in the heat-inactivated microsome controls, the results show a greater decrease in MNF levels when incubated with rat and human liver microsomes.
  • HLM active and heat-inactivated human
  • RLM rat
  • FIG. 11 demonstrates the results from the co-incubation of MNF, 1 or 10 ⁇ M, with a cocktail of model CYP substrates, human liver microsomes and CYP cofactors for 20 minutes at 37° C. Incubations containing known CYP inhibitors were also included as positive controls. Formation of the model substrate metabolites was measured by LC-MS/MS and compared to control incubations (incubation of substrates with microsomes and cofactors, no test articles or inhibitors). CYP activity in presence of test compound (MNF) that was less than 70% of control was considered significant. MNF (10 ⁇ M) inhibited CYP2D6 and CYP3A4 to less than 70% of the control.
  • MNF test compound
  • MNF O-demethylated-MNF.
  • the concentrations of MNF and its metabolites in plasma and brain tissue samples obtained from male Sprague-Dawley rats were determined after administration of a single IV dose of 10 mg/kg of MNF.
  • the assays were conducted using an Eclipse XDB-C 18 guard column (4.6 mm ⁇ 12.5 mm) and an Atlantis HILIC column (150 ⁇ 2.1 mm ID, 5 mm).
  • the mobile phase consisted of water containing 0.1% formic acid as Component A and acetonitrile as component B.
  • a linear gradient was run as follows: 0 minutes 95% B; 5 minutes 60% B; 6 minutes 80% B; 10 minutes 95% B at a flow rate of 1.0 ml/minutes.
  • the total run time was 15 minutes per sample. Identification and quantification of the analytes was accomplished using an API-4000 LC-MS/MS in positive electrospray ionization mode and data was acquired employing multiple reaction monitoring (MRM) and the following MRM transitions: MNF(369-200); MNF-Gluc (545-200).
  • MRM multiple reaction monitoring
  • FIG. 12 illustrates the analysis of a plasma sample obtained 30 minutes post-IV administration of 10 mg/kg MNF.
  • MNF and Gluc-MNF are shown with no interfering peaks being present in the control plasma matrix.
  • FIG. 13 illustrates the analysis of brain tissue obtained 30 minutes post-IV administration of 10 mg/kg MNF. The peak at 6.39 minutes is an unidentified compound present in control brain matrix (see insert of FIG. 13 ).
  • FIG. 14 is a MNF time-course in plasma and brain of Sprague-Dawley male rats and the calculated pharmacokinetic parameters are presented in Table 3 below.
  • MNF Ten mg/kg MNF was administered IV to male Sprague-Dawley rats and the plasma concentrations of MNF were determined between 10 minutes and 24 hours post administration with the levels measured in 3 animals per time point.
  • concentration of MNF in brain tissue was measured between 10 and 60 minutes after administration with 3 animals per time point.
  • the MNF concentration in brain tissue was 200 ng/mg tissue at 10 minutes after administration and peaked at 30 minutes at 800 ng/mg tissue.
  • the relative distribution between the concentration of MNF in blood (measured as ng/ml) and brain tissue (measured as ng/mg tissue) was 0.2 at 10 minutes and 1.0 at 30 minutes and 60 minutes reflecting an equivalent distribution between both the central and peripheral body compartments.
  • MNF is capable of passing through the blood brain barrier and that administration of such compound as well as likely other related fenoterol analogues and/or fenoterol by IV is an effective means of delivering these compounds to the brain, such as to treat a brain tumor regulated by CB receptor activity.
  • MNF and MF are capable of inhibiting additional types of cancer growth, including liver, lung and colon cancer.
  • fenoterol analogues such as other naphthylfenoterol analogues
  • NSCLC H460 750 >50 uM 11.59 0.02 2.90 12.78 10
  • NSCLC A549 750 >50 uM 23.14 0.11 3.27 7.96 11
  • Colon C HT29 375 >50 uM 10.71 0.26 5.85 12.46 12
  • Colon C HCT116 375 >50 uM 12.91 0.06 5.75 10.87 13
  • Panel 15 CNS GL261 375 Caliperls N.E.? 21.32 0.17 7.54 4.61 16 CNS LN-18 1125 N.E.?
  • This example demonstrates antitumor activity of MNF in a rat C6 glioma xenograft model in the mouse.
  • MNF methoxy-1-naphthylfenoterol
  • Dulbecco's modified Eagle Medium (DMEM), trypsin solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), 100 ⁇ solution of L-glutamine (200 mM), and penicillin/streptomycin (a mixture of 10,000 units/ml penicillin and 10,000 ⁇ g/ml streptomycin) were obtained from Quality Biological (Gaithersburg, Md., USA).
  • DMEM Dulbecco's modified Eagle Medium
  • PBS phosphate-buffered saline
  • FBS fetal bovine serum
  • penicillin/streptomycin a mixture of 10,000 units/ml penicillin and 10,000 ⁇ g/ml streptomycin
  • the rat-derived C6 glioma cell line was obtained from the American Type Culture Collection (Manassas, Va.). The cells were routinely maintained in DMEM supplemented with L-glutamine, 1% penicillin/streptomycin solution, and 10% FBS in a humidified CO2 incubator at 37° C.
  • C6 glioma cells were seeded in 12-well plates at ⁇ 5 ⁇ 104 cells/well and incubated for 24 hours followed by a second 24-hour incubation with various concentrations of MNF.
  • Radiolabeled thymidine (10 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, Mass.) was added at 1 ⁇ Ci per well for 16 hours and its incorporation into DNA was then measured. Each treatment group was performed in triplicate and three independent studies were carried out.
  • rat C6 glioma cells were trypsin-collected at confluency and were used to generate tumor xenografts.
  • Athymic female nude mice (SWISS nu+/nu+) were obtained from Charles Rivers (L'Arbresle, France) and maintained under pathogen-free conditions with a 12 hours light/12 hours dark cycle. Animals were fed ad libitum with normal chow (supplier).
  • Athymic nude mice were inoculated subcutaneously with 100 ⁇ l of culture medium containing 0.5 ⁇ 106 C6 glioma cells in the left flank and then were randomly divided into two groups of 10 animals each.
  • mice received daily intraperitoneal injection (10 ⁇ l ⁇ g-1 body weight) of vehicle or MNF (2 mg ⁇ kg-1) in 100 ⁇ M ascorbic acid in saline (vehicle) five days a week for 19 days.
  • Animal survival was monitored daily, and tumor size was determined with the use of a caliper to measure the length (a) and width (b) and estimated as 4/3 ⁇ r12 ⁇ r2, where r1 is the smaller and r 2 the larger radius.
  • the mice were monitored up to 19 days after MNF injection or euthanized earlier if the tumor size was superior to 2 cm3 or the mouse was lethargic, sick and unable to feed, which caused the body weight to drop below 20% of initial weight.
  • the mice were euthanized by cervical extension, and tumor masses were removed, weighed and washed with cold PBS before being snap-frozen in liquid nitrogen. A second set of studies with 8-9 animals in both groups was repeated.
  • the accumulation of MNF in vivo in C6 tumor xenografts in athymic mice was assessed in comparison with vehicle-treated tumor-bearing animals.
  • the frozen tumor samples were thawed, and then homogenized.
  • the concentration of MNF and its metabolites was determined by HPLC followed by LC-MS/MS.
  • the assays were conducted using an Eclipse XDB-C18 guard column (4.6 mm ⁇ 12.5 mm) and an Atlantis HILIC column (150 ⁇ 2.1 mm ID, 5 mm).
  • the mobile phase consisted of water containing 0.1% formic acid as component A and acetonitrile as component B.
  • a linear gradient was run as follows: 0 minutes 95% B; 5 minutes 60% B; 6 minutes 80% B; 10 minutes 95% B at a flow rate of 1.0 ml/minute. The total run time was 15 minutes per sample.
  • Identification and quantification of the analytes was accomplished using an API-4000 LC-MS/MS in positive electrospray ionization mode and data was acquired employing multiple reaction monitoring (MRM) and the following MRM transitions: MNF (369-200); MNF-Gluc (545-200). Tumor tissues from vehicle-injected mice were used as controls.
  • MRM multiple reaction monitoring
  • RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules.
  • the double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP.
  • cRNA single-stranded RNA
  • a total of 0.75 ⁇ g of biotin-labeled cRNA was hybridized at 58° C. for 16 hours to Illumina's Sentrix Rat Ref-12 Expression BeadChips.
  • Each BeadChip has ⁇ 22,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy.
  • the arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3.
  • Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using IIlumina's GenomeStudio software, version 1.6.1. For statistical analysis, the expression data were filtered to include only probes with a consistent signal on each chip and an Illumina detecton p value ⁇ 0.02.
  • Gene set enrichment analysis use gene expression values or gene expression change values for all of the genes in the microarray.
  • Parametric analysis of gene set enrichment was used using the WEB-PAGE GSA tool for gene set analysis.
  • Gene Sets include the MSIG database, Gene Ontology Database, GAD human disease and mouse phenotype gene sets were used to explore functional level changes.
  • Gene-gene interaction was also analyzed using the INGENUITY® Pathway Analysis (IPA) system (INGENUITY® Systems).
  • IPA INGENUITY® Pathway Analysis
  • RNA including the DNase treatment step
  • RNA concentration and quality was measured using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Del.).
  • 2 ⁇ g total RNA was reverse-transcribed to cDNA using the qSCRIPTTM cDNA SuperMix (Quanta Biosciences, Gaithersburg, Md.).
  • Quantitative real-time PCR (qRT-PCR) reactions were performed to validate the expression of 6 genes that were selected from the microarray analysis.
  • the reactions were carried out with SYBR® Green PCR master mix on an ABI Prism 7300 sequence detection system (Applied Biosystems) using commercially available target probes for Sox4, Olig1, Galnt3, Cdkn3, Ccna2, and Bub1b (PrimeTime qPCR Assays and Primers, IDT DNA Technologies, Coralville, Iowa).
  • the data was analyzed using the 2- ⁇ Ct method with Gapdh and vehicle-treated tumors as internal controls. Controls consisting of reaction mixture without cDNA were negative in all runs.
  • Frozen tumor tissues were lysed with radioimmune precipitation buffer containing EGTA and EDTA (Boston BioProducts, Ashland, Mass.) supplemented with a phosphatase inhibitor cocktail (EMB-Calbiochem), and protease inhibitor cocktail (Sigma-Aldrich), according to standard protocols. Equal amounts of protein from the clarified lysates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions (Invitrogen, Carlsbad, Calif.), and electrotransferred onto polyvinylidene difluoride membranes using the iBlot system (Invitrogen).
  • Western blots were performed according to standard methods, which involved blocking the membrane in 5% non-fat milk, followed by sequential incubation method with the primary antibody of interest and secondary antibody conjugated with the enzyme horseradish peroxidase.
  • the detection of immunoreactive bands was performed by chemiluminescence using the ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, N.J.). Quantitation of the protein bands was done by volume densitometry using ImageJ software (National Institutes of Health, Bethesda, Md.).
  • MNF Reduces Tumor Growth In Vivo in a Rat C6 Glioma Xenograft Model.
  • a rat C6 glioma xenograft model was developed in immune-deficient mice. Tumor-bearing female nude mice were treated intraperitoneally with MNF daily for 19 days. Significant reduction in tumor volume was observed in MNF-treated animals compared with the vehicle-treated group (P ⁇ 0.008; FIG. 19A ). These studies were performed in a second independent cohort of mice, and showed similar results (see FIG. 19B for combined results). Tumors were excised after the last day of treatment and snap frozen in liquid nitrogen for subsequent analyses.
  • FIG. 20B The intensity of this signature from the two cohorts of animals is represented in FIG. 20B and a partial list of 10 gene sets influenced most by MNF treatment is depicted in Table 9.
  • Table 9 a partial list of 10 gene sets influenced most by MNF treatment is depicted in Table 9.
  • MMP matrix metalloproteinase
  • FIG. 20C Treatment with MNF also sensitized C6 glioma tumor xenograft to growth arrest via the down-regulation of Galnt3 and other cell cycle regulators, such as Ccna2, Cdkn3 and Bub1b ( FIG. 20C ).
  • Ccna2, Cdkn3 and Bub1b FIG. 20C .
  • apoptosis-associated transcripts such as Casp1, Casp11 and Casp12 were upregulated by MNF ( FIG. 20C ).
  • Quantitative RT-PCR analysis confirmed that MNF decreased the expression of Bub1b, Cdkn3 Ccna2, Olig1, Sox4, and Galnt3 as compared to control ( FIG. 20D ), thus validating the microarray data.
  • these results indicate routes by which MNF might negatively affect glioma growth and progression.
  • biomarkers which can be used to determine the efficiency of MNF treatment as well as glioma growth and progression in general.
  • these studies disclose methods of diagnosing, prognosing and determining the efficiency of MNF as well as other treatments of tumor growth and progression in general by using the disclosed biomarkers as indicators.
  • Oligodendrocyte transcription factor 1 (Olig1) has been identified as a novel glioblastoma marker with diagnostic and prognostic value.
  • SRY-box 4 (Sox4) is a transcription factor that has been implicated in the determination of the cell fate and in tumorigenesis.
  • Olig1 and Sox4 mRNA levels were reduced in MNF-treated C6 glioma tumors compared with the control group is consistent with decreased activation of molecular pathways leading to gliomagenesis.
  • the involvement of Cdkn3, Bub1b and Olig-1 in gliomagenesis is established.
  • MNF is readily transported across the blood-brain barrier and can accumulate in the rat brain.
  • This example demonstrates MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and provides a mechanism for its anticancer action in vivo. Also, diagnostic markers and method of determining the efficiency of tumor treatment were revealed.
  • R,R′ -4′-Methoxy-1-naphthylfenoterol
  • CBR cannabinoid receptor
  • AM251 The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis.
  • GPR55 in MNF signaling in human HepG2 and PANC-1 cancer cell lines in culture was investigated, with a focus on internalization of the fluorescent ligand Tocrifluor 1117 (T1117), reorganization of actin cytoskeleton, and cell motility as measured by scratch assay. Results indicated that GPR55 knockdown by RNA interference markedly reduced cellular uptake of T1117, a process that was sensitive to MNF inhibition. GPR55 internalization mediated by the atypical cannabinoid O-1602 was blocked by MNF in GPR55-expressing HEK293 cells.
  • MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251, O-1602 and fetal bovine serum, known to contain bioactive lipids. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including change in cell morphology and migration using scratch wound healing assay. This study shows that MNF impairs GPR55-mediated signaling and has therapeutic potential in the management of cancer by facilitating future research on GPR55.
  • ⁇ 2-adrenoreceptor ( ⁇ 2-AR) agonists have been associated both with a decrease and increase in the mitogenic response of cancer cell lines in culture.
  • the mechanisms that determine cell type-specificity of ⁇ 2-AR-mediated antitumor activity are poorly understood and raise questions about possible biased agonism of the ⁇ 2-AR, whereby the molecular structure and stereochemistry of the agonist and the cellular environment of the receptor stabilizes one or more ligand-specific active conformations of ⁇ 2-AR.
  • the main consequence of biased agonism at the ⁇ 2-AR [and other G protein-coupled receptors] is the activation of multiple G-protein isoforms and modulation of various downstream signal transduction pathways that can lead to dramatic differences in biological outcomes.
  • (R,R′)-4′-methoxy-1-naphthylfenoterol is an analog of fenoterol with a 573-fold greater selectivity for the ⁇ 2-adrenergic receptor ( ⁇ 2-AR) than ⁇ 1-AR. It enhances cAMP accumulation with EC50 value of 3.90 nM in human ⁇ 2-AR-overexpressing cells and attenuates proliferation of 1321N1 astrocytoma cells with IC 50 value of 3.98 nM. In contrast to (R,R′)-fenoterol, MNF activates both G ⁇ s and G ⁇ i proteins and potently stimulates cardiomyocyte contractility, consistent with its role as a ⁇ 2-AR agonist.
  • MNF treatment of the human-derived HepG2 hepatocarcinoma cell line causes growth arrest and apoptosis via a ⁇ 2-AR-independent route.
  • the MNF response was found to be insensitive to the ⁇ 2-AR antagonist ICI 118,551, and U87MG cells, which lack ⁇ 2-AR binding activity, were responsive to the antimitogenic effects of MNF.
  • the presence of the naphthyl moiety in MNF led us to speculate that it may share structural similarities with other ligands and, therefore, behave as a dually acting compound with unique affinity and selectivity profile.
  • Cannabinoid receptors are often co-expressed with ⁇ 2-AR in many tissues and various cell types, and their propensity to heterodimerize demonstrates the potential for crosstalk between the two receptors.
  • CBRs can modulate ⁇ 2-AR activity.
  • the engagement of CBRs by endogenous and synthetic cannabinoid ligands results in the regulation of proliferation and apoptosis of cancer cells, including HepG2 cells. It is interesting that treatment with selective pharmacological inverse agonists of CBRs blocks the antiproliferative actions of MNF in HepG2 cells, consistent with the potential role of CBRs in MNF signaling.
  • rimonabant (SR141716A; N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride), interact with CB1R as inverse agonists, there is growing body of evidence to suggest that they also act as agonists for the recently deorphanized GPR55.
  • GPR55 is a G protein-coupled receptor with lipid-sensing properties whose upregulation contributes to the aggressive behavior of various cancer types. A role for ERK/MAP kinase signaling during microglial activation and the promotion of cancer cell proliferation by GPR55 has been proposed.
  • AM251 also promotes neutrophil chemotaxis by acting as a GPR55 agonist.
  • the actions of AM251 and rimonabant, which have been widely interpreted as being mediated by CB1R, may, in fact, include off-target effects of GPR55 by this class of compounds.
  • the current example was designed to investigate the contribution of GPR55 in MNF actions in two human cancer cell lines in culture, HepG2 and PANC-1 cells.
  • Tocrifluor 1117 T1117
  • a fluorescent ligand that binds to endogenous GPR55 with low affinity for CB1R
  • pharmacodynamic studies were performed and the data indicate that MNF significantly delays T1117 incorporation via impairment in the internalization/recycling of GPR55.
  • Treatment with MNF also resulted in impaired ligand-mediated activation of downstream GPR55 signaling pathways in both tumor cell lines and their cell migration using the wound-healing assay.
  • the present data show that cell exposure to MNF leads to impairment in GPR55 signaling.
  • HepG2 hepatocarcinoma cells and human PANC-1 cells were purchased from ATCC (Manassas, Va., USA). HepG2 cells were maintained in Eagle's minimum essential medium supplemented with 1% L-glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin, and 10% FBS (Hyclone, Logan, Utah, USA). PANC-1 cells were cultured in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L glucose and 1.5 g/L sodium bicarbonate together with glutamine, pyruvate, penicillin/streptomycin and 10% FBS.
  • DMEM Dulbecco's modified Eagle's medium
  • HEK293 cells stably expressing the HA-tagged human GPR55 were a gift of Maria Waldhoer (Medical University of Graz, Graz, Austria) (Henstridge et al., Br. J. Pharmacol. 160: 604-614, 2010).
  • the cells were maintained in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 0.2 mg/ml G418, and penicillin/streptomycin. All cell lines were maintained in culture at 37° C. in 5% CO2, and the medium was replaced every 2-3 days.
  • Cells were grown in 35-mm glass bottom culture dishes (MatTek Corp., Ashland, Mass., USA) for 48 hours until reaching ⁇ 70% confluence. Serum-depleted cells were incubated either with DMSO (vehicle, 0.1%), MNF (1 ⁇ M), or synthetic cannabinoid compounds (AM630, AM251, O-1602, CP 55,940) for 30 minutes prior to the addition of the novel fluorescent diarylpyrazole cannabinoid ligand, Tocrifluor T1117 (10-100 nM). Cells were imaged with a Zeiss LSM 710 confocal microscope equipped with a temperature-controlled and humidified CO2 chamber and a definite focus system.
  • a 561 nm DPSS laser was used for the excitation of the 5-TAMRA conjugate.
  • the time-series function of the Zeiss Zen software was used to collect images with a 40 ⁇ 1.3 NA objective every 30 s for up to one hour, with all confocal settings remaining the same throughout the studies. Still images, movies and fluorescent intensity quantitation were obtained from these series using the Zeiss Zen software. Studies were repeated at least two to three times.
  • HepG2 cells were transfected with siRNA oligos (1.25 ⁇ g) against CB1R, CB2R, or GPR55 or a non-silencing siRNA control (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 48 hours using 10 ⁇ l of siRNA Transfection Reagent (Santa Cruz Biotechnology) following the manufacturer's protocol. siRNAs have been validated to perform efficient knockdown with minimal off-target effects. Following 48 hours of siRNA treatment, cells were washed with PBS, and maintained in serum-free medium before the addition of T1117.
  • hGPR55-HEK293 cells were grown on Lab-Tek II CC2 chamber slides (Thermo Scientific Nunc, Rochester, N.Y.) for 48 hours in regular media and then were serum starved for 1 hour. A pre-incubation with 1:1000 rabbit HA antibody (Covance, Md.) was performed in the presence of vehicle (0.1% DMSO) or 1 ⁇ M MNF in serum-free media for 45 minutes at 37° C. in the CO 2 incubator.
  • the cells were washed in PBS, nuclear counterstaining was performed with DAPI (4′,6-diamidino-2-phenylindole) added to the Prolong Gold antifade mounting medium (Invitrogen) and cured for 24 hours at room temperature in the dark. Images were acquired with a Zeiss LSM 710 confocal microscope (Thornwood, N.Y.) using Carl Zeiss LSM software.
  • Western blots were performed according to standard methods, which involved blocking in 5% non-fat milk and incubated with the antibody of interest, followed by incubation with a secondary antibody conjugated with the enzyme horseradish perodixase.
  • the detection of immunoreactive bands was performed by chemiluminescence using the ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, N.J.).
  • the quantification of bands was done by volume densitometry by using ImageJ software.
  • the rabbit polyclonal antibodies against EGFR were obtained from Cell Signaling Technology, Inc. (Beverly, Mass.) and the monoclonal anti-Hsp90 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
  • Serum-starved cells were pretreated in the absence or presence of 1 ⁇ M MNF for 10 minutes followed by a 10 minute incubation with 0, 2.5 and 10 ⁇ M O-1602 or 10% FBS, after which the levels of total ERK2 and phosphorylated forms of ERK1/2 (pErk1/2, Thr202/Tyr204), were determined by Western blotting technique. All primary antibodies were purchased from Cell Signaling Technology and used at a dilution recommended by the manufacturer.
  • Prism 4 (GraphPad Software, Inc., La Jolla, Calif.) running on a personal computer was used to perform all the statistical data analysis.
  • T1117 acts as a ligand for GPR55 with low affinity for CB1R.
  • serum-depleted cells were maintained on a confocal microscope stage equipped with a temperature-controlled and humidified CO 2 chamber. Studied herein were the characteristics of T1117 incorporation in human HepG2 and PANC-1 cells at 37° C. The amount of cellular T1117 levels increased in a dose-dependent manner, with a maximal incorporation at 100 nM and a half-maximal effect at approximately 8 nM ( FIGS. 21A , 21 B).
  • Additional events downstream of GPR55 internalization may be impaired by cell treatment with MNF, as the redistribution of ligand-bound receptors from the cell surface to endosomal compartment differentially regulates various signaling pathways and their associated biological outcomes.
  • spatio-temporal activation of extracellular signal-regulated kinase (ERK)-MAP kinase plays a role in the dynamic control of complex cellular functions.
  • ERK extracellular signal-regulated kinase
  • MNF pretreatment abrogated O-1602 responsiveness ( FIGS. 25A and 25B ).
  • PANC-1 cells exhibited the same behavior as HepG2 cells, and displayed extraordinar sensitivity to MNF with regard to O-1602-mediated ERK phosphorylation ( FIGS. 25C and 25D ). Similar findings were observed following cell stimulation with AM251 with and without MNF pretreatment.
  • Bioactive concentrations of endocannabinoids are present in fetal bovine serum (250-700 nM of 2-arachidonoylglycerol), which strongly influence monocytes/macrophage responses. As shown in FIGS. 26A-26D , pretreatment with MNF potently inhibited serum-induced ERK phosphorylation in both cell lines.
  • FIGS. 27A and 29A MNF alone had no effect on the motility of HepG2 cells at the concentration used throughout the study (1 ⁇ M). This is in contrast, however, to its significant inhibitory effect toward AM251-mediated increase in cell motility ( FIGS. 27A , 29 A).
  • the relative wound surface area after 24-hour treatment of HepG2 cells with each condition depicted in FIG. 27B Similar to its effects in HepG2 cells, MNF also produced significant decrease in AM251-induced motility of PANC-1 cells, but did not alter the constitutive rate of gap filling ( FIGS. 27C , 27 D and 29 B).
  • a cell type-selective effect was observed in the presence of MNF.
  • the ability of MNF to inhibit the wound closure evoked by O-1602 in PANC-1 cells was absent in HepG2 cells ( FIG. 29C ), indicative of a complex mode of antagonism.
  • MNF displays a number of characteristics associated with selective attenuation in GPR55 signaling, including 1) delayed cellular entry of a fluorescent GPR55 ligand, 2) inhibition of the internalization of the ligand-occupied GPR55, and 3) a significant reduction in GPR55 agonist efficacy with regard to a number of biological readouts.
  • T1117 (5′-TAMRA-PPA-conjugated AM251) suitable for in vivo imaging approaches aimed at assessing occupancy and internalization of GPR55.
  • the compound T1117 has been shown previously to measure the distribution of GPR55 in small mouse arteries.
  • siRNA-based gene silencing method it was determined that GPR55 was a main molecule responsible for T1117 entry in intact cells.
  • human HepG2 cells the presence of GPR55 and the classical CB1R was evidenced by PCR and functional assays.
  • MNF prevents the recruitment of ⁇ -arrestin to the GPR55, thereby providing a negative impact on internalization and recycling of this GPCR after agonist exposure.
  • ⁇ -arrestin is required for activation of downstream signaling (e.g., ERK activation).
  • GPR55 is thought to bind predominantly G-protein G13, where it promotes Rho-dependent signaling in endothelial cells. Additional events downstream of GPR55 include activation of ERK and Ca2+ release from internal stores.
  • MNF may interact with a putative allosteric binding site on GPR55 and elicit negative allosteric modulation of GPR55 agonists. It is noteworthy that an allosteric binding site has been reported at the CB1R. MNF may inhibit signaling downstream of GPR55 to disrupt the binding of T1117, receptor internalization and induction of the signaling cascade leading to ERK activation.
  • This example indicates MNF reduced proliferation and increased apoptosis in human HepG2 hepatocellular carcinoma cells and PANC-1 pancreatic cancer cell line in culture.
  • the role of GPR55 in MNF signaling in HepG2 and PANC-1 cells was investigated with a focus on internalization of the fluorescent ligand Tocrifluor 1117 (T1117), reorganization of actin cytoskeleton, and cell motility as measured by scratch assay. Results indicated that GPR55 knockdown by RNA interference markedly reduced cellular uptake of T1117, a process that was sensitive to MNF inhibition.
  • GPR55 internalization mediated by the atypical cannabinoid O-1602 was blocked by MNF in GPR55-expressing HEK293 cells.
  • MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251, O-1602 and fetal bovine serum, known to contain bioactive lipids. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including change in cell morphology and migration, using scratch-wound healing assay. Thus, these studies show for the first time that MNF impairs GPR55-mediated signaling and has therapeutic use in the management of cancer.
  • This example describes a method that can be used to treat a tumor in a human subject by administration of a composition comprising fenoterol, a fenoterol analogue or a combination thereof at a therapeutically effective amount to reduce or inhibit on or more signs or symptoms associated with the tumor, such as a glioblastoma or hepatocellular carcinoma.
  • a composition comprising fenoterol, a fenoterol analogue or a combination thereof at a therapeutically effective amount to reduce or inhibit on or more signs or symptoms associated with the tumor, such as a glioblastoma or hepatocellular carcinoma.
  • a subject with a glioblastoma or hepatocellular carcinoma is selected based upon clinical symptoms.
  • a biological sample is isolated from the subject and CB receptor expression, including GPR55, and ⁇ 2-AR expression are determined by microarray, Western blotting or histological studies.
  • a positive result indicates that the tumor may be treated by administration of fenoterol, a disclosed fenoterol analogue or a combination thereof.
  • a tissue biopsy is obtained from a subject with a primary brain tumor.
  • Expression of ⁇ 2-AR and GPR55 is determined in the sample.
  • the absence of ⁇ 2-ARs and the presence of GPR55 in the sample indicates that the primary brain tumor can be treated by administration of a composition including (R,R′)-MNF.
  • the composition including the desired compounds is intraperitoneally administered to the subject at a concentration of 30 mg/kg/day for the first 10 days and 50 mg/kg/day for the remaining 32 days. Tumor growth is then assessed 7 days, 14 days, 21 days, 30 days, and 42 days following treatment.
  • the effectiveness of the treatment is determined by imaging methods, including non-invasive, high-resolution modalities, such as computed tomography (CT) and especially magnetic resonance imaging (MRI).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • contrast agent uptake is monitored to determine the effectiveness of the treatment.
  • a decrease in permeability to the blood-brain barrier marked by an at least twenty percent (20%) decrease in uptake of a contrast agent as compared to reference value or that measured prior to treatment indicates the treatment is effective.
  • a twenty-percent (20%) reduction in tumor size as compared to tumor size prior to treatment is considered to be an effective treatment.
  • the therapeutic effectiveness is determined by measuring expression or activity of one or more molecules demonstrated herein to be regulated by MNF (see for example, Table 10).
  • a subject is administered an intravenous formulation of MNF used with a cGMP-produced (R,R′)-4-methoxynaphthylfenoterol (such as a cGMP-produced (R,R′)-4-methoxynaphthylfenoterol 2 Kg formulation).
  • a subject is administered an intravenous formulation of MNF at a concentration ranging from 0.1 to 10 mg/kg for 4 days as a single agent or in combination with other fenoterol analogs or standard agents used in cancer chemotherapy over a two week period as a continuous or pulsed therapy.
  • a subject is administered orally a 25 mg/kg dose of MNF formulated as a single agent or as a combination of (R,R′)-MNF and other MNF stereoisomers or fenoterol stereoisomers on a daily basis for a certain period of time, such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months followed by additional periods if desired, based upon regression of or inhibition of tumor growth.
  • This example describes a method that can be used to reduce, prevent, or retard tumor growth in a human subject that has been treated for a malignant astrocytoma.
  • a subject with an astrocytoma is selected based upon clinical symptoms and determined to have an astrocytoma expressing CB-receptors.
  • the primary form of treatment of the malignant astrocytoma is open surgery.
  • either radiation or chemotherapy is used as the initial treatment.
  • a subject is administered a pharmaceutical composition containing (R,R′)-MNF and/or (R,R′)—NF orally daily for an indefinite period of time.
  • the reoccurrence of tumor growth is monitored by imaging methods, including non-invasive, high-resolution modalities, such as CT and MRI.
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