WO2005120474A2

WO2005120474A2 - Phophodiesterase 10a inhibitors to amplify the action of glp1-mimetics or dpp-iv inhibitors in diabetes

Info

Publication number: WO2005120474A2
Application number: PCT/BE2005/000091
Authority: WO
Inventors: Frans Schuit
Original assignee: K.U.Leuven Research And Development
Priority date: 2004-06-05
Filing date: 2005-06-06
Publication date: 2005-12-22
Also published as: GB0412526D0; WO2005120474A3

Abstract

The present invention relates to a new method for prevention and treatment of type 2 diabetes based on administration of inhibitors of phosphodiesterase 10A (PDE l0A) expression or PDEl0A activity in combination with agents that enhance or mimic GLP-1 signalling in the beta cell, long-acting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPP-IV inhibitors). The invention is based upon the original findings that the cAMPphosphodiesterase (PDE) isoform 10A, an enzyme that degrades cAMP with high affinity and that so far was mainly associated with brain function is not only expressed in brain but also in endocrine cells like pancreatic islets of Langerhans, the site of insulin secretion, as well as in pancreatic beta cell lines (MIN6 and INS 1). This expression was detected both at the Mrna level (real-time quantitative RT-PCR or microarray analysis), and protein as evidenced by Western blots with an affinity-purified rabbit polyclonal antibody. The high abundance of PDEl0A in neuro-endocrine cell types (brain, adrenal gland, pituitary, islets of Langerhans) which demonstrates a clear role for PDElOA in hormone secretion. Furthermore it was found that PDElOA is induced by food intake in mouse islets and severely (70%) lowered by overnight fasting. As PDE l0A was specifically down-regulated (mRNA-level) in genetically modified mice that do not express functional GLP-1 or GIP receptors in beta cells (DIRKO mice), we claim in~retin receptors induce the mRNA in beta cells. This was further demonstrated in INS1 cells, where PDEl0A is induced by 6-24 exposure to agents that raise cAMP in the cells, such as forskolin, IBMX and GLP-l. Based on these observation a combination medicament has been developed combining the PDEl0A inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to remarka~ y enhance the effect of agents that enhance or mimic GLP-1 signalling in the beta cell, Longacting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPP-IV inhibitors) to treat a disorder of impaired insulin secretion such as type 2 diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes adult (LADA), impaired glucose tolerance (MT), impaired fasting glucose (IFG), gestational diabetes, and metabolic syndrome X and more particularly type 2 diabetes.

Description

Type 2 diabetes

FIELD OF THE INVENTION

This invention relates to the control of glucose homeostasis in a mammalian subject, preferably a human. More specifically, the invention relates to use of inhibitors of phosphodiesterase 10A (PDEIOA) to improve glucose homeostasis in type 2 diabetic patients and the use of PDEIOA as a tool to screen for new pharmaceuticals to control glucose homeostasis in type 2 diabetes .

BACKGROUND OF THE INVENTION

Release of insulin from pancreatic beta cells is a cornerstone of mammalian glucose homeostasis and therefore a target for pharmacological intervention in type 2 diabetes.

The mechanism through which beta cells develop a large plasticity in their insulin secretory competence is currently believed to depend on altered gene expression, which is chronically regulated by glucose and the incretin hormones GLP-1 and GIP (1). Of importance is the idea that chronic incretin stimulation leads to a better insulin secretory competence (2) and protects against beta cell apoptosis (3). Long-acting GLP-1 analogs like exendin-4 and inhibitors of GLP-1 breakdown (DPP-IV inhibitors) are currently tested by the industry and academic partners for their usefulness in type 2 diabetes.

Agents that raise cyclic AMP (cAMP) in beta cells can be considered from the beta cell viewpoint as safe drugs, because they only trigger insulin release when the extracellular glucose concentration is above basal. For this reason, they are better suited than the currently used sulfonylurea drugs (closing K⁺ _Aτp-channels), as those replace part of the glucose stimulation; this mode of action can cause dangerous hypoglycemia when the drug continues to act in a patient whose blood glucose is low (4).

For the reasons summarized above, the pharmaceutical industry has investigated the types of phosphodiesterase expressed in pancreatic beta cells with the goal to develop specific PDE- inhibitors for type 2 diabetes. This initiative was abandoned some years ago after finding that PDE3b was a major isoform not only in beta cells (5) but also in adipocytes (ref. 6) and in liver. Therefore, PDE3b inhibitors would not only stimulate insulin release, but would also counteract insulin action in the liver and in adipose tissue, causing hyperglycemia and lipolysis, thereby actually predisposing to diabetes (7).

By present invention, however, it has been demonstrated that PDEIOA can be used as a novel drag target for type 2 diabetic patients. It has been demonstrated that 1) PDEIOA has a favourable expression profile in primary tissues as compared to other PDE's, particularly PDE3b, 2) that the PDEIOA mRNA abundance is responsive to chronic changes in cellular cAMP levels and might thus influence gene expression in the beta cell. On basis of these observations it can be proposed that inhibitors of PDEIOA will mimic the chronic effect of GLP1 on the beta cell. It is claimed that this novel pharmacodynamic principle will be applicable in concert with other strategies such as long acting incretin hormones or inhibitors of incretin breakdown to improve beta cell function in type 2 diabetes.

ILLUSTRATIVE EMBODIMENT OF THE INVENTION

Some terms and definitions in the present invention will be defined first.

The term "pharmaceutically acceptable" is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product. The term "treatment" refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly.

The term "a compound that inhibits the expression" refers here to gene expression and thus to the inhibition of gene transcription and/or translation of a gene transcript (mRNA) such as for example the PDEIOA gene. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher.

The term "a compound that inhibits the activity" refers to the protein that is produced such as PDEIOA. The inhibition of activity leads to a diminished interaction of PDEIOA. Preferably said inhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even higher. The term 'medicament to treat' relates to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat diseases as indicated above. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'medicament' may be administered by any suitable method within the knowledge of the skilled person. The preferred route of administration is orally. In case of parental administration, the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual.

Legend the graphics

Figure 1 demonstrates the PDEIOA messenger RNA tissue expression profile in random fed C57B16 male mice. Data represent means + SD of 3-5 experiments. mRNA analysis was performed with Affymetrix 430 20 mRNA expression profiles and could be confirmed by real-time RT-PCR (not shown). The data show that PDEIOA is highly expressed in neurotransmitter/hormone secreting cells in brain, islets of Langerhans, pituitary and adrenal glands. On the contrary, PDEIOA mRNA abundance is 10-30 fold lower in fat, muscle and liver, the main insulin target tissues. As cAMP is a well known stimulus for hormone/neurotransmitter release, it can be claimed that the neuroendocrine-specific mRNA expression of PDEIOA reflects a role in hormone/neurotransmitter release.

Figure 2 demonstrates the PDEIOA messenger RNA profile in fasting or fed C57B16 male wild type mice and which have a double incretin receptor null mutation (DLRKO mice).

PDEIOA messenger RNA is lowered by fasting in C57B16 male wild type mice and in random fed mice that have a double incretin receptor null mutation (DLRKO mice). Data represent means + SD ■ of 4-5 experiments performed with Affymetrix 430 20 mRNA expression profiles as can be confirmed by real-time RT-PCR (not shown). The decrease in PDEIOA expression is of similar magnitude during fasting (70%) as in the islets of DIRKO mutant mice (60%; random fed condition), indicating that incretins induce the PDEIOA gene during food intake. Significance of differences in PDEIOA mRNA between fed and fasted wild type mice or between fed wild type and DLRKO mice was calculated using the unpaired two-taild Student's t-test.

The decrease in PDEIOA mRNA in the DLRKO mutation is specific as more than 99% of all transcripts expressed in islets of Langerhans are unaltered by the mutation (data not shown).

Figure 3 is a graphic display of PDEIOA mRNA in LNS1 -cells is induced by agents that raise intracellular cAMP. The cell line LNSlwas exposed for 6 to 24 h to basal medium (control) or cultrae medium supplemented with 300 nM forskolin or 25 microM LBMX. Data are shown for 12 h exposure. Similar data ere obtained at 6 and 24 h. The incretin GLP-1 enhances PDE 10 A mRNA at the 6h time point (data not shown) .

Figure 4 demonstrates the expression of PDEIOA (top) and PDE3B (middle) in mouse primary tissues and the clonal beta cell line INS1. The affinity purified rabbit anti-PDE antiserum was produced at the KU-Leuven.

Examples

Example 1: mRNA expression profile of PDEIOA in mouse tissues and in INS1 cells

The pharmaceutical industry is currently exploring novel anti-diabetic drags that enhance insulin release from beta cells during a meal. Compounds that enhance or mimic the glucagon-like peptide 1 (GLP-l)or glucose-dependent insulinotropic peptide (GLP) - stimulated, cyclic AMP (cAMP)-mediated, signalling pathway in beta cells deserve particular attention, as cAMP -induced insulin release only occurs when glucose is rising at the same time, i.e. during a meal. Drags that act via the GLP-1 / cAMP pathway are therefore considered safer than sulfonylurea or other K+atp-channel blockers, as these drags also induce insulin release at low glucose, potentially causing hypoglycemia. Examples of new drags acting on the cAMP pathway in beta cells are: inhibitors of dipeptidylpeptidase-LV (DPPrV, the enzyme that degrades GLP-1) and exendin-4 (a potent GLP-1 analog). In a systematic research effort to study cAMP mediated and food-intake-related gene expression in beta cells, we discovered that the cAMP-phosphodiesterase (PDE) isoform 10A, an enzyme that degrades cAMP with high affinity (ref 9) and that so far was mainly associated with brain function (ref. 10) is linked to hormone secretion and induced by conditions that are relevant for novel type 2 diabetic therapies. The evidence is threefold: (1) PDEIOA is not only expressed in brain but also in endocrine (hormone-secreting) cells in the pancreatic islets of Langerhans, pituitary and adrenal glands (figure 1). In the islets one expressing cell type is the insulin producing beta cell as the mRNA is high in clonal beta cells (MLN6 and LNS1); this expression was detected both at the mRNA level (real-time quantitative RT-PCR or microarray analysis), and protein as evidenced by Western blots with an affinity-purified rabbit polyclonal antibody. On the contrary, expression is low in the main insulin target tissues (adipose tissue, liver and skeletal muscle). (2) PDEIOA mRNA is induced by regular food intake in mouse islets in vivo; up to 70% decrease in mRNA level is observed after overnight fast (figure 2-left); (3) PDEIOA mRNA is specifically down-regulated at the mRNA-level (figure 2-right) and protein level in genetically modified mice that do not express functional GLP-1 or GLP receptors in beta cells (DLRKO mice; mice obtained form Dr. D. Dracker, University of Toronto, Canada; ref 11). (4) A further relationship between incretins, food intake and PDEIOA gene expression is documented by the fact that mRNA is induced by 6-24 exposure of the clonal beta cell line LNSl (obtained from Dr. C. Newgard, Duke University, Durham, NC, USA; ref. 12) to agents that raise cAMP in the cells, such as forskolin, LBMX (figure 3) as well as and GLP-1 (data not shown). Genome-wide profiling of messenger RNA (mRNA) prepared from the clonal beta cell line INS-1 identifies PDE as the highest expressed PDE isoform.

From these observations we claim that PDEIOA is a neuroendocrine phosphodiesterase that regulates hormone/neurotransmitter release. As the encoded enzyme degrades cAMP, a potent inhibitor of hormone release, the induction of the PDEIOA gene by food intake/incretins is claimed to dampen the efficacy of GLP-1 analogs or DP-LV inhibitors. It is furthermore predicted that the clinical efficacy of new drags acting on the cAMP pathway in beta cells (inhibitors of DPPLV, or analogs of GLP-1 / GLP) will be significantly curtailed by this phenomenon as the desired pharmacological effect (a rise of cAMP in the beta cell) will be counteracted by induction of an enzyme that degrades cAMP.

The patent claims the use of specific PDEIOA antagonists based upon dipyridamole or derivates thereof to synergize with inhibitors of DPPLV, or with analogs of GLP-1 / GLP. The rationale of such synergism is that it allows reduction of required optimal dose, preventing costs and risk for side effects.

Example 2: Regulation of mRNA PDEIOA expression by cAMP The relatively low Km of PDEIOA for cAMP and a nuclear translocation signal in the protein sequence (ref. 9) was earlier interpreted as being an argument against a role of PDEIOA as a signal generator in stimulated beta cells (ref. 13, page 1182). However it can be argued that the low Km may be anticipated that this isoform carries the unique potential to regulate (together with influx rates through nuclear pores) cAMP levels in the nucleoplasm. Such regulation would balance the effect of protein kinase A and allow another level of control by which cAMP initiates, suppresses, modulates or regulates gene transcription. We demonstrated that the abundance of PDEIOA mRNA in the beta cell is affecting cAMP- responsive genes, as discovered by a genome-spanning microarray analysis and that two different agents known to raise cAMP in beta cells, namely 3-isobutyl-l-methylxanthine, 50 μM, a well known non-specific PDE-inhibitor (14), and forskolin, 0.3 μM, a well known activator of adenylyl cyclases (14) induce PDEIOA mRNA as measured after 6 h exposure to these agents in tissue culture. Even with a low number of experiments (N=3) this induction is significant (Figure 3) and is accompanied by induction of other cAMP-responsive genes such as ICER (CREM). These data suggest that induction of PDEIOA allows cells chronically exposed to high cAMP levels to accelerated cAMP breakdown in the nucleus in order to desensitize cAMP-induced gene expression. These experiments could also indicate that the proposed chronic beneficial effects of GLP-1 on beta cell function (1-3), a desired pharmacological action in diabetes, can be mimicked by treating patients with PDEIOA - inhibitors.

This invention also relates to compositions for treatment (particularly the prevention or suppression) of type 2 diabetes in a subject, more specifically a composition comprising a molecule selected from the list consisting of an antisense or gene silencing molecule, a ribozyme (all targeted at PDEIOA expression), a small molecule PDEIOA inhibitor, an antibody in an effective amount to inhibit the expression and/or activity of a PDEIOA gene (PDEIOA inhibitor). The compositions for treating type 2 diabetes, comprising an effective quantity of a PDEIOA inhibitor can be in admixture with pharmaceutically acceptable diluents, carriers or excipients.

The invention provides compositions and methods useful for treating type 2 diabetes in mammals, including humans, particularly when in combination with low doses of agents that enhance or mimic GLP-1 signalling in the beta cell. The invention applies to human and veterinary applications. The inventive composition and method have been shown to be especially effective in to treating type 2 diabetes.

Compounds useful for exercising the type 2 diabetes treatment or the manufacturing of a medicament for type 2 diabetes of present invention are thus compounds that inhibit the activity of PDEIOA. For instance several compounds are known to be PDEIOA antagonists as for instance the compounds of the group consisting of the pyrrolo (2.1 - A) dihydroisoquinolines. Certain pyrrolo[2.1-a] isoquinoline derivatives are known from literature as, for example , hypotensive or psychotropic agents 'e.g. GB-A 1, 153, 670, U.S. 4,694,085; Meyer, Liebigs Ann. Chem. 9, 13_534 - 1544 (1981). Pyrrolo[2.1-a]isoquinoline derivatives for the treatment of dermatologic diseases such as psoriasis are disclosed in WO 98/55118. Pyrrolo [2.1 -a] isoquinoline derivatives have been also described in J. Med. Chem 27, 1321 (1984) and in J.Med. Chem. 31, 2097 (1988). Tetra compounds containing Pyrrolo[2.1-a]isoquinoline moiety have been described in Arch. Pharm 321, 481 (1988). And WO0248144 discloses Pyrrolo[2.1-a]isoquinolines, that specifically inhibit PDE 10a for use in cancer therapy.

Dipyridamole (Persantin) has a relatively high affinity for PDEIOA. Dipyridamole (Persantin) has been used for decades in humans and is known to be a relatively safe drag (15). It inhibits isolated PDEIOA enzyme at 1 μM concentration. Thus some analysis of existing chemical structures seems indicated and it can be proposed at this moment that a dipyridamole-related molecule could fit into a new clinical indication, namely type 2 diabetes with a sufficient beta cell reserve to modify the beta cell secretory competence.

The invention is directed to the usage of molecules that act as selective inhibitors (or antagonists) of PDEIOA such as antibodies and functional fragments derived thereof, anti- sense RNA and DNA molecules (e.g. polynucleotide sequences), ribozymes that function to inhibit the translation of PDEIOA.

One such inhibitor of PDEIOA is Dipyridamole, Registry Number: 58-32-2 of the Formula: C24 H40 N8 04, of CA Index Name: Ethanol, 2,2',2",2'"-[(4,8-di-l-piperidinylpyrimido[5,4- d]pyrimidine-2,6-diyl)dinitrilo]tetrakis- (9CI) and with the structure formula:

and which has been also named by Ethanol, 2,2',2",2'"-[(4,8-dipiperidinopyrimido[5,4- d]pyrimidine-2,6-diyl)dinitrilo]tetra- (6CI,8CI); Pyrimido[5,4-d]pyrimidine, ethanol deriv.; 2,2',2",2'''-[[4,8-Dipiperidinopyrimido(5,4-d)pyrimidine-2,6-diyl]dinitrilo]tetraethanol; 2,6- Bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrimidine; Anginal; Apricor; Cardioflux; Cardoxil; Cardoxin; Cleridium; Coribon; Coridil; Coronarine; Corosan; Coroxin; Curantyl; Dipyridamine; Dipyridamol; Dipyridamole; Dipyridan; Gulliostin; Kurantil; NSC 515776; NSC 619103; Natyl; Peridamol; Persantin; Persantine; Piroan; Prandiol; Protangix; RA 8; Stenocardil; Stimolcardio

Small molecules can also interfere by binding on the promoter region of PDEIOA and inhibit binding of a transcription factor on said PDEIOA promoter region so that no PDEIOA mRNA is produced.

Also, the molecules in this invention comprise antagonists of PDElOA-gene action such as anti- PDEIOA antibodies and functional fragments derived thereof, anti-sense oligonucleotides, small interfering RNA and ribozymes that function to inhibit the synthesis of PDEIOA protein.. Small molecules can bind on the promoter region of PDEIOA and inhibit binding of a transcription factor or said molecules can bind said transcription factor and inhibit binding to the PDEIOA -promoter. By PDEIOA it is meant also its variant forms which may occur as a result of alternative RNA splicing such as PDE10A1 and PDE10A2.

In another the invention provides the use of molecules that inhibit the expression and/or activity of PDEIOA for the manufacture of a medicament for treatment of type 2 diabetes. Thus more specifically the invention relates to the use of molecules that neutralize the activity of PDEIOA by interfering with its synthesis, translation and enzymatic activity (the hydrolysis of cAMP and cGMP). By molecules it is meant peptides, tetrameric peptides, proteins, organic molecules, having the same neutralizing effect as stated above. Also, in this invention the molecules comprise antagonists of PDEIOA such as anti- PDEIOA antibodies and fab's and single chains or other functional fragments derived thereof, anti-sense RNA and DNA molecules and ribozymes that function to inhibit the transcription of the PDElOA-gene or translation of the encoded PDElOA-mRNA, all capable of interfering/or inhibiting the PDEIOA activity.

If the medicament comprises a molecule that neutralizes the activity of PDEIOA is a protein, polypeptide, or peptide the medicament is generally administered so that protein, polypeptide, or peptide is given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg /kg. Preferably it is given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 5 and 20 μg/kg/minute, more preferably between 7 and 15 μg/kg/minute.

The term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against PDEIOA or any functional derivative thereof, with said antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab')2, F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof. These antibodies of the invention, including specific polyclonal antisera prepared against PDEIOA or any functional derivative thereof, have no cross-reactivity to others proteins. The monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse immunized against PDEIOA or any functional derivative thereof, and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing PDEIOA or any functional derivative thereof which have been initially used for the immunization of the animals. The monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Alternatively the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCLD) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in US patent 5,545,806. Also fragments derived from these monoclonal antibodies such as Fab, F(ab)'2 and ssFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. An appropriate label of the enzymatic, fluorescent, or radioactive type can label the antibodies involved in the invention.

Small molecules, e.g. small organic molecules, and other drag candidates can be obtained, for example, from combinatorial and natural product libraries. Random peptide libraries, such as the use of tetrameric peptide libraries such as described in WOO 185796, consisting of all possible combinations of amino acids attached to a solid phase support may be used in the present invention.

Also within the scope of the invention is the use of oligonucleotide sequences that include anti-sense RNA and DNA molecules, small-interfering RNA, and ribozymes that function to inhibit the translation PDEIOA mRNA. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site. Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of PDEIOA RNA sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors, which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducible, depending on the promoter used, can be introduced stably into cell lines.

Another aspect of administration for treatment is the use of gene therapy to deliver the above mentioned anti-sense gene or functional parts of the PDEIOA gene or a ribozyme directed against the PDEIOA mRNA with preferential targeting to pancreatic beta cells. Gene therapy means the treatment by the delivery of therapeutic nucleic acids to patient's cells. This is extensively reviewed in Lever and Goodfellow 1995; Br. Med Bull.,51, 1-242; Culver 1995; Ledley, F.D. 1995. Hum. Gene Ther. 6, 1129. To achieve gene therapy there must be a method of delivering genes to the patient's cells of interest and additional methods to ensure the effective production of any therapeutic genes. There are two general approaches to achieve gene delivery; these are non- viral delivery and virus-mediated gene delivery.

Another embodiment of present invention is the use of a the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to remarkably enhance the effect of agents that enhance or mimic GLP-1 signalling in the beta cell, incretin mimetics, long-acting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPP-LV inhibitors) on secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. The PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE 10 inhibitor, papaverine can be combined with a agents that enhance or mimic GLP-1 signalling in the beta cell, Long-acting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPP-LV inhibitors) to manufacture a medicament to stimulate the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin

This is particularly suitable to treat - critical ill patients, disorders of polycystic ovary syndrome, obesity, and related conditions, disorders of insulin resistance, diabetes, maturity- onset diabetes of the young (MODY), latent autoimmune diabetes adult (LAD A), impaired glucose tolerance (MT), impaired fasting glucose (LFG), gestational diabetes, metabolic syndrome X, premature birth conditions and puberty disorders, in particular precocious puberty or accelerated puberty.

A particular embodiment of present invention is the use of the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to enhance the effect of a DPP-LV inhibitor on the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. Examples of such DPP-LV inhibitors that can be combined with the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to increase the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin are MK-0431, which is a specific DPP-LV inhibitors developed by Merck Research Laboratories or Vildagliptin-DPP-LV inhibitor (Vildagliptin (LAF 237) of Novartis), PT 630 or PT-630 (of Point Therapeutics), TA 6666 (815541 A, T 6666. of Tanabe Seiyaku), SSR 162369 (of Sanofi-aventis), isoleucine thiazolidide, valine- pyrrolidide.

Other DPP-LV suitable for present invention have been described in US20050038020 (adamantylglycine-based inhibitors of dipeptidyl peptidase LV), in US20040152745, in US20040132713 (Fluorinated cyclic amides dipeptidyl peptidase LV inhibitors), WO2005025554, WO2005012249 (adamantylglycine-based inhibitors of dipeptidyl peptidase LV), WO2004099185 (2-cyanopyrrolidine derivatives DPP-LV inhibitors),

Other examples of DPP-LV inhibitors that can be combined with the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to increase the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin is the DPP-LV inhibitor such as Saxaglipin (Synonyms: BMS 477118, Dipeptidyl peptidase LV inhibitors research programme - Bristol-Myers Squibb, DPPLV-BMS Chemical name: (lS,3S,5S)-2-{(2S)-Amino-2-(3-hydroxyadamantan-l- yl)acetyl }-2-azabicyclo(3.1.0)hexane- 3-carbonitrile and with the structure formule:

or the DPP-LV inhibitor Vildagliptin (Synonyms: LAF 237, LAF 237A, NVP LAF 237 Chemical name: l-(2-(3-Hydroxyadamant-l-ylamino)acetyl)pyrrolidine-2(S)-carbonitrile Novartis (Switzerland), Novartis (USA)) and with the structure formula:

or the DPP-LV inhibitor SDZ 272070 (Chemical name: l-(L-Valyl)pyrrolidine), with the structure formula:

The DPP-LV inhibitor to be combined with PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine may be a compound selected from the group of compounds consisting of Talabostat, PSN 9301 (P 9301, P93/01), SYR 619, SYR 322, SSR 162329, FE 999011, P 3298, TSL 225, NVP DPP 728, SDZ 272070, Saxaglipin, Vildagliptin, 823093 and TA 6666.

A particular embodiment of present invention is the use of a the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to enhance the effect of an insulinotropic agent on secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. The PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine can be combined with an insulinotropic agent to manufacture a medicament to stimulate the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. Insulinotropic agents include peptides such as incretins, which promote insulin secretion and beta cell development. Insulinotropic peptides may be synthesized by conventional means as detailed below, such as solid-phase peptide synthesis. Solid phase peptide synthesis is described in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. Insulinotropic peptides may also be produced through the use of recombinant DNA technology (for example, see Sambrook "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press 3rd Edition (2001), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, (1994)).

Yet another particular embodiment of present invention is the use of a the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine, to enhance the effect of incretins, for instance GLP-1, GLP or their mimetics on secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. The PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDE10 inhibitor, papaverine can be combined with incretins, for instance GLP-1, GLP or their mimetics to manufacture a medicament to stimulate the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. Incretins include glucagon-like peptide-1 (GLP-1) (P01275 GL45644939 residues 7 to 36 or 37), glucose-dependent insulinotropic polypeptide (GLP) (P09681 GL121194 residues 52 to 93) and derivatives or agonists of these peptides, including exendin 3 (P20394 GI: 119677), exendin 4 (AAB22006.1 GL248418) and NN2211 (Agersø H, et al. Diabetologia 2002; 45: 195-202). A particular embodiment of present invention is the use of a the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDEIO inhibitor, papaverine, to enhance the effect of exendin-4, in particular the synthetic exendin-4, Exenatide, on secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. The PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDEIO inhibitor, papaverine can be combined with exendin-4, in particular the synthetic exendin-4, Exenatide, to manufacture a medicament to stimulate the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin.

Yet another embodiment of present invention is the use of a the PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDEIO inhibitor, papaverine, to enhance the effect of a incretin mimetics with a DPP-LV inhibitor on secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin. The PDEIOA inhibitor, Dipyridamole or derivatives thereof or the PDEIO inhibitor, papaverine can be combined with a incretin mimetics with a DPP-LV inhibitor to manufacture a medicament to stimulate the secretion of an neuroendocrine factor or endocrine factor, in particular the secretion of insulin.

Summary of the invention

The present invention relates to a new method for prevention and treatment of type 2 diabetes based on administration of inhibitors of phosphodiesterase 10A (PDEIOA) expression or PDEIOA activity in combination with agents that enhance or mimic GLP-1 signalling in the beta cell, Long-acting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPPLV inhibitors). The invention is based upon the original findings that the cAMP- phosphodiesterase (PDE) isoform 10A, an enzyme that degrades cAMP with high affinity and that so far was mainly associated with brain function is not only expressed in brain but also in endocrine cells like in the pancreatic islets of Langerhans, the site of insulin secretion; this expression was detected both at the mRNA level (real-time quantitative RT-PCR or microarray analysis), and protein as evidenced by Western blots with an affinity-purified rabbit polyclonal antibody. The specific expression of PDEIOA in neuro-endocrine cell types (brain, adrenal gland, pituitary, islets of Langerhans) suggests an important and thus far unrecognized role for PDEIOA in hormone secretion. He low expression in muscle, adipose tissue and liver gives tis isoform a more favorable expression prodile than, for instance PDE3B. Second, we claim that PDEIOA expression level is maintained in the beta cell by food intake (at least in the mouse islet), since the expression of PDEIOA is lowered by 70% during overnight fasting. Incretin receptors are likely responsible for this effect as PDEIOA was specifically down-regulated in genetically modified mice that do not express functional GLP-1 or GLP receptors in beta cells (DLRKO mice - ref. 11). Finally a cyclic AMP signalling cascade is proposed for this effect, as PDEIOA mRNA is induced severalfold by 6- 24 exposure of the beta cell line LNS1 (ref. 12) to agents that raise cAMP in the cells, such as forskolin, LBMX and GLP-1. Based on these observations we claim that PDEIOA ihnibitors will be clinically useful to reinforce therapy based upon incretin signalling in the beta cell. For instance, a combination of the PDEIOA inhibitor, dipyridamole or derivatives thereof, or of the PDE10 inhibitor, papaverine togetehr with long-acting GLP-1 analogs like exendin-4 or inhibitors of GLP-1 breakdown (DPP-LV inhibitors) is expected to be particularly effective to treat impaired insulin secretion such as is present in type 2 diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes adult (LAD A), impaired glucose tolerance (MT), impaired fasting glucose (IFG), gestational diabetes, and metabolic syndrome X and other forms of diabetes with a residual beta cell mass.

The present invention involves the use of a Dipyridamole or a pyrrolo (2.1 - A) dihydroisoquinoline derivative a pharmaceutically acceptable composition comprising at least one of these compounds for the manufacture of a medicament to stimulate the insulin production and release in beta cells of a mammal or for the manufacture of a medicament to stimulate the insulin production and release in beta cells of a mammal to treat type 2 diabetes.

Yet another embodiment of present invention is a compound that inhibits the expression of the mammalian phosphodiesterase lOA-gene selected from the list consisting of an antisense molecule, a RNAi and a ribozyme, a small molecule modulator of a transcription factor for the manufacture of a medicament to stimulate the insulin production and insulin release in beta cells of a mammal.

The present invention may also involve the use of a compound that selectively inhibits the activity of phosphodiesterase 10A selected from the list consisting of an small molecule, an aptamer, an antibody, a transdominant ligand, a tetrameric peptide for the manufacture of a medicament to stimulate the insulin production and release in beta cells of a mammal. An alternative embodiment of present invention method of stimulating the insulin production and release in the beta cells of a mammal comprising administering a compound that selectively inhibits the enzymatic activity of phosphodiesteraselOA protein or a method of stimulating the insulin production and release in the beta cells of a mammal comprising administering a compound that that inhibits the expression of the phosphodiesterase 10 A-gene.

References to the present application

1. Hinke SA, Hellemans K, Schuit F (2004). Plasticity of the beta cell insulin secretory competence. Preparing the pancreatic beta cell for a next meal. Topical Review: J. Physiol. (London) In Press

2. Delmeire D, Flamez D, Moens K, Hinke SA, Van Schravendijk CFH, Pipeleers D, Schuit F (2004). Prior in vitro exposure to GLP-1 with or without GLP can influence the subsequent beta cell responsiveness. Biochem. Pharmacol. 68, 33-39

3. Dracker DJ (2003). Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol 17,161-171.

4. Katz ELM, Bissel G (1965). Blood sugar lowering effects of chlorpropamide and tolbutamide. A double blind cooperative study. Diabetes 14, 650-657. 5. Ahmad M, Abdel-Wahab YH, Tate R, Flatt PR, Pyne NJ, Furman BL (2000). Effect of type-selective inhibitors on cyclic nucleotide phosphodiesterase activity and insulin secretion in the clonal insulin secreting cell line BRIN-BDll. Br J Pharmacol. 129, 1228-1234.

6. Onuma H, Osawa H, Yamada K, Ogura T, Tanabe F, Granner DK, Makino H. (2002) Identification of the insulin-regulated interaction of phosphodiesterase 3B with 14-3-3 beta protein. Diabetes 51,3362-7.

7. Tang Y, Osawa H, Onuma H, Nishimiya T, Ochi M, Makino H (1999). Improvement in insulin resistance and the restoration of reduced phosphodiesterase 3B gene expression by pioglitazone in adipose tissue of obese diabetic KKAy mice. Diabetes 48,1830-1835.

8. Pipeleers DG, In 't Veld PA, Van de Winkel M, Maes E, Schuit FC & Gepts W (1985). A new in vitro model for the study of pancreatic A- and B-cells. Endocrinology 117, 806-816. 9. Soderling SH, Bayuga SJ, Beavo JA (1999). Isolation and characterization of a dual- substrate phosphodiesterase gene family: PDEIOA. Proc Natl Acad Sci U S A. 96,7071-7076.

10. Fujishige K, Kotera J, Michibata H, Yuasa K, Takebayashi S, Okumura K, Omori K. (1999) Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDEIOA). J Biol Chem. 274(26): 18438-45.

11. Hansotia T, Baggio LL, Delmeire D, Hinke SA, Yamada Y, Tsukiyama K, Seino Y, Hoist JJ, Schuit F, Dracker DJ. (2004) Double incretin receptor knockout (DLRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-LV inhibitors. Diabetes 53:1326-35.

12. Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB (2000). Isolation of LNS-1 -derived cell lines with robust ATP-sensitive K+channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49,424-30.

13. Pyne NJ, Furman BL (2003). Cyclic nucleotide phosphodiesterases in pancreatic islets. Diabetologia 46,1179-1189.

14. Schuit FC & Pipeleers DG (1985). Regulation of adenosine 3', 5'-monophosphate levels in the pancreatic B-cell. Endocrinology 117, 834-840.

15. Diener HC (1998). Dipyridamole trials in stroke prevention. Neurology. 51(3 Suppl 3),S17-S19.

Claims

I) The use of the PDEIOA inhibitor, Dipyridamole, and an incretin or a DPP-LV inhibitor for the manufacture of a medicament for treatment of an impaired secretion or production of a regulatory protein. 2) The use of claim 1) wherein the regulatory protein is a hormone.

3) The use of claim 1) wherein the regulatory protein is an endocrine factor.

4) The use of claim 1) wherein the regulatory protein is a neuroendocrine factor

5) The use of claim 1) wherein the regulatory protein is insulin

6) The use of claim 1) wherein the incretin is an incretin mimetic 7) The use of claim 1) wherein the incretin is an agent that enhances or mimics GLP-1 signalling

8) The use of claim 1) wherein the incretin is a GLP-1 analog 9) The use of claim 1) wherein the incretin is exendin-4 or a synthetic or recombinant analogue thereof.

10) A pharmaceutical composition comprising the PDEIOA inhibitor, Dipyridamole, and an incretin or a DPP-IV inhibitor for use in treatment to enhance secretion of a regulatory protein.

I I) The pharmaceutical composition of claim 10, wherein the regulatory protein is a hormone.

12) The pharmaceutical composition of claim 10, wherein the regulatory protein is an endocrine factor.

13) The pharmaceutical composition of claim 10, wherein the regulatory protein is a neuroendocrine factor 14) The pharmaceutical composition of claim 10, wherein the regulatory protein is insulin

15) The pharmaceutical composition of claim 10, wherein the incretin is an incretin mimetic

16) The pharmaceutical composition of claim 10, wherein the incretin is an agent that enhances or mimic GLP-1 signalling

17) The pharmaceutical composition of claim 10, wherein the incretin is a GLP-1 analog 18) The pharmaceutical composition of claim 10, wherein the incretin is exendin-4 or a synthetic or recombinant analogue thereof.

19) The use of an antisense, a ribozyme, RNAi molecule inhibiting the expression and/or activity of a PDEIOA gene, and an incretin or a DPP-LV inhibitor for the manufacture of a medicament for treatment of an impaired secretion or production of a regulatory protein.

20) The use of an aptamer, an antibody, a transdominant ligand, a tetrameric peptide inhibiting the activity of PDEIOA, and an incretin or a DPP-LV inhibitor for the manufacture of a medicament for treatment of an impaired secretion or production of a regulatory protein.