WO2007006544A2

WO2007006544A2 - Cyclic adenosine monophosphate compounds for the treatment of immune-related disorders

Info

Publication number: WO2007006544A2
Application number: PCT/EP2006/006761
Authority: WO
Inventors: Ron Kooijman; Sarah Gerlo; Peggy Verdood
Original assignee: Vrije Universiteit Brussel
Priority date: 2005-07-12
Filing date: 2006-07-11
Publication date: 2007-01-18
Also published as: WO2007006544A3

Abstract

The present invention is related to the finding that certain analogues of cAMP such as 8-(4-chlorophenylthio)- 2'- O- methyladenosine- 3', 5'- cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) specifically blocks the production of IL-10 in human T cells. It is also related to the finding that the production of other cytokines (e.g. IL-2, IL-4, IL-5, IL-6, IL-12 and IFN-gamma) is not affected by these analogues. The finding may be used as a treatment of conditions which respond to a reduced level of IL-10 and/or by a change in the balance of the Th1 and Th2 responses. It may be used in research and in testing for dysfunctional EPAC protein and IL-10 producing pathways.

Description

CYCLIC ADENOSINE MONOPHOSPHATE COMPOUNDS FOR THE TREATMENT OF IMMUNE-RELATED DISORDERS

BACKGROUND OF THE INVENTION

B lymphocytes (B cells), which originate in the bone marrow, and T lymphocytes (T cells) which originate in the thymus constitute the two major classes of lymphocytes in the mammalian immune system. B cells are mostly responsible for antibody production (humoral immunity) and T cells are mostly responsible for cell-mediated immunity.

There are considered to be two subclasses of T cells: helper T cells and cytotoxic T cells. Helper T cells activate other lymphocytes, including B cells and cytotoxic T cells, and macrophages, by releasing cytokines which are involved in cell-mediated immunity. Helper T cells are also generally considered to fall into two subclasses, Th1 and Th2.

Th1 cells (also known as Type 1 cells) produce interleukin 2 (IL-2), tumor necrosis factor (TNF-alpha) and interferon gamma (IFN-gamma), and are responsible primarily for cell- mediated immunity such as delayed type hypersensitivity and antiviral immunity. Th2 cells (also known as Type 2 cells) produce interleukins, IL4, IL-5, IL-6, IL-9, IL-IO and IL-13, and are primarily involved in assisting humoral immune responses, for example, in response to allergens, e.g. IgE and lgG4 antibody isotype switching (Mosmann,1989, Annu Rev lmmunol,7:145-173).

Certain disease pathologies are manifest by an imbalance in the TM and Th2 response. By Th1 and Th2 responses is meant the range of effects resulting from induction of TM and Th2 lymphocytes, respectively. Such effects include variation in production of the corresponding cytokines through transcription, translation, secretion and possibly other mechanisms, increased proliferation of the corresponding lymphocytes, and other effects associated with increased production of cytokines, including motility effects.

The mechanisms by which certain diseases cause an imbalance in the TM and Th2 response is unclear, however, selective modulation of TM and Th2 responses to redress the imbalance can be useful in treating a wide variety of conditions and diseases, ranging from infections, infestations, tumors and hypersensitivities to autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis.

Compounds of the art which attempt to target the production of a cytokine may not exclusively modulate the intended response. Thus, mixtures of inhibitors may be required to effectively adjust cytokine levels. In particular, compounds such as cAMP analogues affect the production of many cytokines, which secondary effect is a complex attenuation of Th1 and Th2 response which is difficult to delineate. Thus, treatments of autoimmune disease may stimulate multiple pathways, leading to undesirable side-effects.

The present invention addresses problems in the art of treating patients where a modulation of the balance of Th1 and Th2 response, or of a specific interleukin is needed.

SUMMARY OF SOME EMBODIMENTS OF THE INVENTION One embodiment of the present invention is a use of a compound of formula (I) or formula (II) for the preparation of a medicament for the treatment of a disorder which responds to a decrease in the production of IL-10 and/or a change in the balance of the Th1 and Th2 response which increases the Th1 response, wherein formula (I) is:

and formula (II) is

and wherein:

R1 can be independently H, halogen, azido, alkyl, aryl, amido-alkyl, amido-aryl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, SeH, Se-alkyl, Se-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N- bisaryl, cycloalkylamino;

R2 can be independently H, halogen, azido, O-alkyl, S-alkyl, Se-alkyl, NH-alkyl, N-bisalkyl, alkyl-carbamoyl, cycloalkylamino, silyl ;

R3 can be independently H, halogen, OH, azido, amido-alkyl, amido-aryl, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl, NH-alkyl-carbamoyl, cycloalkylamino; and wherein

R4 is O (H) or S (H); and

R5 is O (H), S (H), amino, H, alkyl, O-alkyl, O-aryl, S-alkyl, S-aryl, NH-alkyl, NH-aryl, N- bisalkyl, N-bisaryl; or R4 is O (H), S (H), amino, H, alkyl, O-alkyl, O-aryl, S- alkyl, S-aryl, NH-alkyl, NH-aryl, N- bisalkyl, N-bisaryl; and

R5 is O (H) or S(H) ;

R6 and R7 can be independently H, halogen, alkyl, nitro, amino, and/or alkoxy, and pharmaceutically acceptable salts, esters, and/or solvates thereof.

Another embodiment of the present invention is a use as described above, wherein R1 is H, halogen, azido, O-alkyl, O-aryl, S-alkyl, S- aryl, NH-alkyl, NH-aryl, or Se-aryl.

Yet another embodiment of the present invention is a use as described above, wherein R1 is O-aryl, S- aryl, NH-aryl, or Se-aryl. Yet another embodiment of the present invention is a use as described above wherein R1 is H, Br, Cl, I, azido, 2-chlorophenylthio, 3-chlorophenylthio, 4-chlorophenylthio, 2- bromophenylthio, 3-bromophenylthio, 4-bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n-hexylthio, 2-fluorophenylthio, 3- fluorophenylthio, A- fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 2- aminophenylthio, 3-aminophenylthio, 4-aminophenylthio, benzylthio, phenylethylamino,3- phenyl-propylamino, 2- methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, 2-aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3-methylphenylthio, 4-methylphenylthio, 2- isopropylphenylthio, 4-isopropylphenylthio, 2,3,5,6- tetrafluorophenylthio, A- hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, ethoxy, propioxy, butoxy, benzyloxy, A- methylbenzyloxy, 4-methoxybenzyloxy, 4-fluorobenzyloxy, 4-chlorobenzyloxy, A- bromobenzyloxy, phenyloxy, cyclohexylamino, benzylamino, phenylseleno, A- isopropyloxyphenylthio, 4-methylthiophenylthio, 6-aminohexylamino, 2,3-dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5-dimethoxyphenylthio, 2,5- dimethylthiophenylthio, 2,6- dimethylthiophenylthio, or 2,6-dichlorophenylthio.

Yet another embodiment of the present invention is a use as described above wherein R1 is H, Br, Cl, azido, 2-chlorophenylthio, 3-chlorophenylthio, 4-chlorophenylthio, 2- bromophenylthio, 3-bromophenylthio, 4-bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n-hexylthio, 2-fluorophenylthio, 3-fluorophenylthio, A- fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 2- aminophenylthio, 3-aminophenylthio, 4-aminophenylthio, benzylthio, phenylethylamino, 2- methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, 2- aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3-methylphenylthio, 4-methylphenylthio, 2-isopropylphenylthio, A- isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, A- isopropyloxyphenylthio, 4-methylthiophenylthio, 6-aminohexylamino, 2,3-dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5-dimethoxyphenylthio, 2,5- dimethylthiophenylthio, 2,6-dimethylthiophenylthio, 2,6- dichlorophenylthio. Yet another embodiment of the present invention is a use as described above wherein R1 is H, Br₁ Cl, azido, 4-chlorophenylthio, methylamino, methylthio, 4-fluorophenylthio, 4- methylcumarinyl, naphtyl-2-thio, phenylthio, 4- nitrophenylthio, 2-aminophenylthio, benzylthio, n-hexylthio, phenylethylamino, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2- hydroxyethylthio, ethylthio, 2-aminoethylthio, pyridinylthio, benzothiazolylthio, 4- methylphenylthio, 3- methoxyphenylthio, 4-isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, 4-isopropyloxyphenylthio, 4- methylthiophenylthio, or 6- aminohexylamino.

Yet another embodiment of the present invention is a use as described above wherein R1 is Cl, Br or S-aryl.

Yet another embodiment of the present invention is a use as described above wherein R1 is Cl, Br, 4-chlorophenylthio, 4-fluorophenylthio, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 4- methoxyphenylthio, pyridinyl-2-thio, 4-methylphenylthio, 4- isopropylphenylthio, 2,3,5,6- tetrafluoro-phenylthio, 4-hydroxyphenylthio, or 2,4-dichloro-phenylthio.

Yet another embodiment of the present invention is a use as described above wherein R1 is 4-chlorophenylthio, 4-fluorophenylthio, 4-methoxyphenylthio, 4-methylphenylthio, or 4- hydroxyphenylthio.

Yet another embodiment of the present invention is a use as described above, wherein R2 is H, halogen, azido, O-alkyl, or S-alkyl.

Yet another embodiment of the present invention is a use as described above, wherein R2 is H, Cl, Br, I, O-alkyl, S-methyl, or S-ethyl

Yet another embodiment of the present invention is a use as described above, wherein R2 is H, Cl, Br, O-methyl, O-ethyl, O-propyl, O-butyl, O-isobutyl, or S-methyl

Yet another embodiment of the present invention is a use as described above, wherein R2 is O-methyl, O-ethyl, O- propyl, O-butyl, or O-isobutyl. Yet another embodiment of the present invention is a use as described above, wherein R2 is O-methyl.

Yet another embodiment of the present invention is a use as described above, wherein R3 is amino, NH-alkyl, N-bisalkyl, NH-aryl, NH-alkyl-carbamoyl, N-bisalkyl-carbamoyl, amido-alkyl, amido-aryl, OH

Yet another embodiment of the present invention is a use as described above, wherein R3 is amino, NH- phenyl, NH-tert-butyl, NH-tert-butylcarbamoyl, NH-phenylcarbamoyl, NH-acetyl, NH-propionyl, NH-butyryl, NH-benzoyl, NH-benzyl, NH-phenylethyl, N H-phenyl propyl, N- bismethyl, N-bisethyl, or OH

Yet another embodiment of the present invention is a use as described above, wherein R3 is amino, NH-phenyl, NH-tert-butyl, NH-tert-butylcarbamoyl, NH-phenylcarbamoyl; NH-butyryl, NH-benzoyl, NH-benzyl, N- bismethyl, N-bisethyl, or OH

Yet another embodiment of the present invention is a use as described above, wherein R3 is amino, NH-phenyl, NH-tert-butyl, NH-tert-butylcarbamoyl, NH-benzoyl, N-bismethyl or OH

Yet another embodiment of the present invention is a use as described above, wherein R3 is amino or NH-tert-butyl

Yet another embodiment of the present invention is a use as described above, wherein R4 and R5 are independently O (H) or S (H).

Yet another embodiment of the present invention is a use as described above, wherein R4 and R5 are O(H).

Yet another embodiment of the present invention is a use as described above, wherein compound of formula (I) is selected from the group consisting of Preferably, the compound is selected from the group consisting of 8-bromo-2¹-deoxyadenosine-3',5'-cyclic monophosphate; 8-(4-chloro-p'henylthio)-2'-deoxyadenosine-3\ 5'-cyclic monophosphate; 8- (4-chloro-phenylthio)- N6-phenyl-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 8-bromo-2'- O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-chloro-phenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate;8-methylamino-2'-0-methyladenosine-3',5'- cyclic monophosphatejδ-methylthio-Z-O-methyladenosine-S', 5'-cyclic monophosphate; 8- (4- fluoro-phenylthio)-2'-O-methyladenosine-3^l, 5'-cyclic monophosphate; 8- (4-methyl-cumarinyl- 7-thio)-2'-O-methyladenosine-3', 5'- cyclic monophosphate; 8-(naphtyl-2-thio)-2'-0- methyladenosine-3', 5'-cyclic monophosphate; 8-phenylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-nitro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (2-amino-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-benzylthio-2'-O- methyladenosine-3',5¹- cyclic monophosphate; 8-n-hexylthio-2'-O-methyladenosine-3', 5'- cyclic monophosphate; 8-phenylethylamino-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-methoxy-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-isopropylthio-2'-O-methyladenosine-3¹,5'-cyclic monophosphate; 8- (benzimidazolyl-2-thio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2-hydroxy- ethylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-ethylthio-2'-O- methyladenosine-3',5¹- cyclic monophosphate; 8- (2-amino-ethylthio)-2'-O-methyladenosine- 3', 5'-cyclic monophosphate; 8-(pyridinyl-2-thio)-2'-0-methyladenosine-3',5'-cyclic monophosphate; 8-(benzothiazolyl-2-thio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate^- (4-methyl-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(3-methoxy-phenylthio)-2¹-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-isopropyl- phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2,3,5,6-tetrafluoro- phenylthio) -2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-hydroxy-phenylthio)-2'- O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2,4-dichloro-phenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate; 8-(4-chloro-phenylthio)-2'-(N, N-dimethyl) - carbamoyl-adenosine-3', 5'-cyclic monophosphate;8-methoxy-2'-O-methyladenosine-3', 5'- cyclic monophosphate; 8-benzyloxy-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- bromo-2¹-O-methyladenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer; 8-bromo-2'-O- methyladenosine-3'-5'-cyclic monophophorothioate, Rp- isomer, 8-(4-chloro-phenylthio)-2'-0- methyladenosine-3¹, 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'- deoxyadenosine-3¹, δ'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'-deoxyadenosine- 3¹, 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5- cyclic monophosphorothioate, Sp- isomer; and 8-cyclohexylamino-2'-deoxyadenosine-3', 5'- cyclic monophosphate;8-chloro-2'-O-methyladenosine-3', 5'-cyclic monophosphate; N⁶-tert- butyl-8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 5,6-Dichloro-l- /?-D-ribofuranbsyl-2'-O-methylbenzimidazole-3', 5'-cyclic monophosphate.

Yet another embodiment of the present invention is a use as described above where, in formula (II):

R4 and R5 are independently O or S R6 and R7 are independently F, Cl, Br or I.

Yet another embodiment of the present invention is a use as described above wherein: R6 and R7 are Cl.

Yet another embodiment of the present invention is a use as described above where, in formula (II): R2 is O-alkyl.

Yet another embodiment of the present invention is a use as described above, wherein the compound of formula (II) is selected from the group consisting of 1-deaza- adenine analogues, 3-deaza-adenine analogues, 7-deaza-adenine analogues, 1 ,3-dideaza-adenine analogues, 1 ,7- dideaza-adenosine analogues and benzimidazole analogues, preferably 5,6- dichlorobenzimidazole.

Yet another embodiment of the present invention is a use as described above wherein said disorder characterised at least in part by an imbalance in the Th1 and Th2 response where the Th1 response is low.

Yet another embodiment of the present invention is a use as described above wherein said disorder characterised at least in part by a high level of IL-10 production. Yet another embodiment of the present invention is a use as described above, wherein said disorder is allergic asthma, atopic dermatitis, cancer and chronic infection.

Yet another embodiment of the present invention is a use as described above, wherein said disorder is systemic lupus erythematosus.

Yet another embodiment of the present invention is a use as described above, wherein said disorder is rheumatoid arthritis.

Yet another embodiment of the present invention is a use as described above, comprising monitoring the levels of IL-10 and optionally IL-12 before and after treatment.

Yet another embodiment of the present invention is a use as described above, wherein further treatment is given if level of IL-10 or optionally, an IL-12/IL-10 ratio show insufficient change from the level or ratio present before treatment.

Yet another embodiment of the present invention is a method of decreasing the level of IL-10 production in an in vitro mammalian system comprising contacting said system with a compound of formula (I) or formula (II) as defined above.

Yet another embodiment of the present invention is a method as described above for further not affecting the level of IL-2, IL-4, IL-5, IL-6, IL-12 or IFN-gamma production.

Yet another embodiment of the present invention is a use of a compound of formula (I) or formula (II) as defined above for the preparation of a medicament for decreasing the level of IL-10 production.

Yet another embodiment of the present invention is a use as described above for further not affecting the level of IL-2, IL-4, IL-5, IL-6, IL-12 or IFN-gamma production.

Yet another embodiment of the present invention is a method of changing the balance of the Th1 and Th2 response in an in vitro mammalian system towards an increased TM response, comprising contacting said system with a compound of formula (I) or formula (II) as defined above.

Yet another embodiment of the present invention is a use of a compound of formula (I) or formula (II) as defined above for the preparation of a medicament for changing the balance of the Th1 and Th2 response towards an increased Th1 response.

Yet another embodiment of the present invention is a method of stimulating immune responses in an in vitro mammalian system comprising contacting said system with a compound of formula (I) or formula (II) as defined above.

Yet another embodiment of the present invention is a use of a compound of formula (I) or formula (II) as defined above for the preparation of a medicament for stimulating immune responses.

Yet another embodiment of the present invention is a method of diagnosing a dysfunction of

EPAC and/or the IL-10 production pathway in a mammalian system, the method comprising the steps: a) contacting a sample with a compound of formula (I) or formula (II) as defined above; b) measuring the level of IL-10 production; and c) comparing the level measured in step (b) with a standard, wherein a difference in production relative to the standard is diagnostic of a dysfunction of EPAC and/or the IL-10 production pathway.

Yet another embodiment of the present invention is a method or use as described above, wherein said compound is a stereoisomer, tautomer, racemate, prodrug, metabolite, pharmaceutically acceptable base, structurally related derivative of said compound.

Yet another embodiment of the present invention is a use as described above, wherein said compound is comprised in a pharmaceutical composition.

Yet another embodiment of the present invention is a method as described above, wherein said compound is comprised in a pharmaceutical composition. Yet another embodiment of the present invention is a use as described above, wherein said compound is a modulator of EPAC activity or a pharmaceutical composition thereof.

Yet another embodiment of the present invention is a method as described above, wherein said compound is a modulator of EPAC activity or a pharmaceutical composition thereof.

LEGENDS TO FIGURES Figure 1. Effect of cAMP on endogenous PRL expression and activation of the extrapituitary PRL promoter in primary human T lymphocytes. A. T lymphocytes from 12 donors were treated with 250 μM cptcAMP for 6 hrs. PRL mRNA levels were quantified by real time PCR and normalized to GAPDH mRNA expression. Data are shown as fold induction (mean ± SD) relative to unstimulated controls. PRL mRNA levels were on average induced 12.94 times (SD=11.18). This induction was significant (p < 0.01) as determined by the Wilcoxon Signed Rank Test. B. T lymphocytes from 6 donors were transiently transfected by nucleofection with equimolar amounts of PRL promoter constructs and stimulated for 6 hrs with cptcAMP (250 μM). Data are shown as fold induction (mean ± SD) relative to unstimulated controls. # : significantly different from control with p < 0.05 as determined by the Wilcoxon Signed Rank Test.

Figure 2. The effect of cAMP on PRL expression in primary T lymphocytes requires de novo mRNA synthesis. A. T lymphocytes were preincubated for 1 hr with vehicle or the transcriptional inhibitor actinomycin D (5 μg/ml) and next stimulated with vehicle or cptcAMP (250 μM) for 6 hrs. PRL mRNA levels were quantified by real time PCR and normalized to GAPDH mRNA expression. # : significantly different from control without actinomycin D with p < 0.01. B. Decay of PRL mRNA levels as measured by real time PCR. T lymphocytes were preincubated for 1 hr with actinomycin D (5 μg/ml) and next stimulated with vehicle or cptcAMP (250 μM) for 0, 4, 6 and 18 hrs. PRL mRNA levels are represented as percentage of control (actinomycin D 5 μg/ml, no cptcAMP at time 0).

Figure 3. Effect of inhibition of PKA on cAMP-induced PRL mRNA expression and CREB phosphorylation in human T lymphocytes. A. T lymphocytes were pretreated for 2 hrs with vehicle, H89 (10μM) or SB203580 (50 μM), before stimulation with 250 μM cptcAMP for 6 hrs. PRL mRNA levels were quantified by real time PCR and normalized to GAPDH mRNA expression. Control conditions are always significantly different from corresponding cptcAMP- stimulated conditions (p<0.01 ). B. T lymphocytes were preincubated for 2 hrs with increasing doses (0, 2 and 10 μM) of H89, before stimulation with cptcAMP (250 μM) for 20 minutes. P- CREB and total CREB were monitored by western blotting.

Figure 4. Effect of cAMP on MAPK activation in human T lymphocytes and role of PKA in this effect. Cells were stimulated for 20 min with the indicated doses of cptcAMP. MAPK activation was assessed by western blotting. A. P-ERK, ERK, P-JNK and JNK in T lymphocytes stimulated with 250 μM of cptcAMP. B. P-p38 and total p38 in T lymphocytes stimulated with increasing doses (25, 250 and 500 μM) of cptcAMP. C. P-p38 and total p38 in T lymphocytes that were preincubated for 2 hrs with increasing doses of H89, before stimulation with cptcAMP (250 μM).

Figure 5. Expression of EPAC1 in primary T-lymphocytes. EPAC1 expression was determined by real-time PCR in T cells (1, 4, 7, 10, 13, 16, 19, 22), peripheral blood mononuclear cells (2, 5, 8, 11 , 14, 17, 20, 23), and non-T cells (3,6,9,12,15,18,21,24) from 8 different donors. PCR products were monitored by agarose gel electrophoresis and subsequent staining with ethidium bromide.

Figure 6. Role of EPAC in the effects of cAMP on PRL expression in human T lymphocytes.

A. T cells were stimulated for 20 min with increasing doses (25, 250 and 500 μM) of Me- cptcAMP. p38 activation was assessed by western blotting. B. T lymphocytes were stimulated for 6 hrs with increasing doses of cptcAMP or Me-cptcAMP. PRL mRNA levels were quantified by real time PCR and normalized to GAPDH mRNA expression. C. P-p38 and total p38 as assessed by western blotting in Jurkat cells stimulated for 20 min with vehicle or 250 μM of cptcAMP.

Figure 7. Effects of cAMP analogues on cytokine production in unstimulated and PHA- activated T cells. Cytokine concentrations (pg/ml) in conditioned media of cultured T cells.

Values are mean ± SD (n=4). The results from two independent experiments using different donors (TC-A and TC-B) are presented. A: IL-2 levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

B: IL-2 levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

C: IL-4 levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml). D: IL-4 levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

E: IL-5 levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

F: IL-5 levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml). G: IL-6 levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

H: IL-6 levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml). I: IL-10 levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

J: IL-10 levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me-cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me- cptcAMP (250μM) / PHA (2μg/ml).

K: IFN-gamma levels, Donor TC-A, C - control, 1 - cptcAMP (250μM) control, 2 -Me- cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5 - Me-cptcAMP (250μM) / PHA (2μg/ml). L: IGN-gamma levels, Donor TC-B, C - control, 1 - cptcAMP (250μM) control, 2 -Me- cptcAMP (250μM) control, 3 - PHA (2μg/ml) control, 4 - cptcAMP (250μM) / PHA (2μg/ml), 5

- Me-cptcAMP (250μM) / PHA (2μg/ml).

Figure 8: Effects of cAMP analogues on cytokine production in unstimulated and LPS- activated non-T cells

Cytokine concentrations (pg/ml) in conditioned media of cultured non-T cells (mainly monocytes and B cells). Values are mean ± SD (n=4). The results from one representative experiment are presented. A: IL-6 concentrations, C - control, 1 - cptcAMP (250μM) control, 2- Me-cptcAMP (250μM) control ,3 - PHA (2μg/ml) control, 4- cptcAMP (250μM) / PHA (2μg/ml), 5 - Me-cptcAMP (250μM) / PHA (2μg/ml), 6 - LPS (10μg/ml) control, 7- cptcAMP (250μM) / LPS (10μg/ml), 8

- Me-cptcAMP (250μM) / LPS (10μg/ml). B: IL-10 concentrations, C - control, 1 - cptcAMP (250μM) control, 2 - PHA (2μg/ml) control, 3 - cptcAMP (250μM) / PHA (2μg/ml), 4 - LPS (10μg/ml) control, 5 - cptcAMP (250μM) / LPS (10μg/ml). C: IL-12 concentrations, C - control, 1- cptcAMP (250μM) control, 2- Me-cptcAMP (250μM) control, 3 - LPS (10ng/ml) control, 4- cptcAMP (250μM) / LPS (10ng/ml), 5 - Me-cptcAMP (250μM) / LPS (10ng/ml), 6 - LPS (1000ng/ml) control, 7- cptcAMP (250μM) / LPS (1000ng/ml), 8 - Me-cptcAMP (250μM) / LPS (1000ng/ml).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. All publications referenced herein are incorporated by reference thereto. All United States patents and patent applications referenced herein are incorporated by reference herein in their entirety including the drawings.

As used herein, "a" includes both the singular and plural. By way of example, "a sample" means one sample or more than one sample.

The recitation of numerical ranges by endpoints, includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1, 2, 3, 4 when referring to, for example, a number of samples, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, concentrations). The present invention is based around the finding by the inventors that a compound of formula (I) or (II) defined below, selectively blocks the production of IL-10, whereas the production of other cytokines (e.g. IL-2, IL-4, IL-5, IL-6, IL-12 and IFN-gamma) is not affected. This is in contrast with cAMP, for example, which affects the production of several cytokines.

This exquisite control permits the treatment of diseases which selectively respond to levels of IL-10, and which disease may be aggravated by a change in the level of other cytokines. For example, patients with chronic inflammation do not all show improvement by a regulation of general cytokine activity. SLE suffers, for instance, benefit from a reduction in IL-10 activity. By contrast, such reduction would be non-beneficial or harmful for inflammatory bowel disorder suffers wherein an increase in IL-10 levels is advantageous. Therefore, the finding that a compound of formula (I) or (II) selectively regulates the production of IL-10 can be used in the precise treatment of a selection of patients, whose conditions respond to IL-10 modulation. Thus, the invention can be applied to any disorder that is susceptible to treatment by the down-regulation of the levels of IL-10.

The present inventors have also found that a compound of formula (I) or (II) defined below, changes the balance of the Th1 and Th2 response, so that the Th1 response is increased. Such control is useful in the treatment of diseases which exhibit an upset in the balance of the Th1/Th2 response during the progress of an inflammatory disorder. For example, during the advancement of rheumatoid arthritis, the cytokine profile of certain T cells indicate a shift in the Th1 and Th2 balance towards Th2. Therefore, the finding that a compound of formula (I) or (II) changes the balance of the Th1 and Th2 response, so that the Th1 response is increased can be used in the precise treatment of a selection of patients, whose conditions respond to Th1/Th2 balance modulation. Thus, the invention can be applied to any disorder that is susceptible to the treatment by increase of the Th1/Th2 balance.

The present invention is related to compounds of formula (I) and deaza-analogues thereof which specifically block the production of IL-10 in human T-cells and/or change the balance of the TH1 and Th2 response which increases the Th1 response,

wherein:

R4 is O (H) or S (H); and

R5 is O (H) or S(H) ; and pharmaceutically acceptable salts, esters, and/or solvates thereof,

According to the present invention novel compounds, in particular cAMP analogues with a modified 2'-O-ribose group, were thus identified, such as 8-(4- chlorophenylthio)-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) which specifically specifically blocks the production of IL-10 in human T-cells.

In particular analogues such as 8-pCPT-2'-O-Me-cAMP not only blocked the production of IL- 10 in human T-cells, but they did not affect the production of other cytokines (e.g. IL-2, IL-4, IL-5, IL-6, IL-12 and IFN-gamma). In a preferred embodiment of the invention

R1 is H, halogen, azido, O-alkyl, O-aryl, S-alkyl, S- aryl, NH-alkyl, NH-aryl, Se-aryl, more preferably R1 is O-aryl, S- aryl, NH-aryl, or Se-aryl.

In a further preferred embodiment of the invention R1 is H, Br, Cl, I, azido, 2-chlorophenylthio, 3-chlorophenylthio, 4-chlorophenylthio, 2-bromophenylthio, 3-bromophenylthio, A- bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n- hexylthio, 2-fluorophenylthio, 3- fluorophenylthio, 4-fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 2- aminophenylthio, 3-aminophenylthio, A- aminophenylthio, benzylthio, phenylethylamino,3-phenyl-propylamino, 2- methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2- hydroxyethylthio, 2-aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3- methylphenylthio, 4-methylphenylthio, 2-isopropylphenylthio, 4-isopropylphenylthio, 2,3,5,6- tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, ethoxy, propioxy, butoxy, benzyloxy, 4-methylbenzyloxy, 4-methoxybenzyloxy, 4-fluorobenzyloxy, A- chlorobenzyloxy, 4-bromobenzyloxy, phenyloxy, cyclohexylamino, benzylamino, phenylseleno, 4-isopropyloxyphenylthio, 4-methylthiophenylthio, 6-aminohexylamino, 2,3- dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5-dimethoxyphenylthio, 2,5-dimethylthiophenylthio, 2,6- dimethylthiophenylthio, 2,6-dichlorophenylthio.

Preferably, R1 is H, Br, Cl, azido, 2-chlorophenylthio, 3-chlorophenylthio, 4-chlorophenylthio, 2-bromophenylthio, 3-bromophenylthio, 4-bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n-hexylthio, 2-fluorophenylthio, 3- fluorophenylthio, 4-fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, A- nitrophenylthio, 2-aminophenylthio, 3-aminophenylthio, 4-aminophenylthio, benzylthio, phenylethylamino, 2-methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, 2- aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3-methylphenylthio, 4-methylphenylthio, 2- isopropylphenylthio, 4-isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4-dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, A- isopropyloxyphenylthio, 4-methylthiophenylthio, 6-aminohexylamino, 2,3-dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5-dimethoxyphenylthio, 2,5- dimethylthiophenylthio, 2,6-dimethylthiophenylthio, 2,6- dichlorophenylthio.

In another preferred embodiment of the invention, R1 is H₁ Br₁CI, azido, 4-chlorophenylthio, methylamino, methylthio, 4-fluorophenylthio, 4- methylcumarinyl, naphtyl-2-thio, phenylthio, 4- nitrophenylthio, 2-aminophenylthio, benzylthio, n-hexylthio, phenylethylamino, 4- methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, ethylthio, 2- aminoethylthio, pyridinylthio, benzothiazolylthio, 4-methylphenylthio, 3- methoxyphenylthio, 4- isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, 4- isopropyloxyphenylthio, 4- methylthiophenylthio, 6-aminohexylamino.

According to a further preferred embodiment of the invention, R1 is Cl, Br or S-aryl; preferably R1 is Cl, Br, 4-chlorophenylthio, 4-fluorophenylthio, naphtyl-2-thio, phenylthio, 4- nitrophenylthio, 4-methoxyphenylthio, pyridinyl-2-thio, 4-methylphenylthio, 4- isopropylphenylthio, 2,3,5,6-tetrafluoro-phenylthio, 4-hydroxyphenylthio, or 2,4-dichloro- phenylthio; and more preferably R1 is 4-chlorophenylthio, 4-fluorophenylthio, 4- methoxyphenylthio, 4-methylphenylthio, 4-hydroxyphenylthio.

In a further preferred embodiment R2 is H, halogen, azido, O-alkyl, S-alkyl ; preferably R2 is H, Cl, Br, I, O-alkyl, S-methyl, S-ethyl; more preferably R2 is H, Cl, Br, O-methyl, O-ethyl, O- propyl, O-butyl, O-isobutyl, S- methyl; and most preferably R2 is O-methyl, O-ethyl, O- propyl, O-butyl, O-isobutyl.

According to another preferred embodiment of the invention R2 is O-methyl.

R3 is preferably amino, NH-alkyl, N-bisalkyl, NH-aryl, NH-alkyl-carbamoyl, N-bisalkyl- carbamoyl, amido-alkyl, amido-aryl, OH; more preferably R3 is amino, NH- phenyl, NH-tert- butyl, NH-tert-butylcarbamoyl, NH-phenylcarbamoyl, NH-acetyl, NH-propionyl, NH-butyryl, NH-benzoyl, NH-benzyl, NH-phenylethyl, NH-phenylpropyl, N-bismethyl, N-bisethyl, OH; and most preferably R3 is amino, NH-phenyl, NH-tert-butyl, NH-tert-butylcarbamoyl, NH- phenylcarbamoyl; NH-butyryl, NH-benzoyl, NH-benzyl, N- bismethyl, N-bisethyl, OH. According to another preferred embodiment R3 is amino, NH-phenyl, NH-tert-butyl, NH-tert- butylcarbamoyl, NH-benzoyl, N-bismethyl, OH, preferably R3 is amino, NH-tert-butyl.

According to a further preferred embodiment of the invention R4 and R5 are independently O (H) or S (H)₁ preferably R4 and R5 are O(H).

Preferably, the compound is selected from the group consisting of 8-bromo-2'- deoxyadenosine-3',5'-cyclic monophosphate; 8-(4-chloro-p'henylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 8- (4-chloro-phenylthio)- N6-phenyl-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 8-bromo-2'-O-methyladenosine-3\ 5'-cyclic monophosphate; 8- (4-chloro- phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate;8-methylamino-2'-O- methyladenosine-3',5'- cyclic monophosphate;8-methylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-fluoro-phenylthio)-2'-O-methyladenosine-3\ 5'-cyclic monophosphate; 8- (4-methyl-cumarinyl-7-thio)-2'-O-methyladenosine-3', 5'- cyclic monophosphate; 8-(naphtyl- 2-thio)-2'-0-methyladenosine-3\ 5'-cyclic monophosphate; 8-phenylthio-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate; 8-(4-nitro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2-amino-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; δ-benzylthio^'-O-methyladenosine-S'.δ¹- cyclic monophosphate; 8-n- hexylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-phenylethylamino-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate; 8-(4-methoxy-phenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate; 8-isopropylthio-2'-O-methyladenosine-3',5'- cyclic monophosphate; 8-(benzimidazolyl-2-thio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2-hydroxy-ethylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-ethylthio-2'-O-methyladenosine-3',5'- cyclic monophosphate; 8- (2-amino-ethylthio)-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate; 8-(pyridinyl-2-thio)-2¹-O-methyladenosine-3',5'- cyclic monophosphate; 8-(benzothiazolyl-2-thio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate^- (4-methyl-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(3-methoxy-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-isopropyl- phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2,3,5,6-tetrafluoro- phenylthio) -2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-hydroxy-phenylthio)-2'- O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2,4-dichloro-phenylthio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate; 8-(4-chloro-phenylthio)-2'-(N, N-dimethyl) - carbamoyl-adenosine-3¹, 5'-cyclic monophosphatejβ-methoxy^'-O-methyladenosine-S¹, 5'- cyclic monophosphate; 8-benzyloxy-2'-O-methyladenosine-3', 5-cyclic monophosphate; 8- bromo-2'-0-methyladenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer; 8-bromo-2'-O- methyladenosine-3'-5'-cyclic monophophorothioate, Rp- isomer, 8-(4-chloro-phenylthio)-2'-0- methyladenosine-3¹, 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-O- methyladenosine-3¹, 5'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'- deoxyadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'-deoxyadenosine- 3', 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'- cyclic monophosphorothioate, Sp- isomer; and δ-cyclohexylamino^'-deoxyadenosine-S¹, 5'- cyclic monophosphateiδ-chloro^'-O-methyladenosine-S¹, 5'-cyclic monophosphate; N6-tert- butyl-8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 5,6-Dichloro-l- /?-D-ribofuranbsyl-2'-O-methylbenzimidazole-3\ 5'-cyclic monophosphate.

The present invention also embraces deaza-analogues of the compounds, which refer to analogues of the compounds as defined wherein the purine lacks one or more ring nitrogen atoms, such as, but not limited to 1-deaza-adenine, 3-deaza-adenine, 7-deaza- adenine, dideaza-adenines, and benzimidazole.

According to another preferred embodiment of the invention, the deaza-analogue is a compound having the structural formula (II):

("I) wherein R1 to R5 are as defined above, and wherein R6 and R7 can be independently H, halogen, alkyl, nitro, amino, and/or alkoxy.

Preferably, R1-R3 are as defined above;

R4 and R5 can be independently O or S; and R6 and R7 can be independently F, Cl, Br or I₁ more preferably R6 and R7 are Cl.

In a preferred embodiment of the invention, R2 is O- alkyl.

According to a further preferred embodiment of the invention R1 and R3 are hydrogen; R2 is O-alkyl ; and R6 and R7 are Cl.

Preferably, the deaza- analogue is selected from the group consisting of 1-deaza- adenine analogues, 3-deaza-adenine analogues, 7-deaza-adenine analogues, 1 ,3-dideaza-adenine analogues, 1 ,7- dideaza-adenosine analogues and benzimidazole analogues, preferably 5,6- dichlorobenzimidazole.

The inhibition of IL-10 proceeds via the exchange protein directly activated by cAMP (EPAC) and not via the normal cAMP target which is protein kinase (PKA). IL-10 is mainly considered as an anti-inflammatory cytokine, because it impairs the production of inflammatory cytokines by macrophages. IL-10 also stimulates B cell proliferation and antibody production and as such may exert stimulatory actions on immune responses. Furthermore, as a Th2 cytokine which inhibits production of Th1 cytokines, IL-10 contributes to development of the Th2 response. Since the compounds of the present invention selectively inhibit IL-10, production of both Th1 and Th2 cytokines and not other cytokines - unlike cAMP - it may be used to inhibit Th2 mediated immune responses and to skew the balance of TM and Th2 response towards an increase in the Th1 response.

Uses

The present invention finds usage in applications relating to the levels of IL-10 and/or the balance of the Th1/Th2 response. It can be used to treat disorders, modulate cytokine levels, used in in vitro investigations and for the diagnoses of disorders. The present invention is especially applicable to mammalian systems, which includes human, pig, sheep, cow, ox, rat, mouse, dog, cat, rabbit, horse, goat, lama, camel, monkey, elephant, whale, dolphin, hamster, tiger, fox, bear, and preferably human. - Treatment of disorders

The present finding by the inventors can be used to treat a wide variety of disorders, which are manifest at least in part by a high level of IL-10 and/or by an imbalance in the Th1 and Th2 response with a low level of Th1 response. It may also be used to treat disorders which respond to a decrease in the production of IL-10 and/or change in the balance of the Th1 and Th2 response, which increases the TM response. It may also be used to treat disorders mediated by IL-10. It may also be used to treat disorders which respond to a modulation of EPAC activity.

One embodiment of the present invention is a method for the treatment of a disorder which responds to a decrease in the production of IL-10 and/or a change in the balance of the TM and Th2 response which increases the TM response, comprising administering an effective amount of a compound of the present invention or derivative thereof.

One embodiment of the present invention is a method for the treatment of a disorder characterised at least in part by a high level of IL-10 production comprising administering an effective amount of a compound of the present invention or derivative thereof. Another embodiment of the present invention is a method for the treatment of a disorder characterised at least in part by an imbalance in the TM and Th2 response where TM response is low, comprising administering an effective amount of a compound of the present invention or derivative thereof.

Another embodiment of the present invention is a method for the treatment of an anti-immune disorder in a subject in need thereof where said disorder is characterised at least in part by a higher than normal level of IL-10 and/or by an imbalance in the TM and Th2 where TM response is lower than normal, comprising administering an effective amount of a compound of the present invention or derivative thereof.

The normal amount of IL-10 and TM and Th2 response levels can be determined from a standard sample taken from a healthy mammal, using known methods and assays such as describe in the examples. The standard is preferably taken from a closely related phenotypic group to the subject. IL-10 levels in serum or other body fluids can be measured by Enzyme- linked Immunosorbent Assays (ELISAs). IL-10 levels in different leukocyte subsets can be determined by flow cytometry using anti-IL-10 monoclonal antibodies and subset-specific monoclonal antibodies. In addition, IL-10 mRNA levels can be measured in leukocyte subsets after cell sorting using RT-PCR (real time polymerase chain reaction). Measurement of other Th2 cytokines (IL-4, IL-4, IL-6, IL13) or Thi cytokines (IFN-gamma, IL-2) can be performed as described for IL-10 in order to measure Th1 and Th2 responses, respectively.

Disorders according to the invention are those recognised as being mediated at least in part IL-10 and/or by the balance of the TH1/Th2 response. They generally fall into the category of autoimmune disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis. They include allergic atopic disorders such as allergic asthma, atopic dermatitis. Other disorders include chronic infection or other infection which necessitates an increase in the immune response. For example, chronic infection may be treated by reducing IL-10 levels in order to increase the inflammatory response. In particular, SLE is a Th2-mediated disease in which IL-10 plays an important role in the production of autoantibodies. Another disorder that may be treated by the present invention is cancer. Cancer may be treated by reducing IL-10 levels in order to increase T cell responses or inflammatory responses against cancer cells.

The treatment can be monitored by assessing one or more parameters before treatment, and comparing them during treatment. Such parameters can include the IL-10 levels in serum, and the IL-12/IL-10 ratio in serum. These can be monitored using the techniques already mentioned above such as ELISA. Alternatively, the production of IL-12 and IL-10 can be monitored by drawing peripheral blood mononuclear cells from subjects before and after treatment, which cells are stimulated in vitro with antibodies against CD3 and CD28 or with lipopolysaccharide. The in vitro production of IL-10 and IL-12 can then assessed using an ELISA assay. A repeat treatment is indicated when IL-10 production or when the IL-12/IL-10 ratio have not changed sufficient compared with before treatment. For example, IL-10 may not be sufficiently reduced or the IL-12/IL-10 ratio may not be sufficiently increased.

Another embodiment of the present invention is a method for the treatment of SLE in a subject in need thereof comprising administering an effective amount of a compound of the present invention or derivative thereof. Another embodiment of the present invention is a method for the treatment of rheumatoid arthritis in a subject in need thereof comprising administering an effective amount of a compound of the present invention or derivative thereof.

IL-10 inhibits the production of inflammatory cytokines, but stimulates immunoglobulin synthesis by B cells. Accordingly, the present invention is especially effective during stages of the abovementioned diseases where autoantibody production is high. Thus, the present invention is particularly applicable to SLE and rheumatoid arthritis.

Another embodiment of the present invention is a method for the treatment of a disorder which responds to a modulation of the EPAC response, comprising administering an effective amount of a compound of the present invention or derivative thereof.

The methods of medical treatment described herein are equivalent to the use of a present compound for the preparation of a medicament for said treatment.

- Method of decreasing the level of IL-10 production in a mammalian system Another embodiment of the present invention is a method of decreasing the level of IL-10 production in a mammalian system comprising contacting said system with an effective amount of a compound of the present invention or derivative thereof. A mammalian system may be a mammalian animal, tissue or a collection of cells comprising T-cells. It may be in an in vitro system. A further aspect of the present invention is a method for decreasing the level of IL-10 production in a mammalian system, and not affecting the level of IL-2, IL-4, IL-5, IL-6, IL-12 or IFN-gamma production comprising contacting said system with an effective amount of a compound of the present invention or derivative thereof. Another embodiment of the present invention is a use of c a compound of the present invention or derivative thereof for the preparation of a medicament for decreasing the level of IL-10 production. Another embodiment of the present invention is a use of a compound of the present invention or derivative thereof for the preparation of a medicament for decreasing the level of IL-10 production, while not affecting the level of IL-2, IL-4, IL-5, IL-6, IL-12 or IFN-gamma production. Selectively controlling the level of IL-10 production in a system permits research into the function of pathways in physiological and pathological studies, and the development of new therapeutic compounds. For example, cellular conditions associated with a deficiency of IL-10 may be created in an in vitro system by the addition of a compound of the present invention, facilitating testing of candidate treatments.

- Method of changing the balance of the Th1 and Th2 response towards a higher Th1 response

Another embodiment of the present invention is a method of changing the balance of the Th1 and Th2 response towards a higher Th1 response in a mammalian system comprising contacting said system with an effective amount of a compound of the present invention or derivative thereof. Another embodiment of the present invention is a use of a compound of the present invention or derivative thereof for the preparation of a medicament for changing the balance of the TM and Th2 response towards an increased Th1 response. The method may be used, for example to mimic in vitro cellular states where the Th1 response relative to the Th2 response is lower than in normal cells, or for the treatment of disorders which response to a change in the balance of the Th1/Th2 response.

- Diagnoses of dysfunction of EPAC and/or IL-10 production pathway The finding that a compound of the present invention specifically inhibits the production of IL- 10 via binding to EPAC allows the diagnosis in a mammalian system of dysfunction of EPAC and/or the IL-10 production pathway.

Therefore, the present invention further encompasses a method of diagnosing a dysfunction of EPAC and/or the IL-10 production pathway, the method comprising: a) contacting a sample with a compound of the present invention or derivative thereof; b) measuring the level of IL-10 production; and c) comparing the level measured in step (b) with a standard, wherein a difference in production relative to the standard is diagnostic of a dysfunction of EPAC and/or the IL-10 production pathway.

A sample as used herein means any material that contains one or more components to be tested. Samples may originate from a collection of cells, tissue, organ. A cell, tissue or organ sample can comprise any cell type or tissue type present in a subject, organism, or biological system. Preferably the biological system is a mammalian system. The sample may also be derived from a synthetic in vitro system. The sample may be in a purified or unpurified form. Non-limiting examples of biological fluids include blood, serum, urine, plasma, cerebrospinal fluid (CSF), optic fluid (vitrius), semen, milk, interstitial fluid, saliva, sputum and/or synovial fluid. The sample can include a mixture of cellular and other components, including drug compounds and compositions, excipients, delivery vehicles, and/or assay reagents. The sample can include other drugs, nucleic acid molecules, infectious agents and/or components thereof. The sample can be assayed directly or can be processed, extracted, or purified to varying degrees before being assayed. The sample can be derived from a healthy subject or a subject suffering from a condition, disorder, disease or infection. For example, the subject is a human who has an auto-immune disease, an inflammatory disease an immune disorder, metabolic disease, CNS disease, neurodegenerative disease, or genetic disease.

- Modulators of EPAC activity The present invention relates to the finding of a link between modulating EPAC, and IL-10 production and/or the balance of TM / Th2 response. Any suitable EPAC modulator may, therefore, be used to selectively change the level of IL-10 production and/or the balance of Th1 / Th2 response. The modulator does not necessarily need to be the compound as disclosed herein. The embodiments described here can apply also when the compound described herein is substituted or supplemented with any suitable EPAC modulator. One embodiment of the present invention is a use as described in the embodiments herein, wherein said compound or derivative thereof is a modulator of EPAC activity or a pharmaceutical composition thereof. Another embodiment of the present invention is a method as described in the embodiments herein, wherein said compound or derivative thereof is a modulator of EPAC activity or a pharmaceutical composition thereof.

Methods of screening suitable modulators of EPAC activity can be readily determined by the skilled person. For example, a library of candidate compounds which bind to EPAC can be screening using well known array techniques which rely on binding constants, for example. The response induced by EPAC in the presence of the candidate compounds can be readily assessed by determining cytokine levels in the presence and absence of candidate compound. Derivatives

Stereoisomer, tautomers, racemates, prodrugs, metabolites, pharmaceutically acceptable salts, bases, esters, structurally related compounds or solvates of a compound of the present invention are within the scope of the invention.

The pharmaceutically acceptable salts of the compounds according to the invention, i.e. in the form of water-, oil-soluble, or dispersible products, include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3- phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such a sarginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl-bromides and others. Other pharmaceutically acceptable salts include the sulfate salt ethanolate and sulfate salts.

The term "stereoisomer", as used herein, defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable, which the compounds of the present invention may possess. Unless otherwise mentioned or indicated, the chemical designation of a compound herein encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the invention either in pure form or in admixture with each other are intended to fall within the scope of the present invention.

The compounds according to the invention may also exist in their tautomeric forms. Such forms, although not explicitly indicated in the compounds described herein, are intended to be included within the scope of the present invention.

For therapeutic use, the salts of the compounds according to the invention are those wherein the counter-ion is pharmaceutically or physiologically acceptable.

As used herein and unless otherwise stated, the term "solvate" includes any combination which may be formed by a compound of this invention with a suitable inorganic solvent (e.g. hydrates) or organic solvent, such as but not limited to alcohols, ketones, esters and the like.

The term "pro-drug" as used herein means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug. The reference by Goodman and Gilman (The Pharmacological Basis of Therapeutics, 8th Ed, McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", p 13-15) describing pro-drugs generally is hereby incorporated. Pro-drugs of the compounds of the invention can be prepared by modifying functional groups present in said component in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent component. Typical examples of pro-drugs are described for instance in WO 99/33795, WO 99/33815, WO 99/33793 and WO 99/33792 all incorporated herein by reference. Pro-drugs are characterized by increased bio-availability and are readily metabolized into the active inhibitors in vivo.

Pharmaceutical compositions

The invention provides for pharmaceutical compositions comprising a compound of the present invention. In addition to the compound, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carrier preparations described below which can be used pharmaceutically. An embodiment of the invention is a pharmaceutical composition comprising one or more compounds of the invention and one or more pharmaceutically acceptable carriers. Another embodiment of the invention is a use or method as described above wherein one or more compounds of the invention is comprised in a pharmaceutical composition.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of a compound of the present invention with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterise the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain a compound of the present invention mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilisers. Pharmaceutical formulations for parenteral administration include aqueous solutions of compounds of the present invention. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer' solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee- making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc... Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.

After pharmaceutical compositions comprising a compound of the invention formulated in a acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition with information including amount, frequency and method of administration. Dose

The present invention is generally administered to a subject in need thereof in a therapeutically effective amount. One of ordinary skill in the art will recognise that a therapeutically effective amount will vary with the disease to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient (animal or human) treated. Thus, effective dosages may range from 1 mg/kg of body weight, or less, to 25 mg/kg of body weight or more, depending upon the compound used, the condition or infection treated and the route of administration. This dosage range generally produces effective blood level concentrations of active compound ranging from about 0.04 to about 100 micrograms/cc of blood in the patient. It is contemplated, however, that appropriate patient-specific regimens will be developed by administering a small amount, and then increasing the amount until either the side effects become unduly adverse, or the intended effect is achieved.

The present invention is demonstrated by the following non-limiting examples

EXAMPLE 1

Materials and Methods

Reagents RMPI-1640 (with glutamax) was purchased from Life Technologies (Merelbeke, Belgium). Bovine serum albumin (BSA) ), H89, 8-(4-chloro-phenyl-thio)-cAMP (cptcAMP) and 8-(4- chloro-phenyl-thio)-2-O-methyl cAMP (Me-cptcAMP) (formula (III)) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

SB203580 was from Biomol (Plymouth, PA, USA). Except for cptcAMP and Me-cptcAMP, which were dissolved in LPS-free water (Baxter), all inhibitors were dissolved in DMSO. The rabbit antibodies against P-p38, ERK1/2, P-JNK, JNK, P-CREB, CREB, and the mouse monoclonal against P-ERK1/2 were obtained from Cell Signaling (Beverly, MA, USA). P-p38 antibody detects p38 only when activated by dual phosphorylation at Thr 180 and Tyr 182, P- ERK1/2 antibody detects ERK1/2 only when doubly phosphorylated at Thr 202 and Tyr 204 and P-JNK antibody detects JNK only when activated by phosphorylation at Thr 183 and Tyr 185. P-CREB antibody detects CREB only when phosphorylated at ser 133 and this antibody also detects the phosphorylated form of CREB-related protein ATF-1. Rabbit antibodies against p38 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase- conjugated donkey anti-rabbit IgG was obtained from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). The PRL-332-Luc and PRL-32-Luc plasmid constructs were described in Pohnke Y., Kempf R., Gellersen B. (1999) J. Biol. Chem. 274: 24808-24818.

Cell preparation and cell culture Jurkat cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). T lymphocytes were isolated from buffy coats obtained from normal donors. First, PBMC were isolated by centrifugation on Ficoll-lsopaque (Pharmacia & Upjohn, Uppsala, Sweden) density gradients (1.077 g/ml) at 1000 g for 20 minutes. Subsequently, T lymphocytes were isolated by sheep red blood cell resetting (Gmelig-Meyling F. and Ballieux R.E. (1977) Vox. Sang. 33: 5-8). Purity of the T lymphocyte fraction was always over 90% as assessed by flow cytometric analysis. T lymphocytes were resuspended in RPMI-1640 supplemented with 1% BSA, at a concentration of 5 x 106/ml. To measure the effects of cAMP analogues on PRL mRNA expression, cells were cultured for 6 hrs in a humidified 5% CO2 atmosphere at 37^CC. To address cAMP-induced protein phosphorylation cells were cultured for 20 min in 2 ml reaction tubes (Eppendorf, Hamburg, Germany) in a waterbath at 37°C.

Transfection of human T lymphocytes.

Freshly isolated human T lymphocytes were transfected using the Nucleofector Technology (Amaxa, Cologne, Germany) according to the manufacturer's instructions. Briefly, 2O x 106 freshly isolated T-lymphocytes were resuspended in 100 μl human T-cell nucleofector solution, supplemented with 10 μg of promoter constructs or 1 μg of pmaxGFP control vector (delivered with the nucleofector kit) and transferred to a cuvette. Nucleofection was performed using program U14. Transfection efficiency with the control vector was between 1 and 10% as assessed by flow cytometric analysis.

RT-PCR Isolation of total RNA and reverse transcription were performed as described before (46). Briefly, for PRL mRNA detection a real time cDNA amplification was performed using the Applied Biosystems (Nieuwerkerk a/d Ussel, The Netherlands) Assay-On-Demand for human prolactin (hs 00168730_m1 ) and a 5-point standard curve (0.02-200 ng total RNA from Jurkat cells). This assay uses a specific TaqMan® MGB probe with a FAMTM reporter dye at the 5¹ end and a non-fluorescent quencher at the 3' end. Fluorescence was monitored using the Taqman 7700 Sequence detector (Applied Biosystems). For detection of EPAC1 and EPAC2 transcripts, cDNA samples were subjected to 35 rounds of PCR cycling using the following primersets: 5'-CTT CCT CCA GAA ACT CTC AG-3¹ and 5'-TCA GCT CAT GCG CTT CCT G-3' (sense and antisens, respectively for EPAC1); 5'-CTC ATT GAA CCT CAC GTT CC-3' and 5'-AGT CAT CTC CTT CAT GCA GG-3' (sense and antisense, respectively for EPAC2)

Western blotting

Preparation of cellular extracts and western blotting were performed as described earlier (28). All primary antibodies were used at a 1 :1000 dilution. Before reprobing, blots were stripped by washing in ddH20 for 10 minutes, followed by a 5 minutes incubation in 0.2 M NaOH and another washing in ddH20.

Statistics

Unless stated otherwise in the figure legend, statistical difference between groups was determined by ANOVA, followed by Tukey's post-test. Data represented are means±SD of quadruplicate incubations. The presented experiments are representative of at least three independent experiments.

Experiment 1 : cAMP stimulates PRL transcription in primary human T lymphocytes.

To assess whether cAMP induces PRL expression in primary T lymphocytes, we investigated the effect of cAMP on PRL mRNA levels in purified primary human T lymphocytes. As shown in Fig. 1A, the long-acting cAMP analogue cptcAMP on average stimulated PRL mRNA levels in primary T lymphocytes by 12.94 (± 11.18) fold. Whereas PRL mRNA levels were positively affected by cAMP in all investigated donors (N=12), the size of the response to cAMP was very much donor dependent, with a 34,67 fold induction in the best responder versus only a 3,64 fold induction in the worst responder.

In Jurkat T cells the effect of cAMP on PRL transcription is mediated via a consensus site for binding of the CAAT/enhancer binding protein (C/EBP) at -214 in the extrapituitary PRL promoter (Gerlo S., Verdood P., Gellersen B., Hooghe-Peters E. L. and Kooijman R. (2004) J Immunol 173: 5952-5962.), which probably acts in concert with a cAMP-responsive element (CRE) at -25 (Reem G.H., Ray D.W. and Davis J.R. (1999) J MoI Endocrinol 22: 285-292.). To investigate whether this promoter region is also responsible for mediating the effect of cAMP on PRL transcription in primary T lymphocytes, we transfected primary T lymphocytes with a promoter construct carrying 332 bp of the extrapituitary PRL promoter coupled to a luciferase reporter gene. As shown in Fig. 1 B, cptcAMP was only a weak inducer (1.46 ± 0.26 fold) of the 332 bp PRL promoter construct. The small effect of cptcAMP on promoter activity was retained using a construct carrying only the 32 proximal bp of the extrapituitary PRL promoter and harbouring the CRE shown to be functional in Jurkat cells (Reem G. H. et al. 1999). Because of the discrepancy between the potent effect of cAMP on endogenous PRL mRNA levels versus its small effect on promoter activation, we assessed whether perhaps cAMP stimulates PRL mRNA levels by enhancing the stability of the PRL message. As shown in Fig. 2A₁ preincubation of primary T lymphocytes with the transcriptional inhibitor actinomycin D completely blocked the effect of cptcAMP on PRL mRNA expression. In addition, cptcAMP did not affect the rate of decay of the PRL message in T cells that were treated with actinomycin D (Fig. 2B). These findings indicate that cAMP enhances PRL expression in primary T lymphocytes by stimulating transcription.

Experiment 2: cAMP-induced PRL expression is partially mediated via PKA in primary human T lymphocytes.

PKA is the best known effector of cAMP signalling. We therefore investigated the effect of PKA inhibition, using H89, on cAMP-induced PRL expression in primary T lymphocytes. Fig. 3A shows that, at maximal subcytotoxic doses (10 μM), H89 only partially blocked cptcAMP- induced PRL expression, whereas it completely abolished cptcAMP-induced phosphorylation of the principal PKA target, CREB (Fig. 3B). Experiment 3: Role of MAPKs in cAMP-induced PRL expression.

It has been shown in leukocytes and other cell types that cAMP mediates some of its effects via activation or inhibition of the mitogen-activated protein kinase (MAPK) pathways (Stork P.J.S. and Schmitt J. M. (2002) Trends Cell Biol 12: 258-266). Therefore, since inhibition of PKA did not completely block cAMP-induced expression, we addressed the effect of cptcAMP on activation of p38, extracellular signal-regulated kinase (ERK) and c-jun N- terminal kinase (JNK). We found that ERK and JNK phosphorylation were undetectable both in unstimulated and cptcAMP-stimulated T lymphocytes (Fig. 4A) but could be induced by TPA and anisomycin respectively (data not shown). However, cptcAMP dose-dependently stimulated p38 phosphorylation (Fig. 4B). To address the role of PKA in the effect of cAMP on p38 activation, we investigated the effect of H89 on the phosphorylation of p38. As shown in Fig. 4C, H89 did not affect cptcAMP-induced p38 phosphorylation, indicating cptcAMP phosphorylates p38 in a PKA-independent manner. The role of the p38 MAPK in the effect of cptcAMP on PRL expression in T lymphocytes was indicated by the finding that SB203580, a specific p38 inhibitor, partially abolished the effect of cptcAMP on PRL expression (Fig. 3A).

Experiment 4: Role of EPAC in PRL expression in human T lymphocytes.

Whereas PKA is the best known mediator of cAMP effects, recently the activation of alternative, PKA-independent, signalling routes through EPAC by cAMP have been described. We therefore addressed the expression of the novel cAMP receptors EPAC1 and EPAC2 by RT-PCR in primary T lymphocytes of eight donors. As shown in Fig. 5, primary T cells from all investigated donors expressed EPAC1 transcripts, whereas EPAC2 was detected in 3 out of 8 donors (data not shown). Using a methylated cptcAMP analog, which is unable to activate PKA, but instead specifically activates EPAC, we investigated the role of EPAC in regulating PRL expression in T cells. As shown in Fig. 6A, Me-cptcAMP stimulated p38 activity in T lymphocytes, whereas it did not affect CREB phosphorylation (data not shown). However, unlike cptcAMP, which dose-dependently stimulated PRL mRNA levels in human T lymphocytes, Me-cptcAMP had no effect on PRL expression (Fig. 6B). We previously demonstrated that the stimulatory effect of cptcAMP on PRL expression in the human T leukemic cell line Jurkat is mediated by PKA (28). Indeed, as depicted in Fig. 6C, in these cells cptcAMP did not induce p38 phosphorylation. EXAMPLE 2

Materials and Methods

Reagents

CptcAMP was purchased from Sigma (Bornem, Belgium) and Me-cptcAMP was from Biolog (Bremen, Germany). SB203580 was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA). LPS

Cell preparation and cell culture Human PBMC were purified from heparinised venous blood drawn from healthy donors between 20 and 60 years of age. Informed consent was obtained from all blood donors, and the research protocol has been approved by the local ethical committee. Mononuclear cells were purified by centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway), and T cells were isolated from PBMC by rosetting with sheep erythrocytes that were pre-treated with AET. The contamination of T cell preparations with other cells, as assessed by flow cytometry, was less than 10%, and cell viability as assessed by trypan blue exclusion was always higher than 95%. Freshly isolated cells were suspended at a density of 106 cells/ml in serum-free medium (RPMI 1640 with glutamax-l, supplemented with 0.02% BSA, 100 U/ml penicillin and 100 //g/ml streptomycin), and cultured in 5 ml polystyrene Falcon tubes (Becton Dickinson, Erembodegem, Belgium) in a humidified 5% CO₂ atmosphere at 37⁰C. After culture, cells were separated from the culture medium by centrifugation for 10 min at 40Og at room temperature. PBMC were cultured in the absence or presence of 2 μg/ml PHA. In all experiments, PHA and cAMP analogues were added directly at the start of the culture period.

Cytokine measurements in culture supernatants Culture media were frozen and stored at -2O⁰C until use, and the cytokine levels were quantified by ELISAs using commercial antibody pairs (Cytosets™) from Biosource International (Nivelles, Belgium) as described in Kooijman R, Coppens A 2004 Insulin-like Growth Factor-I Stimulates IL-10 Production in Human T Cells. Journal of Leukocyte Biology 76:862-867.

Real-time PCR

Total RNA was isolated by extraction with Trizol using the standard procedure supplied by the manufacturer (Invitrogen, Merelbeke, Belgium). Reversed-transcription of 250 ng total RNA was performed using a reverse-transcription kit (Applied Biosystems, Lennik, Belgium) containing random hexamers. The real-time PCR amplification of cDNA (undiluted) from PBMC was performed on an ABI prism 7700 Sequence Detector (Perkin Elmer, Boston, MA) using an IL-10 assay-on-demand kit from Applied Biosystems (Lennik, Belgium). The results of a series of standards prepared by successive dilutions (30-2500 ng) of total RNA and plotted against the logarithm of the concentration were used to estimate the relative amount of specific mRNA initially present in the various samples. The relative IL-10 mRNA levels compared to GAPDH controls are shown.

Statistical analysis

Values are presented as the mean ± SEM. Statistical comparisons were made using the unpaired two-tailed Student's Mest when means of two groups were compared. A one way analysis of variance (ANOVA) with a Dunnett post-test was used when means of more than two groups were compared. A probability value less than 0.05 was considered significant.

Experiment 5: Effects of cptcAMP and Me-cptcAMP on cytokine secretion by T cells Purified human T cells were cultured for 24 hours in the presence or absence of the T cell activating lectin PHA. Addition of cptcAMP to the culture medium resulted in strong reduction of IL-4, IL-5, IL-10, IL-12 and INF-gamma production in PHA-activated T cells (Fig. 7). The results are shown in Table 1.

8-pCPT-2'-O- 8-pCPT-2'-O-

Th1 cpt-cAMP Me-cAMP Th2 cpt-cAMP Me-cAMP cytokines cytokines

IL-2 0 IL-4 — 0

IFNγ 0 IL-5 - 0

IL-12 0 IL-6 0 0

IL-10 - -

Table 1. Overview of differential effects of cptcAMP and Me-cptcAMP on cytokine production in Th1 and Th2 subsets. Key: - small inhibition; -- strong inhibition; 0 no effect; + stimulation.

The production of IL-2 was only slightly reduced, whereas the production of IL-6 was not affected. The cAMP analogue Me-cptcAMP which does not activate PKA, markedly reduced the production of IL-10, but had no effect on other cytokines. In the non-T cell fraction, IL-10 was undetectable in the absence of PHA nor in the presence of PHA, indicating that the effects of Me-cptcAMP were not due to contamination of non-T cells in the T cell fraction (data not shown).

To address the effect of cAMP on IL-10 production by monocytes or B cells, we stimulated the non-T cell fraction with the bacterial cell wall product LPS. The results are shown in Table and in Figure 8. They indicate that cptcAMP did not influence LPS-stimulated IL-10 production in this fraction, while it stimulated IL-6 production and strongly inhibited the secretion of IL-12. The latter two effects were not observed when the non-T cells were treated with Me-cptcAMP.

8-pCPT-2'-O- Non-T cpt-cAMP Me-cAMP cells

IL-6 + 0

IL-10 0

IL-12 0

Table 2: Overview of differential effects of cpt-cAMP and cpt-Me-cAMP on non-T cell cytokine production. Key: - small inhibition; — strong inhibition; 0 no effect; + stimulation.

Example 2 provides evidence that cAMP impairs IL-10 production in human T cells through a PKA-independent pathway, which does not affect the production of other Th2 or Th1 cytokines including IL-4, IL-5, IL-6, IL-2, IFN-gamma or IL-12.

Taken together, the results of Example 2 would indicate that Me-cptcAMP or other derivatives activating PKA-independent signalling pathways in T cells may be used to treat IL-10- mediated diseases or diseases with a Th2-mediated component.

EXAMPLE 3

Therapeutic avenues for 8-pCPT-2'-O-Me-cAMP in the treatment of IL-10-mediated diseases are explored in a mouse model for SLE. New Zealand Black (NZB)ΛΛ/ F1 mice spontaneously develop a lupus-like disease. Like humans with SLE, they develop anti-ds-DNA auto- antibodies and glomerulonephritis. Treatment with anti-IL-10 antibodies increased the 34- week survival of these mice from 10 to 80%, and decreased the level of auto-antibodies and the incidence of glomerulonephritis. In NZB/W F1 mice, we assess the effects of 8-pCPT-2'- O-Me-cAMP treatment on the levels of IL-10 and other cytokines in blood, the development of glomeral lesions, the production of anti-ds-DNA antibodies and survival.

A hallmark of SLE is B cell hyperactivity and autoantibody production. Anti-IL-10 treatment has been applied in a small clinical trial with six patients. A 3-week treatment resulted in a abatement of symptoms which was sustained for 6 months. The cutaneous and arthricular symptoms were particularly sensitive to the treatment, and endothelial and T cell activation were decreased. Although IL-10 markedly increased the in vitro antibody production in human B cells, this phenomenon has not been observed in mice, indicating that the role of IL-10 in regulation of antibody production in mice may not completely reflect its role in humans.

To overcome this problem, transfer of mononuclear cells from patients with SLE to severe combined immunodeficient (SCID) mice has been used as a model to address the in vivo role of IL-10 in autoantibody production by human cells. In this model, treatment with anti-IL-10 resulted in a impaired production of autoantibodies. We use this model to investigate the in vivo effects of 8-pCPT-2'-O-Me-cAMP on the production of autoantibodies and IL-10 by human lymphocytes.

PBMC from SLE patients produce higher levels of IL-10 than PBMC from normal donors. In these patients we investigate the in vitro effects of 8-pCPT-2'-O-Me-cAMP on IL-10 production by purified T cells, B cells and monocytes.

T-B cell interaction through cell surface molecules is crucial for regulating antibody production in B cells. B cells interact through CD40 with the CD40 ligand (CD154) on T helper cells. In addition, T cell-derived cytokines including IL-10 are also pivotal in the regulation of antibody production by B cells. However, B cells also produce IL-10. In SLE patients CD154 expression was not only confined to T cells. Patients with SLE exhibited CD154+ B cells that produce IL-10. Experiments to pinpoint the effects of 8-pCPT-2'-O-Me-cAMP on IL-10 production in different lymphocyte subsets from SLE patients and normal donors are performed. We address the effects of 8-pCPT-2'-O-Me-cAMP on the regulation of IL-10 expression in T and B cell subsets by flow cytometry using cell-specific markers and anti-IL-10 antibodies for detection of intracellular IL-10. We focus on CD154+ B cells (CD20+/CD154+) and the following T cell subsets: Effector T cells (CD8+), Regulatory T cells (CD4+/CD25+) and T helper cells (CD4+/CD154+).

Since fibroblasts and endothelial cells have also been implicated in the increased levels of IL- 10, we will also assess the effects of cAMP analogues on the IL-10 production in these cell types.

The mechanism responsible for the effects of 8-pCPT-2'-O-Me-cAMP on reduction of IL-10 production in lymphocytes using transfectable cell lines are investigated. In these cell lines we address the role of EPAC or other targets of 8-pCPT-2'-O-Me-cAMP in the regulation of IL-10. Identification of the signalling pathways leading to modulation of IL-10 in lymphocytes may lead to additional therapeutic strategies to stimulate or inhibit IL-10 production.

Claims

1. Use of a compound of formula (I) or formula (II) for the preparation of a medicament for the treatment of a disorder which responds to a decrease in the production of IL-10 and/or a change in the balance of the Th1 and Th2 response which increases the Th1 response, wherein formula (I) is:

and formula (II) is

and wherein:

R1 can be independently H, halogen, azido, alkyl, aryl, amido-alkyl, amido-aryl, OH, O-alkyl, O-aryl, SH₁ S-alkyl, S-aryl, SeH, Se-alkyl, Se-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N- bisaryl, cycloalkylamino;

R2 can be independently H, halogen, azido, O-alkyl, S-alkyl, Se-alkyl, NH-alkyl, N-bisalkyl, alkyl-carbamoyl, cycloalkylamino, silyl ; R3 can be independently H, halogen, OH, azido, amido-alkyl, amido-aryl, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, amino, NH-alkyl, NH-aryl, N-bisalkyl, N-bisaryl, NH-alkyl-carbamoyl, cycloalkylamino; and wherein R4 is O (H) or S (H); and R5 is O (H), S (H), amino, H, alkyl, O-alkyl, O-aryl, S-alkyl, S-aryl, NH-alkyl, NH-aryl, N- bisalkyl, N-bisaryl; or

R4 is O (H), S (H), amino, H, alkyl, O-alkyl, O-aryl, S- alkyl, S-aryl, NH-alkyl, NH-aryl, N- bisalkyl, N-bisaryl; and R5 is O (H) or S(H) ; R6 and R7 can be independently H, halogen, alkyl, nitro, amino, and/or alkoxy, and pharmaceutically acceptable salts, esters, and/or solvates thereof.

2. Use according to claim 1 , wherein R1 is H, halogen, azido, O-alkyl, O-aryl, S-alkyl, S- aryl, NH-alkyl, NH-aryl, or Se-aryl.

3. Use according to claim 1 or 2, wherein R1 is O-aryl, S- aryl, NH-aryl, or Se-aryl.

4. Use according to any of claims 1 to 3 wherein R1 is H, Br, Cl, I, azido, 2-chlorophenylthio, 3-chlorophenylthio, 4-chlorophenylthio, 2-bromophenylthio, 3-bromophenylthio, 4- bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n- hexylthio, 2-fluorophenylthio, 3- fluorophenylthio, 4-fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 2- aminophenylthio, 3-aminophenylthio, 4- aminophenylthio, benzylthio, phenylethylamino,3-phenyl-propylamino, 2- methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2- hydroxyethylthio, 2-aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3- methylphenylthio, 4-methylphenylthio, 2-isopropylphenylthio, 4-isopropylphenylthio, 2,3,5,6- tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, ethoxy, propioxy, butoxy, benzyloxy, 4-methylbenzyloxy, 4-methoxybenzyloxy, 4-fluorobenzyloxy, 4- chlorobenzyloxy, 4-bromobenzyloxy, phenyloxy, cyclohexylamino, benzylamino, phenylseleno, 4-isopropyloxyphenylthio, 4-methylthiophenylthio, 6-aminohexylamino, 2,3- dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5-dimethoxyphenylthio, 2,5-dimethylthiophenylthio, 2,6- dimethylthiophenylthio, or 2,6-dichlorophenylthio.

5. Use according to any of claims 1 to 4 wherein R1 is H, Br, Cl, azido, 2-chlorophenylthio, 3- chlorophenylthio, 4-chlorophenylthio, 2-bromophenylthio, 3-bromophenylthio, A- bromophenylthio, methylamino, methylthio, ethylthio, n-propylthio, n-butylthio, n-pentylthio, n- hexylthio, 2-fluorophenylthio, 3-fluorophenylthio, 4-fluorophenylthio, 4-methylcumarinyl, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 2-aminophenylthio, 3-aminophenylthio, A- aminophenylthio, benzylthio, phenylethylamino, 2-methoxyphenylthio, 3-methoxyphenylthio, 4-methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, 2- aminoethylthio, pyridinylthio, benzothiazolylthio, 2-methylphenylthio, 3-methylphenylthio, A- methyl phenylthio, 2-isopropylphenylthio, 4-isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4-dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, 4-isopropyloxyphenylthio, 4-methylthiophenylthio, 6- aminohexylamino, 2,3-dichlorophenylthio, 2,5-dichlorophenylthio, 2,4-difluorophenylthio, 2,5- dimethoxyphenylthio, 2,5-dimethylthiophenylthio, 2,6-dimethylthiophenylthio, 2,6- dichlorophenylthio.

6. Use according to any of claims 1 to 5 wherein R1 is H₁ Br, Cl, azido, 4-chlorophenylthio, methylamino, methylthio, 4-fluorophenylthio, 4- methylcumarinyl, naphtyl-2-thio, phenylthio, A- nitrophenylthio, 2-aminophenylthio, benzylthio, n-hexylthio, phenylethylamino, A- methoxyphenylthio, isopropylthio, benzimidazolyl-2-thio, 2-hydroxyethylthio, ethylthio, 2- aminoethylthio, pyridinylthio, benzothiazolylthio, 4-methylphenylthio, 3- methoxyphenylthio, A- isopropylphenylthio, 2,3,5,6-tetrafluorophenylthio, 4-hydroxyphenylthio, 2,4- dichlorophenylthio, methoxy, benzyloxy, cyclohexylamino, benzylamino, phenylseleno, A- isopropyloxyphenylthio, 4- methylthiophenylthio, or 6-aminohexylamino.

7. Use according to claim any of claims 1 to 3 wherein R1 is Cl, Br or S-aryl.

8. Use according to any claim 7 wherein R1 is Cl, Br, 4-chlorophenylthio, 4-fluorophenylthio, naphtyl-2-thio, phenylthio, 4-nitrophenylthio, 4-methoxyphenylthio, pyridinyl-2-thio, A- methylphenylthio, 4- isopropylphenylthio, 2,3,5,6-tetrafluoro-phenylthio, 4-hydroxyphenylthio, or 2,4-dichloro-phenylthio.

9. Use according to claim 7 or 8 wherein R1 is 4-chlorophenylthio, 4-fluorophenylthio, A- methoxyphenylthio, 4-methylphenylthio, or 4-hydroxyphenylthio.

10. Use according to any of claims 1 to 9, wherein R2 is H, halogen, azido, O-alkyl, or S-alkyl.

11. Use according to any of claims 1 to 10, wherein R2 is H, Cl, Br, I, O-alkyl, S-methyl, or S- ethyl

12. Use according to any of claims 1 to 11 , wherein R2 is H, Cl, Br, O-methyl, O-ethyl, O- propyl, O-butyl, O-isobutyl, or S-methyl

13. Use according to any of claims 1 to 12, wherein R2 is O-methyl, O-ethyl, O- propyl, O- butyl, or O-isobutyl.

14. Use according to any of claims 1 to 13, wherein R2 is O-methyl.

15. Use according any of claims 1 to 14, wherein R3 is amino, NH-alkyl, N-bisalkyl, NH-aryl, NH-alkyl-carbamoyl, N-bisalkyl-carbamoyl, amido-alkyl, amido-aryl, OH

16. Use according to any of claims 1 to 15, wherein R3 is amino, NH- phenyl, NH-tert-butyl, NH-tert-butylcarbamoyl, NH-phenylcarbamoyl, NH-acetyl, NH-propionyl, NH-butyryl, NH- benzoyl, NH-benzyl, NH-phenylethyl, NH-phenylpropyl, N-bismethyl, N-bisethyl, or OH

17. Use according any of claims 1 to 16, wherein R3 is amino, NH-phenyl, NH-tert-butyl, NH- tert-butylcarbamoyl, NH-phenylcarbamoyl; NH-butyryl, NH-benzoyl, NH-benzyl, N- bismethyl, N-bisethyl, or OH

18. Use according any of claims 1 to 17, wherein R3 is amino, NH-phenyl, NH-tert-butyl, NH- tert-butylcarbamoyl, NH-benzoyl, N-bismethyl or OH

19. Use according to any of claims 1 to 18, wherein R3 is amino or NH-tert-butyl

20. Use according to any of claims 1 to 19, wherein R4 and R5 are independently O (H) or S (H).

21. Use according to claim 1 wherein R4 and R5 are O(H).

22. Use according to any of claims 1 to 21 , wherein compound of formula (I) is selected from the group consisting of Preferably, the compound is selected from the group consisting of 8- bromo-2'-deoxyadenosine-3',5'-cyclic monophosphate; 8-(4-chloro-p'henylthio)-2'- deoxyadenosine-3¹, 5'-cyclic monophosphate; 8- (4-chloro-phenylthio)- N6-phenyl-2'- deoxyadenosine-3¹, 5'-cyclic monophosphate; 8-bromo-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-chloro-phenylthio)-2'-O-methyladenosine-3\ 5'-cyclic monophosphate;8-methylamino-2'-O-methyladenosine-3',5'- cyclic monophosphate;8- methylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- (4-fluoro-phenylthio)-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate; 8- (4-methyl-cumarinyl-7-thio)-2'-O- methyladenosine-3¹, 5'- cyclic monophosphate; 8-(naphtyl-2-thio)-2'-0-methyladenosine-3', 5'- cyclic monophosphate; 8-phenylthio-2'-0-methyladenosine-3', 5'-cyclic monophosphate; 8-(4- nitro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2-amino-phenylthio)- 2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-benzylthio-2'-O-methyladenosine-3',5'- cyclic monophosphate; 8-n-hexylthio-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8- phenylethylamino-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-methoxy- phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-isopropylthio-2'-O- methyladenosine-3',5'-cyclic monophosphate; 8-(benzimidazolyl-2-thio)-2'-O- methyladenosine-3¹, 5'-cyclic monophosphate; 8-(2-hydroxy-ethylthio)-2'-O-methyladenosine- 3', 5'-cyclic monophosphate; δ-ethylthio^'-O-methyladenosine-S'.δ'- cyclic monophosphate; 8- (2-amino-ethylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(pyridinyl-2-thio)- 2¹-O-methyladenosine-3',5'-cyclic monophosphate; 8-(benzothiazolyl-2-thio)-2'-O- methyladenosine-3', 5'-cyclic monophosphate^- (4-methyl-phenylthio)-2'-O-methyladenosine- 3', 5'-cyclic monophosphate; 8-(3-methoxy-phenylthio)-2'-O-methyladenosine-3', 5-cyclic monophosphate; 8- (4-isopropyl-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(2,3,5,6-tetrafluoro-phenylthio) -2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-(4-hydroxy-phenylthio)-2'-O-methyladenosine-3¹, 5'-cyclic monophosphate; 8-(2,4-dichloro-phenylthio)-2'-O-methyladenosine-3^I, 5'-cyclic monophosphate; 8-(4-chloro-phenylthio)-2'-(N, N-dimethyl) -carbamoyl-adenosine-3', 5'-cyclic monophosphate;8-methoxy-2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-benzyloxy- 2'-O-methyladenosine-3', 5'-cyclic monophosphate; 8-bromo-2'-O-methyladenosine-3\ 5'- cyclic monophosphorothioate, Sp-isomer; δ-bromo^'-O-methyladenosine-S'-δ'-cyclic monophophorothioate, Rp- isomer, 8-(4-chloro-phenylthio)-2'-0-methyladenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'- deoxyadenosine-3¹, 5'-cyclic monophosphorothioate, Rp-isomer;8-bromo-2'-deoxyadenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3\ 5'-cyclic monophosphorothioate, Rp-isomer; 8-(4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphorothioate, Sp- isomer; and 8-cyclohexylamino-2'-deoxyadenosine-3', 5'-cyclic monophosphatejβ-chloro^'-O-methyladenosine-S', 5'-cyclic monophosphate; N⁶-tert-butyl-8- (4-chloro-phenylthio)-2'-deoxyadenosine-3', 5'-cyclic monophosphate; 5,6-Dichloro-l-/?-D- ribofuranbsyl-2'-O-methylbenzimidazole-3', 5'-cyclic monophosphate.

23. Use according to any of claims 1 to 21 where, in formula (II): R4 and R5 are independently O or S

R6 and R7 are independently F, Cl, Br or I.

24. Use according to claim 23 wherein: R6 and R7 are Cl.

25. Use according to claim 23 or 24 where, in formula (II): R2 is O-alkyl.

26. Use according to any of claims 1 to 21 , 23 to 25 wherein the compound of formula (II) is selected from the group consisting of 1-deaza- adenine analogues, 3-deaza-adenine analogues, 7-deaza-adenine analogues, 1,3-dideaza-adenine analogues, 1 ,7- dideaza- adenosine analogues and benzimidazole analogues, preferably 5,6-dichlorobenzimidazole.

27. Use according to any of claims 1 to 26 wherein said disorder characterised at least in part by an imbalance in the Th1 and Th2 response where the Th1 response is low.

28. Use according to any of claims 1 to 26 wherein said disorder characterised at least in part by a high level of IL-10 production.

29. Use according to any of claims 1 to 28, wherein said disorder is allergic asthma, atopic dermatitis, cancer and chronic infection.

30. Use according to any of claims 1 to 28, wherein said disorder is systemic lupus erythematosus.

31. Use according to any of claims 1 to 28, wherein said disorder is rheumatoid arthritis.

32. Use according to any of claims 1 to 31 , comprising monitoring the levels of IL-10 and optionally IL-12 before and after treatment.

33. Use according to claim 32, wherein further treatment is given if level of IL-10 or optionally, an IL-12/IL-10 ratio show insufficient change from the level or ratio present before treatment.

34. A method of decreasing the level of IL-10 production in an in vitro mammalian system comprising contacting said system with a compound of formula (I) or formula (II) as defined in any of claims 1 to 26.

35. Method according to claim 34 for further not affecting the level of IL-2, IL-4, IL-5, IL-6, IL- 12 or IFN-gamma production.

36. Use a compound of formula (I) or formula (II) as defined in any of claims 1 to 26 for the preparation of a medicament for decreasing the level of IL-10 production.

37. Use according to claim 36 for further not affecting the level of IL-2, IL-4, IL-5, IL-6, IL-12 or IFN-gamma production.

38. A method of changing the balance of the Th1 and Th2 response in an in vitro mammalian system towards an increased Th1 response, comprising contacting said system with a compound of formula (I) or formula (II) as defined in any of claims 1 to 26.

39. A use of a compound of formula (I) or formula (II) as defined in any of claims 1 to 26 for the preparation of a medicament for changing the balance of the Th1 and Th2 response towards an increased Th1 response.

40. A method of stimulating immune responses in an in vitro mammalian system comprising contacting said system with a compound of formula (I) or formula (II) as defined in any of claims 1 to 26.

41. A use of a compound of formula (I) or formula (II) as defined in any of claims 1 to 26 for the preparation of a medicament for stimulating immune responses.

42. A method of diagnosing a dysfunction of EPAC and/or the IL-10 production pathway in a mammalian system, the method comprising the steps: a) contacting a sample with a compound of formula (I) or formula (II) as defined in any of claims 1 to 26; b) measuring the level of IL-10 production; and c) comparing the level measured in step (b) with a standard, wherein a difference in production relative to the standard is diagnostic of a dysfunction of EPAC and/or the IL-10 production pathway.

43. Method or use according to any of claims 1 to 42, wherein said compound is a stereoisomer, tautomer, racemate, prodrug, metabolite, pharmaceutically acceptable base, structurally related derivative of said compound.

44. Use according to any of claims 1 to 33, 36, 37, 39, 41 and 43, wherein said compound is comprised in a pharmaceutical composition.

45. Method according to any of claims 34, 35, 38, 40 and 43, wherein said compound is comprised in a pharmaceutical composition.

46. Use according to any of claims 1 to 33, 36, 37, 39, 41 and 43, wherein said compound is a modulator of EPAC activity or a pharmaceutical composition thereof.

47. Method according to any of claims 34, 35, 38, 41 and 43, wherein said compound is a modulator of EPAC activity or a pharmaceutical composition thereof.