Patent Application
20200179335
The present invention relates to methods and compositions useful for preventing and treating diseases associated with a dysregulation of the mTOR pathway, such as tuberous sclerosis.
Tuberous sclerosis is an autosomal dominant genetic disorder which affects multiple organs with the formation of benign tumors principally in the brain, heart, kidney, intestine, skin and lungs. The incidence of this disorder is approximately 1 per 6000 individuals. Tuberous sclerosis mainly occurs through mutations in the tumor suppressor genes TSC1, which codes for hamartin, or TSC2, which codes for tuberin.
The tumor suppressor genes TSC1 and TSC2 act as negative regulators of the mammalian Target Of Rapamycin (mTOR) pathway which plays an essential role in cellular regulation processes, including cell growth, proliferation and survival, as well as protein translation. As a consequence, the loss of function of either TSC1 or TSC2 may lead to a hyperactivity of the mTOR pathway, which itself leads to improperly regulated cell growth, abnormal differentiation, cell proliferation and tumorigenesis.
The tumors created by the dysregulation of the mTOR pathway are generally hamartomas which do not metastasize. However, morbidity and mortality associated with hamartomas may be significant depending on their location.
Current therapeutic agents prescribed to individuals with tuberous sclerosis principally aim at alleviating or suppressing the symptoms, but do not affect the course of these disorders. These agents notably include rapamycin and everolimus (Serra et. Al. (2013) Forum Med. Suisse, 13: 696-702).
Thus, there is still a need for alternative therapeutic agents to address mTOR dysregulation and in particular tuberous sclerosis or its cause.
Stiripentol (Diacomit, 1-penten-3-ol, 1-(1,3-benzodioxol)-4,4-dimethyl or 4-dimethyl-1-[3,4-methylenedioxy-3,4)-phenyl]-1-penten-3-ol) is a racemic allylic alcohol that is structurally unrelated to other antiepileptic drugs.
Stiripentol has shown anticonvulsant activity in several animal models but its spectrum of clinical activity is relatively narrow. Stiripentol has exhibited a high response rate in SMEI patients at a dose of 50 mg/kg/day. Recently, stiripentol has shown high efficacy in two double blind controlled clinical trials and has received approval from the European Medicines Agency (Chiron (2000) Lancet 356:1638, 2000).
The present invention arises from the unexpected finding that stiripentol can inhibit pharmacologically activated mTOR pathway in adult rats.
The present invention thus relates to a compound of the following formula (I):
wherein:
The present invention also relates to a method for the prevention or treatment of a disease associated with a dysregulation of the mTOR pathway in an individual, comprising administering to the individual a prophylactically or therapeutically effective quantity of at least one compound of formula (I) as defined above or a pharmaceutically acceptable salt, hydrate or solvate thereof.
The present invention also relates to the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt, hydrate or solvate thereof, for the manufacture of a medicament intended for the prevention or treatment of a disease associated with a dysregulation of the mTOR pathway.
The present invention also relates to the compound of formula (I) as defined above or a pharmaceutically acceptable salt, hydrate or solvate thereof for use as defined above, in combination with at least one other inhibitor of the mTOR pathway.
The present invention also relates to a pharmaceutical composition, comprising as active substance at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and optionally at least one pharmaceutically acceptable carrier or excipient, for use in the prevention or treatment of a disease associated with a dysregulation of the mTOR pathway in an individual.
In an embodiment of the invention, the pharmaceutical composition for use as defined above, further comprises at least one other inhibitor of the mTOR pathway, as an active ingredient.
The present invention also relates to a pharmaceutical composition comprising as active substance at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one other inhibitor of the mTOR pathway selected from the group consisting of wortmannin, rapamycin and the analogs of rapamycin, such as temsirolimus and everolimus, optionally in association with a pharmaceutically acceptable carrier or excipient.
The present invention also relates to products containing:
As intended herein, the term “comprising” has the meaning of “including” or “containing”, which means that when an object “comprises” one or several elements, other elements than those mentioned may also be included in the object. In contrast, when an object is said to “consist of” one or several elements, the object is limited to the listed elements and cannot include other elements than those mentioned.
Preferably, the above-defined compound of formula (I) is represented by the following formula (II):
in which n, A1, A2, A3 and R1 are as defined above.
More preferably the above-defined compound of formula (I) or (II) is represented by the following formula (III), i.e. stiripentol:
Preferred alkyl groups according to the invention encompass the methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl and t-butyl groups.
The Cl, I, Br or F atoms are preferred halogen atoms according to the invention.
French patent FR 2 173 691, which is incorporated herein by reference, describes the synthesis of stiripentol, in particular starting from methylenedioxy-3,4-phenyl-1-dimethyl-4,4-penten-1-on-3. It is well within the ordinary skills of one of skill in the art to synthesize the other compounds of formula (I) from this teaching.
As will be clear to one of skill in the art, the above-defined formulas (I), (II), and (III) represent either the various stereoisomers encompassed by these formulas or mixtures thereof, in particular racemic mixtures thereof.
Thus, the compound of formula (III) can be a compound of formula (IIIa) a compound of formula (IIIb), or a mixture of a compound of formula (IIIa) and a compound of formula (IIIb), in particular the racemic mixture thereof.
As intended herein the “mTOR pathway” relates to an intracellular signaling pathway regulating the cell cycle and involving the mTOR protein. “mTOR” relates to the mechanistic or mammalian Target Of Rapamycin and is also known as the FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1). mTOR is encoded by the MTOR gene. As is well known to one of skill in the art, mTOR links with other proteins and serves as a core component of two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which regulate different cellular processes. In particular, as a core component of both complexes, mTOR functions as a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. As a core component of mTORC2, mTOR also functions as a tyrosine protein kinase that promotes the activation of insulin receptors and insulin-like growth factor 1 receptors. mTORC2 has also been implicated in the control and maintenance of the actin cytoskeleton. Examples of upstream regulators of the mTOR pathway, i.e. regulators upstream of mTOR, notably encompass P13K-AKT. Examples of downstream effectors of the mTOR pathway, i.e. effectors downstream of mTOR, notably encompass the S6 kinase which phosphorylates ribosomal protein S6.
As intended herein the expression “disease associated with a dysregulation of the mTOR pathway” relates to any disease in which the regulation of the mTOR pathway is impaired or absent. Preferably, the dysregulation of the mTOR pathway according to the invention relates to an activation, in particular a constitutive activation or a hyper activation of the mTOR pathway, or to a lack of inhibition, in particular a constitutive lack of inhibition, of the mTOR pathway. Preferably also the disease associated with a dysregulation of the mTOR pathway is a disease due to or caused by a dysregulation of the mTOR pathway.
Preferably, the disease associated with a dysregulation of the mTOR pathway according to the invention is a hamartoma syndrome. More preferably, the disease associated with a dysregulation of the mTOR pathway according to the invention is selected from the group consisting of tuberous sclerosis, PTEN-related hamartoma syndrome and Peutz-Jeghers syndrome.
“Tuberous sclerosis” (TS) is well known to one of skill in the art. It is also known as “Bourneville tuberous sclerosis” (BTS), “Bourneville's disease” or “tuberous sclerosis complex” (TSC).
By way of example tuberous sclerosis is classified in paragraph Q85.1 of 10th revision of the international classification of disease of the World Health Organization (ICD-10).
Besides, the diagnosis of tuberous sclerosis, which can be classified as definite, probable or possible diagnostic, can be effected as follows:
Tuberous sclerosis is an autosomal dominant disease. As such, tuberous sclerosis according to the invention is preferably associated to a mutation in the TSC1 gene and/or in the TSC2 gene.
As intended herein “PTEN” relates to Phosphatase and TENsin homolog. As is known in the art, the “PTEN-related hamartoma syndrome” is an autosomal dominant disease resulting from a mutation of the tumor suppressor gene PTEN. The PTEN-related hamartoma syndrome according to the invention in particular includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related proteus syndrome (PS), and proteus-like syndrome.
The Peutz-Jeghers syndrome is well known in the art and results from a mutation in the STK11 tumor suppressor gene.
The individual according to the invention is preferably a mammal, more preferably a human. Preferably also, the individual according to the invention is a child or an infant.
The individual according to the invention, preferably has one or more symptoms of a disease associated with a dysregulation of the mTOR pathway.
Preferably, the individual according to the invention presents a dysregulation of the mTOR pathway.
Preferably, the individual according to the invention presents at least one mutation in a gene selected from TSC1, TSC2, PTEN and SK11 genes.
Preferably also, the individual according to the invention preferably presents at least one symptom of tuberous sclerosis. Most preferably, the individual according to the invention is diagnosed with a definite tuberous sclerosis, or a probable tuberous sclerosis, or a possible tuberous sclerosis.
Preferably also, the individual according to the invention presents at least one symptom of a PTEN-related hamartoma syndrome. More preferably, the individual according to the invention has at least one symptoms selected from the group consisting of Cowden syndrome (CS) and/or at least one symptom of Bannayan-Riley-Ruvalcaba syndrome (BRRS) and/or at least one symptom of PTEN-related proteus syndrome (PS) and/or at least one symptom of proteus-like syndrome.
Preferably, the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof, is to be administered at a unit dose of from 100 mg to 1000 mg or from 5 mg/kg to 100 mg/kg. Preferably also, the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof is to be administered with a dosage regimen of from 10 mg/kg/d to 200 mg/kg/d.
Preferably, the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof, the pharmaceutical composition for use as defined above, the pharmaceutical composition as defined above or the medicament as defined above, is in a form suitable for administration by the oral route. Preferably also, the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof, the pharmaceutical composition for use as defined above, the pharmaceutical composition as defined above or the medicament as defined above, is in the form of a powder, sachets, tablets or capsules.
As intended herein, “pharmaceutically acceptable carrier or excipient” refers to any material suitable with a pharmaceutical composition. Preferably, the pharmaceutically acceptable carrier or excipient according to the invention is suitable for an oral administration. Preferably, the pharmaceutically acceptable carrier or excipient according to the invention includes but is not limited to any of the standard of pharmaceutical composition known to one of skill in the art such as water, glycerin, alcohol, oil emulsion, water emulsion, buffered saline solution, preservative, stabilizer and wetting agents.
As intended herein, the expression “other inhibitor of the mTOR pathway” relates to any compound intended to alleviate one or more of the symptoms or to treat or prevent a disease associated to a dysregulation of the mTOR pathway. Preferably, the other inhibitor of the mTOR pathway is selected from the group consisting of wortmannin, rapamycin and the analogs of rapamycin, such as temsirolimus and everolimus.
As intended herein “combined” or “in combination” means that the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof, in particular stiripentol, are administered at the same time than the additional compound, either together, i.e. at the same administration site, or separately, or at different times, provided that the time period during which the compound of formula (I) as defined above or the pharmaceutically acceptable salt, hydrate or solvate thereof exerts its pharmacological effects on the individual and the time period during which the additional compound exerts its pharmacological effects on the individual, at least partially intersect.
The invention will be further described by the following non-limiting Figures and Example.
The star symbol (*) represents p<0.05 for pentylenetetrazol versus NaCl within either tween or stiripentol (two-way ANOVA followed by a Holm-Sidak multiple comparisons).
The hash symbol (#) represents p<0.05 for pentylenetetrazol-wortmannin compared to pentylenetetrazol-tween (Student t-test).
The star symbol (*) represents p<0.05 for pentylenetetrazol versus NaCl within the tween group and stiripentol versus vehicle within pentylenetetrazol group (two-way ANOVA followed by a Holm-Sidak multiple comparisons).
The hash symbol (#) represents p<0.05 for pentylenetetrazol-wortmannin compared to pentylenetetrazol-tween (Student t-test).
The modulation of the mTOR pathway by the compound according to the invention, in particular stiripentol, was studied and compared with wortmannin, a pharmacological inhibitor of the mTOR pathway.
The mTOR pathway has been studied at two levels (i) an upstream level of regulation with the P13K-Akt, a modulator of the mTOR pathway and (ii) a downstream level with the ribosomal protein S6, the substrate of the S6 kinase, a direct downstream effector of the mTOR pathway.
1. Animals
Adult male Sprague-Dawley rats (Janvier, 220-250 g, n=24, 7 weeks old) were used in this study. They were housed 2 per cage (Techniplast ref. 1291), and maintained in a 12 h light/dark cycle (light ON at 7 AM). Food and water were provided ad libitum. The experiments were conducted in accordance with the European Recommendations (directive 2010/63/EU) for the use and care of laboratory animals. The experimental protocol has been approved by the ethics committee.
2. Pharmacological Treatment
Pentylenetetrazol (batch MKBV0751V, SIGMA), 80 mg/kg in 0.9% NaCl was administered subcutaneously in an injection volume of 5 mL/kg.
3. Test Compounds
Wortmannin (Sigma), 2.4 mg/kg in DMSO 10% v/v in 0.9% NaCl or Stiripentol (Biocodex), 300 mg/kg in tween80 5% v/v, were injected intraperitoneally (10 mL/kg) 30 min before pentylenetetrazol administration.
4. Western Blot
Thirty minutes after pentylenetetrazol administration, animals were sacrificed, the two hemi-hippocampi were isolated, homogenized in RIPA buffer (Abcam, ab 156034) containing a phosphatase and protease inhibitors cocktails (Abcam, ab201119) and frozen in liquid nitrogen.
The protein concentration of each sample was determined by a BCA protein assay. Equal amounts of total protein extract (20 μg) were separated by gel electrophoresis (Biorad 10% precast gel, ref 5671035, 14 μL deposit) and transferred to nitrocellulose membranes (Biorad ref. 1620167).
Membranes were incubated overnight at 4° C. with primary antibodies to S6 Ribosomal Protein (5G10) (Rabbit mAb, 1:1000, Cell Signaling Technology, Danvers, Mass.), or Phospho-S6 Ribosomal Protein (D68F8) (Ser240/244) (Rabbit mAb, 1:1,000, Cell Signaling Technology, Danvers, Mass.), Akt (pan) (C67E7) (Rabbit mAb, 1:1000, Cell Signaling Technology, Danvers, Mass.), Phospho-Akt (Ser473) (D9E) (XP Rabbit mAb, Cell Signaling,).
Secondary Anti-Rabbit IgG, HRP-linked Antibody was then used.
Signals were detected by enzyme chemiluminescence (Vilber Lourmat Fusion FX5 system) and quantitatively analyzed with the Bio-1 D software (Vilber Lourmat, France). The signal of phosphoprotein levels was normalized to the total protein.
Each membrane was also stripped (Restore™ western blot stripping buffer, Thermoscientific) and reprobed to detect beta-actin protein levels, a control for the total quantity of protein deposited.
5. Data Analysis
Data are represented as the mean±standard error of the mean (S.E.M.). The difference between groups was assessed by a two-way ANOVA followed by a Holm-Sidak multiple comparison (factor pretreatment=tween or STP, factor treatment=NaCl or PTZ).
The effect of wortmaninn was assessed by a t-test comparison between tween-pentylenetetrazol (n=5) versus wortmannin-pentylenetetrazol groups (n=3).
For all tests, significance was set at p<0.05 (Sigma Stat, v3.5, SPSS, Chicago, USA).
1. Effect of an Acute Stiripentol Administration on Akt Protein Phosphorylation Levels in the Hippocampus
Western blots analysis revealed a strong band of phosphorylated Akt in either the pentylenetetrazol pretreated with vehicle groups or pentylenetetrazol pretreated with stiripentol group (
A pretreatment with stiripentol did not modify the pentylenetetrazol-induced increase in the ratio of phosphorylated Akt related to total Akt (no statistical difference between stiripentol-pentylenetetrazol (2.46) and tween-pentylenetetrazol (1.81), two-way ANOVA followed by a Holm-Sidak multiple comparisons), whereas the wortmannin treatment statistically significantly decrease the ratio of phosphorylated Akt related to total Akt in the pentylenetetrazol groups (p<0.05, Wortmannin-pentylenetetrazol (0.40) versus Vehicle-pentylenetetrazol (1.81) treated animals, Student t-test,
2. Effect on an Acute Stiripentol Administration on S6 Protein Phosphorylation Levels in the Hippocampus
Western blots analysis revealed a band of phosphorylated S6 protein in the PTZ group (
A pretreatment with STP statistically significantly blocked the increase in the ratio of phosphorylated S6 related to total S6 protein (p<0.05, STP (0.36) versus Veh-treated (0.79) animals within the PTZ group, two-way ANOVA followed by a Holm-Sidak multiple comparisons).
Similarly, a pretreatment with wortmannin blocked S6 phosphorylation level increase in the PTZ groups (p<0.05, Wortmannin-PTZ (0.30) versus Veh-PTZ (0.79) treated animals, Student t-test,
These results suggest that stiripentol could inhibit the mTOR pathway at a specific level, downstream from the Akt level.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17305620.1 | May 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/064282 | 5/30/2018 | WO | 00 |
