JPH11302194A - Retinoic acid activity enhancer - Google Patents

Abstract

(57) [Problem] To provide a substance that enhances the physiological activity of an RXR ligand such as 9-cis retinoic acid. SOLUTION: The medicament which contains difaranisol A or a physiologically acceptable salt thereof as an active ingredient and has an action of enhancing the physiological activity of RXR ligand. For example, it is useful as a therapeutic agent for leukemia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a medicament capable of enhancing the physiological activity of an RXR ligand such as retinoic acid and useful for treating leukemia, for example.

[0002]

2. Description of the Related Art Retinoic acid (vitamin A acid) is an active metabolite of vitamin A, and acts to differentiate developing immature cells into mature cells having specific functions.
It has extremely important physiological actions such as cell growth control action and life support action. For example, all-tra
ns) retinoic acid (ATRA) is a natural morphogen (morphogen) in spinal development and is known to induce myeloid leukemia cell differentiation. The above compounds having retinoic acid and retinoic acid-like biological activity are collectively referred to as "retinoids".

[0003] For example, all-trans retinoic acid is a nuclear receptor superfamily present in the cell nucleus (Evans, RM, Science, 240, p.88).
9, 1988) by binding as a ligand to retinoic acid receptor (RAR), which regulates the growth and differentiation of animal cells or cell death (Petko
vich, M., et al., Nature, 330, pp. 444-450, 1987).
The administration of all-trans retinoic acid can lead to complete remission in most acute promyelocytic leukemia (APL) patients. 9-cis (9-cis) retinoic acid, which is a stereoisomer of all-trans retinoic acid, is superior to all-trans retinoic acid in inhibiting proliferation and differentiation of myeloid leukemia cells in vitro (Saka
shita, A., et al., Blood, 81, pp.1009-1016, 1993)
It is known that the differentiation induction therapy for APL patients is less effective than all-trans retinoic acid in terms of side effects.

Heretofore, there have been few reports on substances that enhance the action of retinoids. For example, JP-A-8-59511
The publication suggests that a compound which is a specific ligand for retinoid X receptor (RXR) has an action of enhancing the action of Am80 which is a specific ligand compound for RAR-α receptor. In this publication,
4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-
Naphthyl) carbonyl] benzoic acid-ethylene acetal is a synthetic retinoid Am80 (4-[(5,6,7,8-tetrahydro
-5,5,8,8-tetramethyl-2-naphthalenyl) carbamoyl] benz
It has been suggested that oic acid) enhances the differentiation inducing action. Also, 4- [5H-2,3- (2,5-dimethyl-2,5-hexano) -5-methyldibenzo [b, e] [1,4] diazepin-11-yl]
Benzodiazepine compounds such as benzoic acid (HX600) are also known to enhance the action of retinoids (Umemiya e
t al., Chem. Pharm. Bull., 43, pp.1827-1829, 199
Five). The action of this compound is believed to activate RXR, which forms an RXR-RAR heterodimer.

On the other hand, difaranisole A (3,5-dichloro-2-hydroxy-4-methoxy-6-n-propylbenzoic acid) was isolated from a culture of a microorganism belonging to the genus Chaetomium. (Japanese Patent Publication No. 4-32816). Since this substance exerts a differentiation inducing effect on mouse erythroid leukemia cells and an effect of inducing normal cells, it is expected to be used as an antitumor agent. Although difaranisol A exhibits antitumor activity against mice inoculated with neuroblastoma C-1300 cells, it has a problem that its activity in inducing differentiation of myeloid leukemia cells is weak (Suzuki, T., etal., J. Antibiotics, 39, p
p.869-871, 1986).

[0006]

DISCLOSURE OF THE INVENTION The present inventors have made intensive studies to develop a drug capable of exerting an excellent growth inhibitory action and differentiation inducing action on human myeloid leukemia cells. As a result, when difaranisol A was combined with a compound having an affinity for RXR such as 9-cis retinoic acid or a salt thereof,
It has been found that the physiological action of RXR ligand is significantly enhanced. Further, when both are used in combination, it is possible to achieve a synergistic growth inhibitory action and differentiation inducing action on leukemia cells, and that a drug containing these substances as an active ingredient can exhibit high efficacy in treating leukemia. I found it. The present invention has been completed based on these findings.

That is, the present invention relates to difaranisol A
Or a physiologically acceptable salt thereof as an active ingredient
It is intended to provide a physiological activity enhancer of an RXR ligand (preferably 9-cis retinoic acid or a physiologically acceptable salt thereof). Further, according to the present invention, difaranisol A or a physiologically acceptable salt thereof is contained as an active ingredient, and an RXR ligand (preferably 9-cis retinoic acid or a physiologically acceptable salt thereof) is used as an active ingredient. The present invention provides a medicament for use in enhancing the action of a medicament, preferably a therapeutic agent for leukemia.

[0008] In another aspect, the present invention relates to a compound of the present invention, wherein RX contains difaranisol A or a physiologically acceptable salt thereof.
R-ligand (preferably 9-cis retinoic acid or a physiologically acceptable salt thereof) as an active ingredient, preferably difaranisol A or a physiologically active substance for the manufacture of a medicament for the treatment of myeloid leukemia. Use of a salt thereof, which is acceptable for the treatment of myeloid leukemia,
Administering to the patient a therapeutically effective amount of difaranisol A or a physiologically acceptable salt thereof and a therapeutically effective amount of an RXR ligand (preferably 9-cis retinoic acid or a physiologically acceptable salt thereof). Are provided. From yet another aspect, a medicament comprising as an active ingredient difaranisol A or a physiologically acceptable salt thereof and an RXR ligand (preferably 9-cis retinoic acid or a physiologically acceptable salt thereof) is provided. Provided. This medicine
It is useful for treating various leukemias such as myeloid leukemia.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Difaranisol A can be produced, for example, by the method described in Japanese Patent Publication No. 4-32816. The kind of the physiologically acceptable salt of difaranisol A is not particularly limited. For example, metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt; ammonium salts: organic amines such as triethylamine salt and morpholine salt Salt: arginine salt,
Base addition salts such as amino acid salts such as lysine salts can be used.

As used herein, the term "RXR ligand" refers to a ligand affinity for retinoid X receptor (RXR), and represents the typical biological activity of retinoids (eg, cell differentiation, cell growth control). And its salts (generally referred to as “RXR agonists”), but not only specific ligands for RXR but also RXR and RAR. It is to be understood that compounds or salts thereof which exhibit an affinity are also included. For example, Ro47-5944 (Ap
fel, CM et al., J. Biol. Chem., 270, pp. 30765-307
72, 1995). Ligands that show affinity for both RAR and RXR include 9-cis retinoic acid (Levin, AA, et al., Nature, 3
55, pp. 359-361, 1992; Zhang, XK, etal., Nature,
358, pp. 587-591, 1992).

[0011] Further, all-trans retinoic acid is known as a ligand that substantially only binds to RAR (Levin et al. And Zhan et al., Supra). • May produce retinoic acid, which can act as an RXR ligand in vivo. It is to be understood that the RXR ligand in the present invention also includes a compound or a salt thereof capable of acting as an RXR ligand in vivo as described above. Whether or not a compound or a salt thereof acts as a ligand for RXR and / or RAR can be easily assayed according to the method described in the above-mentioned literatures of Levin et al. And Zhan et al. RX
As the R ligand, not only a compound in a free form but also a physiologically acceptable salt thereof may be used. For example, the base addition salts exemplified above can be used as the physiologically acceptable salts of 9-cis retinoic acid.

[0012] Difaranisol A or a salt thereof has a growth inhibitory effect on leukemia cells and a differentiation-inducing effect per se, but has a typical biological activity of RXR ligand (eg, cell differentiation action, cell growth control action). , And life sustaining action). More specifically, difaranisol A or a salt thereof has an action of enhancing the growth inhibitory action and differentiation-inducing action of 9-cis retinoic acid or a salt thereof on leukemia cells, and the enhancing action is synergistic. It is a target.

Accordingly, diffaranisol A or a salt thereof can be used to convert RXR ligand into vitamin A deficiency; skin diseases such as keratosis of epithelial tissue and psoriasis; allergic diseases; immunological diseases such as rheumatism; Diseases; Alzheimer's disease; Huntington's chorea; can be used as an agent for enhancing the action of the RXR ligand when administered as a medicament for the prevention and / or treatment of diseases such as leukemia and other cancers. Further, since difaranisol A or a salt thereof enhances the action of an RXR ligand (for example, retinoic acid) already present in a living body, even when RXR ligand is not administered for treatment and / or prevention of the above-mentioned diseases, The treatment and / or prevention of the above diseases can be achieved by enhancing the action of RXR ligand in vivo. Typically, diffaranisol A or a salt thereof and RX
It can be used as a pharmaceutical composition in the form of a so-called combination, which contains an R ligand (preferably 9-cis retinoic acid or a salt thereof) as an active ingredient. This combination can be applied as a therapeutic agent to any of myeloid leukemia and lymphocytic leukemia.

Pharmaceutical compositions suitable for oral administration include:
For example, tablets, capsules, powders, fine granules, granules, solutions, syrups and the like can be mentioned. Examples of the pharmaceutical composition suitable for parenteral administration include injections, drops, suppositories, Examples include inhalants, eye drops, nasal drops, ointments, creams, patches, and the like. Examples of pharmacologically and pharmaceutically acceptable additives for pharmaceuticals used in the production of the above pharmaceutical composition include, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents Pigments, diluents, bases, solubilizers or solubilizers, tonicity agents, pH regulators, stabilizers, propellants, adhesives and the like.

The dose of difaranisol A or a salt thereof is not particularly limited. When administered in combination with an RXR ligand, the dose of difaranisol A or a salt thereof is in the range of about 0.01 to 1,000 mg per adult per day. Administer RX
It is desirable to reduce the dose of the R ligand appropriately from the usual dose. By referring to the dosage in the above dosage form, the mixing ratio of difaranisol A or a salt thereof to the RXR ligand can be appropriately selected even when administered as a combination drug.

[0016]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to the following examples. (1) Materials and methods (a) Reagents etc. Diffaranisol A (3,5-dichloro-2-hydroxy-
4-methoxy-6-n-propylbenzoic acid: may be abbreviated as "Dif". ) Was prepared by the method described in the literature (Iimu
ra, Y. et al., Acta Cryst., C40, pp.2058-2061, 198
4; Mori, K. etal., Liebigs Ann. Chem., Pp. 303-305,
1989; Oka et al., J. Antibiotics, 38, pp. 1100-110
2, 1985). All-trans retinoic acid (hereinafter “ATR
A ". ) And nitroblue tetrazolium (NBT) are available from Sigma Chemicals (St. Louis,
MO). 1α, 25-dihydroxyvitamin D ₃
(Hereinafter sometimes abbreviated as “VD3”) and 9-cis retinoic acid (hereinafter sometimes abbreviated as “9cisRA”).
Are Wako Pure Chemical Industries, Ltd. and BIOMOL Research Laboratories (Premouth Meeting, PA,
USA).

Retinoic acid receptor (RAR) -specific ligand Am80 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramet
hyl-2-naphthalenyl) carbamoyl] benzoic acid; Hashimo
to, Y., et al., Biochem. Biophys. Res.Commun., 16
6, pp.1300-1307, 1990), using retinoid X receptor (RXR) ligand Ro47-5944 (Apfel, CM et al., J. Biol. Che), donated by Professor Shuto (The University of Tokyo).
m., 270, pp. 30765-30772, 1995) used was donated by F. Hoffman-La Roche.

(B) Cell line and cell culture Human myeloid leukemia cell line was supplemented with 10% fetal bovine serum.
The cells were suspended in RPMI-1640 medium and cultured in humidified air at 37 ° C. containing 5% CO ₂ . Peripheral blood was collected from a 28-year-old male patient with a t (15; 17) -positive APL (M3 variant according to the criteria proposed by the French, American and English committees) with informed consent before starting treatment. To purify leukemia cells, mix heparinized blood cells with an equal volume of RPMI-1640 medium,
Centrifuged on Ficoll-Hypaque (Pharmacia).

(C) Cell Proliferation and Assay of Differentiated Cell Properties The number of cells was determined using a ZM type Coulter counter (Coulter®
Electronics, Luton, UK). To assay differentiation-inducing ability, leukemia cells (5 × 10 ⁴ cells / m
l) was cultured for a predetermined period in the presence or absence of the test compound. When 12-O-tetradecanoylphorbol-13-acetate (TPA) was given to cells, oxidase that generated superoxide was measured using the ability to reduce NBT as an index. The NBT reducing ability was as follows: NBT (1 mg / ml) and TPA (100 ng
per ml of RPMI 1640 medium containing 1 × 10 ⁶ cells was incubated at 37 ° C. for 60 minutes. The percentage of cells containing intracellular blue-black formazan precipitate was measured under a microscope and at least 200 cells were counted.

[0020] Morphological differentiation was examined on cell smears stained with May-Grunwald-Giemsa. CD11b expression was determined by indirect immunofluorescence staining and flow
Analyzed by cytometry (Kanatani Y. et al.,
Cell Growth Differ. 7, pp.187-196, 1996). Briefly, leukemia cells (2 × 10 ⁶ ) were placed in phosphate buffered saline (P
BS), 50 μl of mouse anti-CD11b (Mac-1; Nichirei Co., Ltd.) in PBS containing 0.1% bovine serum albumin
And incubated at 4 ° C for 30 minutes. Wash cells with PBS, FIT in PBS containing 0.1% bovine serum albumin
After incubation in 50 μl of C-conjugated anti-mouse IgG (Tago) for 30 minutes, washing with PBS, analysis was performed by Epix XL flow cytometry (Coulter Electronics). Cell cycle was analyzed on a fluorescence activated cell sorter using nuclei stained with propidium iodide as described above.

(D) Transactivation assay of retinoid receptor CV-1 cells were subjected to receptor plasmid (pCMX-h) using Lipofectin reagent (GIBCO BRL, Gaithersburg, MD).
(RAR-α, pCMX-hRXR-α) 200 ng, receptor plasmid
Transfected with 500 ng and 300 ng of pCMX-β-gal (Umesono, K., et al., Cell, 65, pp. 1255-1266,
1991). The receptor used is TK-TREpx for RAR
2-LUC (Umesono, K., et al., Cell, 65, pp. 1255-1266,
1991) and RXR for TK-CRBPII-LUC (Mangelsdor
f, DJ, et al., Cell, 66, pp. 555-561, 1991). The cells were transfected for 18 hours, the medium containing DNA was removed, and then incubated with the test compound in medium containing 10% resin-charcoal-strept fetal bovine serum for 24 hours. Luciferase and β-galactosidase activities were analyzed using a luminometer (BLR-201; Aloka) and a spectrophotometer (Hitachi), respectively. All transfection data were normalized using an internal β-galactosidase marker and expressed as the average of three experiments.

(2) Results (a) Effect of Diffaranisol A on Proliferation and Differentiation of Human Leukemia Cell Line Diffaranisol A inhibited the proliferation of myeloid leukemia cells in a dose-dependent manner. When cells are treated for 5 days, ML-1,
IC for HL-60, NB4, THP-1, U937, and KU812 cells
₅₀ was 89-130 μM. The change in cell cycle distribution of HL-60 cells treated with various concentrations of difaranisol A for 24-72 hours was then measured. The results are shown in Table 1 (the results in the table show the mean ± SD of three measurements).

[0023]

[Table 1]

There was no significant change in the DNA histograms of cells treated with 60 μM difaranisol A,
The cultures treated with μM had accumulated cell number of ₁ phase G. When HL-60 cells (5 × 10 ⁴ / ml) were treated with difaranisol A for 7 days, the cells underwent slightly significant morphological differentiation (Table 2: three separate experiments). Mean ± SD). Functional differentiation (NBT reducing ability) was also induced by difaranisol A, but the differentiation inducing ability of difaranisol A was weak. In addition, difaranisol A also showed weak differentiation-inducing activity on monocyte leukemia U937 cells and erythroleukemia K562 cells.

[0025]

Table 2 ─────────────────────────────────── Diffaranisol A cell proliferation Morphological changes ( %) (ΜM) (% of control value) Bla + Pro Mye Met Neu ────────────── ───────────────────── 0 100 ± 7 87 ± 5 10 ± 3 3 ± 1 0 60 85 ± 5 65 ± 4 17 ± 3 5 ± 3 13 ± 1 120 40 ± 5 45 ± 4 25 ± 2 8 ± 3 22 ± 5 180 15 ± 3 34 ± 4 23 ± 2 10 ± 3 33 ± 2 ───────────────── ──────────────────

(B) Effect of Combination of Diffaranisol A and Differentiation Inducing Agent The effect of diffaranisol A on proliferation and differentiation of leukemia cells in the presence of ATRA, 9cisRA, or VD3 was examined. Diffaranisol A enhances the growth inhibition of HL-60 cells and the induction of NBT reduction ability induced by these substances, and the effects are synergistic, and 9cisRA has a particularly remarkable synergistic effect ( (Fig. 1). Similar results were obtained for the expression of the CD11b surface antigen, another marker for myeloid monocyte-like differentiation (FIG. 2). The activity of inhibiting proliferation and inducing differentiation of promyelocytic leukemia NB4 cells with (15; 17) chromosomal translocation by difaranisol A and 9cisRA was synergistically induced (FIG. 3).

Next, difaranisol A and NF4 in the NB4 cell growth-inhibiting and differentiation-inducing action (NBT reducing ability)
The time effect of cisRA was examined. Pre-incubation of NB4 cells with difaranisol A for 24 hours
Had essentially no effect on the growth-inhibiting and differentiation-inducing activity of, and the preincubation of 9cisRA did not promote the growth-inhibiting effect of difaranisol A (Table 3). Similar results were obtained when pre-treated for 72 hours. On the other hand, when co-treated with 4 nM 9cisRA and 60 μM difaranisol A, remarkably high growth inhibitory activity and differentiation induction activity were observed. Experiments were performed on NB4 cells 6
Treat with 0 μM difaranisol A or 4 nM 9cisRA for 1 day, and then treat with 60 μM difaranisol A or 4 nM 9cisRA.
Further, the cells were cultured in a fresh medium containing 5 days, and the results were shown in Table 3. The average values ± SD of three experiments are shown in Table 3.

[0028]

[Table 3] ─────────────────────────────────── Test treatment Cell proliferation NBT reduction ability ──── ──── (% of control value) (% of positive cells) 0-1 1-6 days 日──────── 1 None None 100 ± 2 5.2 ± 0.7 2 None Dif 83 ± 4 14.2 ± 0.9 3 None 9cisRA 113 ± 9 47.4 ± 3.7 4 None Dif + 9cisRA 42 ± 5 89.2 ± 7.2 5 Dif None 105 ± 7 4.4 ± 0.6 6 Dif 9cisRA 115 ± 3 44.5 ± 3.6 7 9cisRA None 109 ± 3 4.9 ± 0.5 8 9cisRA Dif 95 ± 1 7.8 ± 0.6 ────────────────── ─────────────────

(C) RXR or RAR of diffaranisol A
9cisRA is a ligand for both RAR and RXR,
ATRA is a ligand that binds only to RAR (Levin, A.
A., et al., Nature, 355, pp. 359-361, 1992; Zhang,
XK, et al., Nature, 358, pp. 587-591, 1992). However, ATRA may change to 9cisRA to some extent by metabolism. In order to understand the properties of diffaranisol A, the synergistic effect of diffaranisol A with RAR-specific or RXR-specific ligands was examined. Am47 was used as the RAR-specific ligand, and Ro47-5944 was used as the RXR-specific ligand.
Was used. The results of treating HL-60 cells with various concentrations of Am80 or Ro47-5944 in the presence of difaranisol A are shown in FIG. The results show that the growth inhibitory effects of Am80 and difaranisol A are additive, while Ro47-59
It is clear that the effects of 44 and difaranisol A are synergistic. ATRA also synergistically inhibited the growth of HL-60 cells in the presence of Ro47-5944.

Peripheral blood mononuclear cells (> 80% blasts or promyelocytes) were isolated from APL patients (white blood cell count of 20,000 / μl). The leukemia cells reacted with ATRA and 9cisRA but not with difaranisol A (FIG. 5). However, difaranisol A
Significantly enhanced the retinoid-induced NBT-reducing ability of these cells (0.4 nM 9cisRA plus 60 μM difaranisole A, 88% were NBT-positive and 0.4 nM 9cisRA
Alone was 40%). The promoting effect of difaranisol A was also observed in the morphological changes of cells treated with 9cisRA.

(D) Effect of Diffaranisol A on Transactivation of Retinoid Receptor It is possible that diffaranisol A may partially act as a RAR agonist. Therefore, CV-1 cells were transfected with the receptor plasmids (pCMX-hRARα and pCMX-hRXR-α) and the luciferase reporter plasmid, and the effect of diffaranisol A on receptor-mediated transactivation was examined. No addition of CV-1 cells transfected with plasmid, or
Incubated for 24 hours in the presence of 10 ^-4 M diffaranisol A (DIF), 10 ^-6 M ATRA, or 10 ^-7 M 9cisRA. The RARα and RXRα genes were introduced into the pCMX vector. The results are shown in Table 4 (the values in the table indicate the ratio to the luciferase activity in unstimulated cells, and are shown as the average of three times. NT indicates that it was not tested). Diffaranisol A is TK-TREpx2-LUC
It did not stimulate the transactivating activity of RARα in the reporter, but did significantly increase ATRA. Difaranisol A also showed no effect on RXR. These results show that difaranisol A
Does not act as an agonist for RAR or RXR.

[0032]

[Table 4] 還 元 Receptor reporter luciferase reduction (ratio) ──── ─────────────── DIF ATRA 9cisRA ──────────────────────────────── ─── pCMX TK-TREx2-LUC 1.0 4.6 NT pCMX-RARα 1.2 40.8 NT pCMX-RXRα TK-CRBPII-LUC 0.9 NT 4.6 ───────────────────── ──────────────

[Brief description of the drawings]

FIG. 1 shows the effect of difaranisole A on proliferation and differentiation of leukemia cells HL-60 in the presence of all-trans retinoic acid, 9-cis retinoic acid, or 1α, 25-dihydroxyvitamin D ₃ (cultured for 5 days). FIG. In the figure, (a) is 0 (○), 3 (□), and 30 (▲) n
M shows the results in the presence of 1α, 25-dihydroxyvitamin D ₃ (VD3); (b) 1 (△), 4 (□), 8 (（), and 10 (■) nM all-trans retinoin The results in the presence of acid (ATRA) are shown; (c) is 1 (△), 4 (□), 8
(●) and 10 (■) nM of 9-cis retinoic acid (9cisR
A) shows the results in the presence. Results are shown as the average of four independent experiments.

FIG. 2 All-trans retinoic acid, 9-cis retinoic acid, or 1α, 25-dihydroxyvitamin D ₃ (VD3)
HL of difaranisol A in the presence of HL
FIG. 3 shows the effect of -60 on the expression of CD11b surface antigen, a myeloid monocyte-like differentiation marker. In the figure, (a) shows 3 nM 1α, 25-dihydroxyvitamin D ₃ (V
D ₃ ) shows the results in the presence; (b) shows the results in the presence of 4 nM all-trans retinoic acid (RA); (c) shows the results in the presence of 4 nM.
The results are shown in the presence of nM 9-cis retinoic acid (9cisRA). The solid line represents 3 in the presence of 60 μM difaranisol A.
The results obtained after culturing for one day are shown, and the dashed line shows the results in the absence of difaranisol A. Marker expression is anti-CD11
b Performed by immunofluorescence staining using an antibody. In the figure, a thin line indicates an untreated control, and a dotted line indicates a negative control for immunofluorescence measurement.

FIG. 3. Effect of difaranisole A on proliferation and differentiation of promyelocytic leukemia NB4 cells in the presence of all-trans retinoic acid, 9-cis retinoic acid, or 1α, 25-dihydroxyvitamin D ₃ (cultured for 5 days) FIG. In the figure, (a) is 0 (○), 3 (△), and 30
(▲) shows the results in the presence of nM of 1α, 25-dihydroxyvitamin D ₃ (VD3); (b) shows 4 (□) and 40 (■) nM
Shows the results in the presence of all-trans retinoic acid (ATRA); (c) shows the results in the presence of 4 (□) and 40 (■) nM 9-cis retinoic acid (9cisRA). Results are shown as the average of four independent experiments.

FIG. 4 shows the results of treating HL-60 cells with various concentrations of Am80 or Ro47-5944 for 5 days in the presence of difaranisol A. In the figure, (a) is 0 (o), 30
(△), 60 (□), and 90 (■) show the effect of Am80 in the presence of μM difaranisol A; (b) shows 0 (○),
(C) shows the effect of Ro47-5944 in the presence of 30 (、), 60 (□), and 90 (■) μM of difaranisol A;
(○), 40 (▲), and 400 (◆) show the effect of Ro47-5944 in the presence of nM all-trans retinoic acid. Results are shown as the average of four independent experiments.

FIG. 5 is a graph showing the effects of difaranisol A and retinoids in a primary culture system of peripheral blood mononuclear cells isolated from an APL patient. In the figure, (a) is 0 (o), 30
(△) and 60 (□) show the effect of all-trans retinoic acid (ATRA) in the presence of μM difaranisol A; (b) shows 0 (○), 30 (△), and 60 (□) 9) 9-cis retinoic acid in the presence of μM difaranisol A
(9cisRA). Culture was performed for 7 days. The result is 3
Shown as the average of two experiments.

Claims (5)

[Claims] 1. An RXR ligand bioactivity enhancer comprising as an active ingredient difaranisol A or a physiologically acceptable salt thereof. 2. A medicament comprising difaranisole A or a physiologically acceptable salt thereof as an active ingredient and used for enhancing the action of a medicament comprising an RXR ligand as an active ingredient. 3. The medicament according to claim 2, wherein the RXR ligand is 9-cis retinoic acid. 4. A therapeutic agent for leukemia, comprising as active ingredients difaranisol A or a physiologically acceptable salt thereof and an RXR ligand. 5. The therapeutic agent according to claim 4, wherein the RXR ligand is 9-cis retinoic acid.