JP2002542268A - Compositions and methods for treating hyperproliferative diseases - Google Patents

Abstract

  (57) [Summary] The present invention provides a pharmaceutical composition comprising a retinoid X receptor agonist and an agent capable of activating protein kinase A. The present invention also provides a method for treating a hyperproliferative disease by administering a retinoid X receptor agonist and an agent capable of activating protein kinase A.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a composition comprising a retinoid X receptor (receptor) agonist and a substance capable of activating protein kinase A. The present invention also relates to a method for treating a hyperproliferative disease by administering a retinoid X receptor agonist and a substance capable of activating protein (protein) kinase A.

Related Art Retinoids and Receptors Several studies have revealed that retinoids (vitamin A derivatives) are essential for normal growth, vision, tissue homeostasis, reproduction, and overall survival. (For a review and references, see Sporn et al., The Retin.
oids, Vols. 1 and 2, edited by Sporn et al., Academic Press, Orlando, Florida (1984)
Please refer to).

[0003] Except for those involved in vision (Wald, G. et al., Science 162: 230-239 (1968)), the molecular mechanisms underlying the very diverse actions of retinoids have remained unknown until recently. . Discovery of a nuclear receptor for retinoic acid (RA) (Petkovich et al., Nature 330: 444-
450 (1987); Gigue, et al., Nature 33: 624-629 (1987)) has greatly advanced the understanding of how retinoids exert their pleiotropic effects (Leid
, M. et al., TIBS 17: 427-433 (1992); Linney, E., Current Topics in Dev. Biol.
27: 309-350 (1992)). The effects of the RA signal are indicated by two receptor families belonging to the superfamily of ligand-inducible transcriptional regulators, including the steroid / thyroid hormone and vitamin D3 receptors: the RAR family and the RXR
(See REID, M. et al., TIBS for a review.)
17: 427-433 (1992); Chambon, P., Semin. Cell Biol. 5: 115-125 (1994); Cha.
mbon, P. FASEB J. 10: 940-954 (1996); Giguere, V. Endocrinol. Rev. 15: 6.
1-79 (1994); Mangelsdorf, DJ. And Evans, RM, Cell 83: 841-850 (1995);
See Gronemeyer, H. and Laudet, V. Protein Profile 2: 1173-1236 (1995)).

[0004] Receptors belonging to the retinoic acid receptor family (RARα, β and γ and their isoforms) are activated by both all-trans and 9-cis-RA (Leid, M. et al., TIBS 17: 427-433 (1992); Semin. Cell Bio of Chambon, P.
l. 5: 115-125 (1994); Dolle, P. et al., Mech. Dev. 45: 91-104 (1994).
)). Unlike RAR, members of the retinoid X receptor family (RXRα,
β and γ) are exclusively activated by 9-cis-RA (Chambon, P. Sem).
in. Cell Biol. 5: 115-125 (1994); Dolle (Aksan), P. et al., Mech. Dev. 45:
91-104 (1994); Linney, E. Current Topics in Dev. Biol. 27: 309-350 (1992
); Leid, M. et al., TIBS 17: 427-433 (1992); Kastner et al., In: Vitamin A in Healt.
h and Disease, R. Blomhoff eds., Marcel Dekker, New York (1993)).

[0005] Nuclear receptors (NRs) are members of the superfamily of ligand-inducible transcription factors, including receptors for steroid hormones, thyroid hormones, vitamin D3 and retinoids (Leid, M. et al., Trends Biochem. Sci. 17: 427-433 (1992); Le
id, M. et al., Cell 68: 377-395 (1992); and Linney, E., Curr. Top. Dev. Biol.
27: 309-350 (1992)). NR indicates a modular structure reflecting the presence of several autonomous domains. Based on the amino acid sequence similarity between the chicken estrogen receptor, human estrogen and glucocorticoid receptor, and the v-erb-A oncogene, Krust, A. et al. (EMBO J. 5: 891-897 (1986)) Have determined six regions, A, B, C, D, E and F, that exhibit different degrees of evolutionary conservation among various members of the nuclear receptor superfamily. The highly conserved region C contains two zinc fingers and is responsible for the specific recognition of allogeneic response elements, a DNA binding domain (
DBD). Region E is complex in function because it contains a ligand-dependent activation functional domain (AF-2) and a dimerization interface in addition to the ligand-inducible domain (LBD). Autonomous transcriptional activation functional domain (AF-
1) is present in the non-conserved N-terminal A / B region of the steroid receptor. Interestingly, both steroid receptors AF-1 and AF-2 exhibit differential transcriptional activation properties that appear to be specific for both cell type and promoter context (Gronem
Eyer, H. Annu. Rev. Genet. 25: 89-123 (1991)).

It has been shown that activation of RA-responsive promoters is more likely to occur with RAR / RXR heterodimers than with homodimers (Yu, VC et al., Cell 67:12).
51-1266 (1991); Leid, M. et al., Cell 68: 377-395 (1992b); Durand et al., Cell 71: 7.
3-85 (1992); Nagpal, S. et al., Cell 70: 1007-1019 (1992); Zhang, XK, et al., Natu.
re 355, 441-446 (1992); Kliewer et al., Nature 355: 446-449 (1992); Bugge et al., E
MBO J. 11: 1409-1418 (1992); Marks et al., EMBO J. 11: 1419-1435 (1992); Yu, V.
Biotech. 3: 597-602 (1992); Leid, M. et al., TIBS 17: 427-433 (1
992); Lauret and Stehelin, Curr. Biol. 2: 293-295 (1992); Green, S., Nat.
361: 590-591 (1993)). The RXR portion of these heterodimers has been proposed to be silent in retinoid-induced signaling (Kurosawa, R. et al., Nature 371: 528-531 (1994); Forman, BM et al., Cell 81: 541-). 550 (1995); Man
Gelsdorf, DJ and Evans, RM, Cell 83: 835-850 (1995); Vivat, V. et al., EM.
BO J. 16: 5697-5709 (1997)) have reported conflicting results as far as the ability of RXR to bind the ligand in the heterodimer is described (Kurosawa, R. et al., Nature 3).
71: 528-531 (1994); Chen, J.-Y. et al., Nature 382: 81-822 (1996); Kersten, S.
Biochem. 35: 3816-3824 (1996); Chen, Z. et al., J. Mol. Biol. 275: 55-65 (1
998); Li, C. et al., Proc. Natl. Acad. Sci. USA 94: 2278-2283 (1997). From these and the results of the genetic studies, the RAR / RXR heterodimer is indeed R
It is strongly suggested that it is a functional unit that converts A signal (Chambon, P. Sem
in. Cell Biol. 5: 115-125 (1994); Kastner, P. et al., Cell 83: 859-869 (1995);
Mascrez, B. et al., Development 125: 4691-4707 (1998)). Thus, the basis for the highly pleiotropic effects of retinoids is that the retinoid-responsive promoters differ, at least in part, by a combination of heterodimers of the RAR / RXR subtypes (and isoforms) expressed in a cell-specific manner. It is thought to be in the control of a subset, and the activity of the heterodimer is in turn regulated by cell-specific levels of all-trans and 9-cis-RA (Leid, M. et al., TIBS 17: 427). -433
(1992)).

[0007] RXR receptors may also be involved in RA-independent signaling. For example, the observation of ectopic lipid metabolism in Sertoli cells of RXRβ ^{− / −} mutant animals suggests that a functional interaction may also occur between RXRβ and the peroxisome proliferator-activated receptor signaling pathway. (WO 94/26100; Kastner, P. et al., Genes & Dev. 10: 80-92 (1996)).

Summary of Therapeutic Use of Retinoids : Retinic acid is known to regulate the growth and differentiation potential of several mammalian cell types (Gudas, LJ et al., In: The Retinoids, 2nd ed., Sporn, MB et al., Ne
w York: Raven Pres, pp. 443-520 (1994)), retinoids have been used in a variety of chemopreventive and chemotherapy settings. Prevention of oral, skin, head and neck cancers in patients at high risk of these cancers has been reported (Hong, WK et al.
Engl. J. Med. 315: 1501-1505 (1986); Hong, WK et al., N. Engl. J. Med. 32.
3: 795-801 (1990); Kraemer, KH et al., N. Engl. J. Med. 318: 1633-1637 (1988)
Bollag, W., et al., Ann. Oncol. 3: 513-526 (1992); Chiesa, F., et al., Eur. J. Canc.
er B. Oral Oncol. 28: 97-102 (1992); Costa, A. et al. Cancer Res. 4: Supp. 7,
2032-2037 (1994)). Retinoids are also used for squamous cell carcinoma of the cervix and skin (Verma,
AK Cancer Res. 47: 5097-5101 (1987); Lippman SM et al., J. Natl Cancer.
Inst. 84: 235-241 (1992); Lippman SM et al., J. Natl Cancer Inst. 84: 241-2.
45 (1992)) and Kaposi's sarcoma (Bonhomme, L. et al., Annu. Oncol. 2: 234-235 (1
991)) and has been widely used in the treatment of acute promyelocytic leukemia (Huang, ME et al. Blood 72:56).
7-572 (1988); Castaigne, S. et al. Blood 76: 1704-1709 (1990); Chomienne, C.
Blood 76: 1710-1717 (1990); Chomienne, C. et al., J. Clin. Invest. 88: 2150.
-2154 (1991); Chen Z. et al., Leukemia 5: 288-292 (1991); Lo Coco, F. et al., Blood.
77: 165-1659 (1991); Warrell, RP et al., N. Engl. J. Med. 324: 1385-1393 (1
991); Chomienne, C. et al., FASEB J. 10: 1025-1030 (1996)). Retinoids have also been used to treat hyperproliferative skin disorders such as psoriasis. 13-cis retinoic acid (isotretinoin) is commonly used as a dermatological drug.

Acute promyelocytic leukemia (APL) Balanced chromosomal translocation, t (15 ), in most acute promyelocytic leukemia (APL) cells.
17) has been confirmed (Larson, AR et al., Am. J. Med. 76: 827-841 (1984).
). The breakpoint of this translocation is in the second intron of the RARα gene (Acalay, MD et al.
Natl. Acad. Sci. USA 88: 177-1981 (1991); Chang, KS et al., Leukemia 5
: 200-204 (1991); Chen, S. et al. Blood 78: 2696-2701 (1991) and within two loci of the gene encoding the putative zinc finger transcription factor PML (Goddard, A.
Science 254: 1371-1374 (1991)). This mutual t (15; 17) translocation is associated with PML-RAR co-expressed with PML and RARα in APL cells.
causes the production of alpha fusion proteins (Warrell, RP et al., N. Engl. J. Med. 329: 17
7-189 (1993); Grigna ni, F. et al. Blood 83: 10-25 (1994); Lavau,
C. and Dejean, A., Leukemia 8: 1615-1621 (1994); de The (Axan), H.
FASEB J. 10: 955-960 (1996)). The PML-RARa fusion is clearly responsible for blocking differentiation at the promyelocytic stage. (I) This was observed in almost all APL patients (Warrell, RP et al., N. Engl. J. Med. 329: 177-189).
(1993); Grigna (Aksan) ni, F. et al. Blood 83: 10-25 (1994); Lavau, C. and Dejean, A. Leukemia 8: 1615-1621 (1994)), (ii) U937 or HL
Inhibits bone marrow differentiation when overexpressed in 60 myeloblastic leukemia cells (Grignani,
F. et al., Cell 74: 423-431 (1993)), and (iii) all-trans-retinoic acid (A
Complete remission of leukemia cells by differentiation into mature granulocytes after treatment with TRA) is closely related to the expression of PML-RARa (Warrell, RP et al., N. En.
gl. J. Med. 329: 177-189 (1993); Lo Coco R. et al. Blood 77: 1657-1659 (1991).
Chomienne, C. et al., FASEB J. 10: 1025-1030 (1996)). Multiple tests were performed on cell growth of PML-RARα fusion protein production (Mu, XM et al., Mol. Cell. Biol.
14: 6858-6867 (1994)) and apoptosis (Grignani, F. et al., Cell 74: 423-43).
1 (1993)), AP1 transcriptional repression (Doucas, V. et al., Proc. Natl. Acad. Sci. USA 90:
9345-9349 (1993)), and vitamin D3 signaling (Perez, A. et al., EMBO).
J. 12: 3171-3182 (1993)), but PML-R
The mechanism (s) by which ARα blocks bone marrow cell mutations remains unknown. Consistent with the ectopic nuclear compartmentalization of PML-RARα that assumes the “PML-type” position by RA treatment (Dyck, JA et al., Cell 76: 333-343 (1994)), the currently dominant hypothesis is That PML-RARa has altered transcription properties as compared to PML or RARa and / or may act in a dominant negative manner (Perez, A. et al., EMBO J. 12: 3171). -3182 (1993); de The (
Aksan), H. et al., Cell 66: 675-684 (1991); Kastner, P. et al., EMBO J. 11: 629-.
642 (1992)).

Acute promyelocytic leukemia (APL) is the prototype of cancer that is treated by differentiation therapy with ATRA (Fenaux, P. et al., Semin Oncol. 254: 92-102 (1997)). However, adjuvant chemotherapies required to improve tumor cell remission include, for example, treatment-induced ATR by mutation of the PML-RARa ligand binding domain.
There is an inherent risk of A resistance. Such mutations are in fact recurrent (Imaizumi
, M. et al. Blood 92: 374-382 (1998)) and ATRA resistant cell lines (Ruchaud, S. et al., Proc. Natl. Acad. Sci. USA 91: 8428-8432 (1994); Shao, W. et al. Blood 8
9: 4282-4289 (1997); Kizaki, M. et al., Blood 88: 1824-1833 (1996); Robertson,
KA et al. Blood 80: 1885-1889 (1992); Kitamura, K. et al. Leukemia 11: 1950-195.
6 (1997)).

[0011] The early detection of the primary tumor at the time of breast cancer diagnosis is early and despite its small size (Nystro
em, L. et al. Lancet 341: 973-978 (1993); Fletcher, SW et al., J. Natl. Cancer I.
nst. 85: 1644-1656 (1993)), and concomitant metastases remain the leading cause of death from breast cancer (Frost, P. and Levin, R. Lancet 339: 1458-1461 (1992)).
The first step in transformation, characterized by the escape of malignant cells from normal cell cycle control, is facilitated by the expression of dominant oncogenes and / or deletion of tumor suppressor genes (Hunter, T. and Pines, J. Cell 9: 573-582 (1994)
).

Tumor progression can be thought of as the ability of malignant cells to leave the primary tumor site, migrate through the lymph or blood vessels, then proliferate at distant sites in the host tissue and form a secondary tumor (Fidler , IJ Cancer Res. 50: 6130-6138 (1990); Liotta, L. et al., C.
ell 64: 327-336 (1991)). Progression to metastasis depends not only on transformation but also on the outcome of the interaction cascade between malignant cells and host cells / tissues. These interactions are thought to reflect molecular modifications of the synthesis and / or activity of different gene products in both malignant and host cells. Several genes involved in tumor progression have been identified and found to be involved in cell adhesion, extracellular matrix degradation, immune surveillance, growth factor synthesis and / or angiogenesis (reviewed by Hart
, IR and Saini, A. Lancet 339: 1453-1461 (1992); Ponta, H. et al., BBA.
1198: 1-10 (1994); Curr. Opin. Oncol. Of Bernstein, LR and Liotta, LA.
6: 106-113 (1994); Brattain, MG et al., Curr. Opin. Oncol. 6: 77-81 (1994);
And Fidler, IJ and Ellis, LM, Cell 79: 185-188 (1994)).

However, determining the mechanisms involved in the formation and growth of metastases is still a major challenge in breast cancer research (Rusciano, D. and Burger, MM.
BioEssays 14: 185-194 (1994); Curs. Opin. On of Hoskins K. and Weber, BL.
col. 6: 554-559 (1994)). The process that causes the formation of metastases is complex (Fid
ler, IJ Cancer Res. 50: 6130-6138 (1990); Liotta, L. et al., Cell 64: 327-33.
6 (1991)), and therefore identifying relevant molecular events is very important for selecting the optimal treatment.

SUMMARY OF THE INVENTION In one aspect, the invention is a method of treating a hyperproliferative disease in a subject, comprising:
(A) administering to the subject a pharmaceutically effective amount of a retinoid X receptor (RXR) agonist, and then (b) administering to the subject a pharmaceutically effective amount of a substance capable of activating protein kinase A (PKA). It is directed to a method of treatment characterized by administering an amount. The method may further comprise (c) administering to the subject a pharmaceutically effective amount of a retinoic acid receptor (RAR) agonist. The method can further be characterized in that (d) administering to the subject a pharmaceutically effective amount of the cytokine, with or without a pharmaceutically effective amount of the RAR agonist.

[0015] Also provided are kits useful for performing a method of treating a hyperproliferative disease.

In another aspect, the invention relates to RXR agonists and protein kinase A
The present invention is directed to a method for inhibiting the growth of breast cancer cells by administering a substance capable of activating the same. Breast cancer cell lines include, but are not limited to, T47D.

In another aspect, the present invention is directed to a pharmaceutical composition comprising (a ′) a substance capable of activating retinoid X receptor (RXR) and (b ′) protein kinase A (PKA). Is done. The composition further comprises (c ′) retinoic acid receptor (RA
R) An agonist may be included. The composition may further comprise (d ') a cytokine with or without a RAR agonist (c').

RXR agonists include 9-cis retinoic acid, bexarotene, 4- [1- [5,6
-Dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid, and SR11237.

[0019] The substance capable of activating PKA may be a PKA agonist. P
KA agonists include 8-bromo-cAMP, Sp-cAMPS, 8CPT-cA
MP, dibutyryl-cAMP, Sp-5,6-DCl-cBiMPS, adenylate cyclase toxin, horsecholine, L-858051, and Sp-8-pCP
T-cGMPS includes, but is not limited to. Alternatively, the substance may be a compound that increases cAMP levels either by stimulating cAMP synthesis or by inhibiting phosphodiesterase. Compounds that enhance cAMP synthesis include, but are not limited to, adenylate cyclase toxin (adenylate cyclase toxin), horse choline, and L-858051. Compounds that act as phosphodiesterase inhibitors include RO2
Examples include, but are not limited to, 0-1724, lollipran, etazolate, and 3-isobutyl-1-methylxanthine (IBMX).

RAR agonists include RARα, RARβ and RARγ agonists. Of course, a RAR agonist can be selective or specific for one or more RAR subtypes. RARα agonists include 9-cis retinoic acid, all-trans retinoic acid, 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl) carbonyl] Amino] benzoic acid,
AM-80 and AM-580, but are not limited thereto.

[0021] Cytokines include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) Absent.

In this method, steps (a) to (d) can be performed simultaneously or in any order.

[0023] The hyperproliferative disease can be, but is not limited to, cancer and psoriasis.
Cancers include, but are not limited to, acute promyelocytic leukemia and breast cancer. The subject may be resistant to RARα agonist monotherapy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Herein, ATRA-sensitive (Lanotte, M. et al. Blood 77: 1080-1086 (1
991)) and ATRA resistance (Ruchaud, S. et al., Proc. Natl. Acad. Sci. USA 9
1: 8428-8432 (1994)) is a RARα-independent signaling pathway that induces the maturation of both APL NB4 cells, comprising an RXR ligand (“rexinoid”; Mukherje
e, R. et al., Nature 386: 407-410 (1997)) and a signaling pathway based on crosstalk between protein kinase A signaling is provided. The results show that R can be activated in the absence of a known ligand for the RXR heterodimerization partner.
The presence of the XR-dependent promyelocytic maturation pathway is indicated.

The present invention provides alternative therapies for hyperproliferative diseases including APL, especially breast cancer if ATRA resistant, and other hyperproliferative disorders dependent on protein kinase A activation. This alternative treatment provides a treatment with a higher therapeutic index (ratio of efficacy to toxicity) because known RXR agonists are generally less toxic than RAR agonists. Thus, the present invention provides a means to dramatically reduce the incidence of side effects.

The present invention also provides a method for inhibiting the growth of breast cancer cells by administering an RXR agonist and a substance capable of activating protein kinase A.

The RXR and RAR agonist “retinoids” are retinoid receptors (RARα, RARβ, RARγ, RXR
α, RXRβ and RXRγ). Compounds are either “RAR retinoids” or “RXR retinoids” depending on their binding properties (RAR retinoids bind to one or more RARs, and
Retinoids bind to one or more RXRs (also called "lexinoids"). Retinoids that cause transcription through the receptor are examples of "agonists", but those that do not cause transcription but instead block the transcription caused by other agonists are examples of "antagonists" . The RXR and RAR agonists used in the methods of the invention can be peptides, each of which can be natural or synthetic (eg, prepared using synthetic organic and inorganic chemistry methods known in the art). , Carbohydrates, steroids and vitamin derivatives, but are not limited thereto.

A “specific” retinoid for a retinoid receptor refers to a compound that binds only to a specific retinoid receptor. A retinoid that is “selective” for a retinoid receptor is defined as a retinoid that is at least about 5 times, preferably at least 8 times, more preferably at least 10 times, and more specific than other retinoid receptors. Refers to compounds that bind preferentially to the receptor.

Common retinoids known in the art as RAR agonists include:

Embedded image

Embedded image Is mentioned.

[0030] RARα, β-selective agonists include, but are not limited to,

Embedded image (Takeuchi M. et al., "Brit. J. Haematol." (97: 137-140;
1997)).

RARβ, γ-selective agonists include, but are not limited to,

Embedded image (Shroot B. and Michel S., "J. Amer. Acad. Dermatol."
36: S96-S103, 1997)).

RARγ agonists include, but are not limited to,

Embedded image (Schadendorf D. et al., “Intl. J. Oncol.” (5: 1325-1331)
, 1994)); and

Embedded image (See Swann RT EP 747347).

RAR agonists include, but are not limited to,

Embedded image (Benbrook DM et al., "J. Med. Chem." (40: 3567-3584).
, 1997));

Embedded image (Beard RL et al., "Bioorg. Med. Chem. Lett." (7: 2372-
2378, 1997)); and

Embedded image (Diaz P. et al., "Bioorg. Med. Chem. Lett." (7: 2289-229).
4, 1997)).

In addition, the RARα-specific or selective agonist can contain an amide group. RARγ specific or selective agonists can contain a carbonyl group, such as a hydroxyl group or a flavone structure. RARβ-specific or selective agonists can be characterized by the absence of hydroxy and amide groups. It has been determined that an RARβ-specific agonist can be characterized by a dihydronaphthalene nucleus having a 2-thienyl group at C8 (US Patent No. 5559248; Johnson AT et al., “J. Med. Chem. "(3
9: 5029-5030, 1996)).

[0035] General RXR agonists include, but are not limited to,

Embedded image Is mentioned.

[0036] Additional RXR agonists include, but are not limited to,

Embedded image (U.S. Patent No. 567 from Vuligonda V. and RA Chandraratna)
5033);

Embedded image (See Beard RL et al., WO 97/16422);

Embedded image (See Klaus M. et al., EP728742);

Embedded image (Farmer LJ et al., "Bioorg. Med. Chem. Lett." (7: 2393-).
2398, 1997)); and

Embedded image (Farmer LJ et al., "Bioorg. Med. Chem. Lett." (7: 2747-
2752, 1997)).

[0037] RAR or RXR agonists include, but are not limited to,

Embedded image (See WO 97/26237 by Leblond B.).

Other RXR agonists having various structures are described in Boehm M. F. "J. Med
. Chem. (38: 3146-3155, 1995). In addition, many retinoids of different structural types, such as triple RAR agonists, selective RARα agonists, selective RARβ agonists, selective RARγ agonists, selective RARβ, γ agonists, selective RXR agonists and RX
R / RAR pan-agonists are described in Sun S.A. Y. “Cancer Res.” (57
: 4931-4939, 1997). Also, the present invention provides
It can be performed with the agonist bexarotene, the structure and preparation of which are described in Boehm et al., "J. Med. Chem." (37: 2930-29).
41, 1994). Other RXR agonists are also described, for example, in Lehmann et al., Science, (258: 1944-1946, 1992).

Other candidate RAR and / or RXR agonists include, but are not limited to,

Embedded image

Embedded image (See Bernardon JM EP 722,828);

Embedded image (Chandraratna R. WO 96/11686; and "Drugs of the F.
uture "(22: 249-255, 1997));

Embedded image (See Vuligonda S. et al., U.S. Patent No. 5,599,967);

Embedded image (See Chandraratna RA and M. Teng WO 96/06070);

Embedded image (See Klaus M. and E. Weis EP253302);

Embedded image (See Shroot BV et al., EP210929);

Embedded image

Embedded image (See Johnson AT et al., US Patent No. 5,648,514).

That is, preferred RXR agonists that can be used in the present invention include, but are not limited to, 9-cis-retinoic acid, 4- [1- [5,6-dihydro-3,5,5- Trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid [Compound V; structure and synthesis are described in US Pat. S. Patent application no. 60
/ 127796 (filed on April 6, 1999, name: "selective retinoic acid analog")
(Agent docket number: SD128 ^* ); S. Patent application no. (Applied on April 22, 1999, name: "Selective retinoic acid analog") (described in Attorney Docket No .: SD128a ^* )], SR11237 (structure and synthesis are described in US Patent No. 5552271) ), And bexarotene. RARα agonists that can be used in the present invention include, but are not limited to, all-trans-retinoic acid, 4-[[(2,3-dihydro-1,1,3,3).
3-tetramethyl-2-oxo-1H-inden-5-yl] carbonyl] amino] benzoic acid [compound I; structure and synthesis described in WO 98/47861],
AM-80 and AM-580.

For brevity, the notation (compound number, CAS name and structural formula) shown throughout this disclosure is used. Compound number and CAS name 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl] carbonyl] amino] benzoic acid II. 4-[[[5,6 -Dihydro-5,5-dimethyl-8- (3-quinolinyl) -2-naphthalenyl] carbonyl] amino] benzoic acid III. (E) -3-Chloro-4- [2- (5,6-dihydro-5) , 5-Dimethyl-8-phenyl-2-naphthalenyl) ethenyl] benzoic acid IV.3-Fluoro-4-[[(5,6,7,8-tetrahydro-5,5,8,
8-tetramethyl-2-naphthalenyl) hydroxyacetyl] amino] benzoic acid 4- [1- [5,6-Dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid VI. (E) -4- [2- [8- (1,1′-biphenyl-4-yl) -5,6
-Dihydro-5,5-dimethyl-2-naphthalenyl] ethenyl] benzoic acid

[0042]

Embedded image

Embedded image

Embedded image

Other RAR and RXR agonists suitable for use in the present invention can be prepared according to the methods described below or other conventional methods for those skilled in the art.

Screening Methods Several methods for screening for candidates for RXR and RAR agonists are known in the art, and those skilled in the art will be able to determine if a compound is useful in the present invention. .

[0045] Such agonists can be randomly selected and screened, or can be rationally selected or rationally designed using protein modeling techniques.

For random screening, substances such as peptides, carbohydrates, steroids and vitamin derivatives (eg derivatives of retinoic acid) are selected at random and they are either retinoid receptors or functional retinoid receptor heterodimers. Assays for the ability to bind to, but is not limited to, substances using direct and indirect methods routinely used in the art. Alternatively, agents can be assayed for RXR or RAR agonist activity.

The materials can also be selected rationally. As used herein,
A substance is said to be "reasonably selected" if it is selected based on the physical structure of a known ligand for a retinoid receptor or a functional homodimeric or heterodimeric retinoid receptor. . For example, as retinol-like compounds are known to bind to various retinoid receptor heterodimers, assaying a compound having a retinol-like structure may be a reasonable choice.

[0048] Now that highly purified RXR and RAR proteins are available, ligand binding sites on these proteins, and more specifically, ligand binding sites specifically present on retinoid receptors, are identified. X-ray crystallography and NMR imaging can be used to identify structures. Utilizing such information, computer modeling systems are now available that allow to “rationally design” RXR or RAR agonists that can bind to such defined structures (Hodgson Biotechnology 8: 1245-1247 (1990); Hodgson's Biotechnology.
9: 609-613 (1991)).

As used herein, a substance 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 receptors. .

For example, in Chen. J.-Y. et al., EMBO J. 14: 1187-1197 (1995), it selectively induces the AF-2 activating function present in the RBD of LARβ (βAF-2). Three "reporter" cell lines have been used to characterize several RARα-, RARβ-, or RARγ-specific dissociated synthetic retinoids. These cell lines contain the EF region of RARα, RARβ or RARγ (LBD and AF-2
A DNA-binding domain (GAL-RARα, GAL-RARβ or GAL-RAR, respectively) of a yeast transcriptional activator (GAL4) fused to
γ) and a luciferase reporter gene ((17m) 5-GAL-Luc) driven by a pentamer of the GAL4 recognition sequence (“17m”) in front of the β-globulin promoter. In these cell lines, RAR ligands thus induce luciferase activity in whole cells that can be measured using a single-photon counting camera. This reporter system is insensitive to endogenous receptors that cannot recognize the GAL4 binding site. Using similar screening assays, these synthetic retinoids, such as RA, have been reported to inhibit the fixation-independent growth of oncogene-transformed 3T3 cells, while the products are hematopoietic, immune response, and inflammatory. Promoter of the human interleukin-6 (IL-6) gene involved in E. coli (Kishimoto, T. et al., Science).
258: 593-597 (1992)) has been shown to be induced by RA but not by synthetic dissociated retinoids that suppress its activity.

In a similar manner, using a cell line expressing the RXR receptor linked to the TREpal-tk reporter gene activated by both the RAR / RXR heterodimer and the RXR homodimer, the RXR Agonists have been identified (Lehmann,
JM et al., Science 258: 1944-1946 (1992)). Thus, one of ordinary skill in the art would use reporter cell lines that are readily constructed by routine methods to not only identify the specific RXR or RAR type to which a candidate ligand binds, but also activate that binding (
That is, it is possible to discriminate whether to elicit an effect (as an agonist) or an inhibitory (ie, as an antagonist) effect. Although the reporter cell lines cited above contained the luciferase or thymidine kinase genes as reporters, other reporters such as Neo, CAT, β-galactosidase or green fluorescent protein are also known in the art to practice the present invention. Can be used as well. For example, references disclosing a reporter plasmid containing a reporter gene and an expression vector encoding the nuclear receptor LBD include Me
yer et al., Cell 57: 433-442 (1989); Meyer et al., EMBO J. 9 (12): 3923-3932 (1990);
Tasset et al., Cell 62: 1177-1187 (1990); Gronemeyer, H. and Laudet, V. Prot.
ein Profile 2: 1173-1308 (1995); Webster et al Cell 54: 199-207 (1988); Staeh
le et al., EMBO J. 7: 3389-3395 (1988); Seipel et al., EMBO J. 11: 4961-4968 (1992).
And Nagpal, S. et al., EMBO J. 12: 2349-2360 (1993).

Other routine assays have been used to screen compounds for agonist properties on the function of other nuclear receptors, such as steroid receptors. For example, a transient expression / electrophoretic mobility transfer assay system has been used to examine the effects of synthetic steroids RU486 and R5020 on progesterone and glucocorticoid receptor function, respectively (Meyer, M.-E. et al., EMBO). J. 9: 3923-3932 (1990)). Using a similar assay, tamoxifen has been shown to competitively inhibit estradiol binding of Erap160 to the estrogen receptor, suggesting a mechanism for its growth inhibitory effect in breast cancer (Halachimi, S. et al. Science 264: 1455-1458 (1994)).
The RXR and RAR receptors are apparently similar in structure to other nuclear receptors such as steroid receptors (as reviewed in Chambon, P., FASEB J. 10: 940-954 (1996)). Routine assays of this kind are based on the RAR and / or R
It is useful when evaluating the effect as an agonist on the XR receptor.

As another routine method, the ligand-dependent AF-2 modulator TIF1 R
Glutathione-S-transferase (GST) by labeling the LBD with GST to determine the effect of the candidate agonist on binding to XR or RAR LBD
It can be examined using an interaction assay, which is described in Le Douarin et al., EMBO J.
14: 2020-2033 (1995).

In another screening assay, transgenic animals, such as mice and cell lines in which the expression of one or more RAR and RXR receptors has been modified, are described above (Krezel, W. et al., Proc. Natl. Acad. Sci. USA 93:
9010-9014 (1996)) and can be used to identify agonists of particular members of the RAR / RXR receptor class using the methods described above (WO 94/26100). it can. In such an assay, the substance to be tested is incubated with one or more transgenic cell lines or mice or tissues derived therefrom. Then, by techniques that are routine to those skilled in the art,
The level of binding of the substance is measured or the effect of the substance on biological effects or gene expression is monitored. As used herein,
The term "incubating" refers to contacting the compound or substance under study with appropriate cells or tissues, or exposing the substance or compound to a suitable animal, such as a transgenic mouse, intestinal, intravenous, subcutaneous, and intramuscular. Is defined as being administered via any one of the known routes of administration, including:

Other assays can also be used to determine the effect of RXR and RAR ligand as agonists. For example, retinoids as specific agonists are endogenous PML / PML-RARa fusion protein cells from APL patients (Dyck
Cell 76: 333-343 (1994); Weis, K. et al. Cell 76: 345-356 (1994); Ko.
ken, MHM et al., EMBO J. 13: 1073-1083 (1994)) or association with nucleoli in related established cell lines such as NB4 (Lanotte, M. et al., Blood 77: 1080-1086 (1991)). Is induced. These effects of RXR or RAR agonists may include, for example, PM
It can be examined by various immunization techniques, such as immunofluorescence or immunoelectron microscopy, using antibodies specific for the L, RAR and / or PML-RARa fusion protein. RXR or RAR agonist is used for HL-60 myeloblastic leukemia cells (
Nagy, L. et al., Mol. Cell. Biol. 15: 3540-3551 (1995)), NB4 promyelocytic cells (La
notte, M. et al. Blood 77: 1080-1086 (1991), P19 or F9 embryonal carcinoma cells (Ro
Y, B. et al., Mol. Cell. Biol. 15: 6481-6487 (1995); Horn, V., et al., FASEB J. 10: 1.
071-1077 (1996)), or ras-transformed 3T3 cells (Chen et al., EMBO J. 14:
1187-1197 (1995)) and their ability to induce in vitro differentiation (maturation) of certain established cell lines.

Similarly, candidates for antagonists or agonists include, for example, HL-60 cells (Nagy, L. et al., Mol. Cell. Biol. 15: 3540-3551 (1995)) or P19 cells (
Screen by measuring their ability to induce apoptosis (programmed cell death) in Horn, V. et al., FASEB J. 10: 1071-1077 (1996)), or other primary or established cell lines. be able to. Apoptosis is typically assessed by measuring DNA fragmentation by ligands, which include morphology of the plasma membrane, such as gel electrophoresis (the appearance of low molecular weight bands) and microscopy (formation of surface bumps ("bubbles")). Or changes in nuclear morphology, such as nuclear condensation or fragmentation), or expression of the putative apoptosis inhibitory protein BCL-2 (reduction of apoptotic cells). For general methods and discussion of these assays for RXR and RAR biology, see Nagy, L. et al., Mol. Cell. Biol.
15: 3540-3551 (1995); Horn, V. et al., FASEB J. 10: 1071-1077 (1996). Other methods for assaying ligand-induced apoptosis in primary and established cell lines, such as flow cytometry or particle analysis (emergence of smaller particles with different light scattering and / or DNA content characteristics) are known in the art. Known (Telford, WG et al., J. Immunol. Meth. 172: 1-16 (1994); Ca
mpana, D. et al., Cytometry 18: 68-74 (1994); Sgonc, R. and Wick, G., Int. Ar.
ch. Allergy Immunol. 105: 327-332 (1994); Fraker, PJ et al., Meth. Cell Biol.
46: 57-76 (1995); Meth. Cell Biol. 4 of Sherwood, SW and Schimke, RT.
6: 77-97 (1995); Carbonari, M. et al., Cytometry 22: 161-167 (1995); Mastrangel
o, AJ and Betenbaugh, MJ, Curr. Opin. Biotechnol. 6: 198-202 (1995).
).

Agonist screening can be accomplished by an assay known as “in vivo footprinting” (Mueller, PR and Wold, B., Science).
246: 780-786 (1989); Proc. Natl. Acad. Sc of Garrity, PA and Wold, BJ.
USA 89: 1021-1025 (1992)), which has proven to be useful for the analysis of RA-induced transcription of RARβ2 (Dey, A. et al., Mol. Cell. Biol. 14: 8191). -82
01 (1994)).

Other methods routine in the art for examining the activity of a candidate ligand as an agonist can also be used in practicing the present invention. In performing such assays, one of skill in the art would know which RXR or RAR receptor subtype a substance binds, which particular receptor is used by a given compound, It will be possible to determine whether it is an agonist of the receptor.

The substance that activates protein kinase A Cyclic adenosine monophosphate (cAMP) is an intracellular mediator of hormonal action in prokaryotic and animal cells. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogenolysis, bone resorption, and regulation of heart rate and myocardial contractility. Cyclic AMP
Dependent protein kinase A (PKA) is found in all animal cells and is thought to be responsible for all cAMP effects in most of these cells. In its inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. When each regulatory subunit of PKA is associated with two cAMP molecules, the catalytic subunit is activated and can transfer high-energy phosphate from ATP to the substrate protein serine or threonine. Altered PKA expression has been implicated in a variety of disorders and diseases, including thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease.

When a PKA agonist is administered together with an RXR agonist, NB4 cells (APL
Cell lines) and breast cancer cells. PKA agonists known in the art include 8-bromo-cAMP (8-bromoadenosine-3 ′, 5′-cyclic monophosphate sodium salt), Sp-cAMPS (S
p-adenosine-3 ', 5'-cyclic monophosphorothioate), 8CPT-cA
MP (8- (4-chlorophenylthi) -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-1-β-D-ribofuranosylbenzimidazole-3 ′, 5′-monophosphorothioate), adenylate cyclase toxin (AC toxin), forskolin, L-858051 (7 -With acetyl-7β
-([Gamma] -N-methylpiperazino) -butyrylforskolin.2HCl), and Sp-8-pCPT-cGMPS. Such PKA agonists are described, for example, in BIOMOL, PA.
Available from

Therefore, in another embodiment of the present invention, the substance that activates PKA is c
It may be a compound that increases AMP levels. Cyclic AMP levels can be increased by cAMP synthesis. Compounds that enhance cAMP synthesis include, but are not limited to, adenylate cyclase toxin, horsecholine, and L-858051. Phosphodiesterase (PDE) degrades intracellular cAMP. Thus, the substance that activates PKA may be a compound that inhibits degradation, and thus a compound that inhibits cyclic nucleotide PDE activity. Compounds that inhibit PDE include RO20-1724 (4- (3-butoxy-4-methoxybenzyl) -2-imidazolidinone), rolipram, etazolate, and IB
MX (3-isobutyl-1-methylxanthine), but is not limited thereto. Such PDE inhibitors are described, for example, in B. Pennsylvania, USA.
Available from IOMOL.

Other substances suitable for use in the present invention that can activate PKA include:
It can be manufactured by those skilled in the art or is known.

In the present invention, the substance capable of activating PKA is administered in a pharmaceutically effective amount for treating a hyperproliferative disease.

A biological assay for intracellular cAMP elevation uses a cAMP response element (CRE).
) Is the transcriptional activation of a reporter gene. The sequence of events is (1) increased cAMP levels, (2) activation of PKA and its catalytic subunit phosphorylation of the nuclear translocation CRE-binding protein (CREB) by the catalytic subunit and its activation,
(4) Activation of transcription by binding of CREB to the CRE sequence. Using engineered reporter cells, any substance that increases intracellular cAMP levels can be identified by this method (Brindle, PK and Curmin of Montminy, MR).
r. Op. Genet. Devel. 2: 199-204 (1992); Lee, KA and Masson, N. Biochi.
m. Biophys. Acta 1174: 221-233 (1993)).

[0065] Biological assays to identify other PDE inhibitors are described in Endoc, Conti, M. et al.
rine Reviews 16: 370-389 (1995).

Cytokines Cytokines are biological response regulators that coordinate the interaction of antibodies and the T cell immune system and amplify immunoreactivity (Abbas, AK et al., Cell and Molecular Immunology, 2nd edition, (See 1994). Cytokines include, but are not limited to, monokines synthesized by macrophages 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, α and β interferons, and colony stimulating factor. Lymphokines include, but are not limited to, interleukins (eg, IL-1 and IL-2), gamma interferon, granulocyte-macrophage colony stimulating factor, and lymphotoxin. Other common growth factors include, but are not limited to, EGF, TGFβ and bFGF.

Chemokines are a family of structurally homologous 8-10 KD chemotactic cytokines. Chemokines are chemokinetic and chemotactic, stimulating leukocyte movement and directed movement.

Granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), and macrophage colony stimulating factor (M-CSF)
) Are hematopoietic growth factors that stimulate the proliferation and differentiation of normal myeloid and monocyte-macrophage precursors. GM-CSF enhances the function of differentiated cells such as mature peripheral blood neutrophils and mononuclear phagocytes. Its action is mediated by binding to a GM-CSF-specific membrane receptor.

G-CSF has been shown to promote ATRA-induced APL cell differentiation in vitro, and in combination with ATRA, APL patients who have not previously received ATRA therapy (Usuki, K. et al., Intl. J. Hematol. 64: 213-219 (1996)). G-CS
F has been shown to be useful for enhancing the sensitivity of APL cells to cell cycle specific substances (Katayama, N. et al., Am. J. Hematol. 58: 31-35 (1998)).

In the present invention, cytokines including, but not limited to, G-CSF, GM-CSF and M-CSF are further pharmaceutically useful for treating cancer and other hyperproliferative diseases. It can also be administered in an effective amount.

Hyperproliferative disease "Hyperproliferative disease" refers to diseases caused by rapid cell division. Hyperproliferative disorders include, but are not limited to, cancer, psoriasis, actinic keratosis and ichthyosis. Cancer cells are invasive and migrate to adjacent tissues, whereas psoriasis and lamellar ichthyosis are non-invasive.

The cancer can be oral, skin, head and neck cancer. The cancer can be breast cancer. The cancer can be squamous cell carcinoma of the cervix and skin, and Kaposi's sarcoma. Skin cancers include, but are not limited to, chronic sun damage, nevus basal cell carcinoma syndrome, xeroderma pigmentosum, multiple keratokeratoma pericarp, and skin metastases of malignant melanoma There is no.

Psoriasis includes, but is not limited to, psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis and infectious arthritis.

For other retinoid-related diseases, see generally, “Retinoids: Biology, Chemistry, and Medicine,” Ed. Sporn, MB, et al., Raven Press, Ltd., New York, NY (1994).

As used herein, the phrase “insured person” or “patient”
Refers to animals, including humans, preferably mammals. "Patient" refers to a subject in need of treatment for a hyperproliferative disease.

Formulations and Methods of Administration As used herein, "pharmaceutically effective amount" refers to an amount effective to elicit a clinically significant cellular response without undue side effects.

Thus, the present invention comprises one or more RXR agonists and one or more substances capable of activating PKA, such as those described above, and a pharmaceutically acceptable carrier or excipient. There is provided a pharmaceutical composition of the invention. The pharmaceutical composition can further include one or more RAR agonists (RARα, RARβ and / or RARγ), and can further include one or more cytokines.

The pharmaceutical composition (pharmaceutical composition) may be oral, rectal, parenteral, systemic, vaginal, intraperitoneal, topical (by powder, ointment, drops or transdermal patch), buccal or oral Alternatively, it can be administered as a nasal spray. "Pharmaceutically acceptable carrier" refers to, but is not limited to, non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of compounding aid. Absent. The term “parenteral” as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Pharmaceutical compositions of the invention for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile injectable solutions or solutions immediately prior to use. A sterile powder for reconstitution into a dispersion may be included. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol (glycerol, propylene glycol, polyethylene glycol, etc.)
Carboxymethylcellulose and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by use of a coating material such as lecithin, maintenance of the desired particle size in the case of dispersion, and use of a surfactant.

The compositions of the present invention can also include adjuvants such as, but not limited to, preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenolate sorbate. It may also be desirable to include isotonic substances, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable preparations can be achieved by including time delay agents such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This can be achieved by using a suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the drug in that case depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption 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 microencapsulated substrates of the drug with biodegradable polymers such as polylactide-polyglycolide. Depending on 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 (orthoester) and poly (anhydride). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

Injectable preparations incorporate the sterilizing agent, for example, by filtration through a bacteria-retaining filter, or in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Thereby, it can be sterilized.

[0084] Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such dosage forms, the active compound may be at least one pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) starch, lactose, sucrose, glucose, mannitol And fillers or extenders such as silicic acid, b) binders such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) water retention agents such as glycerol; d) agar-agar. Disintegrants such as calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) dissolution retardants such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g)
Wetting agents such as, for example, cetyl alcohol and glycerol monostearate,
h) adsorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof.

[0085] Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0086] Solid formulations of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical arts. These may optionally include opacifying agents, and may even be a formulation of a composition that selectively releases the active ingredient alone or the active ingredient in a particular part of the intestinal tract, optionally in a delayed manner. Good. Examples of embedding compositions that 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 pharmaceutically acceptable emulsions, solutions, suspensions,
Syrups and elixirs include, but are not limited to.
In addition to the active compounds, liquid dosage forms are, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
Propylene glycol, 1,3-butylene glycol, dimethylformamide,
Water or other solvents, solubilizers, such as oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof. And inert diluents commonly used in the art, such as emulsifiers.

In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.

Suspensions may contain, in addition to the active compounds, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth. Suspending agents such as mixtures can be included.

[0091] Topical administration includes administration to the skin or mucous membranes, including the lung surface and the eyes.
Compositions for topical administration, including for inhalation, can be prepared as a dry powder, which can be pressurized or non-pressurized. In non-pressurized powder compositions, the finely divided form of the active component may be employed in admixture with a larger size pharmaceutically acceptable inert carrier, including, for example, particles having a particle size of up to 100 μm. it can. Suitable inert carriers include sugars such as lactose. Desirably, 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 μm.

Alternatively, the composition may be pressurized and contain a pressurized gas such as nitrogen or a liquefied gas propellant. Preferably, the liquefied injection medium, in fact the entire composition, is such that the active ingredient does not dissolve therein to any substantial extent. The pressurized composition can also include a surfactant. The surfactant may be a liquid or solid non-ionic surfactant, or may be a solid anionic surfactant. Preferably, the solid anionic surfactant is used in the form of a sodium salt.

A further form of topical administration is to the eye. The compounds of the present invention may be used to maintain the compound in contact with the surface of the eye for a sufficient period of time to provide the cornea and internal regions of the eye, such as the anterior chamber, posterior chamber, vitreous, aqueous humor, cornea, iris / ciliary body Can be delivered in a pharmaceutically acceptable ophthalmic vehicle, such as to be able to penetrate the 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 can be prepared by subjecting the compounds of the present invention to cocoa butter, polyethylene, which is solid at room temperature but liquid at body temperature, thus melting the rectum or vaginal cavity to release the drug. Suppositories which can be prepared by mixing with a suitable nonirritating excipient or carrier such as glycols or suppository waxes are preferred.

[0095] The compositions of the present invention can also be administered in the form of liposomes. As is known in the art, 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 compounds in liposome form of the present invention can include, in addition to the compounds of the present invention, stabilizers, preservatives, excipients, and the like. Preferred lipids are both natural and synthetic phospholipids and phosphatidylcholine (lecithin). Methods for forming liposomes are known in the art (see, eg, Prescott eds., Meth. Cell Biol. 14:33 et seq. (1976)
).

Kits Kits are useful for practicing the present invention. The kit is a hermetically sealed (partitioned) transport means (support means) for containing two or more container means therein, the kit comprising (1) a therapeutically effective A vehicle having a first container means containing a significant amount of an RXR agonist and (2) a second container means containing a therapeutically effective amount of a substance activating PKA. Optionally, the kit includes
It can have one or more additional container means containing a therapeutically effective amount of a RAR agonist and / or cytokine.

[0097] If administered by those skilled in the art, various materials, RXR agonists, substances capable of activating PKA, RAR agonists, and effective amount of a cytokine of the present invention can be determined empirically, pure It will be appreciated that the form, or if such a form exists, may be used in the form of a pharmaceutically acceptable salt, ester or prodrug. The substance can be administered to a patient in need thereof in combination with the pharmaceutical composition and one or more pharmaceutically acceptable excipients.
It will be appreciated that when administered to a human patient, the total daily usage of the substances or compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dose level for any particular patient will depend on a variety of factors. The factors are the type and degree of cellular response achieved; the activity of the particular substance or composition used; the particular substance or composition used; age, weight, general health, gender and diet of the patient; , Route of administration, and rate of elimination of the substance; treatment period; drugs used in combination with or concurrently with the particular substance; and factors similar to those known in the medical arts. For example, it is within the skill of the art to start the dose of a substance at a level lower than that required to obtain the desired therapeutic effect and to gradually increase the dose until the desired effect is obtained. is there.

For example, approximately 0.05 to 10 mg / kg / day, preferably 0.1 to 7.
Oral administration of the compound at 5 mg / kg / day, more preferably 0.1 to 2 mg / kg / day once daily, or in 2 to 4 divided doses daily, gives satisfactory results. . Approximately 0.01 to 5 mg / kg / day, preferably 0.05 to 1.0 mg / kg, for parenteral administration, eg, by intravenous infusion or infusion.
Per day, more preferably a dose of 0.1 to 1.0 mg / kg / day can be used. An appropriate daily dose for the patient is therefore approximately 2.5 to 5 orally.
00 mg, preferably 5 to 250 mg, more preferably 5 to 100 mg, or approximately 0.5 to 250 mg, preferably 2.5 to 125 mg intravenously,
More preferably, it is 2.5 to 50 mg.

Administration will be in a patient-specific manner, using routine art-recognized techniques (H
(Preferred by PLC) and can be designed to provide a predetermined blood concentration of the substance. Thus, administration to a patient is approximately 50 to 1000 ng / ml, preferably 150 to 500 ng, as measured by HPLC.
/ Ml can also be adjusted to obtain a constant ongoing blood concentration.

It will be apparent to those skilled in the relevant art that other appropriate changes and modifications can be made to the methods and applications described herein without departing from the scope of the invention or any aspect thereof. It will be readily apparent.

As described herein, “compound” refers to a protein, nucleic acid, carbohydrate, lipid or small molecule.

As described herein, the singular forms (“a” or “an”) refer to one or more unless the context clearly indicates otherwise.

The following examples are provided only to illustrate the present invention and are not to be construed as limiting the invention in any manner.

Examples Example 1 As previously reported (Chen, JY et al., Nature 382: 819-822 (1996)).
, All RARA agonists (ATRA, 9-cis RA, 4-[[(2,3-
Dihydro-1,1,3,3-tetramethyl-2-oxo-1H-indene-5
Yl) carbonyl] amino] benzoic acid (compound I)); the activities of all ligands listed are summarized in Table 1) induces maturation of NB4 cells (FIG. 1a, top row; FIG. 1b, lane 7, 12; FIG. 1c, lanes 5, 9). PML-RA
Consistent with the important role of the Rα and / or unaffected RARα alleles, RARβ-((E) -3-chloro-4- [2- (5,6-dihydro-5,5- Dimethyl-8-phenyl-2-naphthalenyl) ethenyl] benzoic acid (
Compound III)) and RARβγ- (3-fluoro-4 [[(5,6,7,8
-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) hydroxyacetyl] amino] benzoic acid (compound IV))
It did not induce differentiation as assessed by morphological criteria (FIG. 1a, top), NBT reduction (FIG. 1b, lanes 18, 20), and CD11c integrin expression (FIG. 1c, lanes 13, 15). RAR pan-agonist activates NB4 through activation of RARα
Inducing maturation can also be achieved by selecting the RARα selective antagonist 4-[[[5,6-dihydro-5,5-dimethyl-8- (3-quinolinyl) -2-naphthalenyl] carbonyl] amino] benzoic acid (Compound II ) Is 9-cis retinoic acid (9-cis RA)
(Fig. 1b) shows that it dramatically reduced the maturation induced by
, Lane 8; FIG. 1c, lane 6; similar data was obtained for ATRA, but not shown). As expected, the differentiation induced by the RARα agonist Compound I was similar to Compound II or the RAR pan-antagonist (E) -4- [2- [8-
(1,1′-biphenyl] -4-yl) -5,6-dihydro-5,5-dimethyl-2-naphthalenyl] ethenyl] benzoic acid (compound VI) (
1b, lanes 13, 14; FIG. 1c, lane 10). In contrast to the RAR agonist, the RXR agonist (SR11237) alone was completely inactive (FIG. 1a, upper row; FIG. 1b, lane 23; FIG. 1c, lane 17; Kitamura, K. et al., Leuk.
emia 11: 1950-1956 (1997)).

However, surprisingly, the SR11237 rexinoid agonist lacking any RAR activity (Chen, JY et al., Nature 382: 819-822 (2996); Lehmann, JM et al., Science 258: 1944-1946 (1992)). Is expressed in the presence of a protein kinase A (PKA) agonist, 8CPT-cAMP, which did not exhibit any differentiation activity by itself.
Full maturation of NB4 cells was induced. Morphological changes, CD11c integrin expression and NBT reduction assays all showed that RXR-PKA crosstalk truly induced granulocyte maturation of NB4 cells as well as ATRA (FIG. 1a, bottom; FIG. 1b, lane 24). FIG. 1c, lane 18). Importantly, both RARα selective (Compound III) and RAR pan-antagonists (Compound VI; the same results were obtained with other RAR pan-antagonists) whether 9-cis RA or SR11237 were used as RXR agonists Regardless, RXR-P for NB4 maturation
It did not significantly affect KA synergy (FIGS. 1b, c). This excludes any contribution by traces of serum-borne retinoic acid, as well as cross-reactivity of the unrecognized weak RAR agonist SR11237, since RAR agonist-induced differentiation is effectively inhibited by these antagonists. (See above).

It is the RAR / RXR heterodimer whose PKA crosstalk induces differentiation
In order to provide additional evidence that they are not
Id, 4- [1- [5,6-dihydro-3,5,5-trimethyl-8- (1-me
Tylethyl) -2-naphthalenyl] ethenyl] benzoic acid (Compound V; April 6, 1999)
U.S. Patent Application No. 127,976 entitled "Selective Retinoic Acid Analogs"
Agent reference number: SD128^*); And “Selective retinoic acid, filed on April 22, 1999”
U.S. Patent Application No. No. (agent reference number: SD128a ^* )). This retinoid is compatible with 17mer and DR1 reporters.
RXR AF-2 (GAL-RXRα, FIG. 2a) and RXR
Monodimer (FIG. 2b, lane 3) acts as an agonist, but RAR AF-2
Is a pan-antagonist (FIG. 2a) and RAR-RX against the DR5 reporter
Quite an exception, being almost inactive in the R heterodimer (FIG. 2b, lane 7)
Characteristic. Despite being substantially inert by itself, compounds
Product V does not inhibit ATRA-induced transcriptional activation of such reporters, but rather
A synergistic effect was shown (FIG. 2b, lane 8). RAR / RXR heterodimer
Consistent with ineffectiveness in activating NF4, Compound V alone inhibited NB4 cells
No differentiation effect was exhibited (FIG. 2c). However, 8CPT-cAM
In the presence of P, this retinoid induced maturation with similar strength as 9-cis RA (
Figure 2c). Importantly, this activity was either RARα selective (Compound II) or pan-A
This could not be inhibited by
It was also observed in the presence of competent RARα (FIG. 2c).

Without being bound by any theory, based on the foregoing data, there are two different pathways for NB4 cell maturation. One involves the RARα / RXR heterodimer and can be induced by the RARα agonist, while the second does not involve the RARα / RXR heterodimer and crosses between the RXR and PKA agonists. Relies on talk. NB4 by multiplex RNase protection assay
By monitoring cellular cytokine signaling, the two pathways were shown to actually trigger different genetic programs. Most notably, P
G-CSF expression is weakly induced by the KA agonist 8CPT-cAMP (FIG. 3a, lane 9), although SR11237 does not affect G-CSF expression by itself (lanes 10-12), but is expressed by SR11237. Dramatically enhanced (lane 15). The synergistic factor was greater than 50-fold, causing a very strong stimulation of G-CSF gene expression that was undetectable in the uninduced state. In particular, ATRA was unable to stimulate G-CSF expression (lanes 1-3)
Induced the expression of M-CSF (lane 3) and CXC chemokine interferon-inducible protein 10 (IP10; FIG. 3b, lane 3), which was
Not observed with 237 and / or 8CPT-cAMP. GM-CSF
Expression is selectively triggered by RXR-PKA crosstalk (FIG. 3a, lane 15), and SR11237 is also a macrophage inflammatory protein of interleukin 8 (IL8), monocyte chemotactic factor 1 (MCP1), and CC chemokines M
Stimulated 8CPT-cAMP induction of IP1α and MIP1β expression (FIG. 3b)
, Lanes 9, 15). IL8 and MCP1 expression was also induced by ATRA (lanes 2, 3). In particular, c-kit ligand stem cell factor (CSF), IL8
And some cytokines such as MCP1 were induced by SR11237 itself (FIGS. 3a and b, lanes 11, 12). The only other test condition that induced SCF induction was that exposure to vitamin D3 (FIG. 3a, lanes 5, 6) also induced IL8 expression (FIG. 3b, lanes 5,6). CC constitutively expressed in NB4 cells
The cytokine RANTES is inhibited by the PKA agonist. This inhibition was compensated by co-exposure to SR11237 and caused only a transient decline at 24 hours (compare lanes 7-9 with lanes 13-15).

Because two different pathways induce NB4 cell differentiation, we investigated whether ATRA-resistant cells still respond to RXR-PKA crosstalk. In fact, NB4-R2 cells (Ruchaud, S. et al., Proc. Natl. Acad. Sci. USA
91: 8428-8432 (1994)) is ATRA resistant by a Gln ₄₁₁ mutation to the in-phase stop codon in the ligand binding domain of PML-RARa (Figure 4a).
), Arrested by morphological criteria, NBT reduction (FIG. 4b), and CD11c integrin expression, arrested growth in the presence of SR11237 or compound V and 8CPT-cAMP (NB4 cells were inhibited by compound I, NB4
Note that -R2 is not performed), granulocyte maturation occurred. In addition, rexinoid-PKA crosstalk induced the same pattern of cytokine expression in NB4 and NB4-R2 cells, whereas ATRA induced IP10, MCP1 or M-CSF expression in resistant cells as induced in parental NB4 cells. (Figure 3
). ATRA or 9 in the presence of a rexinoid or PKA agonist alone
-No differentiation of NB4-R2 was seen even in the presence of cis RA (Fig. 4b). 8
It is noteworthy that Compound I induced some differentiation of NB4-R2 cells in the presence of CPT-cAMP (lane 15), and that the unmodified RARα allele was PK
It is suggested that it synergizes with A agonists to induce some labeling of differentiation even in the absence of cell morphology changes (Fig. 4b, top).

RXR is a promiscuous heterodimerization partner for many nuclear receptors (
Mangelsdorf, DJ et al. Cell 83: 835-839 (1995); Gronemeyer, H. and Lauda,
V. Protein Profile 2: 1173-1308 (1995); Chambon, P. FASEB J. 10: 940-954
(1996)), NB4 differentiation may be mediated by heterodimers other than RAR / RXR. For this reason, we believe that the RXR agonist SR1123
Thyroid hormone, vitamin D in the presence of either 7 or 8-CPT-cAMP
3 and PPAR agonists were tested. Vitamin D3 is itself and SR1
It was inactive in the presence of 1237, but only cross-talked with 8CPT-cAMP at μM concentrations and caused moderate granulocyte maturation, an effect of SR112
37 were less than 20% of the effects exerted in the presence of the PKA agonist. None of the aforementioned heterodimers efficiently induced NB4 cell differentiation in response to the cognate ligand of the RXR partner when co-exposed to 8CPT-cAMP,
It is unlikely that rexinoids acted through one of the aforementioned heterodimers. This is further supported by a distinctly different genetic program initiated by vitamin D3 and rexinoid-PKA agonist synergy (FIG. 3). The RXR selective pan-antagonist HX531 (Vivat, V. et al.
16: 5697-5709 (1997)) and no cross-talk between the rexinoid antagonist and the PKA pathway, indicating that synergism requires a transcriptionally activated conformation, RXR.

Without being bound by any theory, the differentiation of NB4 cells is achieved with either RARα agonist through two different signaling examples, ie through RARα / RXR heterodimer, or through the synergy of rexinoid-PKA agonists It is concluded that. (I) RAR / RX by “RXR subordination”
R heterodimers cannot respond to pure RXR agonists such as SR11237 unless a ligand is bound to RAR (Lehmann, JM et al.
cience 258: 1944-1946 (1992); Vivat, V. et al., EMBO J. 16: 5697-5709 (1997); K.
urokawa, R. et al., Nature 371: 528-531 (1994); Forman, BM et al., Cell 81: 541-55.
0 (1995)). In addition, compound V is an RXR homodimer agonist and RAR antagonist, which by itself failed to efficiently activate RAR / RXR heterodimers. (Ii) However, together with the PKA agonist, SR11237 and Compound V both efficiently induced NB4 cell differentiation, but no induction was observed alone. RAR and RXR co-expressed in the presence of a PKA agonist allow any of the DR5 reporter SR1123
Note that we have ruled out that 8CPT-cAMP simply eliminates "RXR-dependent" in RAR / RXR heterodimers, since no 7-induced transcriptional activation was also observed. (Iii) Cytokine / chemokine expression analysis confirms that SR11237 and 8CPT-cAMP are not merely alternatives to induce the same signaling pathway as RARα agonists. (Iv) Transgenic mice expressing dominant negative RXR in myeloid cells show a lack of maturation at the promyelocytic stage (Sunaga, S. et al., Br. J. Haematol. 96:19).
-30 (1997)) that RXR is not only a silent heterodimerization partner of RAR, but also plays a very important active transcriptional role for myelopoiesis, either hetero or homodimer. Is shown. (V) NB4 mutant cells that do not respond to ATRA differentiate very efficiently through the retinoid-PKA pathway. This RAR / RXR heterodimer-dependent pathway involves the PKA pathway and the RXR
Whether it works by synergy between homodimers or has not yet been identified
Whether it works with an "RXR tolerant" heterodimer must be established in the future.

Importantly, the data of the present invention suggest a way towards alternative therapies for APL patients to inhibit the growth of the ATRA-resistant pool of leukemic blasts and eliminate such cells from relapsed patients. Open. (I) ATRA uses G-CSF (Tkatch, LS et al., J. Leukoc. Biol. 57: 964-971 (1995)) and GM-CSF (de Gentile, A.
Leukemia 8: 1758-1762 (1994)) to increase the expression level of the receptor, (i.
i) G-CSF and GM-CSF are further induced by PKA and rexinoid agonists (FIG. 3), and (iii) these cytokines
From the finding that in combination with RA, hematologic status of APL patients who have not received ATRA treatment in the past is improved (Usuki, K. et al., Int. J. Hematol. 64: 213-219 (1996)). Retinoids, rexinoids and PKA agonists (or cAM
Combination therapy with drugs that increase P levels) is more effective than simple therapy with ATRA and allows less stringent chemotherapy, suggesting that it reduces the risk of treatment-induced ATRA resistance You. Treatment of the third relapse patient with recombinant G-CSF in patients resistant to both cytotoxic drugs and ATRA sensitized the blasts to cell cycle dependent substances and led to complete remission,
Chemotherapy may be even more effective in G-CSF induced conditions (Katayama, N. et al.
Am. J. Hematol. 58: 31-35 (1998)).

Rexinoid-PKA agonist synergy allows ATRA-resistant APL patients to be treated without toxic substances such as arsenic trioxide (Chen, G. et al.
Q. et al. Blood 88: 1052-1061 (1996); Soignet, SL et al., N. Engl. J. Med. 339: 1.
341-1348 (1998)). ATRA resistance of NB4-R2, a cell line from an unsuccessful patient treated with second-line chemotherapy and ATRA therapy (Lanotte, M. et al.
lood 77: 1080-1086 (1991); Duprez, E. et al., Leukemia 6: 1281-1287 (1992)) show that mutations in the ligand binding domain of PKL-RARα result in LBD deletion resulting in ligand binding. It is thought that it is not possible to do it. NB4
-Even though R2 cells do not respond to ATRA, these cells did not show the side effects normally associated with retinoid therapy in human clinical trials (Miller, V. et al.
A. et al., J. Clin. Oncol. 15: 790-795 (1997))) and PKA agonists provide effective maturation. The PKL-RARa LBD mutation is a major cause identified for ATRA resistance in patients and cell lines (Imaizumi, M. et al. Blood 92: 34-382 (1998); Shao, W. et al. Blood 89: 4282). -43289 (1997); Kitamu
ra, K. et al., Leukemia 11: 1950-1956 (1997)), it is believed that these resistant cells can differentiate similarly to NB4-R2 cells upon treatment with rexinoids and PKA agonists. Toward this end, by stimulating cAMP synthesis or inhibiting phosphodiesterase, the stereoisomer of cAMP, Sp-
By applying synthetic cell penetrating PKA agonists such as cAMPS, or by planning tumor cell targeted liposome delivery drugs, we tested novel rexinoid agonists and modes of increasing intracellular cAMP levels for clinical use. There must be.

[0113]

[Table 1] For the transcriptional activity of the synthetic retinoid, R was determined using a reporter cell.
The AF2 function of ARα, β, γ and RXRα was determined (FIG. 2a, and Chen
J. Y. "EMBO J." (14: 1187-1197, 1995)
reference). RXRα homodimer activity on DR1-tk-CAT or DR5
RARα-RXRα heterodimer activity on the tk-CAT reporter
Evaluated by transient RXR transfection. “+” Is agonist, “(+)”
Indicates a weak agonist, "-" indicates an antagonist, "0" indicates no activity, and "nd" is not determined.

Materials and Methods Retinoids: 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-
1H-Inden-5-yl) carbonyl] amino] benzoic acid (Compound I; WO9
8/47861), 4-[[[5,6-dihydro-5,5-dimethyl-8- (3
-Quinolinyl) -2-naphthalenyl] carbonyl] amino] benzoic acid (compound I
I; U. S. Patent No. 5559248, U.S.A. S. Patent No. 5849923),
(E) -3-chloro-4- [2- (5,6-dihydro-5,5-dimethyl-8-
Phenyl-2-naphthalenyl) ethenyl] benzoic acid (Compound III; US Patent No. 5618839), 3-fluoro-4-[[(5,6,7,8-tetrahydro-5,5,8,8 -Tetramethyl-2-naphthalenyl) hydroxyacetyl] amino] benzoic acid (Compound IV; US Patent No. 5624957), 4
-[1- [5,6-Dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid [Compound V; S. Patent application N
o.60 / 1227976 (filed Apr. 6, 1999, name: "Selective retinoic acid analog") (Attorney Docket No .: SD128 ^* ); S. Patent application No. (Filed on April 22, 1999, name: "selective retinoic acid analog") (agent docket number: SD128a ^* )], and (E) -4- [2- [8- (1,
1′-biphenyl-4-yl) -5,6-dihydro-5,5-dimethyl-2-naphthalenyl] ethenyl] benzoic acid (Compound VI; WO 98/46228) was obtained from Bristol-Myers Squibb.

(E) -3-Chloro-4- [2- (5,6-dihydro-5,5-dimethyl-8
-Phenyl-2-naphthalenyl) ethenyl] benzoic acid (compound III) and 4
The structure of-[1- [5,6-dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (compound V) is as follows.

Embedded image

Compound III has the formula:

Embedded image Using an intermediate (X = Cl) of S. Patent No. Synthesized according to the method described in US Pat.

The intermediate was prepared from commercially available 3-chloro-4-hydroxybenzoic acid.
It was synthesized as shown below.

Embedded image After the acid is first esterified, the OH group is replaced with trifluoromethanesulfonic anhydride (T
Activation with f ₂ O) followed by coupling reaction of the trifluoromethanesulfonate group with vinyltributyltin gives the desired intermediate.

Cells and Culture: NB4 and NB4-R2 cells were cultured [Lanotte M. et al. "Blood" (
77: 1080-1086, 1991). Essentially, culture for 3 days without the culture medium supply, in makes it possible to index growth of untreated control culture density (10 ⁵ cells / ml), was established using the log-phase growth cells. Retinoid (supplied by Bristol-Myers Squibb; dissolved in ethanol as a 10 ⁻³ M stock solution) and 8-CPT-cAMP (Sigma; dissolved in saline at 10 ⁻² M) at the indicated concentrations. Was. Cultures were light protected.

Morphological and functional cell maturation analysis: Cell morphology was analyzed after May-Grunwald staining. The tissue NBT response is
[Lanotte M. et al. "Blood" (77: 1080)
-1086, 1991)]. Morphological analysis and NBT reactions were performed after 72 h of treatment. At least 300 cells were analyzed for each treatment culture. The data shown is representative of one experiment and each treatment was repeated at least three times.

Reporter Cell Assay: Synthetic retinoids were prepared using the corresponding GAL-RAR chimera and Chen J. Y. HeLa cells transfected with the allogeneic (17-mer) ₅ -globin-luciferase reporter gene described in "EMBO J." (14: 1187-1197, 1995) were characterized.

Flow cytometric analysis of CD11c cell surface integrins: Expression of CD11c integrins was essentially as described by Ruchaud S. et al. "Proc. Na
Acad. Sci. USA "(91: 8428-8432, 1994). In short, after culture (48h), cells were
S, and washed with an anti-human CD11c FITC mouse monoclonal antibody (Becto
n-Dickinson). The cells are then washed twice in PBS and fixed in a 1% paraformaldehyde / PBS solution. Cells were analyzed using a COULTER flow cytometer.

RPA analysis: Total RNA was extracted with Trizol reagent (Gibco BRL). The ribonuclease protection assay was performed according to the supplier's instructions (Pharmin, San Diego, CA).
gen). In short, human cytokine 4 and 5 templates
The set (45034P; 45035P) was labeled with α- ³² P uridine triphosphate. For hybridization, RNA (4 μg) and a labeled probe of 6 × 10 ⁵ cpm were used. After ribonuclease treatment, the protected probe was replaced with 5% urea-
The gel was resolved on a polyacrylamide-bis-acrylamide gel.

PML-RARa cDNA Sequencing: Purified total RNA from NB4 and NB4-R2 cells; Dynal magnetic beads coupled to biotinylated PML-RARa oligonucleotides (specific for the sequence at the recombination site) (M-280 streptavid
In)), PML-RARa-specific mRNA was isolated. This mRNA preparation is used for RT-PCR with oligod (N) 6 as primer and AMV reverse transcriptase at 42 ° C. for 60 minutes. R
1/20 of the T reaction was used for PCR amplification with R2L primers (CTG CCC CTG
GAG ATG GAT GAT) (SEQ ID NO: 1) and R2H
Primer (GCG GAG GGC GAG GGC TGT GTC) (S
Used with EQ ID NO: 2) at 95 ° C for 5 minutes; 95 ° C for 1 minute, 65
A cycle of 2 minutes at 72 ° C, 1 minute and 30 seconds at 72 ° C, and 10 minutes at 72 ° C. PC
The R product was gel isolated and cloned into the pCR ^™ 2.1AT sequencing vector. Clones were selected and PCR-tested for the presence of the insert. Positive clones were selected and plasmids were purified from these clones and
n Elmer Big Dye Kit and ABI377 Automated Sequencer
er), the DNA was sequenced with the SP6 and T7 primers.

Example 2 In the human breast cancer cell line T47D, 100 μM cAMP analog (8 CPT)
-CAMP) was evaluated for the effect of RXR ligand on cellular lipid accumulation in the absence or presence (FIG. 5). Cells were treated with RXR alone (blue bar) and RXR + 10
Treated with 0 μM 8CPT-cAMP (red bar) for 7 days,
Stained with ile Red (a fluorescent lipid staining dye). The addition of CPT-cAMP is 1
At μM SR11237 and Bexarotene, T4 was greater than RXR ligand alone.
The differentiation of 7D breast cancer cells was increased by 20%. 100 μM C
No effect was seen in the presence of PT-cAMP alone.

The EC ₅₀ for lipid accumulation of cells treated with the RXR compound alone was 62 nM (bexarotene) and 110 nM (SR11237). Addition of CPT-cAMP did not increase the sensitivity of T47D to differentiation by RXR compounds. These results demonstrate that activation of the cAMP pathway can increase the differentiation response of breast cancer to RXR ligand. This biological strategy can serve as a viable combination therapy for the treatment of solid tumor cancers of epithelial origin.

Materials and Methods (Nile Red Staining Method ) Reagents: 1.5% glutaraldehyde / PBS as fixative solution; Stock solution as stain solution (1 mg Nile red and 1 ml acetone mixed well, stored, chilled and 1 ml as working solution
After adding 4 μl of the stock solution to 75% glycerol (PBS), vortex slightly, and then degas the dye solution slightly under reduced pressure to remove the bubbles. Fixation: Cover slides with 1.5% glutaraldehyde for 5 minutes, then wash with buffered saline.

Staining: For fluorescence microscopy, add one drop of dye / glycerol solution to a glass slide,
Covered with glass coverslip. After 5 minutes, the slide glass was viewed under a fluorescence microscope, excitation 450-500 nm, emission> 528 nm. For flow cell fluorometry, the medium was aspirated off the cells and washed twice with PBS (serum in the medium eliminated the dye from the cells). The cells were then trypsinized and washed with PBS to remove trypsin. Spin down the cells (spun do
wn) Resuspend in 1 ml PBS (1-2 × 10 ⁶ / ml). Stock dye was added directly to the cell suspension in PBS at a 1: 100 dilution, incubated for a minimum of 5 minutes at room temperature, and the samples were analyzed immediately.

For the fluorescence spectrum, remove the medium from the 96-well plate and place the cells in PB
Washed once with S. Cells are fixed with 1.5% glutaraldehyde for 5 minutes, then
Washed with BS (can remove cells). Diluted dye / PBS (1: 100 from stock) was added to the cells and incubated for 5 minutes and the plate readings were excitation 488 nm with a 2-nm slit width and emission 540 nm with a 20-nm slit width. Cells: The breast cancer cell line T47D was obtained from the American Type Culture Collection, Manassas, VA [
Freake et al., “Biochem. Biophys. Res. Comm.” (101: 1131-11).
38, 1981); and Sher et al., "Biochem. J." (200: 315-).
320, 1981)].

Example 3 Production of 4- [1- (5,6-dihydro-3,5,5-trimethyl-8-isopropyl-2-naphthalenyl) ethenyl] benzoic acid (Compound V) Synthesis reaction formula: 1) 1,2,3,4-tetrahydro-4,4,6-trimethyl-7-bromo-
Production of 1-oxonaphthalene

Embedded image

Embedded image

2) Production of ethyl 4- (1- (tributylstannyl) -2- (trimethylsilyl) -ethen-1-yl) benzoate

Embedded image

3) Production of 4- [1- (5,6-dihydro-3,5,5-trimethyl-8-isopropyl-2-naphthalenyl) ethenyl] benzoic acid

Embedded image

Embedded image

1.1,2,3,4-tetrahydro-4,4,6-trimethyl-7-bromo-1
Preparation of -Oxonaphthalene Methyl 4- (p-tolyl) butyrate: 10.0 g of 56.4- (p-tolyl) butyric acid in methanol (680 ml).
11 mmol) is treated with concentrated sulfuric acid (5.4 ml). Bring the reaction to room temperature
Stir for 8 hours. Sodium bicarbonate (15 g) is added and the mixture is stirred for 15 minutes and then concentrated. Dissolve the residue in ethyl acetate / water. The organic phase was separated, washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to give the title material (10.8
g, crude 100%) as an oil which is used in the next reaction.

(Equation 1)

2-Methyl-5- (p-tolyl) pentan-2-ol: methyl 4- (p-tolyl) butyrate (10.8 g, 56.2 mmol) in ether (215 ml) and benzene (215 ml) Is treated dropwise with methylmagnesium bromide (3M in ethyl ether, 45 ml, 135 mmol) (15 min). The mixture is stirred at room temperature for 1.5 hours, cooled to 0 ° C. and treated with 10% aqueous ammonium chloride (100 ml). Then, with concentrated hydrochloric acid, pH 6.5-7.
And dilute the mixture with ethyl acetate (200 ml). The organic phase was separated, washed with brine / water (1: 1), brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give the title material (10.4 g, 96%) as a yellowish oil. This is used for the next reaction.

(Equation 2)

1,2,3,4-Tetrahydro-1,1,7-trimethylnaphthalene: 2-methyl-5- (p-tolyl) pentan-2-ol (10 in ethyl ether (100 ml) at 0 ° C. .4 g, 54.1 mmol) in concentrated sulfuric acid (64
ml). The mixture is stirred at 0 ° C. for 1.5 hours, then poured into an ice / water mixture. The mixture is diluted with ethyl ether and the organic phase is separated and washed with water (twice), saturated sodium bicarbonate and brine. The aqueous phase was extracted with ethyl ether, and the combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated to give the title material (9.6 g, 100% crude) as a yellowish oil, Is used for the next reaction.

(Equation 3)

1,2,3,4-tetrahydro-4,4,6-trimethyl-1-oxonaphthalene; 1,2,3,4-tetrahydro-1,1 in water (28 ml) and dioxane (44 ml) , 7-Trimethylnaphthalene (9.60 g, 55 mmol), potassium bromate (9.17 g, 55 mmol), cerium ammonium nitrate (1.
The 50 g) solution is heated at 85 ° C. under argon for 6 hours. Then the mixture is cooled to 0 ° C. and diluted with ethyl acetate and water. Separate the organic phase and extract the aqueous phase with ethyl acetate. Wash the combined organic extracts with water (2 times), saturated sodium bicarbonate and brine, dry over anhydrous magnesium sulfate, filter and concentrate. The residue is purified by silica gel chromatography (6.5 × 17 cm, 10% ethyl acetate / hexane) to give the title material (9.2 g, 89%) as a colorless oil.

(Equation 4)

1,2,3,4-tetrahydro-4,4,6-trimethyl-7-bromo-1-
Oxonaphthalene: To a stirred suspension of aluminum trichloride (1.1 g, 8.1 mmol) in dichloromethane (2.5 ml) at 0 ° C was added 1,2,3,4 in dichloromethane (1 ml).
-Tetrahydro-4,4,6-trimethyl-1-oxonaphthalene (0.564
g, 3 mmol). The mixture is stirred at this temperature for 45 minutes and then at room temperature for 45 minutes. Then bromine (0.185 ml, 3.6 mmol) is added and the resulting mixture is stirred at room temperature for 2 hours. The mixture is poured onto a mixture of ice (50 ml), concentrated hydrochloric acid (1.5 ml) and ethyl ether (50 ml). Separate the organic phase and wash with 1N hydrochloric acid, saturated sodium bicarbonate, aqueous sodium thiosulfate and brine. The aqueous phase is extracted with ethyl ether, and the combined organic extracts are dried over anhydrous magnesium sulfate, filtered, and concentrated.

The residue is purified by silica gel chromatography (2.5 × 17 cm, 0-5% ethyl acetate / toluene) to give the title material, which is triturated in cold hexane (0.700 g, 87%). .

(Equation 5) Elemental analysis (as C ₁₃ H ₁₅ BrO) Calculated: C 58.44, H 5.66 Actual: C 58.12, H 5.78

2.4 Preparation of ethyl 4- (1- (tributylstannyl) -2- (trimethylsilyl) -ethen-1-yl) benzoate Ethyl 4-ethynylbenzoate: 4-iodobenzoate in triethylamine (800 ml) Ethyl acid (55.2 g
, 0.2 mol) is purged with argon. Then copper iodide (1.1 g) and bis (triphenylphosphine) palladium (II) dichloride (7.0 g)
) And purge the mixture again. Then at 0 ° C. trimethylsilylacetylene (42 ml, 0.3 mol) is added for 30 minutes and the resulting mixture is stirred at room temperature for 1 hour. The mixture is concentrated, triturated in hexane and filtered.

The filtrate is concentrated to give ethyl 4- (2-trimethylsilylethen-1-yl) benzoate (51.5 g, 100% crude) as a black oil.

(Equation 6)

The crude is diluted in ethanol (500 ml) and potassium carbonate (2.4 g) is added. Stir the resulting mixture at room temperature overnight. The mixture is concentrated and the residue is triturated in hexane and filtered. The filtrate is concentrated and the residue is Kugelrohr distilled (0.
Purify at 1 mm Hg, bath temperature 70-80 ° C.) to give the title material (27.2 g, 78%) as a colorless oil which solidifies.

[0141]

(Equation 7)

4- (1- (Tributylstannyl) -2- (trimethylsilyl) ethene-1
-Yl) ethyl benzoate: ethyl 4-ethynylbenzoate (27.0 g, 0 in dioxane (270 ml)).
. 155 mol), trimethylsilyltributyltin (65 ml, 0.186 mol)
, A mixture of tetrakis (triphenylphosphine) palladium (0) (2.9 g) is purged with argon and then heated to 85 ° C. for 1.5 hours. Cool the mixture to room temperature and concentrate. The residue was purified by silica gel pad chromatography (10.5
× 15 cm, 0-5% ethyl acetate / hexane) to give the title material (83.0
g, 100%) as a slightly yellowish oil.

[0143]

(Equation 8)

3.4 Preparation of 4- [1- (5,6-dihydro-3,5,5-trimethyl-8-isopropyl-2-naphthalenyl) ethenyl] benzoic acid 4- [1- (5,6,7 , 8-Tetrahydro-3,5,5-trimethyl-8-
Oxo-2-naphthalenyl) 2-trimethylsilyl-ethenyl] ethyl benzoate: 1,2,3,4-tetrahydro-4,4,6-trimethyl-1-oxo-7-
A solution of bromonaphthalene (8.5 g, 31.8 mmol) is purged with argon (twice). Then lithium chloride (4.0 g), copper iodide (0.860 g), tetrakis (triphenylphosphine) palladium (0) (1.8 g, 1.6 mmol) and 4- (1- (tributylstannyl)- 2- (trimethylsilyl)
-Ethen-1-yl) ethyl benzoate (24.0 g, 44.5 mmol) is added and the resulting mixture is again purged with argon.

The mixture is heated to 80 ° C. for 4 hours and then cooled to room temperature. Mix the mixture with cold water (1
L) and dilute with ethyl ether. Separate the organic phase, cold water (1 L × 2),
Wash with saturated sodium bicarbonate (1 L), brine, dry over anhydrous magnesium sulfate, filter, and concentrate. The residue was purified by silica gel chromatography (8 × 15 cm, 0
Purify with ５5% ethyl acetate / toluene) to give the title material, which is triturated in hexane (12.2 g, 88%).

[0146]

(Equation 9) Elemental analysis (as C ₂₇ H ₃₄ O ₃ Si) Calculated: C 74.61, H 7.89 Actual: C 75.08, H 7.94

4- [1- (5,6,7,8-tetrahydro-3,5,5-trimethyl-8-
Ethyl oxo-2-naphthalenyl) ethenyl] benzoate: 4- [1- (5,6,7,8-tetrahydro-3,5,5-trimethyl-8-oxo-2-naphthalenyl) in dichloromethane (900 ml) A solution of ethyl-2-trimethylsilyl-ethenyl] benzoate (12.0 g, 27.6 mmol) is treated with trifluoroacetic acid (100 ml) at 0 ° C. The mixture is stirred for 18 hours and allowed to reach room temperature. The mixture is diluted with toluene (100ml) and concentrated. The residue is purified by silica gel chromatography (8 × 15 cm, 0-5% ethyl acetate / toluene) to give the title material as a yellowish solid, which is triturated in hexane (9.7 g, 97%). The analytical sample is recrystallized in hexane.

[0148]

(Equation 10) Elemental analysis (as C ₂₄ H ₂₆ O ₃ ) Calculated: C 79.53, H 7.23 Actual: C 79.26, H 7.30

4-[(3,5,5-trimethyl-5,6-dihydro-8-isopropyl-2
-Naphthalenyl) ethenyl] ethyl benzoate: In a three-necked flask, cerium (III) chloride heptahydrate (13.5 g, 35
Mmol) is dried under reduced pressure at 145 ° C. for 2 hours [for details of the drying procedure, see “J. Am. Chem. Soc.” (111: 4392-4398, 1989)]. While still hot, argon is introduced, the flask is cooled to 0-5 ° C., and tetrahydrofuran (120 ml) is added quickly with vigorous stirring. Remove the ice bath and stir the solution at room temperature overnight (18 h).

Then, the solution was cooled again to 0-5 ° C., and isopropylmagnesium chloride (
(3M solution in ethyl ether, 12 ml) is added dropwise and the mixture is stirred vigorously for 1.5 hours. The mixture was then added to 4- [1-- in tetrahydrofuran (15 ml).
(5,6,7,8-tetrahydro-3,5,5-trimethyl-8-oxo-2-
A solution of ethyl naphthalenyl) ethenyl] benzoate (prepared above) (9.06 g, 25 mmol) is added dropwise and the resulting mixture is stirred at 0-5 ° C. for 45 minutes. Acetic acid (10%, 100 ml) was slowly added and the mixture was taken up in ethyl ether (100 ml).
Extract in 1).

The organic phase is washed with water, saturated sodium bicarbonate and brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is purified by silica gel chromatography. The title material (6.1 g, 63%) as a white solid was obtained starting material (1.0 g, 11%
) And ethyl 4-[(3,5,5-trimethyl-5,6-dihydro-8-hydroxy-2-naphthalenyl) ethenyl] benzoate (1.0 g, 11%).

[0152]

[Equation 11] Elemental analysis _{(C 27 H} 32 _{O 2)} Calculated value: C83.46, H8.30 Found: C83.27, H7.67

4- [1- (5,6-Dihydro-3,5,5-trimethyl-8-isopropyl-2-naphthalenyl) ethenyl] benzoic acid: 4-[(3,5,5-trimethyl-5, 6-dihydro-8-isopropyl-2
-Naphthalenyl) ethenyl] solution of ethyl benzoate (3.9 g, 10 mmol) is saponified by dropwise treatment with sodium hydroxide (10 N) and stirred at room temperature.
The solution is cooled to 0-5 ° C. and 1N HCl is added dropwise with vigorous stirring. After stirring, the resulting white precipitate is filtered off, washed with water and dried. After work-up, the title compound is obtained as a white solid (3.1 g, 86%).

[0154]

(Equation 12) Elemental analysis (as C ₂₅ H ₂₈ O ₂ ) Calculated values: C83.29, H7.83 Actual values: C83.24, H8.37

[Brief description of the drawings]

FIG. 1 shows evidence of the differential effects of RAR and RXR specific agonists on NB4 cells, a positive co-operation between the retinoid and protein kinase A signaling pathways. The compound was used as follows: ATR
A, 1 μM; other retinoid receptor agonist, 0.2 μM; receptor antagonist, 2 μM; cell-permeable cAMP analog (8CPT-cAMP), 100 μM
. FIG. 1a shows the morphological characteristics of NB4 cells in response to treatment (after 72 hours). FIG. 1b shows the enzymatic reduction of nitroblue tetrazolium in retinoid-treated NB4 cells (percent positive cells after 72 hours). FIG. 1c shows a flow cytometry analysis of CD11c integrin expression after NB4 cell treatment with retinoids and cAMP (after 48 hours). Pan-agonist (
ATRA, 9-cis RA) and RARα agonists (4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl) carbonyl] amino] benzoic Acid (Compound I)) induces maturation of NB4 cells, but RARβ ((E) -3-chloro-4- [2- (5,6-dihydro-5,5-
Dimethyl-8-phenyl-2-naphthalenyl) ethenyl] benzoic acid (Compound II
I)) and RARβγ (3-fluoro-4 [[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) hydroxyacetyl]
Amino] benzoic acid (Compound IV)) agonists are not induced. The RXR pan-agonist SR11237 by itself failed to cause maturation of NB4 cells. However, consistent with RXR ligand-dependent signaling, SR11237
And cAMP, the RAR antagonist 4-[[[5,6-
Dihydro-5,5-dimethyl-8- (3-quinolinyl) -2-naphthalenyl] carbonyl] amino] benzoic acid (compound II) or (E) -4- [2- [8- (
1,1′-biphenyl] -4-yl) -5,6-dihydro-5,5-dimethyl-
Induces maturation of NB4 cells through a pathway that is not inhibited by 2-naphthalenyl] ethenyl] benzoic acid (Compound VI).

FIG. 2 shows 4- [1- [5,6-dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid as a bifunctional rexinoid It is a figure which shows the characteristic of (compound V). FIG. 2a shows a reporter cell assay. RAR and RXR AF-2 agonists of 4- [1- [5,6-dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (compound V) and Antagonist activity was induced by retinoid induction (1 nM, “−9”) from a 96-well plate containing equal amounts of cells.
1 μM, “-6”) after luciferase activity (Chen, JY et al., EMBO J. 14: 1187-1197 (1995)). Monitoring and quantification were performed using a photon counting camera. Open triangles indicate signal in the presence of retinoid (agonist activity, "-" indicates vehicle-only signal); closed triangles indicate both 10 nM ATRA ("-" indicates ATRA only) and increasing amounts of retinoid Signal (or antagonist activity if the ATRA-inducible signal is reduced). Note that the RXRα reporter cell line also contains an inactive dnRXRαΔAB lacking both AF-1 and AF-2, blocking the endogenous RAR that renders the RXR silent in the absence of a RAR agonist. (
Vivat, V. et al., EMBO J. 16: 5697-5709 (1997)). FIG. 2b shows 4- [1- [5,6-
RXRα to DR1 reporter in the presence of dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (compound V)
Homodimer (for 0.1 μM 9-cis RA normalized to 100) and R
FIG. 4 shows transient transcriptional enhancement of ARα-RXRα heterodimer (relative to 10 nM ATRA normalized to 100). Figure 2b, slight activation of DR5 in lane 7 is likely due to a weak interaction of the RXR homodimer with the DR5 element (
Mader, S. et al., J. Biol. Chem. 268: 591-600 (1993)). FIG. 2c shows 4- [1- [5
, 6-Dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (compound V) is very potent in PKA signaling to cause maturation of NB4 cells It is a figure showing that it is a partner. 4- [1- [5,6-Dihydro-3,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (with or without 8-CPT-cAMP) 5 shows morphological and histochemical characteristics of NB4 cells treated with V). CAMP and 9-cis RA or 4- [1- [5,6-dihydro-3] to induce NBT response and CD11c expression in NB4 cells
Comparative analysis of synergy with 5,5,5-trimethyl-8- (1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid (compound V) is shown in the right figure.

FIG. 3 shows that rexinoid-PKA crosstalk induces a different cytokine expression program than that induced by ATRA. Regulation of cytokine expression in NB4 cells was assessed by an RNase protection assay. Cells were exposed to the drugs shown at the top of the figure for 0, 24 and 48 hours (continuous lanes for each treatment). For comparison, the action of vitamin D3 ("D3")
) Was also examined. FIG. 3a shows the complete probe run on the same gel on the left (L32
And GAPDH used for calibration as an unaltered internal standard), showing the location of protected fragments. FIG. 3b shows only the locations of the protected fragments.

FIG. 4 shows that different RAR-ligand independent signaling pathways
FIG. 2 shows that RB4-R2 cells that are resistant to R agonists are matured. FIG. 4 a shows the molecular determination of mutant PML-RARa in NB4-R2 cells. NB4-R2 is derived from cAMP from myeloid leukemia cells of ATRA-resistant patients.
And ATRA resistant subclones isolated by selection in both ATRA and ATRA (Ruchaud, S. et al., Proc. Natl. Acad. Sci. USA 91: 8428-8432 (1994); Du;
prez, E. et al., Leukemia 6: 1281-1287 (1992)). NB4-R2 has a typical t (1
5; 17) had a translocation and expressed PML-RARa mRNA, but 120 KD
Did not express the PML-RARα chimeric protein (Duprez, E. et al., Oncogene 1).
2: 2451-2459 (1996)). By sequencing the PML-RARa cDNA, NB4
PML-RARa (L) of cells (Kastner, P. et al., EMBO J. 11: 629-642 (1992))
A point mutation that results in a stop codon at position 411 in the retinoid binding domain of E. coli, thus resulting in a shortened chimeric protein is evident. FIG. 4b shows morphological and functional maturation of NB4-R2 cells in response to synergistic treatment with retinoids and cAMP. NB4-R2 cells were treated with a protocol similar to that used for NB4 cells (see description of FIG. 1). Note that the cooperation between cAMP and rexinoid signaling allows the maturation of ATRA resistant cells.

FIG. 5 is a diagram showing differentiation of T47D breast cancer cells. Fig. 5a shows 8CP
FIG. 2 shows the effect of SR11237 on lipid accumulation with or without T-cAMP (100 μM). FIG. 5b shows the effect of bexarotene on or without 8CPT-cAMP (100 μM) on lipid accumulation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/357 A61K 31/357 31/4015 31/4015 31/4166 31/4166 31/437 31/437 31 / 496 31/496 31/522 31/522 31/7076 31/7076 38/00 A61P 17/06 A61P 17/06 35/00 35/00 35/02 35/02 43/00 43/00 A61K 37/02 (71) Applicant: Institut Nacional de la Sante e de la Roussélé Medical (I NS E L.E.M) INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICAL (INSERM) 75654 Paris Cedex 13, Rue du Tolbiac 101 (71) Applicant Center Nacional de la Ruche Rousse Cyanificique CENTRE NATIONAL DE LARECHERCHE SCIENTI FIQUE 75794 Paris, Cedex 16, Ré Michelangelo 3rd (71) Applicant Université Louis-Pasteur UNIVERSITE SOURBOURS France Cedex, Bowt Postal 1032F, Ryu Blaze Pascal No. 4, Strasbourg (72) Inventor Gerard Benowa France, F-75010 Paris, Hospital Saint-Louis, Anselm Yu 496 (72) Inventor Hinrich・ Grone Meyer Federal Republic of Germany-77704 Oberkirch, Oullandstrasse 1st (72) Inventor Michel Lanotte France, F-75020 Paris, Passage Gan Betta 28 (72) Inventor Marco Gottterdis United States 08540 Harris Road, Princeton, NJ 9F F-term (reference) 4C084 AA02 AA03 AA20 AA24 DA01 DA19 MA02 NA14 ZA812 ZA892 ZB262 ZB272 ZC412 ZC752 4C086 AA01 AA02 BA08 BA12 BC BC50 CB05 CB07 GA02 GA07 MA02 MA03 MA04 NA14 ZA81 ZA89 ZB26 ZB27 ZC20 ZC41 ZC75 4C206 AA01 AA02 DB15 DB20 GA34 MA02 MA03 MA04 NA14 ZA81 ZA89 ZB26 ZB27 ZC41 ZC75

Claims (36)

[Claims] 1. A method of treating a hyperproliferative disease in a subject, comprising: (a) administering to said subject a pharmaceutically effective amount of a retinoid X receptor (RXR) agonist; A method of treatment comprising administering to said subject a pharmaceutically effective amount of an agent capable of activating protein kinase A (PKA). 2. The method according to claim 1, further comprising (c) administering to said subject a pharmaceutically effective amount of a retinoic acid receptor (RAR) agonist. 3. The method according to claim 1, further comprising (d) administering a pharmaceutically effective amount of the cytokine to the subject. 4. The method according to claim 1, wherein the hyperproliferative disease is cancer. 5. The method according to claim 4, wherein the cancer is acute promyelocytic leukemia (APL). 6. The method of claim 5, wherein the APL resists treatment with a RARα agonist. 7. The method according to claim 4, wherein the cancer is breast cancer. 8. The method according to claim 1, wherein the hyperproliferative disease is psoriasis. 9. An RXR agonist comprising 9-cis retinoic acid, bexarotene (be
xarotene), 4- [1- [5,6-dihydro-3,5,5-trimethyl-8- (
1-methylethyl) -2-naphthalenyl] ethenyl] benzoic acid and SR11
The method according to claim 1, which is selected from the group consisting of 237. 10. The method according to claim 1, wherein the agent is a PKA agonist. 11. The method according to claim 11, wherein the PKA agonist is 8-bromo-cAMP, Sp-cA.
MPS, 8CPT-cAMP, dibutyryl-cAMP, Sp-5,6-DCI-
cBiMPS, adenylate cyclase toxin, horse choline (forskolin)
), L-858051, and Sp-8-pCPT-cGMPS. 12. The method of claim 1, wherein the agent is a compound that increases cAMP levels. 13. The method of claim 12, wherein the compound stimulates cAMP synthesis. 14. The method according to claim 13, wherein the compound is selected from the group consisting of adenylate cyclase toxin, horse choline, and L-858051. 15. The method according to claim 12, wherein the agent is a compound that inhibits phosphodiesterase. 16. The compound as claimed in claim 20, wherein the compound is RO20-1724, Rolipram.
16.) The method of claim 15, wherein the method is selected from the group consisting of), etazolate, and 3-isobutyl-1-methylxanthine. 17. The RAR agonist is 9-cis retinoic acid, all-trans-retinoic acid, 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-indene- 5-yl) carbonyl] amino] benzoic acid, AM-80
And a RARα agonist selected from the group consisting of AM-580.
The method of treatment described in 1. 18. The cytokine may be a granulocyte colony-stimulating factor (G-CSF).
4.) The method of claim 3, wherein the method is selected from the group consisting of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage-colony-stimulating factor (M-CSF). 19. A pharmaceutical composition comprising (a ') a retinoid X receptor (RXR) agonist; and (b') an agent capable of activating protein kinase A (PKA). 20. The pharmaceutical composition according to claim 19, further comprising (c ′) a retinoic acid receptor agonist. 21. The pharmaceutical composition according to claim 19, further comprising (d ') a cytokine. 22. An RXR agonist (a ') comprising 9-cis retinoic acid, bexarotene, 4- [1- [5,6-dihydro-3,5,5-trimethyl-8- (1-
Methylethyl) -2-naphthalenyl] ethenyl] benzoic acid, and SR1123
20. The pharmaceutical composition according to claim 19, which is selected from the group consisting of: 23. The pharmaceutical composition according to claim 19, wherein the active substance (b ′) is a PKA agonist. 24. The PKA agonist is 8-bromo-cAMP, Sp-cA.
MPS, 8CPT-cAMP, dibutyryl-cAMP, Sp-5,6-DCI-
cBiMPS, adenylate cyclase toxin, horse choline, L-858
24. The pharmaceutical composition according to claim 23, wherein the pharmaceutical composition is selected from the group consisting of 051, and Sp-8-pCPT-cGMPS. 25. The pharmaceutical composition according to claim 19, wherein the agent (b ') is a compound that increases cAMP levels. 26. The pharmaceutical composition according to claim 25, wherein the compound stimulates cAMP synthesis. 27. The pharmaceutical composition according to claim 26, wherein the compound is selected from the group consisting of adenylate cyclase toxin, horse choline, and L-858051. 28. The pharmaceutical composition according to claim 25, wherein the active substance (b ′) is a compound that inhibits phosphodiesterase. 29. The pharmaceutical composition according to claim 28, wherein the compound is selected from the group consisting of RO20-1724, lollipran, etazolate, and 3-isobutyl-1-methylxanthine. 30. The RAR agonist (c ′) is 9-cis retinoic acid, all-trans-retinic acid, 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo- 1H-Inden-5-yl) carbonyl] amino] benzoic acid, A
The pharmaceutical composition according to claim 20, which is a RARα agonist selected from the group consisting of M-80 and AM-580. 31. The cytokine (d ′) is a granulocyte colony-stimulating factor (G
22. The pharmaceutical composition according to claim 21, which is selected from the group consisting of: -CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage-colony-stimulating factor (M-CSF). 32. A kit for use in performing the treatment method of claim 1, comprising: (1) a first container means containing a therapeutically effective amount of an RXR agonist (a); A kit comprising at least two container means of a second container containing an active substance (b) capable of activating a PKA in a tightly confined state. . 33. The kit of claim 32, further comprising a container containing a therapeutically effective amount of a RAR agonist. 34. The kit of claim 32, further comprising a container containing a therapeutically effective amount of a cytokine. 35. A method for inhibiting the growth of breast cancer cells, comprising: (a) administering an RXR agonist, and then (b) administering an agent capable of activating protein kinase A. 36. The method of claim 35, wherein the breast cancer cells are derived from a T47D cell line.