DERIVATIVES FOR THE TREATMENT OF DOWN'S SYNDROME

FIELD OF THE INVENTION

The present invention relates to l-(2-fluorobiphenyl-4-yl)-cyclopropanecarboxylic acid derivatives for use in improving the cognitive capacity of patients with intellectual disabilities, an IQ of less than 85, diagnosed with mental retardation, and, most specifically, those with Down's syndrome.

In particular the invention relates to the use of the aforementioned derivatives for the prevention and/or treatment of cognitive symptoms in patients affected by Down's syndrome.

BACKGROUND OF THE INVENTION

Down's syndrome (hereinafter DS) is the most common genetic source of mental retardation. It is caused by the presence of an extra copy of human chromosome 21 (i.e. trisomy 21, HSA 21), which includes the genes SOD-1, BACE-2, APP and SlOOb. The neuropathology of DS is complex and includes decreased brain weight, decreased neuronal number, abnormal neuronal differentiation, and structural changes in dendritic spines.

Nervous system involvement, which affects patients throughout the lifespan, results in deficits involving learning, memory, language, and movement. Besides neurodevelopmental alterations, DS is characterized by increased mortality rates, both during early and later stages of life, and age-specific mortality risk remains higher in adults with DS compared with the overall population of people with mental retardation and with normal populations. Causes of higher mortality rates later in life may be due to a number of factors, two of which are an increased risk for Alzheimer's disease (hereinafter AD) and an apparent tendency toward premature aging.

In fact, a distinct feature of DS is the onset of Alzheimer's disease (AD)-like pathology by middle age. Virtually all individuals with DS develop sufficient neuropathology for a diagnosis of AD by the age of 40 years. It has not yet been fully characterized the gene(s) responsible for the DS neuropathology, however the development of AD in DS may be related to the overexpression of the amyloid beta precursor protein (AbetaPP), being APP gene on the trisomic 21 chromosome. In DS patients plasma levels of Abeta40 and Abeta42 are increased (Schupf N et al Neurosci Lett 2001, 301, 199-203). Furthermore, diffuse deposits of the toxic and less soluble Abeta42 form were observed in some DS brains during childhood (Lott IT et al Neurobiol Aging 2005, 26, 383-389) and, in contrast with AD, amyloid deposition was widespread in all of the cortical areas investigated and was observed much earlier than neurofibrillary tangle formation (Hof PR et al Arch Neurol 1995, 52, 379-391). The time course of neurofibrillary tangle formation in DS displays regional patterns comparable with those observed in aging and AD with layer II of the entorhinal cortex being affected first in DS, followed by the hippocampus proper and neocortex. The oldest patients with DS had neurofibrillary tangle densities sometimes higher than in patients with AD.

Moreover, in adults with DS, several markers of inflammation are present at higher levels than in younger individuals, particularly in association with Abeta deposits, and this may be due in part to gene over expression. Inflammation may be a major contributor to the acceleration phase of AD pathogenesis, in DS typically observed between the ages of 40 and 50 years, but may also make a life-long contribution leading to pathological aging. Several genes encoding for inflammatory factors are present on chromosome 21 and are overexpressed and, as in AD patients, cytokines are significantly increased in DS compared to age-matching subjects (Lott et al. Neurobiol Aging 2005; 26: 383-389).

From a molecular point of view, a marked alteration in APP processing and Αβ trafficking has been demonstrated in cortical DS astrocytes and neurons (Busciglio J et al. Neuron 2002; 33: 677-688) with increased levels of APP and C99, reduced levels of secreted APP (sAPP) and C83, and intracellular accumulation of insoluble Αβ42.

Similar CNS abnormalities have been described in mouse models of DS. In particular, the Ts65Dn mouse, the most widely used model of DS, possess three copies of the segment of mouse chromosome 16 (MMU16) orthologous (80% genes) to the critical region of human chromosome 21 (HSA21) thought to be responsible for the phenotype of DS. The third copy of the distal region of MMU 16 spans from the APP to Mxl genes, resulting in increased APP gene dosage. These mice have abnormal forebrain and cerebellar development, defects in synapse formation and neurophysiology, and behavioural deficits. These mice recapitulate many characteristics of DS and AD, including age-specific cognitive decline, neuronal cell loss, and decreased levels of nerve growth factor (NGF). This partially trisomic mouse also have elevated expression of APP and Αβ forms, but plaque formation and tau accumulation have not always been observed (Kern DS et al. Cell Transplant 2011; 20: 371-379).

Starting at 10 months of age, brain APP levels were increased proportional to the APP gene dosage imbalance reflecting increased APP message levels in Ts65Dn mice, while at 4 months no difference were observed (Choi JHK et al. Nature 2009; 409: 860- 921).

From the point of view of cognitive performance, Down subjects suffer of mental retardation and accelerated aging prone to Alzheimer-type dementia (Schupf N et al. Neurology 2010; 75: 1639-1644). Mental retardation in DS appears to be related to severe neurogenesis abnormalities during critical phases of brain development. Recent lines of evidence of the mouse model for DS have shown a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting involvement of the Shh pathway in the neurogenesis defects of DS (Trazzi S et al. Human Mol Genet 2011; 20: 1560-1573). It is therefore possible that abnormalities in neuronal generation, migration, allocation and terminal differentiation may underlie the cognitive phenotype of DS.

Chakrabarti L et al {Nature Neurosci 2010; 13: 927-936) demonstrated a complex set of neurogenesis defects in multiple brain regions leading to an excitation/inhibition imbalance in the Ts65Dn forebrain. Furthermore, their results implicate two genes that are triplicated in Down syndrome and in Ts65Dn mice, Oligl and Olig2, in the defective ventral neurogenesis. These results also reveal a broader determinative role of Oligl and Olig2 genes on ventral telencephalic neurogenesis. Thus, although it is well established that Oligl and Olig2 regulate multiple steps of neuron and glia formation in the CNS37, these transcription factors also affect the rate of neuron production from the medial ganglionic neuron of eminences (MGE) of the ventral telencephalon, under normal conditions and are therefore necessary factors for generating the proper ratio of excitatory and inhibitory neurons in the neocortex. Moreover, the genesis of the pool of undifferentiated precursor cells in the dentate gyrus of the hippocampus is impaired, so that neurogenesis in adulthood is also compromised (Bartesaghi R et al. Rev Neurosci 2011; 22: 419-455).

Since DS subjects develop AD pathology in younger age compared to AD subjects, this is also part of the cognitive impairment and it worse in middle and old age. The notion that almost 100% of DS subjects are destined to AD pathology, offers the unique opportunity of pre-treatment in order to prevent/delay the onset of AD like pathology.

In summary, notwithstanding that some correlation has been reported in the prior art between DS and AD, there are fundamental genetic differences between an individual with trisomy 21 to non-trisomic people as well as fundamental anatomical and cognitive differences between people with those diseases.

Most evident is that DS is due to a specific genetic anomaly, comprising a third copy of the genetic contents of chromosome 21 (compared to two in the non-DS population) whereas AD is a neurodegenerative disease of largely unknown cause except for the less than 5% of cases caused by variations in one of about 6 genes. In addition, the overall brain morphology of the person with DS is different in many aspects, two of which are the smaller size of many parts of the DS brain and fewer neuronal cells in general. Using positron emission tomography (PET), researchers have shown that the neurophysiology of an aging DS brain differs from that of an AD brain that does not have DS. Specifically, the PET imaging showed higher levels of probe binding in at least two regions of the DS brain relative to the AD brain (Nelson et al. Prog Brain Res 2012; 197: 101-121). Furthermore, DS is a condition present at birth whereas AD is a disease of aging. Finally, DS cognitive defects do not progress in contrast with those associated with AD which typically progress throughout the 6-15 years prior to demise.

In view of the above considerations, because neither the DS genotype nor phenotype is the same as that of AD, it cannot be predicted that their respective cognitive deficits could be rescued by the same class of compounds.

The compound l-(3',4'-dichloro-2-fluorobiphenyl-4-yl)cyclopropane carboxylic acid, also quoted as CHF 5074, was first disclosed in WO 2004/074232 as a therapeutic agent for the treatment of neurodegenerative diseases. It is currently under development for the treatment of the early stages of AD.

It has now been found that CHF 5074 and strictly related compounds can effectively be used for the prevention and/or treatment of cognitive symptoms in patients affected by Down ' s syndrome .

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions, and therapeutic methods that improves the cognitive capacity of patients with intellectual disabilities, an IQ of less than 85, diagnosed with mental retardation, and, most specifically, those with Down's syndrome (DS).

According to a particular aspect, the invention is directed to the compounds of general formula (I)

(I) wherein

R represents one or more groups, which can be the same or different from each other, independently selected from halogen atoms, preferably chlorine;

for use for the prevention and/or treatment of cognitive symptoms in patients affected by Down's syndrome.

Preferably, the compound of formula (I) is l-(3',4'-dichloro-2-fluorobiphenyl-4- yl)cyclopropanecarboxylic acid also known with the code CHF 5074.

The invention is also directed to the use of the compounds of general formula (I) in the manufacture of a medicament for the prevention and/or treatment of cognitive symptoms in patients affected by Down's syndrome.

In a further aspect, the invention provides a therapeutic method for improving the cognitive capacity of patients with intellectual disabilities, an IQ of less than 85, diagnosed with mental retardation, and, most specifically, those with Down's syndrome (DS), said method comprising administering an effective amount of a compound of general formula (I), including polymorphs, pharmaceutically acceptable salts and prodrugs thereof.

DESCRIPTION OF THE FIGURES

Figure 1 shows the design of the study in Ts65Dn mice.

Figures 2 and 3 show the beneficial effects of long-term treatment with CHF 5074 on some of the age-dependent behavioural and motor abnormalities of Ts65Dn mice.

Figure 4 illustrates the effects long-term treatment with CHF 5074 on glial cell population (microglia and astrocytes).

Figures 5 to 9 illustrate the effects of long-term treatment with CHF 5074 on APP metabolism.

Figure 10 illustrates the effects long-term treatment with CHF 5074 on hippocampal synaptology.

DEFINITIONS

It can be appreciated that, within the compounds of general formula (I), the phenyl ring bears one or more halogen atoms therein referred to as R groups. From the above, it is clear to the skilled person in the art that any of the said halogen atoms, the same or different from each other, may be thus present in any possible free position of the phenyl ring itself.

The term "halogen atoms" includes fluorine, chlorine, bromine, and iodine.

The term "polymorphs" refers to a different crystal structure of the same solid substance. They exhibit different melting point, solubility (which affect the dissolution rate of the drug and consequently its bioavailability in the body), X-ray crystal and diffraction pattern.

The term "prodrug ^" refers to a substance administered in an inactive form that is then metabolized in the body in vivo into the active compound with the aim of optimizing absorption, distribution, metabolism., and excretion. In particular, in the context of the present application, prodrugs are used to improve the CNS drug level, with poor crossing of the blood brain barrier usually being the limiting factor.

The term "prevention" refers to the use for progression-slowing and/or onset delaying the cognitive defects.

When the tern "prevention" is used in connection to the "Down's Syndrome", it should be read as referred to the use for progression-slowing and/or onset delaying the cognitive defects associated to said syndrome.

The term "treatment" refers to the use for curing, symptom-allievating, symptom- reducing the cognitive defects.

The term "IQ" refers to the intelligence quotient. It is a score derived from one of several standardized tests designed to assess intelligence. When current IQ tests are developed, the median raw score of the norming sample is defined as IQ 100 and scores each standard deviation (SD) up or down are defined as 15 IQ points greater or less, although this was not always so historically. By this definition, approximately 95 percent of the population scores an IQ between 70 and 130, which is within two standard deviations of the mean. DETAILED DESCRIPTION OF THE INVENTION

The invention refers to compounds of formula (I)

(I)

wherein R has the above reported meaning for use in improving the cognitive capacity of patients with intellectual disabilities, an IQ of less than 85, diagnosed with mental retardation, and, most specifically, those with Down's syndrome (DS).

Advantageously, R represents a chlorine atom, and preferably the compound of formula (I) is l-(3',4'-dichloro-2-fluorobiphenyl-4-yl)cyclopropanecarboxyl ic acid, hereinafter quoted with the code CHF 5074.

The compounds of general formula (I) may be prepared according to the procedures descibed in WO 2009/149797.

Said compounds may advantageously be used in any form, amorphous or crystalline and solvates or hydrates thereof. Preferably, they are used in crystalline form.

In view of the close relationship between the compounds of general formula (I) in free acid form and those on the form of salts, the invention is also directed to the use of pharmaceutically acceptable salts thereof.

Pharmaceutically acceptable salts according to the invention include those formed with both common organic and inorganic bases.

The compounds of formula (I) may also be administered in form of prodrugs.

Suitable prodrugs may be esters with common alcohols such as ethanol or polyalcohols such as sorbitol, with sugars such as glucose, or with sugar acids such as ascorbic acid.

In particular, since in Down's syndrome, CNS is the most severe affected tissue, prodrugs which are able of crossing the blood brain barrier such as those disclosed in WO 2006/016219 may be advantageously utilised.

The compound of formula (I) may be used alone or in combination with other active ingredients such as N-methyl-D-aspartate (NMDA) receptors receptor antagonists, preferably with memantine.

The compounds of formula (I), may be combined with one or more pharmaceutically acceptable carriers or excipients to provide suitable pharmaceutical compositions.

The pharmaceutically acceptable carriers or excipients may be advantageously selected from the group consisting of diluents, wetting agents, emulsifying agents, binders, coatings, fillers, glidants, lubricants, disintegrants, preservatives, stabilizers, surfactants, pH buffering substances, flavoring agents and similar ones. Comprehensive guidance on pharmaceutical excipients is given in Remington's Pharmaceutical Sciences Handbook, XVII Ed. Mack Pub., N.Y., U.S.A.

The pharmaceutical compositions of the invention may be formulated for administration by any convenient route, e.g. by oral, parenteral, topical, inhalation, buccal, nasal, rectal, vaginal, transdermal administration. Suitable dosage forms can include tablets, capsules, caplets, lozenges, suppositories, solutions, emulsions, suspensions, syrups, ointments, creams, oils, and powders. Preferably, the pharmaceutical compositions of the invention will be administered orally using appropriate dosage forms, such as capsules, tablets, caplets, etc.

The dosage of the compounds of formula (I) and of their salts and prodrugs can vary within wide limits depending on the nature of the disease to be treated, the type of patient, and the mode of administration. A person skilled in the art can determine a therapeutically effective amount for each patient and thereby define the appropriate dosage. When the preferred compound of the invention is administered by oral route to humans, a typical daily dosage might fall within the range of 10 mg to 2000 mg advantageously between 50 to 1000 mg, preferably between 100 and 500, mg administered in a single or multiple daily dosage units. Thus, a single dose of the pharmaceutical preparations of the invention conveniently comprises between about 50 and 1000 mg of CHF 5074 or salt or prodrug thereof.

In a preferred embodiment, the compounds of the invention may be of use for improving the cognitive capacity of patients affected by Down's syndrome, preferably young patients having an age of 2-10 years.

They may be also of use for preventing and/or delaying the onset or slowing the cognitive defects in said disease.

The following Example illustrates in detail the invention.

EXAMPLE

The objective of this study was to evaluate if chronic treatment with CHF 5074 could revert or ameliorate the behavioural, histopathological and biochemical abnormalities observed in a transgenic mouse model DS.

The study was performed on Ts65Dn male, using the administration of CHF 5074 in the diet (375 ppm, equivalent to approximately 60 mg/kg/day).

The treatment started at 2-3 months of age and it was chronically administered in the diet for 15 months, until 19 months of age.

Treatment groups were:

Wild-type mice receiving standard diet (N=20)

- Wild-type mice receiving CHF 5074-medicated diet (375 ppm, approximately 60 mg/kg/day) (N=20)

Ts65Dn mice receiving standard diet (N=20)

Ts65Dn mice receiving CHF 5074-mediacted diet (375 ppm, approximately 60 mg/kg/day) (N=20)

The specific objectives of this study were:

1. To evaluate the effects of chronic treatment with CHF 5074 on spatial learning and memory deficit in Ts56Dn mice.

2. To evaluate the effects of chronic treatment with CHF 5074 on locomotion activity abnormalities in Ts56Dn mice.

3. To evaluate the effects of chronic treatment with CHF 5074 on cellular and molecular players for learning and memory in the hippocampus. To verify the possible effect of chronic treatment with CHF 5074 on expression of molecules known to a play critical role in long-term synaptic plasticity and long-term memory in the hippocampus of adult Ts65Dn mice.

4. To evaluate the effects of chronic treatment with CHF5074 on AD-like brain pathology and APP metabolism.

Methods

Animals and Treatments

Animals were provided by Jackson Laboratory, Bar Harbor, ME, USA) in small cohorts. Ts65Dn segmental male trisomic mice were used. Ts65Dn mice were compared with diploid controls from the same litters.

Assessments

- Spatial learning and memory were assessed with the Y-maze test according to Stewart S et al. J Alzheimer Dis 2011; 26: 105-126.

Locomotion deficits were assessed with the CatWalk technology (gait analysis) according to Faizi M et al. Neurobiol Dis 2011; 43: 397-413.

Brain morphology was assessed by immunohistochemistry, immunofluorescence and quantitative morphometry and microdensitometry according to Imbimbo BP et al. J Alzheimer Dis 2010; 20: 159-173; Sivilia S et al. BMC Neurosci 2013; 14: 44; Guidi S et al. Brain 2014; 137: 380-401. The following proteins were investigated:

• Glial fibrillary acidic protein (GFAP) as astrocyte marker in hippocampus;

· Ionized calcium binding adaptor molecule 1 (Ibal), as microglia marker in hippocampus;

• Synaptophysin. It is a pre-synaptic vesicle membrane protein known to mediate vesicle release in hippocampus; • Vesicular glutamate transporter 1 (VGLUT-1), as marker of glutamatergic synapses in hippocampus;

• Vesicular GAB A transporter (VGAT), as marker of GABAergic synapses in hippocampus;

· Αβ40 and Αβ42 were measured by ELISA in plasma and brain extracts

(IBL kit for mouse Αβ40/Αβ42);

• Intraneuronal APP was measured by immunohistochemistry and computerized microdensitometry (Chemicon clone 22C11) in cerebral cortex;

• Amyloid precursor protein intracellular domain (AICD) (cleavage sites C83 and C99) in hippocampus.

Molecular biology markers were measured in hippocampus and basal ganglia by real-time PCR, according to Massella A et al. BMC Neuroscience 2012; 13: 12; D'Intino G et al. J Neuroendocrinal 2011; 23: 778-790.

Results

Figure 2 shows spatial memory, as assessed with the Y maze test before starting

CHF 5074 treatment (3.5 months of age) and after 6, 9 and 15 months of treatment. Animals were tested in a single trial Y maze task, to measure arm entries (A) and alternation (B). The percentage of spontaneous alternations is defined as (total alternations/total arm entries-2) x 100. Before starting treatment (3.5 months of age), Ts65Dn mice showed a significant deficit in spatial memory compared to wild-type mice as evidenced by low spontaneous alternation (panel B). CHF 5074 treatment progressively normalized spontaneous alternation in Ts65Dn mice and after 15 months of treatment this was not significantly different from that of wild-type mice.

Figure 3 shows locomotion performance, as assessed using computerized gait analysis (Cat Walk) in animals after 15 months of treatment (19 months of age). The print area of front and hind paws is presented, as corrected according to the individual animal body weigh. While no differences were observed in front pow print area, the hind paw print area of old Ts65Dn mice is increased. CHF 5074 tretament normalized this gait parameter.

Figure 4 shows quantification of activated microglia (Ibal -immunoreactivity) and reactive astrocytes (GFAP-immunoreactivity) in the CAl/2 regions of the hippocampus. Aged Ts65Dn mice treated with CHF 5074 showed a substantial reduction of both Ibal- and GFAP-immunoreactivity compared to Ts65Dn mice receiving standard diet (*p<0.05, **p<0.01). These results indicate that CHF 5074 reduced cellular markers of neuro inflammation in the brain of Ts65Dn mice.

Figure 5 illustrates the APP mRNA expression levels in the hippocampus and basal ganglia of wild-type and Ts65Dn mice treated with CHF 5074-medicated diet or normal diet for 15 months (19 months of age). There was a substantial increase in APP mRNA expression levels in Ts65Dn mice compared to wild-type animals. Moreover, CHF 5074 treatment slightly but significantly increased APP mRNA levels in wild-type animals (*p<0.05, ****p<0.0001, ^a p<0.1).

Figure 6 illustrates the plasma levels of Αβ40 and Αβ42 peptides in animals after 15months of treatment (19 months of age). There was a substantial increase in Αβ40 and Αβ42 plasma levels in Ts65Dn mice compared to wild-type animals. CHF 5074 further increased plasma level of both fragment, being this effect signiticant for Αβ42 in wild- type animals.

Figure 7 illustrates the brain (cerebral cortex) levels of Αβ40 and Αβ42 in animals after 15months of treatment (19 months of age). Results are expressed as pmol/gr proteins (panels A and B), and as fmol/g wet tissue (panels C and D). There was a substantial increase in Αβ40 and Αβ42 brain levels in Ts65Dn mice compared to wild-type animals. CHF 5074 treatment did not modify significantly brain Αβ40 and Αβ42 levels.

Figure 8 shows intracellular immunoreactivity of total ΑΡΡ/Αβ40/Αβ42 peptides in cortical neurons. There was a substantial increase in intraneuronal immunoreactivity in Ts65Dn mice compared to wild-type animals and a significant decrease in this immunoreactivity in Ts65Dn mice treated with CHF 5074 compared to control transgenic animals. Figure 9 shows the quantification of AICD (amyloid precursor protein intracellular domain) immunoreactivity (IR) in neurons in the CAl/2 hippocampal regions. Quantification was carried out by counting the percentage of neurons having AICD-positivity. There was a three-fold increase in the percentage of AICD-positive neurons in Ts65Dn mice and this increase 20 was significantly attenuated by CHF 5074 treatment (****p<0.0001).

Figure 10 illustrates main markers of synaptic transmission in the CAl/2 regions of the hippocampus after 15months of treatment (19 months of age). Panel A reports synaptophys levels and there were no significant differences between experimental groups. Panel B reports the variation of immuno staining for the glutamate transporter VGLUT1 with a substantial decrease in Ts65Dn mice compared to wild-type animals. Panel C reports the variation of immunostaining for the GAB A transporter VGAT. There was a substantial increase in Ts65Dn mice compared to wild-type animals and this increase was completely reversed by the CHF 5074 treatment.

The results of this study indicated that prolonged oral treatment with CHF 5074 attenuated spatial memory deficit and completely reversed locomotion deficit in Ts65Dn mice. Histological and biochemical analyses showed that these behavioral improvements were associated to reduced neuro inflammation markers and morphological signs of neuroprotection in hippocampus at the synaptic level (vesicular GABA transporter) in the CHF 5074-treated Ts65Dn mice. Surprisingly, CHF 5074 treatment of Ts65Dn mice significantly attenuated the dramatic increase in amyloid precursor protein intracellular domain (AICD) observed in hippocampal neurons of the control Ts65Dn mice treated with standard diet, suggesting a novel molecular mechanism for the behavioral improvements. Thus, our findings provide evidence for memory and locomotion facilitation of CHF 5074 after chronic treatment in this mouse model. On the basis of these findings, it can be reasonably hypothesized that CHF 5074 and close analogs thereof can be utilized for the treatment of subjects with Down syndrome.