WO2004026319A2

WO2004026319A2 - Pharmaceutical compositions increasing camp useful for the treatment of cancers

Info

Publication number: WO2004026319A2
Application number: PCT/EP2003/010280
Authority: WO
Inventors: Hugues De The; Marie-Claude Colette Raymonde Guillemin-Debons; Michel Lanotte; Laurent Degos
Original assignee: Centre National De La Recherche Scientifique; Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2002-09-17
Filing date: 2003-09-16
Publication date: 2004-04-01
Also published as: WO2004026319A3; AU2003270202A8; AU2003270202A1

Abstract

The present invention relates to the use of at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives, for the preparation of a drug for the treatment of cancers.

Description

PHARMACEUTICAL COMPOSITIONS USEFUL FOR THE TREATMENT OF CANCERS

The present invention relates to new pharmaceutical compositions useful for the treatment of cancers.

Cancers are a group of pathologies characterized, in particular, by abnormal cell proliferation. Among the many strategies used to cure cancers, one of them relies on the induction of cell differentiation to halt the multiplication of cancerous cells. As such, certain leukemia, notably acute myeloid leukemia, are characterized by an arrest in differentiation, induction of proliferation and repression of normal hematopoiesis. These malignancies are often associated to recurrent chromosomal translocations, most of which encode fusion proteins derived from transcription factors (1). Functional analyses of several of these fusion proteins have shown that they behave as potent transcriptional repressors (2), often through modifications of chromatin structure by histone desacetylases, blocking expression of unidentified genes that control myeloid differentiation. Transcription therapy attempts to re-express these genes, resulting in restoration of differentiation. Inhibition of desacetylases by a variety of compounds has shown some efficacy in cell culture (3) and in animal models of leukemia (4), but, as yet, there is only little evidence for their beneficial effects in clinical settings (5). To date, the only real clinical success of transcription/differentiation therapy is acute promyelocytic leukemia (APL), for which two drugs, retinoic acid (RA) and arsenic trioxide (As₂O₃) induce remissions (6, 7). Remarkably, these two drugs target the oncogenic PML/RΛRα fusion protein and reverse PML/RARα-mediated repression (8). However, in certain cases resistance to both drugs has arisen.

Cyclic AMP (cAMP, adenosine 3 '-5' cyclic monophosphate), or its derivatives, could also be viewed as a drug of choice for the induction of differentiation. Indeed, ex vivo, activation of the cAMP signal transduction pathway differentiates many acute myeloid leukemia cell-line and strongly synergizes with other differentiating agents

((9-11), reviewed in (12)). Yet, a number of acute toxicities have precluded or severely limited in vivo trials using cAMP, or its derivatives (13), and its potential benefits in the treatment of cancers have never been soundly assessed. Thus, an object of the present invention is to provide new pharmaceutical compositions, comprising at least one compound activating the cAMP signal transduction pathway, useful for the treatment of cancers.

The present invention relates to the use of at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives, for the preparation of a drug for the treatment of cancers. cAMP corresponds to the following formula:

cAMP

The derivatives of cAMP are well known to the man skilled in the art, they notably comprise 8-Cl-cAMP, 8-CPT-cAMP, 8-Br-cAMP and dibutyryl-cAMP, or pharmacologically acceptable salts thereof.

The formulae of several derivatives of cAMP are shown below:

8-Cl-cAMP 8-Br-cAMP

8-CPT-cAMP dibutyryl-cAMP

The "originally present cellular content of cAMP" relates to the cAMP content of cells prior to the addition to said cells of any compound liable to modify the cellular concentration of cAMP .

The cellular content of cAMP, or of its derivatives, can be measured according to methods well known to the man skilled in the art.

The cAMP content of a cell results from an equilibrium between two opposite reaction types, i.e. reaction concurring to the synthesis of cAMP, such as reactions catalyzed by adenylate cyclases, and reactions concurring to the degradation of cAMP, such as reactions catalyzed by phosphodiesterases (PDE). Consequently, a rise in the cellular content of cAMP can be observed following addition of compounds either activating cAMP synthesis or inhibiting cAMP degradation. Thus, an "agent enabling to increase the cellular content of cAMP or derivatives thereof, can be for instance, cAMP or a derivative thereof in itself, or an agent activating the intracellular synthesis of cAMP, or an agent inhibiting the intracellular degradation of cAMP or derivatives thereof, provided it is added to cells in an amount sufficient to lead to an increase of the cAMP content of said cells.

The present invention also relates to the use of:

- at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives and

- at least one cell-differentiation factor or precursors or derivatives thereof and/or

- at least one apoptotic inducer for the preparation of a drug for the treatment of cancers.

A "cell differentiation factor" refers to compounds liable to induce cellular differentiation, such as retinoic acid, interferons, cytokines or growth factors.

An "apoptotic inducer" refers to compounds liable to induce programmed cell death, such as cancer chemotherapeutic agents or arsenic derivatives (As₂O₃, As₄S₄).

According to an advantageous embodiment, the invention relates to the use of:

- at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives and - at least one cellular differentiating factor or precursors or derivatives thereof for the preparation of a drug for the treatment of cancers.

Advantageously this association is synergic. According to another advantageous embodiment, the invention relates to the use of:

- at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP and

- at least one apoptotic inducer for the preparation of a drug for the treatment of cancers. Advantageously this association is synergic. According to another advantageous embodiment, the invention relates to the use of:

- at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives,

- at least one cellular differentiating factor or precursors or derivatives thereof and

Advantageously this association is synergic.

In an advantageous embodiment of the invention, the agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives is selected from the group comprising cAMP, 8-Cl-cAMP, 8-CPT-cAMP, 8-cAMP, dibutyryl-cAMP or pharmacologically acceptable salts thereof. Particular cAMP derivatives can be selected according to their respective properties, well known to the man skilled in the art, such as stability, solubility, efficacity, or toxicity, as compared to cAMP or other cAMP derivatives, for a given use.

The invention relates more particularly to the abovementioned uses, wherein the agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives is a phosphodiesterase inhibitor. cAMP, and other cyclic nucleotides, are respectively hydrolyzed to AMP, and to the corresponding acyclic nucleotide, by phosphodiesterases (PDE); phosphodiesterase inhibitors limit the hydrolysis of cAMP, and of other cyclic nucleotides, and thus enable to increase the cellular content of cAMP, and of other cyclic nucleotides, as discussed above.

The invention also relates to the preceding use, wherein the phosphodiesterase inhibitor is selected from the group comprising methylxanthines such as caffeine or theophylline or aminophylline or isobutyl-methylxanthine, rolipram, sildenafil, vardenafil, zaprinast, or methoxyquinazoline.

Xanthine corresponds to the following formula:

Methylated derivatives of xanthine are phosphodiesterase inhibitors well known to the man skilled in the art and correspond for example to:

- Theophylline (1,3 dimethylxanthine):

Aminophylline (1,3 dimethylxanthine complexed to diaminoethan):

Advantageously aminophylline has an increased solubility as compared to theophylline. - Caffeine (1,3,7 trimethylxanthine):

Isobutyl-methylxanthine (3-isobutyl-l-methylxanthine)

Rolipram, sildenafil (Viagra®), vardenafil, zaprinast, or methoxyquinazoline, are phosphodiesterase inhibitors well known to the man skilled in the art.

Rolipram

Sildenafil

Vardenafil

Zaprinast methoxyquinazoline (MQZ)

The invention more particularly relates the above mentioned use, wherein the phosphodiesterase inhibitor is theophylline or aminophylline.

According to yet another embodiment, the invention relates to the preceding uses wherein the cell-differentiation factor or precursors or derivatives thereof is selected from the group comprising retinoic acid, particularly all-trans retinoic acid or 9-cis retinoic acid or 13-cis retinoic acid, or pharmacologically acceptable salts thereof, vitamin A (retinol), carotene or rexinoids.

All-trans retinoic acid

13-cis retinoic acid

9-cis retinoic acid Retinol (all-trans)

Carotene (β configuration)

Rexinoids are specific ligands of the RXR receptors, they notably comprise LG 100268 (LG268) or LGD 1068 (Targretin) for example.

LGD 1068 LG268 The invention also relates to the abovementioned use wherein the cell- differentiation factor or precursors or derivatives thereof is retinoic acid, particularly all-trans retinoic acid, or pharmacologically acceptable salts thereof. According to another particular embodiment, the invention relates to the above mentioned use, wherein the apoptotic inducer is selected from the group comprising arsenic trioxide (As₂O₃) or arsenic sulfide (As₄S₄).

Arsenic trioxide is notably described in Chen et al. (1996) Blood 88:1052-1061.

Arsenic sulfide is notably described in Lu et al. (2002) Blood 99:3136-3143. . Both compounds have similar properties and act on similar cellular targets.

According to still another particular embodiment, the invention relates to the above mentioned use of theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof, for the preparation of a drug for the treatment of cancers. Advantageously theophylline, or aminophylline, synergizes with retinoic acid.

The present invention also relates to the abovementioned use of theophylline or aminophylline and arsenic trioxide or arsenic sulfide, for the preparation of a drug for the treatment of cancers.

Advantageously theophylline, or aminophylline, synergizes with arsenic trioxide, or arsenic sulfide.

The present invention equally relates to the abovementioned use of theophylline or aminophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, for the preparation of a drug for the treatment of cancers.

Advantageously the association of theophylline, or aminophylline, and retinoic acid, and arsenic trioxide, or arsenic sulfide, is synergic.

According to a further embodiment, the invention relates in particular to abovementioned uses, wherein the cancers are selected from the group comprising solid tumor cancer, neuroblastoma, skin cancer, oral cavity cancer, lung cancer, mammary gland cancer, prostatic cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, ovarian cancer, head and neck cancer, colon cancer, germ cell cancer, leukemia, acute leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, aleukemic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia. According to another aspect, the invention relates to products containing 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment. The invention relates in particular to products as defined above, containing 8-C1- cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

The invention more particularly relates to the above defined products, containing 8- Cl-cAMP or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

The invention further relates to the abovementioned products, containing 8-C1- cAMP or pharmacologically acceptable salts thereof, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

According to another embodiment, the invention relates to products containing aminophylline or theophylline and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

The invention relates in particular to products as defined above, containing aminophylline or theophylline and retinoic acid or pharmacologically acceptable salts thereof, as a combined preparation for simultaneous, separate or sequential use in cancer treatment. The invention also relates to products as defined above, containing aminophylline or theophylline and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

Advantageously, the invention relates to products as defined above, containing aminophylline or theophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

According to yet another aspect, the present invention also relates to a pharmacological composition comprising as active substance 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

The invention relates in particular to a pharmacological composition as defined above, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof, in association with a pharmacologically acceptable vehicle.

The invention also relates to a pharmacological composition as defined above, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

The invention further relates to a pharmacological composition as defined above, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle. The present invention also relates to a pharmacological composition comprising as active substance theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

The invention relates in particular to a pharmacological composition as precedingly defined, wherein the active substance is theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof, in association with a pharmacologically acceptable vehicle.

Advantageously the invention relates to a pharmacological composition as defined above, wherein the active substance is theophylline or aminophylline and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

The invention also relates to an abovementioned pharmacological composition, wherein the active substance is theophylline or aminophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle. The invention relates in particular to a pharmacological composition as defined above, in a form appropriate for the administration of about 0.36 mg/kg/day to about 14.3 mg/kg/day of theophylline or aminophylline, of about 4.5 mg/m²/day to about 135 mg/m²/day of all-trans retinoic acid and of about 0.014 mg/kg/day to about 0.43 mg/kg/day of arsenic trioxide. Brief description of the figures

Figure 1A, Figure IB, Figure 1C, Figure ID, Figure IE, Figure IF, Figure 1G, Figure 1H, Figure II, Figure 1J

Figure 1 A represents the spleen weight (vertical axis, mg) of retinoic acid sensitive mice treated (+) or untreated (-) by 8-Cl-cAMP during 7 days.

Figure IB represents the spleen weight (vertical axis, mg) of retinoic acid sensitive mice treated (+) or untreated (-) by 8-Cl-cAMP during 7 days. Figures 1C, ID, IE et IF represent pictures of bone marrow samples, after May-

Grϋnwald-Giemsa (MGG) staining, taken from retinoic acid sensitive mice treated

(figure ID) or untreated (figure IE) by 8-Cl-cAMP and from retinoic acid resistant mice treated (figure IF) or untreated (figure 1G) by 8-Cl-cAMP.

Figures 1G, 1H, II et 1 J represent pictures of liver samples, after hematoxylin-eosin staining, taken from retinoic acid sensitive mice treated (figure 1G) or untreated

(figure 1H) by 8-Cl-cAMP and from retinoic acid resistant mice treated (figure II) or untreated (figure 1 J) by 8-Cl-cAMP.

In figure IF an apoptotic cell is marked by an arrow.

Figure 2A, Figure 2B, Figure 2C, Figure 2D, Figure 2E, Figure 2F

Figure 2A represents the spleen weight (vertical axis, mg) of a retinoic acid sensitive mouse model of APL treated during 3 days by 8-Cl-cAMP (cAMP), As₂O₃ (As), 8-C1- cAMP and As₂O₃ (cAMP + As), or untreated (0).

Figure 2B represents a western blot of protein extracts of bone of retinoic acid f sensitive APL mice revealed by an anti-p21 antibody (arrow). The APL mice were either treated 24 h in vivo by retinoic acid (RA), As₂O₃ (As), 8-Cl-cAMP (cAMP), or untreated (0). The star (*) denotes to a cross-reactive protein.

Figure 2C represents a picture of a bone marrow sample, after May-Griinwald-Giemsa

(MGG) staining, taken from retinoic acid sensitive APL mice treated by 8-Cl-cAMP (cAMP), As O₃ (As), 8-Cl-cAMP and As₂O₃ (cAMP + As), during 3 days, or untreated (0).

Figure 2D represents a picture of a liver sample, after hematoxylin-eosin staining, taken from retinoic acid sensitive APL mice treated by 8-Cl-cAMP (cAMP), As₂O₃

(As), 8-Cl-cAMP and As₂O₃ (cAMP + As), during 3 days, or untreated (0). Figure 2E represents a picture of a bone marrow sample, after May-Griinwald-Giemsa (MGG) staining, taken from retinoic acid sensitive APL mice treated by theophylline (T), As₂O₃ (As), theophylline and As₂O₃ (T + As), during 3 days, or untreated (0). Figure 2F represents the percentage of NBT positive NB4 cells (vertical axis) treated by As₂O₃ at various concentrations (from left to right) 0, 10^"10, 10^"9, 10^"8, 10^"7, 10^"6 M, in the presence (+) or the absence (-) of 8-Cl-cAMP.

Figure 3A, Figure 3B, Figure 3C, Figure 3D

Figure 3 A represents the spleen weight (vertical axis, mg) of a retinoic acid sensitive mouse model of APL treated during 7 days by retinoic acid (RA), 8-Cl-cAMP

(cAMP), retinoic acid and 8-Cl-cAMP (RA + cAMP), or untreated (0).

Figure 3B represents a picture of a bone marrow sample, after May-Griinwald-Giemsa

(MGG) staining, taken from retinoic acid sensitive APL mice treated by retinoic acid during 7 days. Figure 3C represents a picture of a bone marrow sample, after May-Griinwald-Giemsa

(MGG) staining, taken from retinoic acid sensitive APL mice treated by retinoic acid and 8-Cl-cAMP during 7 days.

Figure 3D represents the number of bone marrow blasts (horizontal axis) marked by an anti-CD 11 antibody (vertical axis) of APL mice treated for 24 hours by cAMP (cAMP), As₂O₃ (As), 8-Cl-cAMP and As₂O₃ (As + cAMP) or untreated (0).

Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E, Figure 4F, Figure 4G, Figure 4H, Figure 41, Figure 4 J

Figure 4A represents the spleen weight (vertical axis, mg) of a retinoic acid resistant mouse model of APL treated during 7 days by 8-Cl-cAMP (cAMP), As₂O₃ (As), retinoic acid (RA), retinoic acid and 8-Cl-cAMP (RA + cAMP), As₂O₃ and 8-C1- cAMP (As + cAMP), retinoic acid and As₂O₃ (RA + As), or untreated (0).

Figures 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, 4J represent pictures of bone marrow samples, after May-Griinwald-Giemsa (MGG) staining, taken from retinoic acid resistant APL mice treated by 8-Cl-cAMP (Figure 4C), As₂O₃ (Figure 4E), retinoic acid (Figure 4D), retinoic acid and 8-Cl-cAMP (Figure 4F, 4G), As₂O₃ and 8-Cl-cAMP (Figure 4H), retinoic acid and As₂O₃ (Figure 41), or untreated (Figure 4B). Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E, Figure 5F, Figure 5G

Figures 5A, 5B and 5C represent pictures of bone marrow samples taken at day 0 (figure 5A), at day 14 (figure 5B) or at day 28 (figure 5C) from a retinoic acid/As2O3 resistant APL patient treated by a combined retinoic acid As2O3 therapy. Figures 5D, 5E and 5F represent pictures of bone marrow samples taken at day 0

(figure 5D), at day 14 (figure 5E) or at day 28 (figure 5F) from the same retinoic acid/As2O3 resistant APL patient treated by a combined retinoic acid/As2O3/theophylline therapy. Figure 5G is a schematic representation of the clinical events of the treatment of a patient by a combined theophylline/ As2O3/RA therapy. The horizontal axis represents the time course in weeks. The left upward vertical axis and the corresponding curves represent hemoglobin concentration (Hb) in gr/1 and the platelet (Pit) count per mm³ (times 10³), the right upward vertical axis and the corresponding curve represent the white blood cells (WBC) count per mm³ (times 10³). The left downward vertical axis and the left bars (dark gray) represent the blast count in percent, the right downward vertical axis and the right bars (light gray) represent the erythroblast count in percent. The upper downward arrows represent red cells transfusions and the lower downward arrows represent platelet transfusions. The three upper horizontal black bars at the bottom of the figure represent the periods of combined As2O3/all-trans retinoic acid treatment (As₂O₃/ATRA), the lower horizontal black bar at the bottom represent the period of theophylline treatment.

EXAMPLES

EXAMPLE 1 cAMP synergizes with AS2O₃ to differentiate APL cells ex vivo cAMP is well-known to greatly enhance RA-induced (retinoic acid) differentiation of many cell lines derived from embryonal carcinoma or myeloid leukemia, in particular APL (acute promyelocytic leukemia) (11). Low concentrations of As₂O can induce incomplete differentiation in an APL cell line (7). The Inventors tested the hypothesis that cAMP would also enhance As O₃-induced differentiation. The APL model cell line NB4 was cultured as described previously in Lanotte et al.

(1991) Blood 77:1080-1086. Morphology and cellular differentiation were evaluated on May-Griinwald-Giemsa-stained cytospins. Differentiation was quantified by reduction of nitroblue-tetrazolium (NBT) according to procedure well known to the man skilled in the art. 8-Cl-cAMP (8-Chloro-adenosine 3-5' cyclic monophosphate) and 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine 3-5' cyclic monophosphate (15), a low toxicity cAMP analogue) were obtained from Biolog Life Research Institute (Bremen, Germany) and Sigma (St. Louis, MI), respectively. 8-CPT-cAMP was used at a concentration of 2.10^"4M.

As shown in Figure 2F, even very low doses of As₂O₃ (Sigma) combined with 8- CPT-cAMP induced NBT reduction in 40% of the cells, while cAMP or As₂O₃ alone had insignificant effects. Higher As₂O₃ concentrations inhibited NBT reduction, as reported. Similarly, only combined As₂O₃ and 8-CPT-cAMP induced morphological differentiation into myelocyte-like cells, consistent with a very recent report (17).

EXAMPLE 2

Antileukaemic effects ofcAMP in two models of APL mice

To assess a possible in vivo efficiency of cAMP, the Inventors turned to a transplantation APL model (14) derived from RA-sensitive PML/RARa transgenics (18). The Inventors similarly developed a transplantation model for RA-resistant APL using leukemic cells from PML/RARa transgenics in which a point mutation in the transgene impairs the binding of RA to PML/RARo: (16). In vivo growth of this leukemia is much slower, liver or spleen invasion is not as pronounced and either RA or As₂O₃ have modest anti-proliferative effects in the absence of significant differentiation (see Figures 4 A, 4D and 4EV Spleen-derived leukemic blasts (10⁷) were serially passaged in syngenic FN/B mouse (6 weeks old, weighting 20 g), as previously described (14). Both RA-sensitive leukemia (strain 935) or RA-resistant ones (strain 4048 (16)) were used. Animals were treated according to institutional guidelines. All experiments involving mice were repeated between 2 and 8 times, usually with two mice in each treatment arm. Alzet pumps (0.5 μl/h, Cupertino, CA) were loaded with 8-Cl-cAMP (20 mg/ml) and implanted subcutaneously on the back of treated mice. Aminophylline, a soluble precursor of theophylline, was injected intraperitonealy (100 μl/day of a 25 mg/ml solution, Renaudin, France). All-trans retinoic acid (Innovative Research of America, Sarasota, FL) and As₂O₃ treatments, autopsies and cellular or tissue analyses were performed as previously described (14). For Western-blot, a p21 monoclonal antibody (Pharmingen, San Diego, CA) was used at a 1/500 dilution. Dosage of plasma 8-C1- cAMP was performed by HPLC using a C18 column (Chromosep Inertil 5 ODS3) with a 15% methanol/50 mM pH 5.85 phosphate buffer as a mobile phase and UN. detection at 254 nm.

Animals bearing established leukemia were treated with 8-Cl-cAMP, As₂O₃, RA or combinations of these drugs and sacrificed 1 to 7 days post-treatment. Continuous 8- Cl-cAMP infusions allowed significant plasma concentrations to be reached (1 μM on average at day 3). Despite its toxicity, this compound induced major anti-leukemic effects in both RA-sensitive and RA-resistant APLs, assessed by the spleen weight

(Figures 1A, IB), liver infiltration (Figures lC-lF) or marrow infiltration (Figures 1G-1J). Differentiated cells were consistently observed in the marrow after 7 days of treatment in RA-resistant APL (Figure IF), and often found in RA-sensitive APL

(Figure ID). In leukemic cells infiltrating the liver, a sharp reduction in the number of j mitosis was observed (3% post-treatment vs 28% pretreatπient) and, although a few condensed nuclei were seen, TUΝEL assays remained negative, suggesting that 8-C1- cAMP mainly triggers growth arrest. Yet, apoptosis was also noted in the bone marrow in some experiments (Figure IF). Furthermore, the treatment restored normal liver architecture for RA-sensitive as well as for RA-resistant animals as evidenced by Figures 1G-1J. Altogether, in these RA-sensitive or RA-resistant mouse models of

APL, cAMP triggers a combination of growth arrest, differentiation and apoptosis, resulting in dramatic regressions of the leukemia. Yet, in most cases, cAMP was unable to eradicate APL. Since cAMP greatly increases As₂O₃-triggered differentiation ex vivo (see Example 1), the Inventors associated 8-Cl-cAMP and As₂O₃ treatments in vivo in a RA- sensitive mouse model of APL. With this combined treatment, the spleen, liver and bone marrow became leukemia-free between days 1 and 3, while animals treated with As₂O₃ alone retained a significant tumor burden consisting of differentiating leukemic cells (Figures 2A, C and D). Interestingly, erythroblasts and megakaryocytes were extremely numerous in a cell-rich marrow (Figure 2C), consistent with the idea that cAMP promotes regrowth of these cells. In keeping with ex vivo results, cAMP synergized with As₂O to trigger differentiation, as was evidenced by CD lib expression on bone marrow blasts at day 1 or 2 by fluo-cytometry (Figure 3D), strongly suggesting that enhanced differentiation of the leukemic blasts contributes to accelerated remissions. The cdk inhibitor p21, a known cAMP target implicated both in growth arrest, differentiation and apoptosis, was sharply induced in bone marrow APL blasts (Figure 2B). Some synergy was also noted with RA with respect to both spleen weight and marrow differentiation, although RAs' much stronger differentiating effect dims the effect (Figure 3A-3C).

The stable cAMP derivative used here induces massive diuresis, precluding any long-term use, and may further be metabolized into potentially cytotoxic nucleotide analogues (15). To ensure that the antileukemic effect indeed results from activation of cAMP signaling, the Inventors used theophylline (under its stabilized form aminophylline), a phosphodiesterase inhibitor which stabilizes pools of endogenous intracellular cAMP, in our RA-sensitive APL model. Theophylline, similar to 8-C1- cAMP, blocked APL growth and induced some apoptosis, accompanied by nonterminal differentiation (Figure 2E). As expected, enhancement of differentiation was more pronounced for As₂O₃ than for RA (Figure 2E). Yet, there was no obvious boost in normal haematopoiesis, possibly reflecting low production of endogenous cAMP in these cells.

8-Cl-cAMP induced even more dramatic regressions in RA-resistant APLs, with some morphologically complete clearances (Figures IB. IE, IF, II, 1J and 4A-4I). Unexpectedly, a major enhancement in leukemia clearance and differentiation was consistently observed when RA was combined to 8-Cl-cAMP (Figure 4A. 4G). Synergistic effects with As₂O₃ were also noted.

EXAMPLE 3 Theophylline induces remission in aRA- and As- resistant APL patient

With these promising results in mouse APLs, a RA/As₂O₃-resistant APL patient was offered an experimental course of combined RA/As₂O₃/theophylline therapy.

The patient gave informed consent for use of theophylline to enhance RA/ As₂O₃ differentiation. The daily treatment was with RA 45 mg/m² P.O., As₂O₃: 10 mg IN., theophylline 250 mg P.O.

Previously, a month of RA/As₂O₃ association had yielded a slow decrease in bone marrow blasts, a peak of differentiating myeloid cells in the blood, but normal haematopoiesis was not restored and the leukemia reappeared 5 weeks later (Figure 5A-5C, 5G). With combined RA/As₂O₃/theophylline, leukemic cells underwent differentiation and normal erythroblasts rapidly appeared (Figure 5D-5G). The patient no longer required transfusions and platelets, white blood cells and hemoglobin levels reached sub-normal levels. The patient remained leukemia-free for 4 months and then the leukemic clone reappeared. Paradoxically with ongoing RA/As₂O₃/theophylline therapy and despite leukemia relapse, normal hematopoiesis was maintained to date (5 months since relapse), a situation highly unusual in APL where cytopenia is the first sign of relapse.

These data demonstrate that, through a combination of growth arrest, apoptosis and differentiation, in vivo activation of cAMP signaling is beneficial in two distinct animal models of APL, as well as in a RA As₂O -resistant APL patient. The relative balance between growth arrest, apoptosis and differentiation likely depends on the dose of the compound, the microenvironment of the APL blasts (marrow vs. liver metastasis, for example) and their nature (RA-sensitive or RA-resistant cells). At present, it is difficult to explain the greater 8-Cl-cAMP sensitivity of RA-resistant

\ APLs, although it might be explained by their slower growth rates. As₂O was initially believed to trigger apoptosis, but in vivo observations (14), as well ex vivo data (Fig. 2)

(17), now implicate differentiation as alternative mechanism (reviewed in (19)). The dramatic in vivo enhancement of As O₃ triggered differentiation by 8-Cl-cAMP further strengthens this point. Cyclic AMP -triggered growth arrest may result from induction of the cell-cycle inhibitor p21, which was previously implicated in RA-induced APL differentiation (20). Cyclic AMP enhancement of RA-, As₂O₃- or rexinoids- triggered differentiation may also result from induction of the G-CSF receptor (21). In F9 embryonal carcinoma cells, cAMP was shown to modulate RA-triggered differentiation through RARαphosphorylation (22). Since RARa plays a critical role in myeloid differentiation, including in IL3 or GM-CSF response (23), such modulation of RARc signaling may also contribute to cAMP response. Therapy-resistant patients only exceptionally exhibit mutations in PML/RARα, particularly in European trials, which does not favor a direct parallelism between the cases of this patient and of the RA-resistant APL mice. The RA/8-Cl-cAMP synergy for differentiation in RA- resistant APL was unexpected. It is possible that this reflects the in vivo conversion of RA to rexinoids, allowing the cAMP/rexinoid triggered differentiation demonstrated in cell-lines (21). Although As₂O alone triggered a modest anti-proliferative effect in the absence of significant differentiation, the RA/As O combination triggered a minor, but reproducible, differentiation (Fig. 4 b).

In the patient, as in the APL mice, cAMP induces the rapid regrowth of normal erythroblasts and megakaryocytes. This could result from a direct positive effect on the normal progenitors, as reported ex vivo (24). Alternatively, APL cells secrete inhibitors of normal haematopoiesis (25, 26) whose synthesis or downstream signaling, could be blocked by cAMP. In the clinical setting, such in vivo stimulatory- effect on normal haematopoiesis could be as important as leukemia inhibition. The low toxicity of theophylline, its ability to accelerate RA- or As₂O₃-triggered remissions favor the use of theophylline in de novo patients.

Cyclic AMP is active both in RA-sensitive and RA-resistant APLs. Moreover, in contrast to RA and As₂O₃, cAMP does not obviously target PML/RARα and therefore might be valuable in malignancies other that APL. Non-APL myeloid cells are also very sensitive to cAMP triggered differentiation, particularly in the presence of other differentiation inducers. Similar to histone desacetylase inhibitors, which unravel RA- induced differentiation in acute myeloid leukemia (3, 27), theophylline may greatly j increase the potency of other differentiation inducers in vivo. ^'

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Claims

1. The use of at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP

^' 5 or said derivatives, for the preparation of a drug for the treatment of cancers.

2. The use of:

- at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said

10 cAMP or said derivatives and

15 3. The use according to claim 2, of:

- at least one cellular differentiating factor or precursors or derivatives thereof 20 for the preparation of a drug for the treatment of cancers.

4. The use according to claim 2, of:

25 cAMP and

5. The use according to claim 2, of:

30 - at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives,

- at least one cellular differentiating factor or precursors or derivatives thereof and - at least one apoptotic inducer for the preparation of a drug for the treatment of cancers.

6. The use according to anyone of claims 1 to 5, wherein the agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives is selected from the group comprising cAMP, 8-Cl-cAMP, 8-CPT-cAMP, 8-Br-cAMP, dibutyryl-cAMP or pharmacologically acceptable salts thereof.

7. The use according to anyone of claims 1 to 5, wherein the agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP or said derivatives is a phosphodiesterase inhibitor.

8. The use according to claim 7, wherein the phosphodiesterase inhibitor is selected from the group comprising methylxanthines such as caffeine or theophylline or aminophylline or isobutyl-methylxanthine, rolipram, sildenafil, vardenafil, zaprinast, or methoxyquinazoline.

9. The use according to claims 7 to 8, wherein the phosphodiesterase inhibitor is theophylline or aminophylline.

10. The use according to claim 2, wherein the cell-differentiation factor or precursors or derivatives thereof is selected from the group comprising retinoic acid, particularly j all-trans retinoic acid or 9-cis retinoic acid or 13-cis retinoic acid, or pharmacologically acceptable salts thereof, vitamin A (retinol), carotene or rexinoids.

11. The use according to claim 10, wherein the cell-differentiation factor or precursors or derivatives thereof is retinoic acid, particularly all-trans retinoic acid, or pharmacologically acceptable salts thereof.

12. The use according to claim 2, wherein the apoptotic inducer is selected from the group comprising arsenic trioxide (As₂O₃) or arsenic sulfide (As S₄).

13. The use according to claim 2, of theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof, for the preparation of a drug for the treatment of cancers.

14. The use according to claim 2, of theophylline or aminophylline and arsenic trioxide or arsenic sulfide, for the preparation of a drug for the treatment of cancers.

15. The use according to claim 2, of theophylline or aminophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, for the preparation of a drug for the treatment of cancers.

16. The use according to anyone of claims 1 to 15, wherein the cancers are selected from the group comprising solid tumor cancer, neuroblastoma, skin cancer, oral cavity cancer, lung cancer, mammary gland cancer, prostatic cancer, bladder cancer, liver cancer, pancreatic cancer, cervical cancer, ovarian cancer, head and neck cancer, colon cancer, germ cell cancer, leukemia, acute leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, aleukemic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia.

17. Products containing 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

18. Products according to claim 17, containing 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

19. Products according to claim 17, containing 8-Cl-cAMP or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

20. Products according to claim 17, containing 8-Cl-cAMP or pharmacologically acceptable salts thereof, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

21. Products containing aminophylline or theophylline and retinoic acid or pharmacologically acceptable salts thereof and or arsenic trioxide or arsenic sulide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

22. Products according to claim 21, containing aminophylline or theophylline and retinoic acid or pharmacologically acceptable salts thereof, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

23. Products according to claim 21, containing aminophylline or theophylline and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

24. Products according to claim 21, containing aminophylline or theophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, as a combined preparation for simultaneous, separate or sequential use in cancer treatment.

25. A pharmacological composition comprising as active substance 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

26. A pharmacological composition according to claim 25, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof and retinoic acid or pharmacologically acceptable salts thereof, in association with a pharmacologically acceptable vehicle.

27. A pharmacological composition according to claim 25, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

28. A pharmacological composition according to claim 25, wherein the active substance is 8-Cl-cAMP or pharmacologically acceptable salts thereof, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

29. A pharmacological composition comprising as active substance theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof and/or arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

30. A pharmacological composition according to claim 29, wherein the active substance is theophylline or aminophylline and retinoic acid or pharmacologically acceptable salts thereof, in association with a pharmacologically acceptable vehicle.

31. A pharmacological composition according to claim 29, wherein the active substance is theophylline or aminophylline and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

32. A pharmacological composition according to claim 29, wherein the active substance is theophylline or aminophylline, retinoic acid or pharmacologically acceptable salts thereof and arsenic trioxide or arsenic sulfide, in association with a pharmacologically acceptable vehicle.

33. A pharmacological composition according to anyone of claims 29 to 32, in a form appropriate for the administration of about 0.36 mg/kg/day to about 14.3 mg/kg/day of theophylline or aminophylline, of about 4.5 mg/m²/day to about 135 mg/m²/day of all- trans retinoic acid and of about 0.014 mg/kg/day to about 0.43 mg/kg/day of arsenic trioxide.