WO2012119949A1 - Moyen et procédés de traitement de troubles neurodégénératifs - Google Patents

Moyen et procédés de traitement de troubles neurodégénératifs Download PDF

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WO2012119949A1
WO2012119949A1 PCT/EP2012/053646 EP2012053646W WO2012119949A1 WO 2012119949 A1 WO2012119949 A1 WO 2012119949A1 EP 2012053646 W EP2012053646 W EP 2012053646W WO 2012119949 A1 WO2012119949 A1 WO 2012119949A1
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alkyl
aryl
hydroxyl
disease
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Peter Carmeliet
Annelies QUAEGEBEUR
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Vib Vzw
Life Sciences Research Partners Vzw
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    • C12Y207/01105—6-Phosphofructo-2-kinase (2.7.1.105)
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    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
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Definitions

  • the present invention relates to the field of neurodegenerative diseases.
  • the invention has found that inhibitors reducing the glycolytic flux can be used for treatment of neurodegenerative diseases.
  • 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.
  • 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.
  • 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).
  • PFKFB3 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3
  • aza chalcone 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).
  • aza chalcones as described in WO2008/156783
  • WO2008/156783 can also be used for the treatment of neurodegenerative diseases.
  • FIG. 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.
  • FIG. 2 Panel A. Glycolytic flux measured via radioactive labeled glucose substrate (5- 3 H- 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- 3 H-glucose) in cultured cortical neurons (control versus 3PO-treated neurons).
  • the invention provides a compound inhibiting the glycolytic flux for treatment of neurodegenerative diseases.
  • the compounds inhibit the glycolytic flux and enhance the pentose-phosphate pathway.
  • a compound is a siRNA with a specificity for prolyl hydroxylase 1 (PHD1) for the treatment of amyotrophic lateral sclerosis.
  • PLD1 prolyl hydroxylase 1
  • 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.
  • siRNA with a specificity for PHD1 is expressed by an expression construct incorporated into an adenoviral-2 associated (AAV-2) vector.
  • 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).
  • RNAi RNA interference
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • GenBank 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 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.
  • 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.
  • 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.
  • 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.
  • the PHD1 and/or PFKFB3 siRNAs of the invention may be chemically modified, e.g.
  • 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).
  • siRNA conjugates which form part of the present invention include cholesterol and alternative lipid-like molecules, such as fatty acids or bile-salt derivatives.
  • 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.
  • regulatory signals promoters, suppressors etc.
  • 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.
  • 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).
  • the compound is a small molecule compound able to inhibit the enzyme PFKFB3 for the treatment of a neurodegenerative disease.
  • a compound is a molecule with the formula (I) for the treatment of a neurodegenerative disease:
  • X, X 2 , and X 3 are each C or CH;
  • X ! is selected from the group consisting of O, S, NR ⁇ C(R 2 )2, OR 3 , SR 4 , NR 5 R 6 , and C(R 7 ) 3 , wherein R ⁇ R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of H, alkyl, aryl, aralkyl, and acyl, and each R 2 and R 7 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 , A 2 , A 3 , A 4 , and A 5 are each independently N or CR12, wherein each R 12 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:
  • X 4 is NR 14 , wherein R 14 is selected from the group consisting of H, alkyl, hydroxyl, aralkyl, and aryl;
  • X 5 is selected from the group consisting of O, S, C(Ri 5 ) 2 , and N R 14 , wherein each R 15 is independently selected from the group consisting of H, hydroxyl, alkoxy, alkyl, aralkyl, and aryl; and
  • X 6 is selected from H, alkyl, aralkyl, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;
  • R 10 and one R 12 are together alkylene
  • Ar 2 is selected from the group consisting of
  • each Yi , Y 2 , Y3, Y 4 , Y5, YQ, Y 7 , Ys, Y9, Y10, Y11 , Y12, Y13, Yi 4 , Y15, Y16, Y17, Y18, and Y 19 is independently selected from the group consisting of N and CR1 3 , wherein each R 13 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:
  • X 4 is N R 14 , wherein R 14 is selected from the group consisting of H, alkyi, hydroxyl, aralkyi, and aryl;
  • X 5 is selected from the group consisting of O, S, C(Ri 5 ) 2 , and N R 14 , wherein each R 15 is independently selected from the group consisting of H, hydroxyl, alkoxy, alkyi, aralkyi, and aryl; and
  • X 6 is selected from H, alkyi, aralkyi, aryl, heteroaryl, alkylamino, dialkylamino, and alkoxy;
  • R 10 and one R 13 are together alkylene
  • A! , A 2 , A 3 , A 4 , A 5 , Yi , Y 2 , Y 3 , Y 4 , Y 5 , Ye, Y 7 , Ye, Yg, Y10, Yi i , Yi2, Yi3, Yi 4 , Vis, Yie, Yi7, Y18, and Y 19 is N; or a pharmaceutically acceptable salt thereof.
  • a compound is according to the previous embodiment wherein X ! is O and X is C.
  • Ar 2 is:
  • X, X 2 , and X 3 are each C;
  • Xi is selected from the group consisting of O, S, NRi . and C(R 2 ) 2 , wherein R ⁇ . is selected from the group consisting of H and alkyi, and each R 2 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):
  • X, X 2 , and X 3 are each C;
  • Xi is selected from the group consisting of O, S, NRi , and C(R 2 )2, wherein is selected from the group consisting of H and alkyl, and each F3 ⁇ 4 is
  • X, X 2 , and X 3 are each C;
  • Xi is selected from the group consisting of O, S, N R ⁇ , and C(R 2 ) 2 , wherein is selected from the group consisting of H and alkyl, and each R 2 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):
  • a compound according to Formula (IV) is:
  • 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.
  • 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.
  • siRNA with a specificity for PFKFB3 is expressed by an expression construct incorporated into a viral vector.
  • siRNA with a specificity for PFKFB3 is expressed by an expression construct incorporated into an adenoviral-2 associated (AAV-2) vector.
  • neurodegenerative disease 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.
  • alkyl refers to Ci_ 2 o 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.
  • alkyl refers, in particular, to Ci_ 8 straight- chain alkyls.
  • alkyl refers, in particular, to Ci_ 8 branched-chain alkyls.
  • 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.
  • 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.
  • 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.
  • aryl group substituent includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, a
  • 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.
  • 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.
  • 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.
  • 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).
  • RCO— substituent
  • 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.
  • alkyl 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.
  • alkoxyl as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, f-butoxyl, and pentoxyl.
  • 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.
  • 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.
  • Alkoxycarbonyl refers to an aralkyl-O-CO- group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an H 2 N-CO- group.
  • Alkylcarbamoyl refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R 1 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.
  • amino refers to the -NH 2 group.
  • halo refers to fluoro, chloro, bromo, and iodo groups.
  • hydroxyl refers to the -OH group.
  • hydroxyalkyl refers to an alkyl group substituted with an -OH group.
  • mercapto refers to the -SH group.
  • oxo refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.
  • aza refers to a compound wherein a carbon atom is replaced by a nitrogen atom.
  • nitro refers to the -N0 2 group.
  • thio refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • R groups such as groups and R 2 , or groups X and Y
  • R groups can be identical or different.
  • R 2 can be substituted alkyls, or can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • compositions containing one or more compounds of the present invention 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.
  • the administration is intrethecally.
  • the administration is intracerebroventricularly.
  • 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.
  • 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.
  • 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 star
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions may be in the form of sterile injectable aqueous suspensions.
  • 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
  • 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.
  • sterile fixed oils are conventionally employed as solvents or suspending media.
  • any bland, fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can be used in the preparation of injectables.
  • 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.
  • 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.
  • 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.
  • transdermal delivery devices 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.
  • a drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier.
  • implantable delivery system used for the transport of agents to specific anatomical regions of the body, is described in US 5,011 ,472.
  • 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
  • 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:
  • 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.
  • the compounds of this invention can be combined with known medicines for neurodegenerative diseases.
  • 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.
  • the 'molecules' may be administered by a method close to the place of onset.
  • a continuous infusion is used and includes the continuous subcutaneous delivery via an osmotic minipump.
  • said close to the onset administration is an intrathecal administration.
  • said close to the onset administration is an intracerebroventricular administration.
  • 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.
  • 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.
  • "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 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.
  • 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
  • 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.
  • 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.
  • 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 ⁇ 10 12 DNAse-resistant particles per milliliter (Zen Z. et al (2004) Hum. Gene Ther. 15: 709- 715).
  • 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 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.
  • 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.
  • PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
  • 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.
  • AAV-2 a recombinant adeno-associated virus
  • 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).
  • 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.
  • 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.

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Abstract

La présente invention concerne l'utilisation d'inhibiteurs qui réduisent le flux glycolytique pour le traitement de maladies neurodégénératives telles que la maladie d'Alzheimer, la maladie de Parkinson et la sclérose latérale amyotrophique. La présente invention concerne en particulier des constructions comprenant des ARNsi dirigés contre PHD1 pour le traitement de la SLA et des ARNsi dirigés contre PFKFB3 pour le traitement de maladies neurodégénératives. L'invention concerne en outre l'utilisation d'une quantité thérapeutiquement efficace d'aza-chalcones ou d'un sel pharmaceutiquement acceptable de celles-ci pour le traitement de maladies neurologiques.
PCT/EP2012/053646 2011-03-04 2012-03-02 Moyen et procédés de traitement de troubles neurodégénératifs WO2012119949A1 (fr)

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