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WO2000064260A1 - Compositions et procedes pour traiter des maladies hyperproliferatives - Google Patents

Compositions et procedes pour traiter des maladies hyperproliferatives Download PDF

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WO2000064260A1
WO2000064260A1 PCT/US1999/008908 US9908908W WO0064260A1 WO 2000064260 A1 WO2000064260 A1 WO 2000064260A1 US 9908908 W US9908908 W US 9908908W WO 0064260 A1 WO0064260 A1 WO 0064260A1
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agonist
rxr
compound
camp
composition
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PCT/US1999/008908
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English (en)
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Gerard Benoit
Hinrich Gronemeyer
Michel Lanotte
Marco Gottardis
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Bristol-Myers Squibb Company
Institut National De La Sante Et De La Recherche Medicale
Centre National De La Recherche Scientifique
Universite Louis Pasteur
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Application filed by Bristol-Myers Squibb Company, Institut National De La Sante Et De La Recherche Medicale, Centre National De La Recherche Scientifique, Universite Louis Pasteur filed Critical Bristol-Myers Squibb Company
Priority to JP2000613263A priority Critical patent/JP2002542268A/ja
Priority to EP99925558A priority patent/EP1173061A1/fr
Priority to PCT/US1999/008908 priority patent/WO2000064260A1/fr
Priority to CA002369910A priority patent/CA2369910A1/fr
Priority to AU41815/99A priority patent/AU773928B2/en
Publication of WO2000064260A1 publication Critical patent/WO2000064260A1/fr

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    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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 invention relates to compositions comprising a retinoid X receptor agonist and an agent capable of activating protein kinase A.
  • the invention also relates to methods of treating hype ⁇ roliferative diseases by administering a retinoid X receptor agonist and an agent capable of activating protein kinase A.
  • retinoids vitamin A derivatives
  • retinoids are essential for normal growth, vision, tissue homeostasis, reproduction and overall survival (for reviews and references, see Sporn et al., The Retinoids, Nols. 1 and 2, Sporn et al. , eds., Academic Press, Orlando, Florida (1984)).
  • RAR family and RXR family — which belong to the superfamily of ligand-inducible transcriptional regulatory factors that include steroid/thyroid hormone and vitamin D3 receptors (for reviews, see Leid, M., etal., TIBS 77:427-433 (1992); Chambon, ?.,Semin. Cell Biol. 5:115-125 (1994); Chambon, P., FASEB J. 70:940-954 (1996); Giguere, N., Endocrinol. Rev. 75:61-79 (1994); Mangelsdorf, D.J., and Evans, R.M., Cell 55:841-850 (1995); Gronemeyer, H., and Laudet, N., Protein Profile 2:1173-1236 (1995)).
  • Receptors belonging to the retinoic acid receptor family are activated by both all-trans- and 9-C/--.-RA (Leid, M., et al. ,
  • Nuclear receptors are members of a superfamily of ligand-inducible transcriptional regulatory factors that include receptors for steroid hormones, thyroid hormones, vitamin D3 and retinoids (Leid, M., et al, Trends Biochem.
  • NRs exhibit a modular structure which reflects the existence of several autonomous functional domains. Based on amino acid sequence similarity between the chicken estrogen receptor, the human estrogen and glucocorticoid receptors, and the v-erb-A oncogene, Krust,
  • Region A A., et al. (EMBOJ. 5:891-897 (1986)) defined six regions - A, B. C, D, E and F — which display different degrees of evolutionary conservation among various members of the nuclear receptor superfamily.
  • the highly conserved region C contains two zinc fingers and corresponds to the core of the DNA-binding domain (DBD), which is responsible for specific recognition of the cognate response elements.
  • Region E is functionally complex, since in addition to the ligand- binding domain (LBD), it contains a ligand-dependent activation function (AF-2) and a dimerization interface.
  • An autonomous transcriptional activation function (AF-1) is present in the non-conserved N-terminal A/B regions of the steroid receptors.
  • AF-1 and AF-2 of steroid receptors exhibit differential transcriptional activation properties which appear to be both cell type and promoter context specific (Gronemeyer, H., Annu. Rev. Genet. 25:89-123 (1991)).
  • the basis for the highly pleiotropic effect of retinoids may reside, at least in part, in the control of different subsets of retinoid-responsive promoters by cell-specifically expressed heterodimeric combinations of RAR/RXR subtypes (and isoforms), whose activity may be in turn regulated by cell-specific levels of Urans- and 9-cw-RA (Leid, M., et al, TIBS 77:427-433 (1992)).
  • the RXR receptors may also be involved in RA-independent signaling. For example, the observation of aberrant lipid metabolism in the Sertoli cells of
  • RXR ⁇ _/" mutant animals suggests that functional interactions may also occur between RXR ⁇ and the peroxisomal proliferator-activated receptor signaling pathway (WO 94/26100; Kastner, P., et al, Genes & Dev. 70:80-92 (1996)).
  • retinoids are used in a variety of chemopreventive and chemotherapeutic settings.
  • the prevention of oral, skin, head and neck cancers in patients at risk for these tumors has been reported (Hong, W. K., et al, N.
  • Retinoids have also been used to treat squamous cell carcinoma of the cervix and the skin (Nerma, A. K., Cancer Res. 47:5097- 5101 (1987); Lippman S. M., et al. J. Natl Cancer Inst. 54:235-241 (1992); Lippman S. M., et al, J. Natl Cancer Inst. 54:241-245 (1992)) and Kaposi's sarcoma (Bon Subscribe, L., et al, Ann. Oncol.
  • APL Acute Promyelocytic Leukemia
  • a balanced chromosomal translocation t(15;17), has been identified in most acute promyelocytic leukemia (APL) cells (Larson, A.R., et al. , Am. J. Med. 76:827-841 (1984)).
  • the breakpoint for this translocation occurs within the second intron of the RAR gene (Alcalay, M.D., et al, Proc. Natl. Acad. Sci.
  • APL Acute promyelocytic leukemia
  • Tumor progression can be considered as the ability of the malignant cells to leave the primary tumoral site and, after migration through lymphatic or blood vessels, to grow at a distance in host tissue and form a secondary tumor (Fidler, I.J., Cancer Res. 50:6130-6138 (1990); Liotta, L. etal, Cell 64:327-336 (1991)).
  • Progression to metastasis is dependent not only upon transformation but also upon the outcome of a cascade of interactions between the malignant cells and the host cells/tissues. These interactions may reflect molecular modification of synthesis and/or of activity of different gene products both in malignant and host cells.
  • the invention is directed to a method of treating a hype ⁇ roliferative disease in a subject, the method comprising: (a) administering to the subject a pharmaceutically effective amount of a retinoid X receptor (RXR) agonist; and (b) administering to the subject a pharmaceutically effective amount of an agent which is capable of activating protein kinase A (PKA).
  • the method can further comprise (c) administering to the subject a pharmaceutically effective amount of a retinoic acid receptor (RAR) agonist.
  • RAR retinoic acid receptor
  • the method can further comprise (d) administering to the subject a pharmaceutically effective amount of a cytokine, with or without a pharmaceutically effective amount of an RAR agonist.
  • a kit useful for carrying out the method of treating a hype ⁇ roliferative disease is also provided.
  • the invention is directed to a method of inhibiting proliferation of breast cancer cells by administering an RXR agonist and an agent capable of activating protein kinase A.
  • Breast cancer cell lines include, but are not limited to, T47D.
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising (a') a retinoid X receptor (RXR) agonist and (b') an agent capable of activating protein kinase A (PKA).
  • the composition can further comprise (c') a retinoic acid receptor (RAR) agonist.
  • the composition can further comprise (d') a cytokine, with or without an RAR agonist (c').
  • RXR agonists include, but are not limited to, the group consisting of 9-cis retinoic acid, bexarotene, 4-[l-[5,6-Dihydro-3,5,5-trimethyl-8-(l-methylethyl)-2- naphthalenyl]ethenyl]benzoic acid, and SRI 1237.
  • the agent capable of activating PKA can be a PKA agonist.
  • PKA agonists include, but are not limited to, 8-bromo-cAMP, Sp-cAMPS,
  • the agent can be a compound that increases cAMP level, either by stimulating cAMP synthesis or by inhibiting a phosphodiesterase.
  • Compounds which increase cAMP synthesis include, but are not limited to, adenylate cyclase toxin, forskolin, and L-858051.
  • Compounds that act as inhibitors of phosphodiesterases include, but are not limited to, RO 20-1724, Rolipram, Etazolate, and 3-isobutyl-l-methylxanthine (IBMX).
  • RAR agonists include RAR ⁇ , RAR ⁇ and RAR ⁇ agonists.
  • RAR ⁇ agonists include, but are not limited to, 9-cis retinoic acid, all-trans retinoic acid, 4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH-inden-5-yl) carbonyl] amino] benzoic acid, AM-80, and AM-580.
  • Cytokines include, but are not limited to, a granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF).
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • M-CSF macrophage colony-stimulating factor
  • steps (a)-(d) can be done concurrently, or in any order.
  • Hype ⁇ roliferative diseases can be, but are not limited to, cancer and psoriasis. Cancers include, but are not limited to, acute promyelocytic leukemia and breast cancer. The subject can be resistant to treatment with an RAR ⁇ agonist alone.
  • FIG. 1 Differential effects on NB4 cells of RAR and RXR specific agonists: evidence for positive cooperation between the retinoid and protein kinase A signaling pathways. Compounds were used as follows: ATRA , 1 ⁇ M; other retinoid receptor agonists, 0.2 ⁇ M; receptor antagonists, 2 ⁇ M; cell permeable cAMP analogue (8CPT-cAMP), 100 ⁇ M.
  • FIG. la Mo ⁇ hological features of NB4 cells in response to treatments (after 72 h).
  • FIG. lb Nitroblue tetrazolium enzymatic reduction in retinoid-treated NB4 cells (after 72 h; percent of positive cells).
  • FIG. Ic is an example of the retinoid and protein kinase A signaling pathways. Compounds were used as follows: ATRA , 1 ⁇ M; other retinoid receptor agonists, 0.2 ⁇ M; receptor antagonists, 2 ⁇ M; cell permeable cAMP
  • FIG.2 Featuresof4-[l-[5,6-Dihydro-3,5,5-trimethyl-8-(l-methylethyl)- 2-naphthalenyl]ethenyl]benzoic acid (Compound V) as a bi-functional rexinoid.
  • FIG.2a Featuresof4-[l-[5,6-Dihydro-3,5,5-trimethyl-8-(l-methylethyl)- 2-naphthalenyl]ethenyl]benzoic acid
  • Reporter cell assays the RAR and RXR AF-2 agonistic and antagonistic activities of 4-[l -[5,6-Dihydro-3,5,5-trimethyl-8-(l -methylethyl)-2- naphthalenyl]ethenyl]benzoic acid (Compound V) are displayed as false color illustration of the luciferase activity emanating after retinoid induction (from InM, "-9" to l ⁇ M, "-6”) from 96 well plates containing equal amounts of cells (Chen, J. Y. et al, EMBOJ. 74:1187-1197 (1995)). Monitoring and quantitation was done by using a photon-counting camera.
  • the open triangle points to signals in the presence of the retinoid (agonist activity; "-" indicates the signal for vehicle alone), the black triangle points to signals in the presence of both 1 OnM ATRA ("-", only ATRA) and increasing amounts of the retinoid (antagonistic activity, if the ATRA-induced signal decreases).
  • the RXR ⁇ reporter cell line contains also the inactive dnRXR ⁇ AB, which lacks both AF-1 and AF-2, to sequester endogenous RARs that silence RXR in the absence of RAR agonists
  • FIG. 2b Transient transactivation of RXR ⁇ homodimers on a DR1 reporter in presence of 4-[ 1 -[5,6- Dihydro-3,5,5-trimethyl-8-(l-methylethyl)-2-naphthalenyl]ethenyl]benzoic acid (Compound V) (relative to O.l M 9-cis RA, standardized to 100) and of RAR ⁇ -RXR ⁇ heterodimers (relative to 10 nM ATRA, standardized to 100).
  • Compound V (relative to O.l M 9-cis RA, standardized to 100)
  • RAR ⁇ -RXR ⁇ heterodimers respectively to 10 nM ATRA, standardized to 100.
  • FIG. 2c 4-[l-[5,6-Dihydro-3,5,5-trimethyl-8-(l- methylethyl)-2-naphthalenyl]ethenyl]benzoic acid (Compound V) is a highly potent partner of PKA signaling to trigger NB4 cell maturation.
  • FIG.3 Rexinoid-PKA crosstalk induces a cytokine expression program that differs from that induced by ATRA. Modulation of cytokine expression in NB4 cells was assessed by RNAse protection assays. Cells were exposed to the agents displayed at the top for 0, 24 and 48 h (subsequent lanes for each treatment). For comparison the effect of vitamin D3 (“D3”) was studied also.
  • FIG. 3a The intact probes run on the same gel are shown on the left (L32 and GAPDH are the invariant internal controls used for calibration) and the positions of the protected fragments are indicated.
  • FIG. 3b Only the positions of the protected fragments are shown.
  • FIG. 4a Molecular definition of the mutated PML-RAR ⁇ in NB4-R2 cells.
  • NB4-R2 is an ATRA-resistant subclone isolated from bone marrow leukemic cells of a ATRA-resistant patient by a selection with both cAMP and ATRA (Ruchaud, S. et al, Proc. Natl. Acad. Sci. U.S.A. 97:8428-8432 (1994); Duprez, E. et al,
  • the NB4-R2 cells carry the typical t(15;17) translocation and expressed PML-RAR ⁇ mRNA, but not the 120 KD PML-RAR ⁇ chimeric protein (Duprez, E. et al , Oncogene 12:2451 -2459 ( 1996)). Sequencing of the PML-RAR ⁇ cDNA reveals a point mutation generating a stop codon at the position 411 in the retinoid-binding domain of PML-RAR ⁇ (L)
  • FIG. 4b Mo ⁇ hological and functional maturation of NB4-R2 cells in response to synergistic treatment by retinoids and cAMP.
  • NB4-R2 cells were treated with protocols similar to those used for NB4 cells (see description of Fig. 1). Note that cooperation between cAMP and rexinoid signaling allows maturation of the ATRA-resistant cells.
  • FIG. 5 Differentiation of T47D breast cancer cells.
  • FIG. 5a Effect of SRI 1237, with or without 8CPT-cAMP (100 ⁇ M), on lipid accumulation.
  • FIG. 5b Effect of bexarotene, with or without 8CPT-cAMP (100 ⁇ M), on lipid accumulation.
  • RAR ⁇ -independent signaling pathway that induces maturation of both ATRA-sensitive (Lanotte, M. et al, Blood 77: 1080- 1086 ( 1991 )) and ATRA-resistant (Ruchaud, S. et al. , Proc. Natl Acad. Sci. U.S.A. 97 :8428-8432 (1994))
  • APL NB4 cells which is based on the crosstalk between RXR ligand ("rexinoid"; Mukherjee, R. et al, Nature 556:407-410
  • the invention provides an alternative therapy for hype ⁇ roliferative diseases, including APL, particularly in case of ATRA resistance, breast cancer and other hype ⁇ roliferative disorders dependent on protein kinase A activation.
  • This alterative therapy provides a treatment method with a higher therapeutic index (the ratio of efficacy to toxicity), as the known RXR agonists are generally less toxic than RAR agonists.
  • the present invention provides a means to dramatically reduce the incidence of side-reactions.
  • the invention also provides a method of inhibiting proliferation of a breast cancer cell by administering an RXR agonist and an agent capable of activating protein kinase A.
  • a “retinoid” is a compound which binds to one or more of the retinoid receptors (RAR ⁇ , RAR ⁇ , RAR ⁇ , RXR ⁇ , RXR ⁇ and RXR ⁇ ).
  • Compounds are either "RAR retinoids” or “RXR retinoids” depending on their binding characteristics (RAR retinoids bind to one or more RARs; RXR retinoids bind to one or more RXRs (also referred to as “rexinoids”)).
  • RXR and RAR agonists to be used in the methods of the present invention can be, but are not limited to, peptides, carbohydrates, steroids and vitamin derivatives, which may each be natural or synthetic (prepared, for example, using methods of synthetic organic and inorganic chemistry that are well-known in the art).
  • retinoids that are "specific” for a retinoid receptor are intended compounds that only bind to a particular retinoid receptor.
  • retinoids that are “selective” for a retinoid receptor are intended compounds that preferably bind to a particular retinoid receptor over others by a magnitude of approximately fivefold or greater than to other retinoid receptors, preferably eight-fold or greater, more preferably, ten-fold or greater.
  • Standard retinoids known in the art as RAR agonists include the following:
  • RAR ⁇ , ⁇ -selective agonists include, but are not limited to,
  • RAR ⁇ , ⁇ -selective agonists include, but are not limited to,
  • RAR ⁇ agonists include, but are not limited to,
  • RAR agonists include, but are not limited to,
  • RAR ⁇ specific or selective agonists can contain an amide group.
  • RAR ⁇ specific or selective agonists can contain a hydroxyl group or a carbonyl group such as a flavone structure.
  • RAR ⁇ specific or selective agonists can be characterized by the absence of a hydroxy and amide groups.
  • RAR ⁇ specific agonists can be characterized by a dihydronaphthalene nucleus bearing a 2-thienyl group at C8 (see, U.S. Patent No. 5,559,248; Johnson, A.T., et al, J. Med. Chem. 59:5029-5030 (1996)).
  • RXR agonists include, but are not limited to,
  • Additional RXR agonists include, but are not limited to,
  • RAR or RXR agonists include, but are not limited to,
  • RXR agonists with a variety of structures, are disclosed in Boehm, M.F., etal, J. Med. Chem. 55:3146-3155 (1995). Further, a number of retinoids of diverse structure types which are triple RAR agonists, selective RAR ⁇ agonists, selective RAR ⁇ agonists, selective RAR ⁇ , ⁇ agonists, selective RXR agonists and RXR/RAR pan-agonists are described in Sun, SN., et al, Cancer Res. 57:4931-4939 (1997). The invention can also be carried out with the RXR agonist bexarotene, the structure and preparation of which are described in Boehm etal, J. Med. Chem. 57:2930-2941 (1994). Other RXR agonists are also described in, for example, Lehmann et al, Science 255:1944-1946 (1992).
  • RAR and/or RXR agonists include, but are not limited to,
  • RXR agonists that can be used in the invention include, but are not limited to, 9-cis retinoic acid, 4-[l -[5,6-Dihydro-3,5,5-trimethyl-8-(l - methylethyl)-2-naphthalenyl]ethenyl]benzoic acid (Compound V; structure and synthesis provided in U.S. Appl. No. 60/127,976, filed April 6, 1999, titled “Selective Retinoic Acid Analogs" (Atty. Docket: SD128*); and U.S. Appl.
  • RAR ⁇ agonists that can be used in the invention include, but are not limited to, all-trans retinoic acid, 4-[[(2,3-Dihydro-l , 1, 3, 3- tetramethyl-2-oxo-lH-inden-5-yl)carbonyl]amino]benzoic acid (Compound I; structure and synthesis provided in WO 98/47861), AM-80 and AM-580.
  • RAR and RXR agonists suitable for use in the present invention may be prepared by the below-cited methods and others routine to those of ordinary skill in the art.
  • Such agonists can be selected and screened at random, or can be rationally selected or rationally designed using protein modeling techniques.
  • agents such as, but not limited to, peptides, carbohydrates, steroids and vitamin derivatives (e.g., derivatives of retinoic acid) are selected at random and assayed, using direct and indirect methods that are routine in the art, for their ability to bind to a retinoid receptor or a functional retinoid receptor heterodimer.
  • agents can be assayed for RXR or RAR agonist activity.
  • Agents can be rationally selected.
  • an agent is said to be "rationally selected” when the agent is chosen based on the physical structure of a known ligand of a retinoid receptor or a functional homodimeric or heterodimeric retinoid receptor. For example, assaying compounds possessing a retinol-like structure would be considered a rational selection since retinol-like compounds are known to bind to a variety of retinoid receptor heterodimers.
  • RXR and RAR proteins Since highly purified RXR and RAR proteins are now available, X-ray crystallography and NMR-imaging techniques can be used to identify the structure of the ligand binding site present on these proteins and, by extension, that which is specifically present on the retinoid receptors. Utilizing such information, computer modeling systems are now available that allows one to "rationally design" an RXR or RAR agonist capable of binding to such a defined structure (Hodgson, Biotechnology 5: 1245- 1247 ( 1990); Hodgson, Biotechnology 9:609-613 (1991)).
  • an agent is said to be “rationally designed” if it is selected based on a computer model of the ligand binding site of one or more retinoid receptor(s).
  • GAL-RAR ⁇ a luciferase reporter gene driven by a pentamer of the GAL4 recognition sequence ("17m") in front of the ⁇ -globin promoter ((17m)5-GAL- Luc).
  • the RAR ligands thus induce luciferase activity that can be measured in the intact cells using a single-photon-counting camera.
  • This reporter system is insensitive to endogenous receptors which cannot recognize the
  • these synthetic retinoids like RA, have been reported to inhibit the anchorage-independent growth of oncogene-transformed 3T3 cells, while the promoter of the human interleukin-6 (IL-6) gene, whose product is involved in the regulation of hematopoiesis, immune responses and inflammation (Kishimoto, T., et al, Science 255:593-597 (1992)) has been shown to be induced by RA but not by the synthetic dissociating retinoids which repressed its activity.
  • IL-6 human interleukin-6
  • RXR agonists have been identified using cell lines that express a RXR receptor linked to a TREpal-tk reporter gene which is activated by both RAR/RXR heterodimers and RXR homodimers (Lehmann,
  • reporter cell lines that are easily constructed, by methods routine to one of ordinary skill, can be used to distinguish not only the specific RXR or RAR types to which a candidate ligand will bind, but also whether that binding induces an activating (i.e., agonistic) or repressive (i.e., antagonistic) effect.
  • activating i.e., agonistic
  • repressive i.e., antagonistic
  • reporter cell lines comprised the luciferase or thymidine kinase genes as reporters
  • other reporters such as Neo, CAT, ⁇ -galactosidase or Green Fluorescent Protein are well known in the art and can be used in a similar fashion to carry out the present invention.
  • references disclosing reporter plasmids containing a reporter gene and expression vectors encoding a LBD of a nuclear receptor include Meyer etal, Cell 57:433-442 (1989); Meyer etal, EMBOJ. 9(12):3923- 3932 (1990); Tasset et al, Cell 62. 1177-1187 (1990); Gronemeyer, H., and Laudet, V., Protein Profile 2: 1173-1308 (1995); Webster et al, Cell 54: 199-207 (1988); Strahle et al, EMBO J. 7:3389-3395 (1988); Seipel et al, EMBO J. 77:4961-4968 (1992); and Nagpal, S., et al, EMBOJ. 72:2349-2360 (1993).
  • RXR and RAR receptors are apparently structurally similar to other nuclear receptors such as the steroid receptors (as reviewed in Chambon, P., FASEB J. 70:940-954 (1996)), routine assays of this type can be useful in assessing compounds for their agonistic activities on RAR and/or RXR receptors.
  • the effect of a candidate agonist on the binding of the ligand-dependent AF-2 modulator TIFl to a RXR or RAR LBD can be studied using glutathione-S-transferase (GST) interaction assays by tagging the LBDs with GST as described in detail in Le Douarin et al, EMBOJ.
  • GST glutathione-S-transferase
  • transgenic animals e.g., mice, and cell lines, that are altered in their expression of one or more of RAR and RXR receptors can be made as described previously (Krezel, W., et al, Proc. Natl. Acad. Sci. USA
  • the agent which is to be tested will be incubated with one or more of the transgenic cell lines or mice or tissues derived therefrom. The level of binding of the agent is then determined, or the effect the agent has on biological effect or gene expression is monitored, by techniques that are routine to those of ordinary skill.
  • the term "incubate” is defined as contacting the compound or agent under investigation with the appropriate cell or tissue, or administering the agent or compound to the appropriate animal, e.g., transgenic mouse, via any one of the well-known routes of administration including enteral, intravenous, subcutaneous, and intramuscular.
  • RXR and RAR ligands can also be used to determine the agonistic effects of RXR and RAR ligands.
  • certain agonistic retinoids will induce the association of endogenous PML/PML-RAR ⁇ fusion protein with nuclear bodies in cells from APL patients (Dyck, J.A., et al, Cell 76:333-343 (1994); Weis, K., et al, Cell 76:345-356 (1994); Koken. M.H.M., et ⁇ /., E RO J 75:1073-1083 ( 1994)) or in related established cell lines such as NB4 (Lanotte, M., et al. , Blood 77:1080-1086 (1991)).
  • These effects of RXR or RAR agonists can be determined, for example, by various immunological techniques such as immunofluorescent or immunoelectron microscopy, using antibodies specific for
  • RXR or RAR agonists can also be identified by their abilities to induce the in vitro differentiation (maturation) of certain established cell lines such as HL-60 myeloblastic leukemia cells (Nagy, L., et al, Mol Cell. Biol. 75:3540-3551 (1995)), NB4 promyelocytic cells (Lanotte, M., etal, Blood 77:1080-1086 (1991), P19 or F9 embryonic carcinoma cells (Roy, B., et al, Mol. Cell Biol. 75:6481-6487 (1995); Horn, V., et al,
  • Ligand-induced differentiation in these and other cell lines can be determined by assaying ligand-treated or -untreated cells for the expression of a variety of well-known markers of differentiation as generally described in the above references.
  • the candidate antagonists or agonists can be screened by measuring their abilities to induce apoptosis (programmed cell death) in, for example, HL-60 cells (Nagy, L., et al, Mol Cell Biol. 75:3540-3551 (1995)) or P19 cells (Horn, V., et al, FASEB J. 70:1071-1077 (1996)), or in other primary cells or established cell lines.
  • HL-60 cells Nagy, L., et al, Mol Cell Biol. 75:3540-3551 (1995)
  • P19 cells Heorn, V., et al, FASEB J. 70:1071-1077 (1996)
  • Apoptosis is typically assessed by measurement of ligand-induced DNA fragmentation, which is accomplished by methods such as gel electrophoresis (appearance of smaller molecular weight bands), microscopy (changes in plasma membrane mo ⁇ hology such as formation of surface protuberances ("blebbing") or in nuclear mo ⁇ hology such as pycnosis or fragmentation) or expression of the putative apoptosis suppressive protein BCL-2
  • Cyclic adenosine monophosphate is an intracellular mediator of hormone action in prokaryotic and animal cells. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone reso ⁇ tion, and regulation of heart rate and force of heart muscle contraction. Cyclic AMP-dependent protein kinase A (PKA) is found in all animal cells and is thought to account for all the effects of cAMP in most of these cells. In its inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits.
  • the catalytic subunit When each regulatory subunit of PKA has bound two molecules of c AMP, the catalytic subunit is activated and can transfer a high energy phosphate from ATP to the serine or threonine of a substrate protein. Altered PKA expression is implicated in a variety of disorders and diseases including thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease.
  • PKA agonists known in the art include, but are not limited to, 8-bromo-cAMP (8-Bromoadenosine-3',5'-cyclic monophosphate, sodium salt), Sp-cAMPS (Sp-Adenosine-3',5'-cyclic monophosphorothioate), 8CPT-cAMP (8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphate, sodium salt), dibutyryl-cAMP (N 6 ,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate, sodium salt monohydrate), Sp-5,6-DCl-cBiMPS (Sp-5,6- dichloro-l- ⁇ -D-ribofuranosylbenzimidazole-3',5'-monophosphorot
  • the agent which activates PKA can be a compound that increases cAMP level.
  • Cyclic AMP level can be increased by cAMP synthesis.
  • Compounds that increase cAMP synthesis include, but are not limited to, adenylate cyclase toxin, forskolin, and L-858051.
  • Phosphodiesterases (PDEs) degrade intracellular cAMP.
  • an agent which activates PKA can be a compound which blocks degradation and thus inhibits cyclic nucleotide PDE activity.
  • Compounds that inhibit PDE include, but are not limited to, RO 20-1724 (4-(3-Butoxy-4-methyoxybenzyl)-2-imidazolidinone), Rolipram, Etazolate, and IBMX (3-isobutyl-l-methylxanthine).
  • PDE inhibitors are available, for example, from BIOMOL. PA, USA.
  • agents capable of activating PKA that are suitable for use in the present invention can be prepared or known by those of ordinary skill in the art.
  • an agent capable of activating PKA is administered in a pharmaceutically effective amount for treatment of hype ⁇ roliferative diseases.
  • a biological assay for elevation of intracellular cAMP is the transcriptional activation of reporter genes containing a cAMP-responsive element (CRE). The sequence of events is: (1) increased cAMP level,
  • Cytokines are biological response modifiers that coordinate antibody and T cell immune system interactions and amplify immune reactivity (see, Abbas, A.K., et al, CELLULAR AND MOLECULAR IMMUNOLOGY, 2nd ed., 1994). Cytokines include, but are not limited to, monokines synthesized by macrophage and lymphokines produced by activated T lymphocytes and natural killer cells and other growth factors. Monokines include, but are not limited to, interleukin-1, tumor necrosis factor, ⁇ and ⁇ interferon, and colony-stimulating factors.
  • Lymphokines include, but are not limited to, interleukins (e.g., IL-1 and IL-2), gamma interferon, granulocyte-macrophage colony-stimulating factor, and lymphotoxin.
  • Other general growth factors include, but are not limited to, EGF, TGF ⁇ and bFGF.
  • Chemokines are a family of structurally homologous 8 to 10 KD chemotactic cytokines. Chemokines are chemokinetic and chemotactic, stimulating leukocyte movement and directed movement.
  • Granulocyte-macrophage colony-stimulating factor GM-CSF
  • G-CSF granulocyte colony-stimulating factor
  • M-CSF macrophage colony- stimulating factor
  • G-CSF which was shown to enhance differentiation of APL cells induced by ATRA in vitro, combined with ATRA can improve the hematological state in APL patients not previously receiving ATRA therapy (Usuki, K. et al, Intl. J. Hematol. 64:213-219 (1996)).
  • G-CSF was shown to be useful for augmenting susceptibility of APL cells to cell-cycle specific agents (Katayama, N. et al, Am. J. Hematol. 55:31-35 (1998)).
  • cytokines including, but not limited to, G-CSF, GM-CSF and M-CSF can additionally be administered in a pharmaceutically effective amount for treatment of cancers and other hype ⁇ roliferative diseases.
  • Hype ⁇ roliferative diseases include, but are not limited to, cancer, psoriasis, actinic keratosis and lamellar ichthyosis. Cancer cells are invasive and move to adjacent tissues whereas psoriasis and lamellar ichthyosis are noninvasive.
  • Cancer can be oral, skin, head and neck cancers.
  • the cancer can be breast cancer.
  • the cancer can be squamous cell carcinoma of the cervix and the skin, and Kaposi's sarcoma.
  • Skin cancers include, but are not limited to, chronic sunlight damage, migrained basal cell carcinoma syndrome, xeroderma pigmentosum, multiple keratoacanthomas, and cutaneous metastasis of malignant melanoma.
  • the cancer is acute promyelocytic leukemia or breast cancer.
  • Psoriasis include, but are not limited to, psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis and psoriatic arthritis.
  • subject or "patient” as used herein is intended an animal, preferably a mammal, including a human.
  • patient is intended a subject in need of treatment of a hype ⁇ roliferative disease.
  • a pharmaceutically effective amount is intended an amount effective to elicit a cellular response that is clinically significant, without excessive levels of side effects.
  • a pharmaceutical composition of the invention is thus provided comprising one or more RXR agonists and one or more agents capable of activating PKA (such as those described above), and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition can further comprise one or more RAR agonists (RAR ⁇ , RAR ⁇ and/or RAR ⁇ ), and can further comprise one or more cytokines.
  • the pharmaceutical composition can be administered orally, rectally, parenterally, intrasystemically, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is intended, but not limited to, a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • a pharmaceutical composition of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • compositions of the present invention can also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents.
  • adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents.
  • Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
  • Prolonged abso ⁇ tion of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay abso ⁇ tion such as aluminum monostearate and gelatin.
  • agents which delay abso ⁇ tion such as aluminum monostearate and gelatin.
  • the rate of abso ⁇ tion of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form.
  • delayed abso ⁇ tion of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by inco ⁇ orating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules.
  • the active compounds are mixed with at least one item pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) abso ⁇ tion accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetylene glyco
  • compositions of a similar type can also be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees. capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacify ing agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzy
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
  • Compositions for topical administration can be prepared as a dry powder which can be pressurized or non-pressurized.
  • the active ingredients in finely divided form can be used in admixture with a larger- sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 ⁇ m in diameter.
  • suitable inert carriers include sugars such as lactose.
  • at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 ⁇ m.
  • the composition can be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant.
  • a compressed gas such as nitrogen or a liquefied gas propellant.
  • the liquefied propellant medium and indeed the total composition is preferably such that the active ingredients do not dissolve therein to any substantial extent.
  • the pressurized composition can also contain a surface active agent.
  • the surface active agent can be a liquid or solid non-ionic surface active agent or can be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
  • a further form of topical administration is to the eye.
  • the compounds of the present invention can be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compounds are maintained in contact with the ocular surface for a sufficient time period to allow the compounds to penetrate the corneal and internal regions of the eye, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.
  • the pharmaceutically acceptable ophthalmic vehicle can be, for example, an ointment, vegetable oil or an encapsulating material.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the drugs.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the drugs.
  • compositions of the present invention can also be administered in the form of liposomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium.
  • any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to the compounds of the invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell Biol. 74:33 et seq (1976)).
  • kits are useful for carrying out the present invention.
  • the kit can have a carrier means being compartmentalized in close confinement to receive two or more container means therein, having (1) a first container means containing a therapeutically effective amount of a RXR agonist and (2) a second container means containing a therapeutically effective amount of an agent which activates PKA.
  • the kit can have additional container mean(s) containing a therapeutically effective amount of a RAR agonist and/or cytokine.
  • agents of the invention can be determined empirically and can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form.
  • the agents can be administered to a patient in need thereof as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents or composition of the present invention will be decided by the attending physician within the scope of sound medical judgement.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
  • satisfactory results are obtained by oral administration of the compounds at dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day.
  • dosages on the order of from 0.01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0 mg/kg/day can be used.
  • Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.
  • Dosaging can also be arranged in a patient specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art (HPLC is preferred).
  • HPLC is preferred
  • patient dosaging can be adjusted to achieve regular on-going blood levels, as measured by HPLC, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
  • compound is intended a protein, nucleic acid, carbohydrate, lipid or a small molecule.
  • RAR ⁇ agonist Compound I-induced differentiation was abrogated by Compound II or the RAR pan-antagonist (E)-4-[2-[8-(l,l'- Biphenyl]-4-yl)-5,6-dihydro-5,5-dimethyl-2-naphthalenyl]ethenyl]benzoic acid (Compound VI) (Fig. lb, lanes 13, 14; Fig. lc, lane 10).
  • RXR agonists SRI 1237) alone were completely inactive (Fig. la, top panel; Fig. lb, lane 23, Fig. lc, lane 17; see also Kitamura, K. et al, Leukemia
  • the SRI 1237 rexinoid agonist which is devoid of any RAR activity (Chen, J. Y. et al, Nature 552:819-822 (1996); Lehmann, J. M. et al, Science 258: 1944- 1946 ( 1992)) induced full maturation of NB4 cells in the presence of the protein kinase A (PKA) agonist 8CPT-cAMP which on its own did not exhibit any differentiating activity.
  • PKA protein kinase A
  • Mo ⁇ hological changes, CDl lc integrin expression and NBT reduction assays all demonstrated that the RXR-PKA crosstalk induced bonafide granulocytic maturation of NB4 cells as does ATRA (Fig.
  • This retinoid has the rather exceptional characteristics that it acts as strong RXR AF-2 (GAL-RXR ⁇ , Fig.2a) and RXR homodimer (Fig. 2b, lane 3) agonist on 17mer- and DR1 -based reporters, respectively, while it is a pan-antagonist for RAR AF-2s (Fig. 2a), and nearly inactive with RAR-RXR heterodimers on a DR5-based reporter (Fig. 2b, lane 7). Despite being virtually inactive on its own, Compound V did not inhibit, but rather synergized with, ATRA-induced transactivation of such reporters
  • G-CSF expression (lanes 10-12).
  • the factor of synergy was >50-fold, leading to a huge stimulation of G-CSF gene expression, which was undetectable in the non-induced state.
  • ATRA was incapable of stimulating G-CSF expression (lanes 1-3), but induced the expressions of M-CSF (lane 3) and the C-X-C chemokine interferon-inducible protein 10 (IP 10; Fig. 3b, lane 3), which were not seen with SRI 1237 and/or 8CPT-cAMP.
  • GM-CSF expression was selectively triggered by the RXR-PKA crosstalk (Fig.
  • IL8 and MCP1 expression were also induced by ATRA (lanes 2, 3). Notably, some cytokines were induced by SRI 1237 on its own, as the c-kit ligand stem cell factor (SCF), IL8 and MCP1 (Fig. 3a and b, lanes 11, 12). The only other tested condition leading to SCF induction was exposure to vitamin D3 (Fig.
  • NB4-R2 cells Rost, S. et al, Proc. Natl. Acad. Sci. USA
  • telomeres induced the same pattern of cytokine expression in NB4 and NB4-R2 cells, whereas ATRA was unable to induce IP 10, MCP1 or M-CSF expression in the resistant cells, as it did in the parental NB4 cells (Fig. 3). No differentiation of NB4-R2 was seen in presence of the rexinoid or PKA agonists alone, nor in presence of ATRA or 9-cis RA (Fig. 4b).
  • RXR is a promiscuous heterodimerization partner for a great number of nuclear receptors (Mangelsdorf, D.J. et ⁇ /., Cell 55:835-839 (1995); Gronemeyer, H.
  • NB4 cell differentiation can be achieved through two different signaling paradigms, i.e., either RAR ⁇ agonists through RAR ⁇ /RXR heterodimers or through rexinoid-PKA agonist synergism.
  • RAR ⁇ agonists through RAR ⁇ /RXR heterodimers or through rexinoid-PKA agonist synergism.
  • RAR Due to "RXR subordination" the RAR/RXR heterodimer cannot respond to pure RXR agonists like SRI 1237 unless RAR is liganded (Lehmann, J. M. etal, Science 255:1944-1946 (1992); Vivat, V. et al, EMBOJ. 76:5697-5709 (1997); Kurokawa, . etal, Nature 577:528-531 (1994);
  • the present data pave the way towards alternative therapies for APL patients to inhibit the outgrowth of ATRA-resistant pools of leukemic blasts and eliminate such cells from relapsed patients.
  • ATRA augments the expression levels of the receptors for G-CSF (Tkatch, L. S. etal, J. Leukoc. Biol. 57:964-971 (1995)) and GM-CSF (de Gentile, A. etal, Leukemia 5:1758-1762 (1994)
  • G-CSF and GM-CSF are superinduced by the PKA and rexinoid agonists (Fig. 3), and (iii) these cytokines combined with
  • ATRA improve the haematological state of APL patients not previously receiving ATRA therapy (Usuki, K. et al, Int. J Hematol. 64:213-219 (1996)), suggest that a combination therapy using retinoid, rexinoid and PKA agonists (or drugs elevating cAMP levels) can be more efficient than a plain therapy with ATRA and allow a less stringent chemotherapy, thereby reducing the risk of therapy-induced ATRA resistance.
  • Chemotherapy can even become more efficient in G-CSF-inducing conditions, as treatment with recombinant G-CSF of a patient in the third relapse resistant to both cytotoxic drugs and ATRA sensitized the blasts to cell-cycle-dependent agents and led to complete remission (Katayama, N. et al, Am. J. Hematol. 55:31-35 (1998)).
  • Rexinoid-PKA agonist synergism can allow treatment of ATRA-resistant
  • APL patients without involving toxic substances like arsenic trioxide Choen, G.Q. et al, Blood 55:1052-1061 (1996); Soignet, S.L. et al, N Engl. J. Med. 339: 1341-1348 (1998)). It is shown herein that the ATRA resistance of NB4-R2, a cell line derived from a patient that was unsuccessfully treated by subsequent chemo- and ATRA therapy (Lanotte, M. et al, Blood 77:1080-1086 (1991);
  • RAR ⁇ -RXR ⁇ heterodimer activity on a DR5-tk-C AT reporter were assessed by transient RXR transfections. "+”, agonist, "(+)”, weak agonist, "-”, antagonist, "0”, no activity, "nd”, not determined.
  • Retinoids 4-[[(2,3-Dihydro- 1,1,3 ,3-tetramethyl-2-oxo- 1 H-inden-5- yl)carbonyl]amino]benzoic acid (Compoundl; WO 98/47861), 4-[[[5,6-Dihydro- 5,5-dimethyl-8-(3-quinolinyl)-2-naphthalenyl]carbonyl]amino]benzoic acid (Compound II; U.S. Patent No. 5,559,248; U.S. Patent No.
  • the acid was first esterified, followed by activation of the OH group with trifluoromethane sulfonic anhydride (Tf 2 O), then the trifluoromethane sulfonate group was coupled with vinyl tributyl tin to give the desired intermediate.
  • Tf 2 O trifluoromethane sulfonic anhydride
  • NB4 and NB4-R2 cells were cultured as previously (Lanotte, M. et al, Blood 77:1080-1086 (1991)). Essentially, cultures were established with log phase growing cells at concentration (10 5 cell per ml) allowing exponential growth of untreated control cultures over 3 days without medium replenishment. Retinoids (provided by Bristol-Myers Squibb; dissolved in ethanol as stock solution at 10 "3 M) and 8-CPT-cAMP (Sigma; dissolved in saline at 10 "2 M) were added at the indicated concentrations. Cultures were light-protected.
  • Synthetic retinoids were characterized using HeLa cells transfected with the corresponding GAL-RAR chimeras and the cognate
  • CDllc cell surface integrin Flow cytometry analysis of CDllc cell surface integrin.
  • the expression of CDl lc integrin was analyzed by direct immunofluorescence, essentially as described (Ruchaud, S. et al, Proc. Natl. Acad. Sci. USA 97:8428-
  • human cytokine 4 and 5 template sets (45034P; 45035P) were labeled with ⁇ - 32 P uridine triphosphate.
  • RNA (4 ⁇ g) and 6xl0 5 cpm of labeled probes were used for hybridization. After RNAse treatments, the protected probes were resolved on a 5% urea-polyacrylamide-bis-acrylamide gel.
  • PML-RAR ⁇ cDNA sequencing Total RNA from NB4 and NB4-R2 cells was purified and PML-RAR ⁇ specific mRNA was isolated using Dynal magnetic beads (M-280 streptavidin) coupled to a biotinylated PML-RAR ⁇ oligonucleotide (specific for the sequence at the recombination site). This mRNA preparation was used for RT-PCR with oligod(N)6 as primer and AMV reverse transcriptase at 42°C for 60 min.
  • PCR product was gel isolated and cloned into the pCR TM 2.1 AT sequencing vector. Clones were selected and PCR-tested for the presence of the insert. Positive clones were selected, plasmids were purified from these clones and DNA was sequenced with
  • Fixing solution 1.5% glutaraldehyde in PBS.
  • Staining Solution Stock Solution ( 1 mg Nile red and 1 ml acetone were mixed thoroughly and stored, chilled and protected from light).
  • Working Solution 4 ul stock solution was added to 1 ml of 75% glycerol (PBS) followed by briefly vortexing; the dye solution was then briefly degassed by vacuum to remove bubble.
  • PBS glycerol
  • the medium was aspirated off from cells and washed with PBS two times (serum in the medium drained the dye out of the cells). Then the cells were trypsinized and washed with PBS to get rid of trypsin. The cells were spun down and resuspended in 1ml PBS (1-2 x 10 6 /ml). The stock dye was added directly to the cells suspension in PBS with
  • Ethyl 4-ethynyl benzoate Ethyl 4( 1 -tributylstannyl- 1 -trimethyl-silyl)- ethen- 1 -yl)-benzoate ) Preparation of 4[l(5,6-dihydro-3,5,5-trimethyl-8-isopropyl-2- naphthalenyl)ethenyl] -benzoic acid
  • Ethyl 4 [l(5,6,7,8-tetrahydro-3,5,5- Ethyl 4[3,5,5-trimethyl-5,6-dihydro-8- trimethyl-8-oxo-2-naphthalenyl)-ethenyI] isopropyl-2-naphthalenyl)-ethenyl] benzoate benzoate
  • Ethyl 4-[l-(5,6,7,8-tetrahydro-3,5,5-trimethyl-8-oxo-2-naphthalenyI)- ethenyl] benzoate A solution of ethyl 4-[l-(5,6,7,8-tetrahydro-3,5,5-trimethyl-8-oxo-2- naphthalenyl)-2-trimethylsilyl-ethenyl]benzoate (12.0 g, 27.6 mmol) in dichloromethane (900 mL) was treated with trifluoroacetic acid (100 mL) at 0 °C. The mixture was stirred for 18 hours and allowed to reach room temperature.
  • cerium(III) chloride heptahydrate (13.5 g

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Abstract

L'invention concerne des compositions comportant un agoniste du récepteur X des rétinoïdes et un agent capable d'activer la protéine kinase A. L'invention concerne aussi des procédés pour traiter des maladies hyperprolifératives par l'administration d'un agoniste du récepteur X des rétinoïdes et d'un agent capable d'activer la protéine kinase A.
PCT/US1999/008908 1999-04-23 1999-04-23 Compositions et procedes pour traiter des maladies hyperproliferatives WO2000064260A1 (fr)

Priority Applications (5)

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JP2000613263A JP2002542268A (ja) 1999-04-23 1999-04-23 過増殖性疾患の処置組成物および方法
EP99925558A EP1173061A1 (fr) 1999-04-23 1999-04-23 Compositions et procedes pour traiter des maladies hyperproliferatives
PCT/US1999/008908 WO2000064260A1 (fr) 1999-04-23 1999-04-23 Compositions et procedes pour traiter des maladies hyperproliferatives
CA002369910A CA2369910A1 (fr) 1999-04-23 1999-04-23 Compositions et procedes pour traiter des maladies hyperproliferatives
AU41815/99A AU773928B2 (en) 1999-04-23 1999-04-23 Compositions and methods for treatment of hyperproliferative diseases

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WO2001078700A3 (fr) * 2000-04-13 2002-05-30 Frederic Geissmann Compositions et procedes utilises pour moduler la fonction du systeme immunitaire
WO2002045718A1 (fr) * 2000-12-08 2002-06-13 Institut Pasteur De Lille Utilisation de composes actifs capables de moduler la voie intracellulaire declenchee par le recepteur de dp dans des cellules de langerhans
JP2003238406A (ja) * 2001-12-11 2003-08-27 Sankyo Co Ltd 医薬組成物
WO2004056351A1 (fr) * 2002-12-20 2004-07-08 Eisai Co., Ltd. Regulateur d'activite pka
WO2004026319A3 (fr) * 2002-09-17 2004-09-02 Centre Nat Rech Scient Compositions pharmaceutiques utilisees pour le traitement de cancers
WO2004035083A3 (fr) * 2002-10-21 2004-11-18 Medical Res Council Compositions et procedes de therapie
US10231947B2 (en) 2017-01-23 2019-03-19 Arizona Board Of Regents On Behalf Of Arizona State University Isochroman compounds and methods of use thereof
US10238655B2 (en) 2017-01-23 2019-03-26 Arizona Board Of Regents On Behalf Of Arizona State University Dihydroindene and tetrahydronaphthalene compounds
US10238626B2 (en) 2017-01-23 2019-03-26 Arizona Board Of Regents On Behalf Of Arizona State University Therapeutic compounds
US10328040B2 (en) 2014-01-17 2019-06-25 Arizona Board Of Regents On Behalf Of Arizona State University Therapeutic methods

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001078700A3 (fr) * 2000-04-13 2002-05-30 Frederic Geissmann Compositions et procedes utilises pour moduler la fonction du systeme immunitaire
WO2002045718A1 (fr) * 2000-12-08 2002-06-13 Institut Pasteur De Lille Utilisation de composes actifs capables de moduler la voie intracellulaire declenchee par le recepteur de dp dans des cellules de langerhans
JP2003238406A (ja) * 2001-12-11 2003-08-27 Sankyo Co Ltd 医薬組成物
WO2004026319A3 (fr) * 2002-09-17 2004-09-02 Centre Nat Rech Scient Compositions pharmaceutiques utilisees pour le traitement de cancers
WO2004035083A3 (fr) * 2002-10-21 2004-11-18 Medical Res Council Compositions et procedes de therapie
WO2004056351A1 (fr) * 2002-12-20 2004-07-08 Eisai Co., Ltd. Regulateur d'activite pka
US10328040B2 (en) 2014-01-17 2019-06-25 Arizona Board Of Regents On Behalf Of Arizona State University Therapeutic methods
US10231947B2 (en) 2017-01-23 2019-03-19 Arizona Board Of Regents On Behalf Of Arizona State University Isochroman compounds and methods of use thereof
US10238655B2 (en) 2017-01-23 2019-03-26 Arizona Board Of Regents On Behalf Of Arizona State University Dihydroindene and tetrahydronaphthalene compounds
US10238626B2 (en) 2017-01-23 2019-03-26 Arizona Board Of Regents On Behalf Of Arizona State University Therapeutic compounds

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AU4181599A (en) 2000-11-10
EP1173061A1 (fr) 2002-01-23
AU773928B2 (en) 2004-06-10
CA2369910A1 (fr) 2000-11-02

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