WO2010084201A1

WO2010084201A1 - Novel derivative of erythromycin for the treatment and diagnosis of prion disease

Info

Publication number: WO2010084201A1
Application number: PCT/EP2010/050848
Authority: WO
Inventors: Marlène REBOUL; Franck Mouthon; Jean-Philippe Deslys; Mathieu Charveriat; Eric Jacquet; Françoise GUERITTE; Jean-Yves Lallemand; Qian Zhu Wang
Original assignee: Commissariat A L'energie Atomique Et Aux Energies Alternatives; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2009-01-26
Filing date: 2010-01-26
Publication date: 2010-07-29

Abstract

The present invention concerns a pharmaceutical composition comprising the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A of formula (I) or pharmaceutically acceptable salts thereof, and a pharmaceutically carrier, excipient or diluent; a method of prophylactically and/or therapeutically treating amyloϊd neurodegenerative diseases by administering to a subject in need thereof an effective amount of said pharmaceutical composition; a method for testing at least one sample obtained from a mammal, for the presence or absence of an aberrant prion protein (PrP^res) in order to diagnose a Prion infection within said mammal, said method using (E)-9-[0-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A; and a kit for detecting the presence or absence of an aberrant prion protein PrP^res, the kit comprising at least the (E)-9-[0-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A.

Description

NOVEL DERIVATIVE OF ERYTHROMYCIN FOR THE TREATMENT AND DIAGNOSIS OF PRION DISEASE

Field of the invention

This invention concerns a novel compound and pharmaceutical compositions containing it that are useful to destabilize the PrP protein, in order to inhibit the formation of protease resistant prion proteins (PrP^res), such as protease resistant prion proteins associated with transmissible spongiform encephalopathies.

Background of the invention

The abnormal accumulation of host proteins in the brain is the putative cause of many neurodegenerative disorders, called amyloidoses. They include Alzheimer's disease, Parkinson's disease, Huntington's disease, Prion diseases (or Transmissible

Spongiform Encephalopathies), and a variety of other disorders (ROSS & POIRIER,

Nat. Med., vol.10 Suppl, p:S10-17, 2004). Amyloid deposits seem central to the observed neuropathogenesis, therefore inhibition of protein misfolding might constitute an essential strategy for therapeutical developments. In order to evaluate new possible treatments, Prion diseases were chosen as a unique and robust model of transmissible amyloidosis. These diseases still constitute a large public health issue, since 4 cases of transmission by blood transfusion have been reported at this time, and since there is currently no available treatment, neither curative, nor preventive, against Prion infection.

Prion diseases are fatal neurodegenerative diseases that include human disorders such as variant, iaotrogenic or sporadic Creutzfeldt-Jakob disease (CJD), kuru, fatal sporadic insomnia (FSI), or genetic familial forms such as familial Creutzfeldt-Jakob disease (fCJD), fatal familial insomnia (FFI) and Gerstmann- Straussler-Scheinker disease (GSS). Animal forms of the diseases include notably scrapie in sheep and bovine spongiform encephalopathy in cattle

These prion diseases in animals and humans have a long incubation period and a long clinical course, and are always fatal within an average period of 7 months (CJD).

Neuropathological features consist of neuronal vacuolization, neuronal death, and gliosis with hyperastrocytosis.-These diseases are characterized by the formation and accumulation in the brain of an abnormal proteinase K resistant isoform (PrP^res) of a normal protease-sensitive host-encoded prion protein (PrP^c). PrP^res is formed from PrP^c by a post-translational process involving conformational changes that convert the PrP^c into a PrP^res having higher β-sheet content. The formation of these macromolecular aggregates of PrP^res is closely associated with the Prion-mediated brain pathology in which amyloid deposits of PrP^res are formed in the brain, which eventually becomes spongiform.

Thus, the presence of a native prion protein (PrP^c) has been shown to be essential to pathogenesis of Prion diseases. The cellular protein PrP is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20. The PRNP gene is expressed in neural and non-neural tissues, with the highest concentration of its mRNA being in neurons. The translation product of the PRNP gene consists of 253 amino acids in cows, 254 amino acids in sheep, 253 amino acids in humans. In Prion protein related encephalopathies, the cellular PrP is converted into the altered PrP^res that is distinguishable from PrP^c in that PrP^res i) aggregates, ii) is insoluble in detergent, iii) is proteinase K resistant in that only approximatively the N- terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrP^c is completely degraded, and iv) has an alteration in protein conformation in which the amount of α-helical conformation for PrP is reduced, and the amount of β- sheet conformation for PrP^res is increased. It also exists genetic familial forms of Prion diseases (FFI, GSS, and fCJD), characterized in that the polynucleotide sequence of PrP^c contains at least one mutation in the open reading frame (ORF), thereby favouring abnormal conformation changes that help converting PrP^c in the abnormal PrP^res (TATEISHI et al, Neurology, vol.40(10), p:1578-81 , 1990 ; HAΪK et al., Ann Neurol., vol. 56(6), p:909-10; author reply 910-1 , 2004).

Classical strategies for Prion treatment are based on destabilizing directly the amyloid protein, i.e. PrP^res, or targeting the level or trafficking of its precursor (GILCH et a/., Vet. Microbiol., vol.123, p:377-386,2007; TAGLIAVINI et al., J. MoI. Biol., vol.300, p:1309-1322, 2000; TREVITT & COLLINGE, Brain, vol.129, p:2241-2265, 2006). Hence, numerous compounds, such as amphotericins, sulfated polyanions, porphyrins, and Congo red dye, have been reported as prospective treatments ( TREVITT & COLLINGE, abovementioned, 2006). However, none of these useful compounds appeared to affect the stability of the precursor protein PrP^c itself. Also, most of these compounds displayed very low anti-Prion in vivo activity, although some extended the incubation period when administered near the time of infection. Clinical trials have been conducted, with the use of quinacrine and pentosan polysulfate (PPS), but none of them appeared to be efficient (STEWART et al., Neurology, vol.70, p:1272-1281 , 2008). Moreover, both active and passive immunotherapies were developed, but for the moment they remained unsuccessful (TREVITT & COLLINGE, abovementioned, 2006), or presented partial but promising protection (GONI et al., Neuroscience, vol.153, p:679-686, 2008).

Thus, the need still remains for agents that will specifically and efficiently inhibit the formation of PrP^res and by extension prevent or slow the deposition of amyloid deposits in the tissues of subjects that have been exposed to Prion disease or are suffering from a neurodegenerative disorder such as amyloidosis.

Detection of the pathogenic isoforms of conformational disease proteins in living subjects, and samples obtained from living subjects has proven difficult. Although detection of amyloids in general can be achieved with Congo red staining, this type of staining is inaccurate and not sensitive. Specific and high affinity detection of a given protein in the presence of other proteins like in cell, tissue, biological fluid, or homogenate is usually done with antibodies that are specific to the targeted protein. Yet, proteins that share the same sequence but differ by conformation complicate discriminatory detection by antibodies. In fact, the majority of antibodies raised against PrP bind PrP^c or to both PrP^c and PrP^res. Several published immunoassay methods were based on comparing the binding level of antibodies that only bind native PrP^c but not native PrP^res to the binding level of antibodies that bind both native PrP^res and PrP^c in order to determine the presence of PrP^res (see e.g. US patent Nos 6,214,565 B1 and 6,406,864 B2). However, there are limitations in these assay methods because effective antibody detection of PrP^res can only be obtained when the amount of PrP^c is very low. In an excess of PrP^c, e.g. when samples are taken from subjects in the early stages of prion disease, it is difficult or even impossible to observe an increase in immunodetection due to the presence of PrP^res.

To overcome this difficulty, samples are often treated with non-specific proteases such as proteinase K (PK) or dispase before the detection of PrP^res (see e.g., US patent No. 7,163,798 and European Patent 1 1 19 773). Because of the structure of the PrP^res isoform, PrP^res is largely resistant to protease digestion. The PrP^c isoform, on the other hand, is theoretically completely degraded by treatment with such proteases.

One major limitation of PK treatment is the degradation of the amino-terminal, or amino-proximal residues of PrP^res, which is useful for immunodetection since this region contains a number of epitopes including epitopes in the octarepeat region. An additional disadvantage of using non-specific proteases like PK is that PrP^res is only partially resistant; given high concentrations and enough time, most or even all residues will be digested as well. It is thus important not to use high concentrations of proteases in order to limit false-negative results.

Conversely, in case of low concentration of proteases, another limitation of this assay arises: the PrP^c might not be completely digested, leading to the irrelevant presence of some PrP regions that may lead to false positive results.

Thus, the simple use of proteases such as protease K is not satisfactory and there remains a need for a specific, sensitive and relatively simple or quick method and a kit using such method to detect the presence of the pathogenic prion proteins in various samples, for example in samples obtained from living subjects, in blood supplies, in farm animals and in other human and animal food supplies. The present invention is also directed to this important ends.

Summary of the invention

In a first aspect, the present invention is directed to a pharmaceutical composition comprising the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A having the formula:

or pharmaceutically acceptable salts thereof, and a pharmaceutically carrier, excipient or diluent.

This pharmaceutical composition will be herein referred to as "the pharmaceutical composition of the invention".

In a second aspect, the present invention discloses a method of prophylactically and/or therapeutically treating amyloϊd neurodegenerative diseases by administering to a subject in need thereof an effective amount of said pharmaceutical composition of the invention.

Preferably, said amyloϊd neurodegenerative disease is chosen among Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Prion disease. Also, following neurodegenerative diseases can be targeted: cortical basal degeneration/ progressive supranuclear palsy, Dementia with Lewy bodies, Multiple system atrophy, Pick's disease, Spinocerebellar ataxia, Famiiia! amyloϊd polyneuropathy, Cerebral amyloϊd angiopathy. Identification of these diseases and the molecular mechanisms underlying them are clearly indicated in SKOVRONSKY et al. {Annu. Rev. Pathol. Mech. Dis., vol.1 , p: 151-70, 2006).

More preferably, said amyloϊd neurodegenerative disease is a genetic familial amyloϊd neurodegenerative disease, in particular a Prion-related disease, such as the familial Creutzfeldt-Jakob disease, the fatal familial insomnia (FFI) and Gerstmann- Straussler-Scheinker disease (GSS).

In another embodiment of the invention, said amyloϊd neurodegenerative disease is a sporadic or infectiously acquired amyloϊd neurodegenerative disease, in particular a Prion-related disease, for example the sporadic Creutzfeldt-Jakob disease (sCJD), the fatal sporadic insomnia (FSI), the variant of CJD (vCJD), kuru or iatrogenic CJD (iCJD).

"Sporadic" amyloϊd neurodegenerative disease are namely the fatal sporadic insomnia (FSI) and the sporadic Creutzfeldt-Jakob disease (sCJD). "Infectiously acquired" amyloϊd neurodegenerative diseases target the variant of Creutzfeldt Jakob diseases (vCJD), kuru, or iatrogenic Creutzfeldt Jakob disease (iCJD).

Moreover, in animal, said Prion-related disease is chosen among Bovine spongiform encephalopathy (BSE), Scrapie disease.

As shown herein, the compound of the invention acts by destabilizing the normal prion protein PrP^c, inhibiting the ability of PrP^c to be converted into PrP^res, and therefore impairing the accumulation of PrP^res.

In a particular embodiment, the pharmaceutical composition of the invention is intended to treat human subjects.

In a third aspect, the present invention concerns a method for testing at least one sample obtained from a mammal, for the presence or absence of an aberrant prion protein (PrP^res) in order to diagnose a Prion infection within said mammal, said method comprising:

a) Preparing said at least one sample from a mammal,

b) Adding the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A in said at least one sample,

c) Adding sufficient amount of protease(s) to the sample in order to digest completely remaining protease-sensitive proteins or regions of protein, without digesting completely the PrP^res protein,

d) Testing said sample for the presence of the prion protein region PrP 27-30, which is protease-resistant in the PrP^res form of the prion protein,

wherein the detection of PrP 27-30 is taken as conclusive evidence indicating the presence of PrP^res in said at least one sample.

Preferably, said at least one sample is a biological fluid or a tissue homogenate.

More preferably it is a blood sample or a brain tissue homogenate.

In a particulate embodiment, the (E)-9-[O-(methyl 2-O-benzyl-4,6-O- benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A is added at step b) at a dose of at least 1 μM, preferably at least 10 μM, more preferably at least 20 μM and even more preferably at least 50 μM.

In another embodiment, step c) is performed in the presence of a chaotropic agent.

In still another embodiment, the testing method according to the invention is intended to diagnose a Prion-related disease such as Bovine spongiform encephalopathy (BSE), Scrapie disease, or Creutzfeldt Jakob Disease (CJD) in a subject, and particularly to diminish the risk of scoring false-positive and/or false- negative results.

In another aspect, the present invention proposes a kit for detecting the presence or absence of an aberrant prion protein PrP^res in at least one sample containing a biological sample, i.e. a biological fluid or a tissue homogenate, said kit comprising at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A.

Brief description of the drawings

Figure 1 highlights the inhibitory effect of the PrP^res protein in the presence of the compound of the invention (MBBAO-erythromycin), by showing the quantification of the p_rp^res p_resence obtained after treatment with serial dilutions of the compound (O) or erythromycin A (•).

Figure 2 highlights the cytotoxicity induced by the compound, and more particularly the estimation of the viability after two-week treatment with the compound of the invention

(MBBAO-erythromycin), in GT1-7 (•) and SN56 (O).

Figure 3 and figure 4 show the destabilisation of the PrP^rec protein in the presence of the compound of the invention:

Figure 3 shows the results of the fluorescence-based thermal shift assay on murine PrP^rec, on 6-plicates experiments, when the recombinant protein is treated by 50μM of the compound of the invention (MBBAO-erythromycin) (■), or Erythromycin A (D), or 1 % (vol/vol) of DMSO(A ). Figure 4 shows the melting temperatures (T_m) of PrP^rec in the presence of 1 % (vol/vol) of DMSO, or 50μM of erythromycin A, or 50μM of the compound of the invention. ^** : significativity of Student's t test: p<0.05.

Detailed description of the invention

Erythromycin A is a macrolide antibiotic that has an antimicrobial spectrum similar to or slightly wider than that of penicillin, and is often used for people who have an allergy to penicillins. In structure, this macrocyclic compound contains a 14- membered lactone ring with ten asymmetric centers and two sugars (L-cladinose and D-desoamine), making it a compound very difficult to produce via synthetic methods. More precisely, Erythromycin A has the following structure:

Erythromycin may possess bactericidal activity, particularly at higher concentrations. The mechanism is not fully elucidated however. By binding to the 5OS subunit of the bacterial 70S rRNA complex, protein synthesis and subsequently structure/function processes critical for life or replication are inhibited. Erythromycin A interferes with aminoacyl translocation, preventing the transfer of the tRNA bound at the A site of the rRNA complex to the P site of the rRNA complex. Without this translocation, the A site remains occupied and thus the addition of an incoming tRNA and its attached amino acid to the nascent polypeptide chain is inhibited. This interferes with the production of functionally useful proteins and is therefore the basis of antimicrobial action.

Erythromycin A has previously been proposed as treatment for Alzheimer's disease (Tucker et al., Curr Alzheimer Res., vol.2(2), p:249-54, 2005). However, it is completely inactive in inhibiting PrP^res formation and accumulation (cf. Figure 1 ).

The present invention takes advantage of the surprising finding that a derivative of Erythromycin A can actually destabilize the PrP^c protein and thereby inhibit the formation of PrP^res under conditions where it would otherwise be formed.

The present invention is therefore based on this particulate derivative of Erythromycin A having the following structure:

This compound is called (E)-9-[0-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A and belongs to the family of erythromycin derivatives with an oxime functionality.

Its synthesis has been described in 1996 (GRANDJEAN & LUKACS, J. of

Carbohydrate Chemistry, vol .15 (7), pp 831-855, 1996, and GRANDJEAN & LUKACS, The Journal Antibiotics, vol.49(10), p:1036-1043, 1996), but it has never been proposed as a medicament, a fortiori in the field of Transmissible Spongiform

Encephalopathies.

In the context of the present application, this particulate compound is referred to as being "the compound of the invention", "MBBAO-erythromycin" or the "present derivative of Erythromycin".

Surprisingly, it is shown in the present invention that this derivative is potentially interesting for Prion treatment, because it interacts with PrP^c: this interaction apparently leads to a peculiar conformation of PrP^c, which can induce the alteration of its amyloidogenic properties, and, consequently, the inhibition of PrP^res conversion (MCCUTCHEN et al., Biochemistry, vol. 32, p:121 19-12127, 1993; PELLARIN & CAFLISCH, J. MoI. Biol., vol.360, p:882-892, 2006).

To date, the classical strategies for Prion treatment were exclusively based on destabilizing the amyloid protein, i.e. PrP^res, or targeting the level or trafficking of its precursor (GILCH et al., abovementioned, 2007; TAGLIAVINI et al., abovementioned, 2000; TREVITT & COLLINGE, abovementioned, 2006). On the contrary, the present invention proposes here a new strategy based on the alteration of the stability of the precursor protein PrP^c and the alteration of its amyloidogenic properties, impairing the formation of PrP^res.

In the present application, "Prion", "prion protein", "PrP protein" and "PrP" are used interchangeably to refer to both the pathogenic prion protein form (also referred to as scrapie protein, pathogenic protein form, pathogenic isoform, pathogenic prion and PrP^res) and the non-pathogenic isoform (also referred to as the normal form, cellular protein form, cellular isoform, non-pathogenic prion protein and PrPc), as well as the denatured and various recombinant forms of the prion protein that may not have either the pathogenic conformation or the normal cellular conformation.

Use of the terms "prion", "prion protein", or "PrP protein" is not meant to be limited to polypeptides having the exact sequences to those described herein. It is readily apparent that the terms encompass conformational disease proteins from any of the identified or unidentified species (e.g. human, bovine,) or diseases (e.g.

Alzheimer's, Parkinson's, etc.).

A first aim of the invention concerns pharmaceutical compositions containing as active principle at least the compound of the invention, namely the (E)-9-[O-(methyl 2-

O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A or any pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable excipients or vehicles.

In the sense of the present invention, the term "pharmaceutically acceptable salt" of a compound is intended to mean the salts which are pharmaceutically acceptable, as defined above, and which have the desired pharmacologic activity of the parent compound. Such salts include: (1 ) acid addition salts formed with inorganic acids such as hydrochloric acid, sulphuric acid, nitric acid, hydrobromic acid, phosphoric acid and the like or formed with organic acids such as ascorbic acid, benzoic acid, aspartic acid, oxalic acid, benzene sulphonic acid, tartaric acid, diatriazoic acid, glutamic acid, lactic acid, maleic acid, succinic acid, fumaric acid, citric acid, edetic acid, malic acid, mandelic acid methanesulfonic acid, mucic acid, pantothenic acid, para-toluenesulfonic acid, acetic acid, gluconic acid, ethanesulfonic acid, propionic acid, salicylic acid and the like; or

(2) salts formed when an acid proton present in the parent compound is either replaced by a metallic ion, for example alkali metal ion, alkaline-earth metal ion or aluminum ion; or is coordinated with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like.

Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

It should be understood that all references to pharmaceutically acceptable salt include solvates and polymorphs of the same acid addition salts.

Preferably, the pharmaceutically acceptable salts are the one that have been already described for Erythromycin A, namely the carbonate, oxalate, aspartate, glutamate, tartrate, ascorbate, or phosphate salts.

The pharmaceutical compositions according to the invention can, for example, be compositions administered by oral, nasal, intrathecal, sublingual, intraventricular, rectal or parenteral means. As an example of compositions able to be administered by oral means, pills, capsules, granules, powders and oral suspensions or solutions can be mentioned.

The appropriate formulations for the chosen method of administration are known and described, for example, in Remington, The Science and Practice of Pharmacy, 19th edition, 1995, Mack Publishing Company.

The effective dose of a compound according to the invention varies in function of numerous parameters such as, for example, the chosen administration method, the weight, age, sex, the substance or substances responsible for the pathology, and the sensitivity of the individual to be treated. Consequently, the optimal dose must be determined individually, in function of the relevant parameters, by a medical specialist. It is foreseen that the effective doses will range between 1 μg/kg to 1 g/kg, preferably between 0.1 mg/kg and 1 g/kg and more preferably between 0.1 mg/kg and 10 mg/kg.

In a second aspect, the present invention concerns the use of the (E)-9-[O- (methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A or its pharmaceutically acceptable salts for the preparation of a pharmaceutical composition for preventing and/or treating amyloϊd neurodegenerative diseases.

In other words, the present invention targets a method of prophylactically and/or therapeutically treating amyloϊd neurodegenerative diseases, by administering to a subject in need thereof an effective amount of the pharmaceutical composition containing at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A or its pharmaceutically acceptable salts.

In the context of the invention, the term "amyloϊd neurodegenerative disease" targets diseases that involve selective neuronal vulnerability with degeneration in specific brain regions, and deposits of abnormal proteins in neurons and other cells or extracellularly. These neurodegenerative diseases can be of familial, infectiously acquired or sporadic forms. They are commonly known as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or Prion disease, all sharing common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibres containing misfolded proteins with a β-sheet conformation, termed amyloid (ROSS & POIRIER, abovementioned, 2004; SKOVRONSKY et al, abovementioned, 2006).

In some cases, amyloϊd neurodegenerative diseases can be induced by genetic alteration(s) which render(s) the amyloϊd precursor proteins sensitive to conformational changes, favour(s) the accumulation and deposit of these abnormal proteins in neurons. These familial forms of the disease are herein called "genetic familial amyloid neurodegenerative disease". Such disease are namely Familial amyloid polyneuropathy, FFI, fCJD, GSS, Huntington's disease, and familial forms of Parkinson's disease, and Alzheimer's disease.

In a preferred embodiment, the present invention provides a way to prevent such genetic familial amyloϊd neurodegenerative diseases. "Sporadic" amyloϊd neurodegenerative disease are namely the fatal sporadic insomnia (FSI) and the sporadic Creutzfeldt-Jakob disease (sCJD), and sporadic form of Parkinson's and Alzheimer's disease.

"Infectiously acquired" amyloϊd neurodegenerative diseases target the variant of Creutzfeldt Jakob diseases (vCJD), kuru, or iatrogenic Creutzfeldt Jakob disease (iCJD).

Preferably, the present invention concerns a method of therapeutically treating amyloϊd neurodegenerative disease, whatever its form is (i.e. infectiously acquired, sporadic and/or genetically acquired).

As it is shown herein, the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A is able to interact with PrP^c protein, and to change its stability, and may therefore affect its amyloidogenicity (MCCUTCHEN et al, abovementioned, 1993; PELLARIN & CAFLISCH, abovementioned, 2006). This may explain why it is such an efficient inhibitor of PrP^res formation and accumulation.

Therefore, in a preferred embodiment, the invention concerns the use of (E)-9-

[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A in order to destabilize the normal prion protein PrP^c. Also, as shown herein, said compound can be used to inhibit the accumulation of the abnormal prion protein PrP^res in prion-infected cells.

Because of these important results, it is thought that a pharmaceutical composition containing at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β- D-allopyranosid-3-yl)oxime] erythromycin A enables to treat and/or prevent Prion- related diseases.

Therefore, the present invention concerns a medicament for preventing and/or treating a Prion infection, said medicament containing the (E)-9-[O-(methyl 2-O-benzyl- 4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A or pharmaceutically acceptable salts thereof.

"Prion infection" or "Prion-related disease" refers to a disease caused in whole or in part by a pathogenic prion protein, or a pathogenic form of prion protein (e.g. PrPr^es). Examples of prion-related disease include, without limitation, scrapie, bovine spongiform encephalopathies (BSE), mad cow disease, feline spongiform encephalopathies, kuru, Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD), chronic wasting disease (CWD), Gerstmann-Strassler-Scheinker Disease (GSS), fatal familial insomnia (FFI), fatal sporadic insomnia (FSI).

Preferably, said Prion-related disease in animal is chosen among Bovine spongiform encephalopathy (BSE), or Scrapie disease.

In human, Prion-related diseases include variant or sporadic or iatrogenic Creutzfeldt-Jakob disease (CJD), kuru, fatal sporadic insomnia (FSI), and also genetic familial forms such as familial Creutzfeldt-Jakob disease, fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker disease (GSS).

Thus, in a preferred embodiment, the present invention targets a method of preventing and/or treating sporadic amyloϊd neurodegenerative disease, in particular sporadic or infectiously acquired Prion-related disease, for example variant Creutzfeldt- Jakob disease (vCJD), sporadic Creutzfeldt-Jakob disease (sCJD), fatal sporadic insomnia (FSI), kuru, or iatrogenic Creutzfeldt-Jakob disease (iCJD) T

In a rather preferred embodiment, the present invention provides a method of preventing and/or treating infectiously acquired Prion-related diseases such as kuru, the variant Creutzfeldt-Jakob disease (vCJD), and the iatrogenic Creutzfeldt-Jakob disease (iCJD).

In another rather preferred embodiment, the present invention provides a method of preventing and/or treating sporadic Prion-related diseases such as sporadic Creutzfeldt-Jakob disease (sCJD), and fatal sporadic insomnia (FSI).

More precisely, the present invention provides a method of therapeutically treating infectiously acquired Prion-related diseases such as kuru, the variant Creutzfeldt-Jakob disease (vCJD), and the iatrogenic Creutzfeldt-Jakob disease (iCJD).

Also, the present invention provides a method of therapeutically treating sporadic Prion-related diseases such as sporadic Creutzfeldt-Jakob disease (sCJD), and fatal sporadic insomnia (FSI). However, in some cases, the convertibility of PrP^c in PrP^res is favoured by familial acquired mutation(s) present in the open reading frame of the PrP^c (VANIK & SUREWICZ, J. Biol. Chem.yo\.277{50), p:49065-70, 2002).

In another preferred embodiment, the present invention provides a method of preventing such genetic familial Prion-related diseases, in a subject presenting an identified mutation in the ORF of the PrP^c protein, said method comprising the administration of an effective amount of the pharmaceutical composition containing at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A or its pharmaceutically acceptable salts to said subject.

In this case, the present invention thus highlights a method of prophylactically treating said genetic familial Prion-related diseases, in particulate familial Creutzfeldt- Jakob disease, fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker disease (GSS).

In all cases, the present invention provides a method of therapeutically treating a Prion-related disease, whatever its form is (i.e. infectiously acquired, sporadic and/or genetically acquired).

As shown in the present invention, the (E)-9-[O-(methyl 2-O-benzyl-4,6-O- benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A is shown to be able to inhibit the accumulation of the amyloϊd abnormal prion protein in prion-infected cells. It is thus likely that this compound may also be able to inhibit the accumulation of the other misfolded proteins having a β-sheet conformation, namely Huntingtin for Huntington's disease (HD), Aβ for Alzheimer's disease (AD), α-synuclein for Parkinson's disease, superoxide dismutase 1 (SOD1 ) for ALS. It has indeed already been shown that therapeutic molecules that are efficient against Prion diseases are also useful in the treatment of the Alzheimer disease (e.g. Red Condo dye, FINDEIS et al., Biochim Biophys Acta., vol. 1502(1 ), p:76-84, 2000). Conversely, some molecules used in the treatment of the Alzheimer disease have been shown to counteract Prion infection in vivo (e.g. curcumin or memantin; RIEMER et al, J. Gen. Virol., vol.89(Pt 2), p:594-7, 2008). Eventually, it has been shown that the proteins PrP^c and Aβ are able to interact (PARKIN et al, Proc Natl Acad Sci U S A., vol.104(26), p:11062-7, 2007). PrP^c destabilisation could therefore affect Aβ accumulation, thereby leading to alleviate the Alzheimer disease as well. In a final embodiment, the method of the invention enables to treat either animals or human beings, and preferably human beings.

In a third aspect of the invention, the present disclosure provides a new, simple, specific and sensitive way, in the form of assay methods and kits, to detect the presence of pathogenic prion proteins, which may be use in connection with methods for diagnosing a prion-related disease (e.g. in human or non-human animal subjects), for ensuring a substantially PrP^res-free blood supply, blood products supply, or food supply, for analysing organ and tissue samples for transplantation, for monitoring the decontamination of surgical tools and equipment, as well as any other situation in which knowledge of the presence or absence of the pathogenic prion is important.

The assay method of the invention takes advantage of the fact that the (E)-9-[O- (methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A is able to interact with PrP^c protein, and may change its stability.

Said compound can thus be used, alone or together with proteases, to destabilize the cellular Prion protein in tested samples in order to enhance the specific and sensitive detection of PrP^res, and reduce the risk of scoring false-positive and false- negative results, especially the one produced by the protein misfolding cyclic amplification (PCMA) detection process.

In this third aspect, the present invention proposes a method for testing at least one sample obtained from a mammal for the presence or absence of an aberrant prion protein (PrP^res) in order to diagnose a Prion infection within said mammal, said method comprising:

a) Preparing said at least one sample from a mammal,

d) Testing said sample for the presence of the prion protein region PrP 27-30, which is protease-resistant in the PrP^res form of the prion protein, wherein the detection of PrP 27-30 is taken as conclusive evidence indicating the presence of PrP^res in said at least one sample.

Said at least one sample can be a tissue homogenate or any biological fluid wherein the abnormal protein can be found (e.g. blood, serum, etc.).

The present invention therefore provides a way to increase the reliability of

Prion diagnosis tests such as those described in US 2006/0121507, US 7,344,842, US 2004/0115752, and WO2008/124098 which are incorporated herein by reference.

In still another embodiment, the testing method according to the invention is intended to diagnose a Prion disease such as Bovine spongiform encephalopathy (BSE), Scrapie disease, or Creutzfeldt Jakob Disease (CJD) in a subject, preferably a mammal, more preferably a human being.

More particularly, the method of the invention enables diminish the risk of scoring false-positive and/or false-negative results, thereby enhancing the reliability of existing Prion diagnosis tests.

In a preferred embodiment of the diagnosis method of the invention, said tissue homogenate is brain tissue homogenate.

In another preferred embodiment of the diagnosis method of the invention, the (E)-9-[O-(methyl2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A is added at a dose of at least 1 μM, preferably at least 10 μM, more preferably at least 20 μM and even more preferably at least 50 μM.

In another preferred embodiment of this method, step c) is performed in the presence of a chaotropic agent, which is a compound that can enhance immunologically the signal of PrP^res. Such agents are well-known from the man skilled in the art and are for example formic acid, guanidium chloride, lithium perchlorate or those described in PRIVAT et al {Microsc. Res. 7ec/?.;vol.50(1 ):26-31 , 2000): the physical treatments (e.g. micro-waves) also known from the man skilled in the art to enhance immunodetection of PrP^res can also be included in the present method (cf. PRIVAT et al, abovementioned, 2000).

In a last aspect, the present invention provides a kit for detecting the presence or absence of an aberrant prion protein PrP^res in a sample, the kit comprising at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A or pharmaceutically acceptable salts.

Again, said kit is intended to help diagnosing a Prion disease such as Bovine spongiform encephalopathy (BSE), Scrapie disease, or Creutzfeldt Jakob Disease (CJD) in a subject, preferably a mammal, more preferably a human being.

The following examples serve to illustrate the pharmacological activity of the compound of the invention and, therefore, its potential utility in the therapeutic aim claimed in the invention.

Examples

Synthesis of the compound of the invention

The compound of the invention has been synthesised according to the process described in GRANDJEAN & LUKACS {J. of Carbohydrate Chemistry, vol .15 (7), pp 831-855, 1996; and The Journal Antibiotics, vol.49(10), p:1036-1043, 1996), in the ICSN (Institute of Chemistry of Natural Substances, CNRS), purified and dissolved in DMSO at 5mg/ml_.

Cell cultures and antibodies

The mouse cholinergic septal neuronal cell line SN56 (MAGALHAES et al., J Neurosci., vol.25, 5207-5216, 2005) was grown in culture medium composed of OptiMem, supplemented with 10% fetal calf serum (FCS) and 1 % Penicillin- Streptomycin (PS). The hypothalamic neuronal GT1-7 cell line (SCHATZL et al., J. Virol., vol.71 , p:8821-8831 , 1997) was maintained in OptiMem, with 5% FCS, 5% horse serum and 1% PS. Cells were inoculated by clarified homogenates from brains of mice infected by Chandler or 22L Prion strains, as described elsewhere (VILETTE et al., Proc. Natl. Acad. Sci. U.S.A., vol.98, p:4055-4059, 2001 ).

Saf70, Saf83 and Bar233 were used as anti-PrP antibodies.

Test of PrP^rΘS inhibitory activity of the compound of the invention in cell cultures

10⁴ Chandler-infected or non-infected SN56 cells were seeded in 96-well plates, with 100 μl of culture medium. The following day, cells were treated with two concentrations of the compound of the invention, 5 μg.ml^"1 and 0.25 μg.ml^"1 (dilution in cell culture medium), and cells were incubated for six days.

The toxicity of the compound was evaluated using the cell proliferation reagent (WST- 1 , Roche).

After the removal of culture medium, cells were washed in phosphate-buffered saline (PBS), and 50 μl of lysis buffer were added (0.5% sodium deoxycholate (wt/vol), 0.5% Triton X-100 (wt/vol), 50 mM Tris-HCI pH 7.4). After 10 min at 4 ⁰C, lysates were treated with 500 ng of proteinase K for 30 min at 37 ⁰C, and PK activity was inhibited by the addition of phenylmethylsulfonyl fluoride (PMSF, 0.8 mM final concentration). Samples were applied under vacuum to nitrocellulose membranes via a 96-well dot- blot apparatus. The membrane was then immersed in guanidium thiocyanate (4.23 M in PBS) for 10 min. After rinses in PBS and PBS-Tween buffer (PBS Tween-20 0.1%), the membrane was blocked in 5% (wt/vol) fat-free milk, and processed with Saf70, Saf83, or Bar233 anti-PrP antibodies, and anti-mouse horseradish-peroxidase coupled secondary antibody. Revelation was made using ECL kit (Amersham Pharmacia Biotech) by autoradiographic method. This technique was validated using PPS-treated cells as positive controls, while non-treated and DMSO-treated cells which were used as negative control, presented similar strong PrP^res signal.

Analysis of toxicity

In 96-well plates, 10⁴ SN56 cells and 2.10⁴ GT1-7 cells were seeded, in 100 μl of culture medium. They were treated with a range of dilutions of the identified compound, and six days later viability was assessed by a WST-1 test. Cytotoxic dose (CD₅₀) was then estimated graphically.

Confirmation of the PrP^rΘS inhibitory activity of the compound of the invention by Western Blot

SN56 and GT1-7 cells were treated with two concentrations of the identified compound (5 μg.ml^"1 and 1 μg.ml^"1), with DMSO (0.5% and 0.1%) and PPS (5 μg.ml^"1). After six days, cells were trypsinized, and lysed for 10 min at 4 ⁰C. Nuclei and cellular remnants were removed by centrifugation at 10⁴ g for 2 min. The amount of protein was determined using bicinchoninic acid protein assay (Uptima). Samples were PK-treated for 30 min at 37 ⁰C, PMSF was added (1 mM) and proteins were centrifuged at 2.10⁴ g for 1 h at 4 ⁰C. The pellets were resuspended and heated for 5 min at 100 ⁰C in 25 μl of Laemmli buffer.

Samples were subjected to sodium dodecyl sulfate 12% polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes in transfer buffer (12 mM Tris; 80 mM glycine; 10% isopropanol), at 20 V overnight. The membrane was blocked and revealed exactly as described for the dot-blot method. After revelation, the intensities of PrP^res signals were assessed by densitometry using the ImageJ freeware

(NIH), and samples were normalized with blank controls, allowing the determination of half-maximal inhibitory concentrations (IC₅₀), using the same method as described for estimation of CD₅₀.

Fluorescence-based thermal shift assay of PrP protein

Ten micrograms of purified recombinant murine PrP (produced in E. CoIi, in MOPS 20 mM, pH 7.4), according to previous publication (REZAEI et al., Eur. J. Biochem., vol.267, p:2833-2839, 2000) were diluted in phosphate buffer with compounds, and the fluorescent dye SYPRO Orange (Invitrogen) (SENISTERRA et al., J. Biomol. Screen., vol.13, p:337-342, 2008). SYPRO Orange is highly fluorescent when bound to hydrophobic sites on unfolded proteins, and non-fluorescent in aqueous solutions, where the fluorescence was quenched. Samples were denaturated, from 25 ⁰C to 95 ⁰C, at 3 ⁰C. min^"1, and SYPRO Orange fluorescence was monitored at real time, on an Applied Biosystems 7900HT Fast Real-Time PCR system. PrP molecules were denaturated by heat, and the exposure of hydrophobic sites was quantified by fluorescence. Therefore, fluorescence increased with temperature, and when the intensity peak had been reached, gradual decreases were observed, indicating possible protein aggregation or precipitation, or a drop of SYPRO efficiency. Diverse quantities of PrP^rec, different buffers (Tris-HCI, MOPS, phosphate buffer, MES), with salts (KCI, NaCI, or MgCI₂) or glycerol, at various concentrations and pH (ranging from 4.5 to 1 1 ) were tested: results were coherent and reproducible notably for phosphate buffer (50 mM, pH 8), with NaCI at physiological concentrations (150 mM) (data not shown). This buffer was used for the presented results.

Results

To evaluate if the compound of the invention presents a therapeutic interest, both cytotoxic dose (CD₅₀) and inhibiting concentration (IC₅₀) were determined. Cellular viability after treatment with serial dilutions of the compound was assessed as presented in figure 2. A dose-response curve, allowing the identification of IC₅O by densitometry, confirmed the concentration-dependant PrP^res inhibitory activity on Chandler-infected SN56 (fig. 1 ).

Results are summarized in the following table 1 :

Studies of PrP^rec stability by fluorescence-based thermal shift assay

To assess recombinant murine Prion Protein (PrP^rec) stability in the presence of the compound of the invention, a thermal shift assay was used. The method was validated, since observed melting temperatures (T_m), which are around 59-60 ⁰C, were in the same range as those identified on other studies, such as for human Prion Protein (121 - 230) by circular dichroism (KNOWLES & ZAHN, Biophys J., vol. 91 , p:1494-1500, 2006), or ovine Prion Protein by differential scanning calorimetry (REZAEI et al., J. Therm. Anal. CaI., vol.71 , p:237-247, 2003).

The effect of the compound on PrP^rec was compared with erythromycin A and DMSO (Figure 4). When in solution with the compound of the invention at 50 μM, PrP protein T_m was reduced significantly by more than 0.8 ⁰C, in comparison with DMSO and erythromycin A. These modifications were not observed for the compound incubated with SYPRO Orange alone, and without PrP^rec (data not shown).

Consequently, contrary to DMSO and erythromycin A, the compound of the invention interacts with PrP protein, and may change its stability.

Claims

1. A pharmaceutical composition comprising the (E)-9-[O-(methyl 2-O-benzyl-4,6- O-benzylidene-β-D-allopyranosid-3-yl)oxime] erythromycin A of formula I:

2. A method of prophylactically and/or therapeutically treating amyloϊd neurodegenerative diseases by administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 1.

3. The method according to claim 2, wherein said amyloϊd neurodegenerative disease is chosen among Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Prion-related disease.

4. The method according to claim 2, wherein said amyloϊd neurodegenerative disease is a genetic familial amyloϊd neurodegenerative disease.

5. The method according to claim 4, wherein said genetic familial amyloϊd neurodegenerative disease is a Prion-related disease, in particulate a familial Creutzfeldt-Jakob disease, fatal familial insomnia (FFI) and Gerstmann-Straussler- Scheinker disease (GSS).

6. The method according to claim 2, wherein said amyloϊd neurodegenerative disease is a sporadic or infectiously acquired amyloϊd neurodegenerative disease.

7. The method according to claim 6, wherein said sporadic amyloϊd neurodegenerative disease is a Prion-related disease, for example sporadic Creutzfeldt-Jakob disease (CJD), fatal sporadic insomnia (FSI).

8. The method according to claim 6, wherein said infectiously acquired amyloϊd neurodegenerative disease is a Prion-related disease, for example the variant of Creutzfeldt Jakob diseases (vCJD), kuru, or iatrogenic Creutzfeldt Jakob disease (iCJD).

9. The method according to claims 5 or 7, wherein said pharmaceutical composition acts by destabilizing the normal prion protein PrP^c.

10. The method according to claim 3, wherein said Prion-related disease is chosen among Bovine spongiform encephalopathy (BSE), Scrapie disease, or Creutzfeldt Jakob Disease (CJD).

1 1. The method according to any one of claims 2 to 9, wherein said subject is a human.

12. A method for testing at least one sample obtained from a mammal, for the presence or absence of an aberrant prion protein (PrP^res) in order to diagnose a Prion infection within said mammal, said method comprising:

a) Preparing said at least one sample from a mammal,

b) Adding the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A as defined in claim 1 in said at least one sample,

13. Method according to claim 12, wherein said at least one sample is a tissue homogenate of said mammal.

14. Method according to claim 12, wherein said at least one sample is a biological fluid of said mammal.

15. Method according to claim 12, wherein step c) is performed in the presence of a chaotropic agent.

16. A kit for detecting the presence or absence of an aberrant prion protein PrP^res, the kit comprising at least the (E)-9-[O-(methyl 2-O-benzyl-4,6-O-benzylidene-β-D- allopyranosid-3-yl)oxime] erythromycin A as defined in claim 1.