WO2020152280A1

WO2020152280A1 - Lsd1 inhibitors for use in the treatment of type 2 diabetes

Info

Publication number: WO2020152280A1
Application number: PCT/EP2020/051654
Authority: WO
Inventors: Benoit Raymond Gauthier; Petra Isabel LORENZO OVEJERO; Esther DE LA FUENTE MARTÍN; Nadia COBO VUILLEUMIER; José Manuel MELLADO GIL; Francisco Javier BERMÚDEZ SILVA; Gemma ROJO MARTÍNEZ; José Carlos REYES ROSA
Original assignee: Consejo Superior de Investigaciones Cientificas CSIC; Servicio Andaluz de Salud; Fundacion Publica Andaluza Progreso y Salud
Current assignee: Consejo Superior de Investigaciones Cientificas CSIC; Servicio Andaluz de Salud; Fundacion Publica Andaluza Progreso y Salud
Priority date: 2019-01-24
Filing date: 2020-01-23
Publication date: 2020-07-30
Anticipated expiration: 2021-07-24

Abstract

The present invention is focused on the use of lysine specific demethylase (LSD1) inhibitors for the treatment of type 2 diabetes, by means of the re-differentiation of pancreatic beta cells once they have been de-differentiated.

Description

LSD1 INHIBITORS FOR USE IN THE TREATMENT OF TYPE 2 DIABETES

FIELD OF THE INVENTION

The present invention refers to the medical field. Particularly, it is focused on the use of Lysine specific demethylase (LSD1) inhibitors for the treatment of type 2 diabetes, by means of the re-differentiation of pancreatic beta cells once they have been de-differentiated.

STATE OF THE ART

Diabetes mellitus type 2 (also known as type 2 diabetes) is a long-term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin. Common symptoms include increased thirst, frequent urination, and unexplained weight loss. Symptoms may also include increased hunger, feeling tired, and sores that do not heal. Often symptoms come on slowly. Long-term complications from high blood sugar include heart disease, strokes, diabetic retinopathy which can result in blindness, kidney failure, and poor blood flow in the limbs which may lead to amputations. The sudden onset of hyperosmolar hyperglycemic state may occur; however, ketoacidosis is uncommon.

Type 2 diabetes primarily occurs as a result of obesity and lack of exercise. Some people are more genetically at risk than others. Type 2 diabetes makes up about 90% of cases of diabetes, with the other 10% due primarily to diabetes mellitus type 1 and gestational diabetes. In diabetes mellitus type 1 there is a lower total level of insulin to control blood glucose, due to an autoimmune induced loss of insulin-producing beta cells in the pancreas. Diagnosis of diabetes is by blood tests such as fasting plasma glucose, oral glucose tolerance test, or glycated hemoglobin (AIC).

Type 2 diabetes is partly preventable by staying a normal weight, exercising regularly, and eating properly. Treatment involves exercise and dietary changes. If blood sugar levels are not adequately lowered, the medication metformin is typically recommended. Many people may eventually also require insulin injections. In that on insulin, routinely checking blood sugar levels is advised; however, this may not be needed in those taking pills. Bariatric surgery often improves diabetes in those who are obese. Rates of type 2 diabetes have increased markedly since 1960 in parallel with obesity. As of 2015 there were approximately 392 million people diagnosed with the disease compared to around 30 million in 1985. Typically it begins in middle or older age, although rates of type 2 diabetes are increasing in young people. Type 2 diabetes is associated with a ten-year- shorter life expectancy.

Loss of the fully differentiated phenotype is a recognized potential mechanism underlying loss of b-cell function in type 2 diabetes. The metabolic stress of chronic nutrient oversupply can lead to reduced expression or nuclear activity of key b-cell transcription factors, including Pdxl, Nkx6.1, and MafA. This results in loss of critical end-differentiated genes, including insulin itself, in parallel with induction of“disallowed genes,” including lactate dehydrogenase and hexokinase. b-Cell dysfunction ensues from a combination of decreased insulin biosynthesis and loss of physiological nutrient-secretion coupling. This has been demonstrated in a number of models of glucotoxicity, including partial pancreatectomy, with changes largely prevented by maintenance of normoglycemia, and in b-cells in vitro after chronic palmitate exposure.

Re-differentiation provides an attractive potential underlying mechanism for recovery of b- cell function after a reduction in islet fat content over the time course observed in vivo in humans. The dedifferentiated state could be considered as providing a“hideaway” until the metabolic insult subsides, offering an opportunity for restoration of the end-differentiated state after alleviation of metabolic stresses.

The potential for re-differentiation as a mechanism underlying restoration of b-cell function in type 2 diabetes is illustrated by the restoration of b-cell function by intensive insulin therapy in a rodent model of hyperglycemia. This restoration in b-cell function was associated with b-cell re-differentiation characterized by increased levels of mature b-cell markers, including insulin, Pdxl, and MafA. These observations support the proposition that after removal of hyperglycemia and change in lipid metabolism achieved by intensive insulin therapy, b-cell re-differentiation can occur and contribute to diabetes reversal.

Specific therapies targeted toward reversal of b-cell dedifferentiation are needed. Identification is underway of candidate pathways that could be targeted to reverse b-cell dedifferentiation associated dysfunction. A small molecule inhibitor of the transforming growth factor-b receptor (Alk5) was shown to be capable of reversing b-cell dedifferentiation in islets isolated from mice with extreme diabetes, with urocortin 3 used as a marker for mature b-cells. Potential translation to human disease was indicated by incubation of isolated human islets with ALK5 inhibitor, bringing about increased expression levels of a number of mature b-cell markers, including insulin, Pdxl, and MafA. However, administration of ALK5 inhibitor to diabetic animals failed to improve glycemic control and caused an overall deterioration in health. This highlights the requirement for development of pathway-specific therapeutics to restore mature, functional b-cells if a successful pharmacological approach to controlling type 2 diabetes is to be developed.

The present invention is focused on solving the above cited problem and provides a new strategy for the treatment of type 2 diabetes, by means of the re-differentiation of pancreatic b-cells, once they have been de-differentiated.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention provides a new strategy for the treatment of type 2 diabetes, preferably by means of the re-differentiation of pancreatic b-cells, once they have been de-differentiated. The first embodiment of the present invention refers to Lysine specific demethylase (LSD1) inhibitors for use in the treatment of type 2 diabetes.

In a preferred embodiment, the present invention refers to LSD1 inhibitors for use in the differentiation of pancreatic b-cells, particularly for the re-differentiation of pancreatic b-cells once they have been de-differentiated. This means that, in a particularly preferred embodiment, type 2 diabetes is treated in the present invention by means of the administration of LSD 1 inhibitors which are able to re-differentiate pancreatic b-cells once they have been de-differentiated.

It is important to note that, in a preferred embodiment, the inhibitors used in the present invention are not only directed to the inhibition of the activity of LSD 1 but they are also directed to inhibit the expression of KDM1A gene which encodes the protein LSD1. This means that, according to a preferred embodiment of the invention, the re-differentiation of pancreatic b-cells and the treatment of type 2 diabetes can be achieved not only by inhibiting the activity of LSD 1 but also by inhibiting the expression of KDM1A gene. On the other hand, it is important to consider that the stimulation of HMG20A by any means known in the prior art, could directly give rise to the re-differentiation of pancreatic b-cells and the treatment of type 2 diabetes. In a preferred embodiment, the stimulation of HMG20A inhibits the activity of LSD 1 and consequently, again, the stimulation of HMG20A could be directly used for re-differentiating pancreatic b-cells and treating type 2 diabetes.

Consequently, LSD1 is defined in the present invention as a therapeutic target whose inhibition would result in improving the health of patients suffering from type 2 diabetes. Consequently, according to the present invention, any compound or molecule able to inhibit LSD1 and /or KDM1A can be used for re-differentiating pancreatic b-cells and treating type 2 diabetes. In a preferred embodiment, said LSD1 inhibitors a compound of Formula I

(I)

wherein: each of Ri, R₂, R₃, R₄ or R₅ is independently chosen from -H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryl oxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido;

wherein ₅ is chosen from -H and alkyl; wherein R₇ is chosen from -H, alkyl, and cycloalkyl; and wherein the ring of said cycloalkyl has from 0-3 substituents chosen from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido.

In a preferred embodiment, the LSD1 inhibitor is the compound Trans-Nl-((lR,2S)-2- phenylcy cl opropyl)cyclohexane- 1,4-diamine (ORY-1001) (CAS Number: 1431304-21-0), characterized by Formula II:

(P)

Moreover, as explained above, the most important technical feature of the present invention is the inhibition of LSD1 and /or KDM1A for treating type 2 diabetes, irrespective of the molecule or compound which is used for reaching said inhibition. In this regard, although compounds of Formula I, preferably the compound of Formula II, is especially preferred in the present invention, any of compound or molecule able to inhibit LSD1 and /or KDM1A could be used according to the present invention for treating type 2 diabetes. In this regard, examples of LSD 1 inhibitors that can be used in the present invention are disclosed in the international patent applications W02010084160 and WO2012072713 which are herein incorporated by reference in its entirety. Particularly, it is herein incorporated the claim 1 of the international patent application WO2010084160 which refers to compounds having the formula

wherein: each of Ri -R5 is independently chosen from -H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryl oxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido; 5 is chosen from -H and alkyl; R7 is chosen from -H, alkyl, and cycloalkyl; Rx is a -L-heterocyclyl wherein the ring or ring system of said -L- heterocyclyl has from 0-3 substituents chosen from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryl oxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido; or R8 is -L-aryl wherein the ring or ring system of said -L-aryl has from 1-3 substituents chosen from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryl oxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O-carbamyl, N- carbamyl, O- thiocarbamyl, N-thiocarbamyl, and C-amido; each L is independently chosen from -(CHi)n-(CHi)n-, -(CHi)nNH(CHi)n-, -(CHi)nO(CHi)n-, and -(CHi)nS(CHi)n-, and where each n is independently chosen from 0, 1 , 2, and 3; or a pharmaceutically acceptable salt thereof.

Moreover, it is herein incorporated the following formula of the international patent application W02010084160:

wherein A is cyclyl optionally having 1 , 2, 3 or 4 substituents A' . Preferably, said cyclyl is aryl or heteroaryl. Said aryl is preferably phenyl. Said heteroaryl is preferably selected from pyridinyl, pyrimidinyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, furanyl or thiazolyl, more preferably said heteroaryl is selected from pyridinyl, pyrimidinyl or thiazolyl, still more preferably said heteroaryl is pyridinyl (in particular, pyridin-2-yl or pyridin-3-yl) or thiazolyl (in particular thiazol-5-yl) and even more preferably said heteroaryl is pyridin-3-yl or thiazol- 5-yl. It is preferred that said cyclyl (or said aryl or said heteroaryl, or any of the above- mentioned specific aryl or heteroaryl groups) is unsubstituted or has 1 or 2 substituents A', and it is more preferred that said cyclyl (or said aryl or said heteroaryl, or any of the above- mentioned specific aryl or heteroaryl groups) is unsubstituted or has 1 substituent A . Said substituent(s) A' is/are each independently selected from -LI -cyclyl (e.g., -L'-aryl, -L' - cycloalkyl or -L'-heterocyclyl), alkyl, alkenyl, alkynyl, alkoxy, amino, amido (e.g., -CO- NH2), -CH2-CO-NH2, alkyl amino, hydro yl, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfonyl, sulfmyl, sulfonamide, acyl, carboxyl, carbamate or urea, wherein the cyclyl moiety comprised in said -LI -cyclyl is optionally further substituted with one or more (e.g., 1 , 2 or 3) groups independently selected from halo, haloalkyl, haloalkoxy, aryl, arylalkoxy, aryloxy, arylalkyl, alkyl, alkenyl, alkynyl, alkoxy, amino, amido (e.g., -CO-NH2), alkylamino, hydroxyl, nitro, -CH2-CO-NH2, heteroaryl, heteroarylalkoxy, heteroaryloxy, heteroarylalkyl, cyano, sulfonyl, sulfmyl, sulfonamide, acyl, carboxyl, carbamate or urea, preferably selected from halo, haloalkyl, N-sulfonamido or cyano. It is preferred that the cyclyl moiety comprised in said -LI -cyclyl is unsubstituted or is substituted with one of the above groups (including, e.g., one of the preferred groups halo, haloalkyl, N-sulfonamido or cyano). In one preferred embodiment, the cyclyl moiety comprised in said -L'-cyclyl is substituted with one of the above groups (including, e.g., one of the preferred groups halo, haloalkyl, N- sulfonamido or cyano). In another preferred embodiment the cyclyl moiety is unsubstituted. Said -LI -cyclyl is preferably -L'-aryl, -L'-cycloalkyl or -L' -heterocyclyl (e.g., -LI - heteroaryl or -LI -heterocycloalkyl), more preferably -L ' -aryl or -LI -heteroaryl, even more preferably -L'-aryl, even more preferably -LI -phenyl. Each LI is independently selected from a covalent bond, -(CH2) i -6-, -(CH2)0-3-0-(CH2)o-3-, -(CH2)o-3-NH-(CH2)o-3- or - (CH2)o-3-S-(CH2)o-3-, preferably from a covalent bond, -(CH2) i 3-, -0-(CH2)o-3- or -NH- (CH2)0-3-, more preferably from a covalent bond, -CH2-, -0-, -O-CH;-, -0-(CH2)2-, -NH- or -NH-CH2-, even more preferably from a covalent bond, -CH2- or -0-CH2-. It is furthermore preferred that the aforementioned groups LI (connecting the moiety A to the cyclyl moiety comprised in -L'-cyclyl) are in the specific orientation indicated above (accordingly, the group "-0-CH2-" as an example for LI is preferably in the orientation ( ..)-A-0-CH2-cyclyl).

Moreover, the following KDM1A/LSD1 inhibitors could be used in the present invention:

GSK2879552:

T-3775440:

In a preferred embodiment of the present invention the LSD1 inhibitor is used for the re- differentiation of pancreatic b-cells once they have been de-differentiated, preferably for the treatment of type 2 diabetes. The second embodiment of the present invention refers to a pharmaceutical composition comprising any of the LSD1 inhibitors described above, for use in the differentiation of pancreatic b-cells, preferably in the re-differentiation of pancreatic b-cells once they have been de-differentiated, more preferably in the treatment of type 2 diabetes.

The third embodiment of the present invention refers to LSD1 for use in the differentiation of pancreatic b-cells, preferably in the re-differentiation of pancreatic b-cells once they have been de-differentiated, more preferably in the treatment of type 2 diabetes, wherein LSD1 activity is inhibited by means of a LSD1 inhibitor.

The fourth embodiment of the present invention refers to an in vitro method for screening, identifying and/or producing LSD1 inhibitors suitable for the differentiation of pancreatic b- cells, for the for the re-differentiation of pancreatic b-cells once they have been de differentiated or for the treatment of type 2 diabetes which comprises: (a) Measuring at least the activity or the concentration level of LSD 1 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if the activity or the concentration level of LSD 1 determined in step (a) is statistically lower than the activity or concentration level of LSD1 determined before the administration of the candidate compound, this is indicative that the candidate compound is a LSD1 inhibitor suitable for the differentiation of pancreatic b-cells, for the re-differentiation of pancreatic b-cells once they have been de-differentiated or for the treatment of type 2 diabetes.

The fifth embodiment of the present invention refers to an in vitro method for monitoring the efficacy or response to LSD1 inhibitors in the treatment of type 2 diabetes, in the differentiation of pancreatic b-cells, or in the re-differentiation of pancreatic b-cells once they have been de-differentiated, which comprises: a) Measuring the concentration level of C- peptide after the administration of the LSD1 inhibitor, wherein if the concentration level of C-peptide after the administration of LSD1 inhibitors is statistically higher than the concentration level of C-peptide determined in non-treated patients suffering from type 2 diabetes, this is an indication that pancreatic b-cells are being differentiated and that type 2 diabetes is being effectively treated with LSD1 inhibitors.

In a preferred embodiment, before measuring the levels of C-peptide in order to determine the efficacy or response to LSD1 inhibitors, it is determined whether the LSD1 inhibitor is actually inhibiting the activity or reducing the concentration level of LSD1. This can be done by measuring the activity or the concentration level of LSD 1 in a biological sample isolated from the patient after the administration of a LSD1 inhibitor; wherein if the activity or the concentration level of LSD1 after the administration of the LSD1 inhibitor is statistically lower than the activity or the concentration level of LSD1 determined before the administration of the LSD1 inhibitor, this an indication that the LSD1 inhibitor is actually inhibiting the activity or reducing the concentration level of LSD1.

The sixth embodiment of the present invention refers to the in vitro use of LSD1 for screening, identifying and/or producing LSD1 inhibitors suitable for the differentiation of pancreatic b-cells, preferably for the re-differentiation of pancreatic b-cells once they have been de-differentiated, more preferably for the treatment of type 2 diabetes.

The seventh embodiment of the present invention refers to the in vitro use of LSD1 for monitoring the efficacy of a treatment with LSD1 inhibitors in the differentiation of pancreatic b-cells, preferably in re-differentiation of pancreatic b-cells once they have been de-differentiated, more preferably in the treatment of type 2 diabetes.

For the purpose of the present invention:

• The term "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

• By "consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. • In some embodiments, when a method for screening, identifying and/or producing LSD1 inhibitors is claimed, the activity or concentration level before the treatment is used as a“reference” or“control level” for assessing whether the candidate compound is a LSD1 inhibitor suitable for the treatment of type 2 diabetes.

• In some embodiments, when a method for monitoring the efficacy of a treatment, for predicting the response to a treatment or for monitoring the efficacy of a treatment is claimed, the concentration level of C-peptide in non-treated patients suffering from type 2 diabetes is used as a“reference” or“control level” for assessing whether the patient is responding to the treatment. If the concentration level of C-peptide determined after the treatment with LSD1 inhibitors is higher than the above cited “reference” or“control level”, this is an indication that the LSD1 inhibitor is properly working in the treatment of type 2 diabetes.

• In some embodiments, when the method for monitoring the efficacy of a treatment, for predicting the response to a treatment or for monitoring the efficacy of a treatment also requires to confirm whether the LSD1 inhibitor is actually inhibiting the activity of LSD 1, the activity or concentration level of LSD 1 before the treatment with LSD1 inhibitors is used as a“reference” or“control level” for assessing whether the LSD1 inhibitor is working properly. If the activity or concentration level of LSD1 determined after the treatment with LSD1 inhibitors is statistically lower than the above cited“reference” or“control level”, this is an indication that the LSD1 inhibitor is properly working in the inhibition of LSD 1.

Brief description of the figures

Figure 1. ORY-1001 is not toxic for the insulinoma INS-1E cell line (insulin- secreting cell lines). Graphical representation of the total cell number in the different conditions used in the experiment, relative to the cell number in vehicle treated cells. Error bars represent standard deviation.

Figure 2. ORY-1001 treatment stimulates the expression of mature beta-cell markers in INS- 1E cell line (insulin-secreting cell lines). Graphical representation of the relative expression of the indicated genes referred to expression in vehicle treated cells. Expression levels were analysed by RT-PCR using specific primer pairs and corrected by the housekeeping gene beta-Actin. Figure 3. ORY-lOOl treatment reverts the effect of HMG20A silencing in INS-1E cell line (insulin- secreting cell lines). Graphical representation of the relative expression of the indicated genes referred to expression in vehicle treated cells. Silencing of HMG20A induces a 50% decrease in the expression of HMG20A mRNA, correlating with a decrease in beta cells markers NeuroD, MafA and Insulin. The treatment of these cells with 50nM ORY-lOOl reverts the effect of HMG20A silencing, restoring the expression of these mature beta-cells markers. Expression levels were analysed by RT-PCR using specific primer pairs and corrected by the housekeeping gene beta-Actin.siRNA specific for HMG20A (siHMG20A). siRNA against luciferase (siCT) used as control.

Detailed description of the invention

Example 1. ORY-lOOl is not toxic for the insulinoma INS-1E cell line.

HMG20A is a chromatin binding factor, member of the High Mobility Group (HMG) box. GW AS studies have linked small nucleotide polymorphisms (SNPs) in the HMG20A gene to type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). In agreement with this, a decrease in HMG20A expression in islets from type 2 diabetic donors has been observed in the present invention compared to normoglycemic individuals. Moreover, HMG20A expression is transiently upregulated in mice during pregnancy, supporting the involvement of this factor in the adaptation process that takes place in islets during periods of increased insulin demand. In order to characterize the mechanisms of action of MHG20A in islets the effect of HMG20A silencing was analysed in the pancreatic cell line INS-1E, observing that the decrease in HMG20A expression correlates with the decrease in mature beta cell markers such as Insulin, MafA, and NeuroD, and increases the plasticity gene Pax4, suggesting that HMG20A is required for full maturation of beta cells. In agreement with this, silencing of HMG20A in both INS-1E cell line and isolated mouse islets in culture blunts glucose stimulated insulin secretion (GSIS).

HMG20A is a member of the LSD 1 -CoREST inhibitory complex. In this context, HMG20A, through the displacement of HMG20B from the complex, relieves the transcriptional repression imposed by the LSD 1 -CoREST. Therefore, the present invention suggests that the action of HMG20A in pancreatic islets is through its modulation of LSDl-CoREST complex. Thus the role of HMG20A onto islet maturity/functionality will rely on the action of LSD 1- CoREST complex. Based on this premise, it is proposed in the present invention that LSD1- CoREST complex inhibition will potentiate the maturation of pancreatic beta cells. This premise was validated in the insulinoma cell line INS-1E. The toxicity of LSD1 inhibitor ORY 1001 was first analysed in this cell line. To do so, cells were incubated in the presence of increasing concentrations of ORY- 1001 for 24, 48 and 72h. At the end of the experiment total cell number was counted and represented relative to the cell number in vehicle treated cells after 72h in culture (Figure 1). Total cell number after ORY-1001 treatment did not significantly change, indicating that ORY-1001 is not toxic for INS-1E cells at the concentrations and time points used.

Example 2. ORY-1001 treatment stimulates the expression of mature beta-cell markers in INS-1E cell line. The effect of ORY-1001 treatment on the expression of HMG20A-regulated genes was analysed. It was initially observed that HMG20A silencing induces a decrease in the expression of mature beta-cell markers such as NeuroD, MafA and insulin, and increased the expression of the transcription factors Pax4 and REST. Thus, the effect of ORY-1001 treatment onto the expression levels of these genes was analysed. Since for silencing experiments the cells were cultured for 72h after transfection, and in order to avoid any alteration due to the time in culture of the cells, INS-1E cells were cultured for 72h adding the inhibitor 6 and 16h before harvesting the cells. As shown in Figure 2, treatment with ORY-1001 at the 2 doses analysed (lOOnM and 250nM) stimulated HMG20A expression after both 6 and 16h. This increase in HMG20A expression correlated with the increase in insulin (at 6 and 16h) and MafA (at 6h). NeuroD expression also showed a slight increase at 6h with lOOnM ORY-1001. On the contrary, Pax4, was strongly downregulated. Altogether, these data support our hypothesis that HMG20A effect onto islet cells functionality/maturation is mediated through LSD 1 -CoREST complex activity.

Example 3. ORY-1001 treatment reverts the effect of HMG20A silencing in INS-1E cell line.

For final confirmation that HMG20A mediated action on beta cell functionality/maturation is through the inhibition of LSD 1 -CoREST complex, it was studied whether it is possible to revert the detrimental effect of HMG20A silencing by direct inhibition of LSD 1 -CoREST complex. Since 6h treatment with ORY-1001 was rendering the strongest regulation, this time point was chosen for the analysis of ORY-1001 effect on INS-1E cells with silenced HMG20A. HMG20A was silenced in INS-1E by transfection of these cells with a siRNA specific for HMG20A (siHMG20A). INS-1E cells transfected with a siRNA against luciferase (siCT) was used as a control. Six hours before the end of the experiment, transfected cells were treated with ORY-1001 50nM or vehicle. As we can see in Figure 3, silencing of HMG20A in vehicle treated cells induced the downregulation of the mature beta cell markers NeuroD, MafA and insulin. However, the treatment of these cells with ORY- 1001 reverted the effect of the downregulation of HMG20A, resulting in the increase of the expression of NeuroD, MafA and insulin. All together these data indicates that the treatment of HMG20A depleted INS-1E cells with ORY-1001 restores the expression of mature beta cell markers, suggesting that the treatment of beta-cells with the LSD1 inhibitors ORY-1001 will likely enhance their maturation to functional beta-cells, being therefore a promising treatment for T2DM.

Claims

1. Lysine specific demethylase (LSD1) inhibitor characterized by Formula I or a

pharmaceutically acceptable salt thereof

(I)

wherein: each of Ri, R₂, R₃, R₄ or R₅ is independently chosen from -H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, and C-amido; wherein ₅ is chosen from -H and alkyl; wherein R₇ is chosen from -H, alkyl, and cycloalkyl; and wherein the ring of said cycloalkyl has from 0-3 substituents chosen from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L- carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, suifmyl, sulfonyl, sulfonamido, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido; for use in the treatment of type 2 diabetes.

2. Lysine specific demethylase (LSD1) inhibitor for use, according to claim 1, wherein the inhibitor is a compound of formula II or a pharmaceutically acceptable salt thereof

(P)

3. Lysine specific demethylase (LSD1) inhibitor for use, according to any of the claims 1 or 2, wherein the treatment is performed by means of the differentiation of pancreatic b-cells.

4. Lysine specific demethylase (LSD1) inhibitor for use, according to any of the claims 1 to 3, wherein the treatment is performed by means of the re-differentiation of pancreatic b-cells once they have been de-differentiated.

5. Pharmaceutical composition comprising a LSD1 inhibitor according to any of the claims 1 to 4, for use in the treatment of type 2 diabetes, preferably in the differentiation of pancreatic b-cells, and more preferably in the re-differentiation of pancreatic b-cells once they have been de-differentiated.

6. LSD1 for use in the treatment of type 2 diabetes, preferably in the differentiation of pancreatic b-cells, more preferably in the re-differentiation of pancreatic b-cells once they have been de-differentiated, wherein LSD1 activity is inhibited by means of a LSD1 inhibitor.

7. In vitro method for screening, identifying and/or producing LSD1 inhibitors suitable for the treatment of type 2 diabetes which comprises: (a) Measuring at least the activity or the concentration level of LSD 1 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if the activity or the concentration level of LSD1 determined in step (a) is statistically lower than the activity or concentration level of LSD 1 determined before the administration of the candidate compound, this is indicative that the candidate compound is a LSD1 inhibitor suitable for the treatment of type 2 diabetes.

8. In vitro method, according to claim 7, for screening, identifying and/or producing LSD1 inhibitors suitable for the differentiation of pancreatic b-cells which comprises:

(a) Measuring at least the activity or the concentration level of LSD 1 in a biological sample isolated from the patient after the administration of the candidate compound;

(b) wherein if the activity or the concentration level of LSD 1 determined in step (a) is statistically lower than the activity or concentration level of LSD 1 determined before the administration of the candidate compound, this is indicative that the candidate compound is a LSD1 inhibitor suitable for the differentiation of pancreatic b-cells.

9. In vitro method, according to any of the claims 7 or 8, for screening, identifying and/or producing LSD1 inhibitors suitable for the for the re-differentiation of pancreatic b-cells once they have been de-differentiated which comprises: (a) Measuring at least the activity or the concentration level of LSD1 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if the activity or the concentration level of LSD 1 determined in step (a) is statistically lower than the activity or concentration level of LSD 1 determined before the administration of the candidate compound, this is indicative that the candidate compound is a LSD1 inhibitor suitable for the re-differentiation of pancreatic b-cells once they have been de-differentiated.

10. In vitro method for monitoring the efficacy or response to LSD1 inhibitors in the treatment of type 2 diabetes which comprises: a) Measuring the concentration level of C-peptide after the administration of the LSD1 inhibitor, b) wherein if the concentration level of C-peptide after the administration of LSD1 inhibitor is statistically higher than the concentration level of C-peptide determined in non-treated patients suffering from type 2 diabetes, this is an indication that pancreatic b-cells are being differentiated and that type 2 diabetes is being effectively treated with LSD1 inhibitors.

11. In vitro method, according to claim 10, for monitoring the efficacy or response to LSD1 inhibitors in the differentiation of pancreatic b-cells which comprises: a) Measuring the concentration level of C-peptide after the administration of the LSD1 inhibitor, b) wherein if the concentration level of C-peptide after the administration of LSD1 inhibitor is statistically higher than the concentration level of C-peptide determined in non-treated patients suffering from type 2 diabetes, this is an indication that pancreatic b-cells are being differentiated.

12. In vitro method, according to any of the claims 10 or 11, for monitoring the efficacy or response to LSD1 inhibitors in the re-differentiation of pancreatic b-cells once they have been de-differentiated which comprises: a) Measuring the concentration level of C-peptide after the administration of the LSD1 inhibitor, b) wherein if the concentration level of C-peptide after the administration of LSD1 inhibitor is statistically higher than the concentration level of C-peptide determined in non-treated patients suffering from type 2 diabetes, this is an indication that pancreatic b-cells are being re-differentiated once they have been de-differentiated .

13. In vitro method, according to any of the claims 10 to 12, which further comprises determining whether the LSD1 inhibitor is actually inhibiting the activity or reducing the concentration level of LSD 1 by measuring the activity or the concentration level of LSD 1, in a biological sample isolated from the patient, after the administration of a LSD1 inhibitor; wherein if the activity or the concentration level of LSD 1 after the administration of the LSD1 inhibitor is statistically lower than the activity or the concentration level of LSD1 determined before the administration of the LSD1 inhibitor, this an indication that the LSD1 inhibitor is actually inhibiting the activity or reducing the concentration level of LSD1.

14. In vitro use of LSD 1 for screening, identifying and/or producing LSD1 inhibitors suitable for the treatment of type 2 diabetes, preferably for the differentiation of pancreatic b-cells, more preferably for the re-differentiation of pancreatic b-cells once they have been de-differentiated.

15. In vitro use of LSD 1 for monitoring the efficacy of a treatment with LSD1 inhibitors in the treatment of type 2 diabetes, preferably in the differentiation of pancreatic b- cells, more preferably in re-differentiation of pancreatic b-cells once they have been de-differentiated.