WO2012119949A1

WO2012119949A1 - Means and methods for the treatment of neurodegenerative disorders

Info

Publication number: WO2012119949A1
Application number: PCT/EP2012/053646
Authority: WO
Inventors: Peter Carmeliet; Annelies QUAEGEBEUR
Original assignee: Vib Vzw; Life Sciences Research Partners Vzw
Priority date: 2011-03-04
Filing date: 2012-03-02
Publication date: 2012-09-13
Also published as: GB201103762D0

Abstract

The present invention has found that inhibitors reducing the glycolytic flux can be used for treatment of neurodegenerative diseases such as Alzheimer's disease, Parkonson's disease and amyotrophic lateral sclerosis. In particular the invention provides constructs comprising siRNAs directed against PHD1 for the treatment of ALS and siRNA's directed against PFKFB3 for the treatment of neurodegenerative diseases. The invention also provides the use of a therapeutically effective amount of aza chalcones or a pharmaceutically acceptable salt thereof for the treatment of neurological diseases.

Description

Means and methods for the treatment of neurodegenerative disorders

Field of the invention

The present invention relates to the field of neurodegenerative diseases. In particular the invention has found that inhibitors reducing the glycolytic flux can be used for treatment of neurodegenerative diseases. In particular the invention provides constructs comprising siRNAs and siRNAs directed against PHD1 and PFKFB3 for the treatment of neurodegenerative diseases. The invention also provides the use of a therapeutically effective amount of aza chalcones or a pharmaceutically acceptable salt thereof for the treatment of neurological diseases.

Introduction to the invention

The brain is particularly sensitive to hypoxia and oxidative stress. This vulnerability is mainly due to the extraordinary metabolic requirements of normally functioning neurons. Vital neuronal processes such as neurotransmission and ion homeostasis are critically depending on a continuous oxygen and glucose supply. Any cerebrovascular deficit or neurovascular uncoupling, even subtle in nature, may therefore lead to metabolic deregulation, ultimately progressing to metabolic collapse and neuronal death. Oxygen radicals are highly reactive species that are generated as normal byproducts of mitochondrial oxidative metabolism. In physiological conditions, antioxidant enzymes keep their levels in check. Given the limited antioxidant defense mechanisms, especially in the brain, excessive ROS formation can result in oxidative damage. It is increasingly recognized that perfusion deficits and oxidative stress are both important and active contributors to the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. In order to maintain the homeostatic energetic balance in conditions of varying oxygen and oxygen radical levels, mammalian organisms are equipped with intricate molecular sensing mechanisms such as the prolyl hydroxylase domain proteins (PHD1-3) and factor inhibiting HIF (FIH). These cellular oxygen sensors activate in an oxygen- dependent manner a major transcriptional pathway governed by the hypoxia-inducible factors, allowing the cell to transduce oxygen levels to adaptive gene expression. In normoxic conditions, PHDs hydroxylate specific proline residues on the HIFa isoforms, thereby targeting them for proteosomal degradation. Hypoxia will lead to the accumulation of HIFa as PHD mediated degradation is prohibited. Transcriptional activity is additionally regulated by FIH, hydroxylating an asparaginyl residue on HIFa, which prevents binding of the coactivator p300/CBP. Oxygen radicals will via oxidation of iron, a cofactor necessary for the hydroxylation reaction, inhibit PHD and FIH activity as well. The resulting transcriptional response involves several biologic processes including angiogenesis, erythropoiesis, glucose metabolism, apoptosis etc.

In the present invention we have found that a deficiency of PHD1 (but not the lower expression of its family members PHD2 or PHD3) in an animal model for amyotrophic lateral sclerosis leads to a reduced paralysis and a significantly improved survival. Moreover, our data show that a reduction of PHD1 leads to a reduction of aerobic glycolysis and an increase pentose-phosphate pathway. In concordance with these data we have also shown that diseased ALS neurons (i.e. neurons isolated from a murine model for ALS) display an enhanced level of aerobic glycolysis. Such an increased glycolytic flux is a documented phenomenon in tumor cells because tumors use this metabolic pathway for the generation of ATP as a main source of energy supply and biosynthesis of macromolecules. This phenomenon, known as the Warburg effect, is considered as one of the most fundamental alterations during malignant transformation. Several glycolytic inhibitors have been described which are currently being used in pre-clinical uses for cancer treatment (Pelicano H et al (2006) Oncogene 25, 4633-4646. Some examples of glycolytic inhibitors include 2- deoxyglucose, lonidamine, 3-bromopyruvate, imatinib, oxythiamine and aza chalcones described in WO2008/156783. Aza chalcones specifically inhibit the enzyme 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). One example of such an aza chalcone is 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) which has been shown to actively suppresses the glycolytic flux in tumors (Clem B et al (2008) Mol. Cancer Ther. 7(1): 110-120). We have surprisingly shown that aza chalcones (as described in WO2008/156783) can also be used for the treatment of neurodegenerative diseases.

Figure legends

Figure 1 : Survival analysis of PHD1^"'^" SOD mice and PHD1^{+ +} SOD mice. It can be observed that PHD1 deficient SOD mice have an extended life span of, on average, 14 days.

Figure 2: Panel A. Glycolytic flux measured via radioactive labeled glucose substrate (5-³H- glucose) in cultured cortical neurons (wild type (WT) versus PHD1 knock-out neurons (KO). Panel B. Similar experiments were set up in wild type neurons (normoxia versus hypoxia 1 % during 14 hours)

Figure 3: Glycolytic flux measured via radioactive labeled glucose substrate (5-³H-glucose) in cultured cortical neurons (control versus 3PO-treated neurons).

Detailed description to the invention Increased aerobic glycolysis is commonly seen in a wide spectrum of human cancers, and hypoxia is present in most tumor microenvironments, the development and the use of glycolytic inhibitors is currently been explored in a variety of pre-clinical tumor models. In the present invention we show that compounds able to inhibit the glycolytic flux (or compounds inhibiting the glycolysis or compounds inhibiting the aerobic glycolysis which are equivalent wordings) can be used for the treatment of neurodegenerative diseases.

Accordingly in a first embodiment the invention provides a compound inhibiting the glycolytic flux for treatment of neurodegenerative diseases. In particular the compounds inhibit the glycolytic flux and enhance the pentose-phosphate pathway.

In a particular embodiment a compound is a siRNA with a specificity for prolyl hydroxylase 1 (PHD1) for the treatment of amyotrophic lateral sclerosis.

In yet another embodiment the siRNA with a specificity for PHD1 is expressed by an expression construct which is further incorporated into a gene therapy vector. A gene therapy vector can be a viral or a non-viral vector. Examples of viral vectors include adenoviral, adeno-associated vectors, lentiviral vectors and the like.

In a specific embodiment the siRNA with a specificity for PHD1 is expressed by an expression construct incorporated into an adenoviral-2 associated (AAV-2) vector.

The term "siRNA" refers to a small interfering RNA(s), which also has been referred to in the art as short interfering RNA and silencing RNA, among others. siRNAs generally are described as relatively short, often 20-25 nucleotide-long, double-stranded RNA molecules that are involved in RNA interference (RNAi) pathway(s). Generally, siRNAs are, in part, complementary to specific mRNAs (such as PHD1 or PFKFB3) and mediate their down regulation (hence, "interfering"). siRNAs thus can be used for down regulating the expression of specific genes and gene function in cells and organisms. siRNAs also play a role in related pathways. The general structure of most naturally occurring siRNAs is well established. Generally, siRNAs are short double-stranded RNAs, usually 21 nucleotides long, with two nucleotides single stranded "overhangs" on the 3 of each strand. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. In vivo, the structure results from processing by the enzyme "dicer," which enzymatically converts relatively long dsRNAs and relatively small hairpin RNAs into siRNAs. The term siNA refers to a nucleic acid that acts like a siRNA, as described herein, but may be other than an RNA, such as a DNA, a hybrid RNA: DNA or the like. siNAs function like siRNAs to down regulate expression of gene products. The term "RNA interference" which also has been called "RNA mediated interference" refers to the cellular processes by which RNA (such as siRNAs) down regulate expression of genes; i.e., down regulate or extinguish the expression of gene functions, such as the synthesis of a protein encoded by a gene. Typically, double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. RNA interference pathways are conserved in most eukaryotic organisms. It is initiated by the enzyme dicer, which cleaves RNA, particularly double-stranded RNA, into short double- stranded fragments 20-25 base pairs long. One strand of the double-stranded RNA (called the "guide strand") is part of a complex of proteins called the RNA-induced silencing complex (RISC). The thus incorporated guide strand serves as a recognition sequence for binding of the RISC to nucleic acids with complementary sequences. Binding by RISC to complementary nucleic acids results in their being "silenced." The best studied silencing is the binding of RISCs to RNAs resulting in post-transcriptional gene silencing. Regardless of mechanism, interfering nucleic acids and RNA interference result in down regulation of the target gene or genes that are complementary (in pertinent part) to the guide strand. A polynucleotide can be delivered to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to affect a specific physiological characteristic not naturally associated with the cell. The polynucleotide can be a sequence whose presence or expression in a cell alters the expression or function of cellular genes or RNA.

In addition, the present invention contemplates polynucleotide-based expression inhibitors of PHD1 or PFKFB3 which may be selected from the group comprising: siRNA, microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes or antisense nucleic acids.

For the avoidance of doubt, PFKFB3 is the 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 enzyme. The nucleotide sequence of the human PFKFB3 can be retrieved from GenBank as NM004566.

PHD1 is the HIF prolyl hydroxylase 1 enzyme, an alternative name is egl nine homolog 2 (EGLN2). The nucleotide sequence of the human PHD1 can be retried from GenBank as AJ310544 or NM080732.

SiRNA comprises a double stranded structure typically containing 15 to 50 base pairs and preferably 19 to 25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small noncoding polynucleotides, about 22 nucleotides long, that direct destruction or translational repression of their mRNA targets. Antisense polynucleotides comprise a sequence that is complimentary to a gene or mRNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-0-methyl polynucleotides, DNA, RNA and the like. The polynucleotide-based expression inhibitor may be polymerized in vitro, recombinant, contain chimeric sequences, or derivatives of these groups. The polynucleotide-based expression inhibitor may contain ribonucleotides, deoxynbonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. Polynucleotides may contain an expression cassette coded to express a whole or partial protein, or RNA. An expression cassette refers to a natural or recombinantly produced polynucleotide that is capable of expressing a sequence. The cassette contains the coding region of the gene of interest along with any other sequences that affect expression of the sequence of interest. An expression cassette typically includes a promoter (allowing transcription initiation), and a transcribed sequence. Optionally, the expression cassette may include, but is not limited to, transcriptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals. An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins. Optionally, the expression cassette may include, but is not limited to, translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non-coding sequences. The polynucleotide may contain sequences that do not serve a specific function in the target cell but are used in the generation of the polynucleotide. Such sequences include, but are not limited to, sequences required for replication or selection of the polynucleotide in a host organism.

Based on the RNA sequence of PHD1 and/or PFKFB3, siRNA molecules with the ability to knock-down PHD1 and/or PFKFB3 activity can be obtained by chemical synthesis or by hairpin siRNA expression vectors. There are numerous companies that provide the supply of costumer-designed siRNAs on a given RNA sequence, e.g. Ambion, Imgenex, Dharmacon. The PHD1 and/or PFKFB3 siRNAs of the invention may be chemically modified, e.g. as described in US20030143732, by phosphorothioate internucleotide linkages, 2'-0-methyl ribonucleotides, 2'-deoxy-2'fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. The sense strand of PHD1 and/or PFKFB3 siRNAs may also be conjugated to small molecules or peptides, such as membrane-permeant peptides or polyethylene glycol (PEG). Other siRNA conjugates which form part of the present invention include cholesterol and alternative lipid-like molecules, such as fatty acids or bile-salt derivatives.

In a further embodiment, the present invention relates to an expression vector comprising any of the above described polynucleotide sequences encoding at least one PHD1 and/or PFKFB3 siRNA molecule in a manner that allows expression of the nucleic acid molecule, and cells containing such vector. The polynucleic acid sequence is operably linked to regulatory signals (promoters, enhancers, suppressors etc.) enabling expression of the polynucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs. A variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral vector systems. Selection of the appropriate viral vector system, regulatory regions and host cell is common knowledge within the level of ordinary skill in the art.

As gene delivery and gene silencing techniques improve, the selective deletion of PHD1 and/or PFKFB3, in particular in neuronal tissues may prove useful in order to limit the impact of protein deletion to a particular system under study. The PHD1 and/or PFKFB3 siRNA molecules of the invention may be delivered by known gene delivery methods, e.g. as described in US20030143732, including the use of naked siRNA, synthetic nanoparticles composed of cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, or bioconjugates including siRNAs conjugated to fusogenic peptides. Delivery of siRNA expressing vectors can also be systemic, such as by intravenous or intramuscular administration or by intrathecal or by intracerebral injection that allows for introduction into the desired target cell (see US 20030143732).

In yet another embodiment the compound is a small molecule compound able to inhibit the enzyme PFKFB3 for the treatment of a neurodegenerative disease.

In a specific embodiment a compound is a molecule with the formula (I) for the treatment of a neurodegenerative disease:

wherein:

X, X₂, and X₃ are each C or CH;

X_! is selected from the group consisting of O, S, NR^ C(R₂)2, OR₃, SR₄, NR₅R₆, and C(R₇)₃ , wherein R^ R₃, R₄, R₅ and R₆ are each independently selected from the group consisting of H, alkyl, aryl, aralkyl, and acyl, and each R₂ and R₇ is independently selected from the group consisting of H, halo, hydroxyl, alkoxy, alkyl, aralkyl, and aryl;

Rio is selected from the group consisting of H, alkyl, halo, cyano, hydroxyl, aryl, and aralkyl; Ri i is selected from the group consisting of H, alkyl, halo, cyano, hydroxyl, aryl, and aralkyl;

Ai , A₂, A₃, A₄, and A₅, are each independently N or CR12, wherein each R₁₂ is independently selected from the group consisting of H , alkyl, halo, nitro, cyano, hydroxyl, mercapto, amino, alkylamino, dialkylamino, carboxyl, acyl, carbamoyl, alkylcarbamoyi, dialkylcarbamoyi, sulfate, and a group having the structure:

wherein:

X₄ is NR₁₄, wherein R₁₄ is selected from the group consisting of H, alkyl, hydroxyl, aralkyl, and aryl;

X₅ is selected from the group consisting of O, S, C(Ri₅)₂, and N R₁₄, wherein each R₁₅ is independently selected from the group consisting of H, hydroxyl, alkoxy, alkyl, aralkyl, and aryl; and

X₆ is selected from H, alkyl, aralkyl, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;

or wherein R₁₀ and one R₁₂ are

together alkylene;

Ar₂ is selected from the group consisting of

wherein:

each Yi , Y₂, Y3, Y₄, Y5, YQ, Y₇, Ys, Y9, Y10, Y11 , Y12, Y13, Yi₄, Y15, Y16, Y17, Y18, and Y₁₉ is independently selected from the group consisting of N and CR1₃, wherein each R₁₃ is independently selected from the group consisting of H, alkyi, halo, nitro, cyano, hydroxyl, mercapto, amino, alkylamino, dialkylamino, carboxyl, acyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, sulfate, and a group having the structure:

wherein:

X₄ is N R₁₄, wherein R₁₄ is selected from the group consisting of H, alkyi, hydroxyl, aralkyi, and aryl;

X₅ is selected from the group consisting of O, S, C(Ri₅)₂, and N R₁₄, wherein each R₁₅ is independently selected from the group consisting of H, hydroxyl, alkoxy, alkyi, aralkyi, and aryl; and

X₆ is selected from H, alkyi, aralkyi, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;

or wherein R₁₀ and one R₁₃ are together alkylene; and

wherein at least one of A! , A₂, A₃, A₄, A₅, Yi , Y₂, Y₃, Y₄, Y₅, Ye, Y₇, Ye, Yg, Y10, Yi i , Yi2, Yi3, Yi₄, Vis, Yie, Yi7, Y18, and Y₁₉ is N; or a pharmaceutically acceptable salt thereof.

In yet another embodiment a compound is according to the previous embodiment wherein X_! is O and X is C.

In yet another embodiment in the compound of formula (I) Ar₂ is:

X, X₂, and X₃ are each C;

Xi is selected from the group consisting of O, S, NRi . and C(R₂)₂, wherein R< . is selected from the group consisting of H and alkyi, and each R₂ is independently selected from the group consisting of H, halo, hydroxyl, alkoxy, and alkyi; and the compound of Formula (!) has a structure of Formula (II):

In yet another embodiment the compound according to formula (I) is wherein Ar₂ is:

X, X₂, and X₃ are each C;

Xi is selected from the group consisting of O, S, NRi , and C(R₂)2, wherein is selected from the group consisting of H and alkyl, and each F¾ is

independently selected from the group consisting of H, halo, hydroxyl, aikoxy, and alkyl; and the compound of Formula (I) has a structure of Formula (II I):

(I I I)

In a specific embodiment the compound of Formula (I I I) is

In yet another embodiment the compound according to formula (I) is wherein Ar₂ is

X, X₂, and X₃ are each C;

Xi is selected from the group consisting of O, S, N R^ , and C(R₂)₂, wherein is selected from the group consisting of H and alkyl, and each R₂ is independently selected from the group consisting of H, halo, hydroxyl, aikoxy, and alkyl; and the compound of Formula (i) has a structure of Formula (IV):

(IV)

In a specific embodiment a compound according to Formula (IV) is:

In specific embodiments the compounds according to the previous embodiments are used for the treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, Frontotemporal lobe dementia and amyotrophic lateral sclerosis. In yet another embodiment the invention provides a siRNA with a specificity for PFKFB3 for the treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, Frontotemporal lobe dementia and amyotrophic lateral sclerosis.

In a specific embodiment said siRNA with a specificity for PFKFB3 is expressed by an expression construct incorporated into a viral vector.

In yet another specific embodiment said siRNA with a specificity for PFKFB3 is expressed by an expression construct incorporated into an adenoviral-2 associated (AAV-2) vector.

The term "neurodegenerative disease" as used herein refers to diseases which suffer from a progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, fronto temporal lobe dementia, Huntington's disease occur as a result of neurodegenerative processes. Still other diseases were a progressive loss of neurons occurs are stroke, multiple sclerosis and Charcot-Marie-Tooth disease.

The term "alkyl" refers to Ci_₂o inclusive, linear (i.e. , "straight-chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e. , alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, te/t-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a Ci_₈ alkyl), e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 1 1 , 12, ... or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to Ci_₈ straight- chain alkyls. In other embodiments, "alkyl" refers, in particular, to Ci_₈ branched-chain alkyls.

An "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

"Aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. The aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents, which can be the same or different, wherein "aryl group substituent" includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene.

Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

The term "aza" refers to a heterocyclic ring structure containing at least one nitrogen atom. Specific examples of aza groups include, but are not limited to, pyrrolidine, piperidine, quinuclidine, pyridine, pyrrole, indole, purine, pyridazine, pyrimidine, and pyrazine.

The term "aza-aryl" refers to a heterocyclic aryl group wherein one or more of the atoms of the aryl group ring or rings is nitrogen.

"Alkylene" refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents," including hydroxyl, halo, nitro, alkyl, aryl, aralkyl, carboxyl and the like. There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as previously described. The term "acyl" refers to an organic carboxylic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO— , wherein R is an alkyl, aralkyl or aryl group as defined herein, including substituted alkyl, aralkyl, and aryl groups). As such, the term "acyl" specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.

"Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. "Alkoxyl" refers to an alkyl-O- group wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, f-butoxyl, and pentoxyl. The term "oxyalkyl" can be used interchangably with "alkoxyl".

"Aryloxyl" refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term "aryloxyl" as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl. "Aralkyl" refers to an aryl— alkyl— group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

"Alkoxycarbonyl" refers to an alkyl-O-CO- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and f-butyloxycarbonyl.

"Aryloxycarbonyl" refers to an aryl-O-CO- group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

"Aralkoxycarbonyl" refers to an aralkyl-O-CO- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl. "Carbamoyl" refers to an H₂N-CO- group.

"Alkylcarbamoyl" refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R¹ is alkyl and/or substituted alkyl.

"Acyloxyl" refers to an acyl-O- group wherein acyl is as previously described. "Acylamino" refers to an acyl-NR- group wherein acyl is as previously described and R is H or alkyl. Thus, the "acylamino" group can have the structure -NR-C(=0)-R', wherein R' is alkyl, aryl, aralkyl, and the like.

The term "amino" refers to the -NH₂ group.

The term "carbonyl" refers to the -(C=0)- group.

The term "carboxyl" refers to the -COOH group.

The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups.

The term "hydroxyl" refers to the -OH group.

The term "hydroxyalkyl" refers to an alkyl group substituted with an -OH group.

The term "mercapto" refers to the -SH group. The term "oxo" refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term "aza" refers to a compound wherein a carbon atom is replaced by a nitrogen atom.

The term "nitro" refers to the -N0₂ group. The term "thio" refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.

The term "sulfate" refers to the -S0₄ group.

When the term "independently selected" is used, the substituents being referred to (e.g., R groups, such as groups and R₂, or groups X and Y), can be identical or different. For example, both and R₂ can be substituted alkyls, or can be hydrogen and R₂ can be a substituted alkyl, and the like.

Medicinal uses:

This invention also relates to pharmaceutical compositions containing one or more compounds of the present invention. These compositions can be utilized to achieve the desired pharmacological effect by administration to a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease. Therefore, the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound, or salt thereof, of the present invention. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The compounds of the present invention can be administered with pharmaceutically- acceptable carriers well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, intrathecally, intracerebroventricularly and the like. In a preferred embodiment the administration is intrethecally. In another preferred embodiment the administration is intracerebroventricularly. For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions. The solid unit dosage forms can be a capsule that can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment, the compounds of this invention may be tableted with conventional tablet bases such as lactose, sucrose and cornstarch in combination with binders such as acacia, corn starch or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, gum tragacanth, acacia, lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example talc, stearic acid, or magnesium, calcium or zinc stearate, dyes, coloring agents, and flavoring agents such as peppermint, oil of wintergreen, or cherry flavoring, intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent or emulsifying agent. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance tablets, pills or capsules may be coated with shellac, sugar or both.

Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example those sweetening, flavoring and coloring agents described above, may also be present.

The pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as, for example, beeswax, hard paraffin, or cetyl alcohol. The suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin. Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, and preservative, such as methyl and propyl parabens and flavoring and coloring agents.

The parenteral compositions of this invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) preferably of from about 12 to about 17. The quantity of surfactant in such formulation preferably ranges from about 5% to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia ; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadeca- ethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that may be employed are, for example, water, Ringer's solution, isotonic sodium chloride solutions and isotonic glucose solutions. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

A composition of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are, for example, cocoa butter and polyethylene glycol.

Another formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (see for example US 5,023,252). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Controlled release formulations for parenteral administration include liposomal, polymeric microsphere and polymeric gel formulations that are known in the art. It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for the delivery of pharmaceutical agents is well known in the art. Direct techniques for, for example, administering a drug directly to the brain usually involve placement of a drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the transport of agents to specific anatomical regions of the body, is described in US 5,011 ,472.

The compositions of the invention can also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al., "Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311 ; Strickley, R.G "Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1 " PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349 ; and Nema, S. et al. , "Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171. Pharmaceutical compositions according to the present invention can be illustrated as follows:

Sterile IV Solution: A 5 mg/mL solution of the desired compound of this invention can be made using sterile, injectable water, and the pH is adjusted if necessary. The solution is diluted for administration to 1 - 2 mg/mL with sterile 5% dextrose and is administered as an IV infusion over about 60 minutes.

Lyophilised powder for IV administration: A sterile preparation can be prepared with

(i) 100 - 1000 mg of the desired compound of this invention as a lyophilised powder,

(ii) 32- 327 mg/mL sodium citrate, and (iii) 300 - 3000 mg Dextran 40. The formulation is reconstituted with sterile, injectable saline or dextrose 5% to a concentration of 10 to 20 mg/mL, which is further diluted with saline or dextrose 5% to 0.2 - 0.4 mg/mL, and is administered either IV bolus or by IV infusion over 15 - 60 minutes.

Intramuscular suspension: The following solution or suspension can be prepared, for intramuscular injection:

50 mg/mL of the desired, water-insoluble compound of this invention

5 mg/mL sodium carboxymethylcellulose

4 mg/mL TWEEN 80

9 mg/mL sodium chloride

9 mg/mL benzyl alcohol

Combination therapies

The compounds of this invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects. The present invention relates also to such combinations. For example, the compounds of this invention can be combined with known medicines for neurodegenerative diseases.

The term "treating" or "treatment" as stated throughout this document is used conventionally, e.g. , the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of, etc., of a disease or disorder, such as a carcinoma.

In a particular preferred embodiment the 'molecules' may be administered by a method close to the place of onset. Preferably a continuous infusion is used and includes the continuous subcutaneous delivery via an osmotic minipump. In another embodiment said close to the onset administration is an intrathecal administration.

In another embodiment said close to the onset administration is an intracerebroventricular administration. Thus in a particular embodiment the infusion with a composition comprising a molecule of the invention is intrathecal. Intrathecal administration can for example be performed by means of surgically implanting a pump and running a catheter to the spine or in the skull.

Dose and administration:

Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of neurodegenerative diseases, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known medicaments that are used to treat these conditions, the effective dosage of the compounds of this invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.

The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 20 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, "drug holidays" in which a patient is not dosed with a drug for a certain period of time, may be beneficial to the overall balance between pharmacological effect and tolerability. A unit dosage may contain from about 0.5 mg to about 150 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, intrathecal, intraceroventricularly and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight. It is evident for the skilled artesan that the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests. Examples

I .The loss of hypoxia-inducible factor prolyl 4-hvdroxylase isoform 1 (PHD1) in a murine model for amyotrophic lateral sclerosis enhances the prolongation of survival.

The role of oxygen sensors (PHD1 , PHD2, PHD3 and FIH) and their downstream targets Hypoxia-Inducible Factors 1a and 2a (HIF-1 a and HIF-2a) was assessed by crossbreeding an established model for ALS (i.e. the SOD^G93A mice) with mice that constitutively or conditionally underexpress each of the HIFs, PHDs and FIH. It was observed that HIF-2a^{+ "} x SOD1^G93A mice ("x" depicts the symbol of crossing) do not show a survival difference when compared to HIF-2a^{+ +} x SOD1^G93A mice (HIF2a^{+ "} SOD^G93A mice (n= 23): 132 ± 2 days; HIF2a^{+ +} SOD^G93A mice (n=23): 131 ± 3 days; p = NS). In addition the data on the HIF1 SOD^G93A intercross did not show a survival difference either (HIF1a^{+ "} SOD^G93A mice (n=8): 120 ± 2 days; HIF1a^{+ +} SOD^G93A mice (n=13): 117 ± 3 days; p = NS.

However, in contrast the analysis of the PHD1 knockout - SOD^G93A crossbreeding showed a significant and substantial prolongation of survival. As shown in figure 1 , SOD^G93A mice live on average 138 days, whereas PHD1 deficient SOD^G93A mice have an extended life span of, on average, 14 days (PHD1^"A SOD^G93A mice (n=7) 152 ± 7 days; PHD1^{+ +} SOD^G93A mice (n=10): 138 ± 3 days; p=0.043). Apart from the enhancement of survival we also measured a reduced paralysis in these mice. This indicates that specific inhibition of PHD1 is a safe therapeutic approach for interfering with the disease progression of ALS. 2. Selective inhibition of PHD1 prolongs the survival of animal models for amyotrophic lateral sclerosis

It has previously been shown that viral delivery of small interfering RNAs to the spinal cord can be accomplished by injection of a recombinant adeno-associated virus (AAV-2) expressing a small interfering RNA, into muscles (Miller TM et al (2005) Ann. Neurol 57: 773-776). We are following essentially the same strategy for specifically targeting PHD-1 in spinal motor neurons. Two siRNA constructs directed against PHD1 are constructed as described (Brummelkamp TR et al (2002) Science 296:550-553) with the pSuper vector using the sequences 5'-GCUGCAUCACCUGUAUCUATT-3' (SEQ ID NO: 1) and 5'- GGUGUUCAAGUACCAGUAUTT-3' (SEQ ID NO: 2) for the siRNA specific for PHD1. Recombinant design of the AAV-2 viral vector comprising the siRNA construct driven by the H1 RNA promoter is produced in HEK293 cells (ATCC) by calcium phosphate transient transfection of vector plasmid, pAAV/Ad8, and pXX6 helper plasmid, as described previously. Xiao X et al (1998) J. Virol. 72:2224-2232). Virus is purified by CsCI density gradient dialysis and heparin affinity column chromatography (Amersham Biotech) using vector services from Virapur, LLC (San Diego, CA). Titers are determined by quantitative polymerase chain reaction (PCR) methods, as described previously, and are diluted to 1 ^χ 10¹² DNAse-resistant particles per milliliter (Zen Z. et al (2004) Hum. Gene Ther. 15: 709- 715).

All surgical procedures are performed in accordance with institutional approval. At aged 45 days, 20 randomly assigned transgenic mice with the G93A human SOD1 mutation (B6SJL- TgN [SOD1-G93A] 1 Gur; Jackson Laboratories, Bar Harbor, ME) are injected with recombinant 25μΙ AAV-siRNA-PHD1 into the right lower hind limb using a Hamilton syringe. Motor function is tested weekly with a Grip Strength Meter (Columbus Instruments, Columbus, OH). Grip strength meter testing is performed by allowing the animals to grasp a platform with one hind limb (left or right), followed by pulling the animal until it releases the platform; the force measurement is recorded in four separate trials.

In a second strategy we are administering modified versions of siRNA oligonucleotides specific for PHD1 in a continuous way to a transgenic rat model for amyotrophic lateral sclerosis via an osmotic pump into the intracerebral ventricles. Thereto two different synthetic oligonucleotides with dTdT- overhangs (CAGCACUACCCAUAGCAGUdTdT (SEQ ID NO: 3) and UCAAGCUCUCCCUCAGUUGdTdT (SEQ ID NO: 4) targeting prolyl hydroxylase 1 (PHD1) were used based on published rat siRNA sequences specific for PHD1 (Siddiq A et al (2009) J. of Neuroscience 29(27):8828-8838). Alternatively SEQ ID NO: 3 and 4 are modified with phosphorothioate modifications throughout and 2'-0-(2- methoxy)ethyl substitutions on the sugars of the first and last 5 nucleotides to increase biological half-lives and binding affinity (McKay RA et al (1999) J. Biol. Chem. 274:1715- 1722); and essentially carried out according to the Methods section on page 2294 of Smith RA et al (2006) J. Clinical Investigation 1 16 (8): 2290-2296). SEQ ID NO: 3 and SEQ ID NO: 4 (or alternatively the chemically modified versions thereof) are continuously pumped into the right lateral ventricle of a transgenic rat carrying the G93A SOD1 mutation via a catheter surgically implanted through the skull and connected to an osmotic pump imbedded subcutaneously. Motor performance of treated vs non-treated rats is quantified via rotarod analysis twice a week, recording the time the mouse stays on the rotarod before falling of; rotarod failure is defined when this time drops below 60 seconds. Survival or age of death is defined as the inability of the rats to right itself within 30 seconds after having been placed on its back. We complete this phenotype analysis by performing nerve conduction studies and electromyography in the hindlimbs. The clinical phenotype is histologically characterized by analyzing the number of motoneurons in the lumbal and cervical spinal cord and brainstem, the number of viable axons and the degree of Wallerian degeneration in ventral root preparations and peripheral nerves.

3. Metabolic reprogramming in neurons lacking PHD1

In this experiment we investigated via which mechanisms PHD1 modulates motor neuron degeneration. It was previously shown that muscle cells deficient in PHD1 were metabolically reprogrammed in their basal metabolism and acquired an induced glycolytic gene expression and a reduced mitochondrial oxidative phosphorylation (Aragones J. et al (2008) Nat. Genet. 40: 170-180). We therefore questioned whether a similar metabolic switch also existed in PHD1 deficient motor neurons which could explain the protection of motor neurons to hypoxia and oxidative stress (i.e. in a background of PHD1 deficiency). We surprisingly found that the glycolytic flux in PHD1 knock-out cortical neurons is 40 % reduced (p<0.05) with as a result less extracellular acidification. In addition, it was observed that the pentose-phosphate pathway (PPP) was upregulated in PHD1 -deficient neurons. The finding of the reduced glycolytic flux is in contrast to the fact that upon hypoxic inhibition of all PHD isoforms, neurons do indeed upregulate their glycolytic flux with accordingly increased lactate production and decreased oxygen consumption (see Figure 2, panel B). Our findings show that whereas a general PHD inhibition results in a shift from oxidative phosphorylation to glycolysis, a specific PHD1 deletion results in an isoform-specific metabolic rewiring. Figure 2 shows the results from representative experiments.

4. Increased glycolytic fluxes in neurodegenerative diseases

In order to confirm our hypothesis (i.e. the inhibition of the glycolytic flux as a viable strategy for the treatment of neurodegenerative diseases) we also investigated whether neurons derived from several neurodegenerative murine disease models also displayed an increased glycolytic flux. It was indeed found that the glycolytic flux was significantly increased in motor neurons derived from SOD1^G93A mice (i.e. an established animal model for amyotrophic lateral sclerosis) with respect to wild type murine motor neurons.

Our experiments are completely in line with published data were it was found that embryonic hippocampal neurons, isolated from a transgenic mouse Alzheimer model, also displayed an increased glycolytic flux (Yao J. et al (2009) Proc. Natl. Acad. Sc. 106(34): 14670-14675) and that amyloid-beta preferentially accumulates in highly glycolytic brain regions (Vlassenko AG et al (2010) Proc. Natl. Acad. Sc. 107(41): 17763-7).

5. Neuroprotective effect of reduced neuronal glycolysis

Given the striking and consistent reduction in glycolytic rate in PHD1 knockout cortical neurons, we wondered whether this metabolic alteration was responsible for the neuroprotective effect. In the art it has been shown that neurons rely for their survival on glucose flux through the pentose phosphate pathway (PPP). This is because the PPP provides the cell with NADPH, necessary for the replenishment of reduced glutathion in neurons and thus anti-oxidant defenses (Herrero-Mendez A. et al (2009) Nature Cell Biology 1 1 (6):747). We therefore hypothesized that a reduced glycolytic rate could be obtained by applying pharmacological inhibitors of the glycolysis. An important class of inhibitors of the glycolytic flux are aza chalcone analogues which are described in WO2008/156783. Said inhibitors are specific for the glycolytic PFKFB3 enzyme.

5.1 Pharmacological inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) reduces the glycolytic flux

Several small molecules able to specifically inhibit the PFKFB3 enzyme have been described in the art and these molecules have been shown to suppress the glycolytic flux (see Clem B et al (2008) Mol. Cancer Ther. 7(1): 110 and also patent application WO2008/156783). One of these compounds, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) has been shown to suppress the glycolytic flux and the compound was shown to be cytostatic to neoplastic cells (Clem B. et al (2008) Mol. Cancer Ther. 7(1): 1 10). We have surprisingly demonstrated that 3PO not only is capable of inhibiting the glycolysis in tumor cells but that this compound also significantly reduces the glycolytic flux in cultured cortical neurons (see Figure 3). Our experiments show that aza chalcones, and in particular 3PO, can be used for the treatment of neurodegeneration.

5.2. Selective inhibition of PFKFB3 can be used to treat neurodegeneration in an animal model

Viral delivery of small interfering RNAs specific for PFKFB3 to the spinal cord of a transgenic mouse model for ALS is accomplished by injection of a recombinant adeno-associated virus (AAV-2) expressing a specific small interfering RNA for PFKFB3 into muscles. The strategy is essentially the same as followed in example 2 except for the specific siRNAs. The siRNA sequence directed against murine PFKFB3 used are the sequence 5'- GGATAGGTGTTCCAACGAA-3' (SEQ ID NO: 5), 5'-CCACATCCAGAGCCGAATT-3' (SEQ ID NO: 6) and 5'-GCTGCCTACTAGCCTACTT-3' (SEQ ID NO: 7).

In a second strategy we are treating a transgenic rat model for ALS by administering siRNA sequences directed against rat PFKFB3, administration is as described in example 2. Thereto three different synthetic oligonucleotides with dTdT- overhangs: GGATAGGTGTTCCAACGAAdTdT (SEQ ID NO: 8), CCAGAGCCGAATCGTGTATdTdT (SEQ ID NO: 9) and GCTGCCTACTAGCCTACTTdTdT (SEQ ID NO: 10) targeting rat PFKFB3 were used. Alternatively SEQ ID NO: 8, 9 and 10 are modified with phosphorothioate modifications throughout and 2'-0-(2-methoxy)ethyl substitutions on the sugars of the first and last 5 nucleotides to increase biological half-lives and binding affinity. SEQ ID NO: 8, 9 and 10 (or alternatively chemically modified versions thereof) are administered in a continuous way via an osmotic pump into the intracerebral ventricles of a rat model for ALS. Administration and clinical analysis of the rats is carried out essentially as described in example 2.

In a third strategy the compound 3PO is continuously pumped into the right lateral ventricle of a transgenic rat carrying the G93A SOD1 mutation via a catheter surgically implanted through the skull and connected to an osmotic pump imbedded subcutaneously. Motor performance of treated vs non-treated rats is quantified as described in example 2.

Claims

1. A compound with a specificity for a PFKFB3 polynucleotide selected from the list consisting of a small interfering RNA (siRNA), an artificial microRNA, an antisense polynucleotide or a ribozyme for the treatment of a neurodegenerative disease.

2. A small interfering RNA according to claim 1 wherein said siRNA is produced by an expression construct incorporated into a viral vector.

3. A viral vector according to claim 2 wherein said viral vector is an adenoviral- associated viral vector.

4. A pharmaceutical composition comprising a compound according to any one of claims 1 , 2 or 3 for the treatment of a neurodegenerative disease.

5. A pharmaceutical composition according to claim 4 wherein said neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal lobe dementia or amyotrophic lateral sclerosis.

6. A compound with a specificity for a PHD1 polynucleotide selected from the list consisting of a small interfering RNA (siRNA), an artificial microRNA, an antisense polynucleotide or a ribozyme for the treatment of a neurodegenerative disease.

7. A small interfering RNA according to claim 6 which is produced by an expression construct incorporated into a viral vector.

8. A viral vector according to claim 7 wherein said vector is an adenoviral-associated viral vector.

9. A pharmaceutical composition comprising a compound according to any one of claims 6, 7 or 8 for the treatment of a neurodegenerative disease.

10. A pharmaceutical composition according to claim 9 wherein said neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal lobe dementia or amyotrophic lateral sclerosis.

1 1. A compound which is a molecule with the formula (I) or a stereoisomer, a tautomer, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of the same for the treatment of a neurodegenerative disease:

(I) wherein:

X, X₂, and X₃ are each C or CH;

X_! is selected from the group consisting of O, S, NR^ C(R₂)₂, OR₃, SR₄, NR₅R₆, and C(R₇)₃ , wherein R^ R₃, R₄, R₅ and R₆ are each independently selected from the group consisting of H, alkyl, aryl, aralkyi, and acyl, and each R₂ and R₇ is independently selected from the group consisting of H, halo, hydroxyl, alkoxy, alkyl, aralkyi, and aryl;

Rio is selected from the group consisting of H, alkyl, halo, cyano, hydroxyl, aryl, and aralkyi;

Rii is selected from the group consisting of H, alkyl, halo, cyano, hydroxyl, aryl, and aralkyi;

Ai , A₂, A₃, A₄, and A₅, are each independently N or CR₁₂, wherein each R₁₂ is independently selected from the group consisting of H, alkyl, halo, nitro, cyano, hydroxyl, mercapto, amino, alkylamino, dialkylamino, carboxyl, acyl, carbamoyl, alkylcarbamoyi, dialkylcarbamoyi, sulfate, and a group having the structure:

wherein:

X₄ is NR₁₄, wherein R₁₄ is selected from the group consisting of H, alkyl, hydroxyl, aralkyi, and aryl;

X₅ is selected from the group consisting of O, S, C(Ri₅)₂, and NR₁₄, wherein each R₁₅ is independently selected from the group consisting of H, hydroxyl, alkoxy, alkyl, aralkyi, and aryl; and

X₆ is selected from H, alkyl, aralkyi, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;

or wherein R₁₀ and one R₁₂ are together alkylene;

Ar₂ is selected from the group consisting of

wherein:

each Υι , Υ2, Υ3, Υ₄, Ys, Υβ, Υ7, Ys, Υ9, Υ10, Υ11 , Υ12, Υ13, Υι₄, Υΐ5, Υΐ6, Υΐ7, Υΐ 8, and Υ₁₉ is independently selected from the group consisting of N and CR1₃, wherein each R₁₃ is independently selected from the group consisting of H, alkyi, halo, nitro, cyano, hydroxyl, mercapto, amino, alkylamino, dialkylamino, carboxyl, acyl, carbamoyl, alkylcarbamoyi, dialkylcarbamoyi, sulfate, and a group having the structure:

wherein:

X₆ is selected from H , alkyi, aralkyi, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;

or wherein R₁₀ and one R₁₃ are together alkylene; and

wherein at least one of A! , A₂, A₃, A₄, A₅, Yi , Y₂, Y₃, Y₄, Ys, Y₆, Y₇, Ys, Y₉, Y10, Y11 , Y12, Yi3, Yi₄, V₁₅, Y16, Yi 7, Y18, and Y₁₉ is N ; or a pharmaceutically acceptable salt thereof.

12. A compound according to claim 1 1 wherein X_! is O and X is C.

13. A compound according to claim 1 1 wherein Ar₂ is:

X, X₂, and X₃ are each C;

Xi is selected from the group consisting of O, S, NF¾ , and C(R₂)2, wherein i¾ , is selected from the group consisting of H and alkyl, and each F¾ is independently selected from the group consisting of H, halo, hydroxy!, aikoxy, and alkyi; and the compound of Formula (I) has a structure of Formula (II):

(l l)

14. A compound according to claim 13 wherein the compound of formula (II) is selected from the group consisting of:

15. A compound according to claim 11 wherein Ar₂ is:

X, X₂, and X₃ are each C;

Xi is selected from the group consisting of O, S, NRi . and C(R₂)2. wherein R< . is selected from the group consisting of H and alkyl, and each R₂ is independently selected from the group consisting of H, halo, hydroxyl, alkoxy, and alkyl; and the compound of Formula (I) has a structure of Formula (III):

(III)