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WO2009120700A2 - Inhibition de dcps - Google Patents

Inhibition de dcps Download PDF

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Publication number
WO2009120700A2
WO2009120700A2 PCT/US2009/038108 US2009038108W WO2009120700A2 WO 2009120700 A2 WO2009120700 A2 WO 2009120700A2 US 2009038108 W US2009038108 W US 2009038108W WO 2009120700 A2 WO2009120700 A2 WO 2009120700A2
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WO
WIPO (PCT)
Prior art keywords
sma
dcps
smn
cpd
compound
Prior art date
Application number
PCT/US2009/038108
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English (en)
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WO2009120700A3 (fr
Inventor
Jill Jarecki
Jasbir Singh
Mark Gurney
Brian Pollok
Gregory Michaud
Shin-Wu Liu
Megerditch Kiledjian
Matthew Butchbach
Arthur Burghes
Original Assignee
Families Of Spinal Muscular Atrophy
Rutgers, The State University Of New Jersey
The Ohio State University
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Application filed by Families Of Spinal Muscular Atrophy, Rutgers, The State University Of New Jersey, The Ohio State University filed Critical Families Of Spinal Muscular Atrophy
Publication of WO2009120700A2 publication Critical patent/WO2009120700A2/fr
Publication of WO2009120700A3 publication Critical patent/WO2009120700A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine

Definitions

  • the technology described herein generally relates to enhancing SMN expression by inhibiting DcpS.
  • the technology more particularly relates to methods and compositions for increasing expression of SMN, and treating or preventing an SMA condition in a subject.
  • SMA Spinal muscular atrophy
  • SSN full-length survival motor neuron
  • Type I (Acute) SMA is also called Werdnig-Hoffmann Disease. SMA type I is evident before birth or within the first few months of life. There may be a reduction in fetal movement in the final months of pregnancy. There is a general weakness in the intercostals and accessory respiratory muscles. The chest may appear concave. Symptoms include floppiness of the limbs and trunk, feeble movements of the arms and legs, swallowing and feeding difficulties, and impaired breathing. Affected children never sit or stand and usually die before the age of 2.
  • Type II (Chronic) SMA is usually diagnosed by 15 months. Children may have respiratory problems, floppy limbs, decreased or absent deep tendon reflexes, and twitching of arm, leg, or tongue muscles.
  • Type III (Mild) SMA often referred to as Kugelberg-Welander or Juvenile Spinal Muscular Atrophy, is usually diagnosed between 2 and 17 years of age. Symptoms include abnormal manner of walking; difficulty running, climbing steps, or rising from a chair; and slight tremor of the fingers. The patient with Type III can stand alone and walk; tongue fasciculations are seldom seen. Types I, II and III progress over time, accompanied by deterioration of the patient's condition.
  • Type I, II and III SMA are caused by a mutation in a part of the DNA called the Survival of Motor Neuron (SMNl) gene, which normally produces a protein called SMN. Because of their gene mutation, people with SMA make less SMN protein, which results in the loss of motor neurons. SMA symptoms may be improved by increasing the levels of SMN protein. Normally the SMNl gene provides instructions for making a protein called Survival of Motor Neuron 1. The SMNl protein helps to assemble the cellular machinery needed to process pre-mRNA. More than 90 percent of individuals with spinal muscular atrophy lack part or all of both copies of the SMNl gene. A small percentage of people with this condition lack one copy of the SMNl gene and have a point mutation in the remaining copy.
  • SMA Survival of Motor Neuron
  • SMN2 gene copy number can help replace the protein needed for the survival of motor neurons.
  • symptoms are less severe and begin later in life in affected individuals with more copies of the SMN2 gene.
  • the SMNl and SMN2 genes provide instructions for making a protein called Survival of Motor Neuron.
  • the SMN2 gene makes full-length, functional SMN protein in much lower amounts than the SMNl gene. This protein is made in four different versions by both the SMNl and SMN2 genes. Only isoform d is full size and functional.
  • SMN2 gene Due to a single point mutation at the beginning of exon 7 in the SMN2 gene, the full length form is made at a much higher proportion in the SMNl gene versus the SMN2 gene.
  • the other isoforms (a, b, and c) are smaller and are not fully functional when made from either gene.
  • the SMN2 gene primarily makes the form of the protein missing exon 7 due to the single point mutations. SMN protein missing exon 7 cannot oligomerize, which is required for function, and is very unstable.
  • additional copies of the SMN2 gene can modify the course of the disorder.
  • the extra SMN2 genes can help replace the protein needed for the survival of motor neurons.
  • Spinal muscular atrophy still occurs, however, because most of the proteins produced by SMN2 genes are isoforms a, b, and c, which are smaller than the SMNl protein and cannot fully compensate for the loss of SMNl genes.
  • the present disclosure is based, at least in part, on methods and compositions for inhibiting the mRNA decapping protein DcpS (a scavenger pyrophosphatase enzyme that hydrolyzes the residual cap structure following mRNA degradation in the 3' to 5' exoribonuclease pathway and the m 7 GDP product of the Dcp2 decapping enzyme) and thereby enhancing the expression of SMN in a cell.
  • DcpS a scavenger pyrophosphatase enzyme that hydrolyzes the residual cap structure following mRNA degradation in the 3' to 5' exoribonuclease pathway and the m 7 GDP product of the Dcp2 decapping enzyme
  • compositions e.g., compounds, pharmaceutical compositions containing the compositions, and articles of manufacture
  • methods and compositions useful for increasing expression of SMN in a cell (e.g., in vitro or in vivo) as well as methods and compositions for use in treating, or preventing, or ameliorating one or more symptoms of an SMA condition in a subject.
  • a method for increasing expression of SMN protein in a cell comprising contacting a cell with a DcpS inhibitor in an amount effective to increase the expression of SMN protein in the cell.
  • the DcpS inhibitor can be a compound having the following structural formula:
  • R and R are each independently H or lower alkyl; n is 0, 1, 2, or 3;
  • X and Y are each independently CH or N, so that the ring that contains X and Y is cyclohexane, piperidine, or piperazine;
  • R 10 , R 11 , R 12 and R 13 are independently H or alkyl;
  • a method for treating an SMA condition in a subject comprising administering to the subject a compound that inhibits DcpS.
  • the compound can have the following structural formula:
  • R 2 and R 3 are each independently H or lower alkyl; n is 0, 1, 2, or 3;
  • X and Y are each independently CH or N, so that the ring that contains X and Y is cyclohexane, piperidine, or piperazine;
  • R 10 , R 11 , R 12 and R 13 are independently H or alkyl;
  • FIG. IA is a schematic diagram depicting the structure of an exemplary C5- substituted quinazoline compound (Cpd. 1 ) shown with a 2-fluoro-benzyl piperidine substituent. Atoms of the quinazoline numbered. The structure of Cpd. 1 is depicted in Table 1, herein.
  • FIG. IB is a line graph depicting the ability of Cpd. 1 to induce ⁇ -lactamase expression from an SMN promoter-driven ⁇ -lactamase reporter construct in NSC-34 cells (mean + SD, triplicate wells).
  • the Y-axis represents the "fold" increase over control of ⁇ -lactamase activity and the X-axis represents the log concentration of Cpd.l ( ⁇ M).
  • the EC 50 was 9.1 nM.
  • FIG. 1C is a bar graph depicting the ability of Cpd. 1 to induce SMN mRNA expression from an endogenous SMN gene in NSC-34 cells.
  • the Y-axis represents the "fold” increase over control of SMN mRNA expression and the X-axis represents the concentration of Cpd.l (50 or 500 nM), trichostatin A (TSA; 100 nM), or a DMSO control.
  • FIG. ID is a bar graph depicting diminution of DcpS levels, which led to an increase in SMN2 that was not further accentuated by Cpd. 1 in human 293T cells.
  • SMN2 mRNA levels are presented relative to beta-actin mRNA levels.
  • FIG. 2 is a photograph of an immunoblot depicting the effect of three exemplary compounds (Cpd. 1 and Cpd. 12), or DMSO control, on SMN protein expression in human fibroblasts obtained from a type I SMA patient (3813).
  • 60 ⁇ g of cell lysate prepared from the treated cells (or from human fibroblasts obtained from an SMA carrier/SMN positive patient, 3814) were subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and SMN protein was detected using a mouse anti-SMN monoclonal antibody (MANSMA2 (8F7)).
  • FIG. 3 is a schematic diagram of a synthetic reaction to produce Cpd. 3.
  • a tin derivative of Cpd. 1 was synthesized (Cpd. 2) and 125 I sodium iodide/chloramine T was used to displace the SnBu 3 of Cpd. 2, yielding the desired radiolabeled C5-quinazoline (Cpd. 3).
  • FIG. 4 A is a pair of photographs of array images identifying DcpS as a potential Cpd. 3 target protein.
  • Arrays comprised of 5,000 immobilized human proteins were probed with I-Cpd. 3 in the presence and absence of unlabeled small molecules.
  • Specific displacement of I-Cpd. 3 at 100 nM concentration
  • Cpd. 3 and Cpd. 1 unlabeled at 10 ⁇ M concentration
  • I-Cpd. 3 at 100 nM concentration
  • control compounds staurosporine and tertbutylquinone was not observed.
  • 19S correspond to the positional mapping reagent I-streptavidin binding to a biotinylated control protein.
  • the lower left box within each enlarge box corresponds to the binding between the 125 I- labeled compound and DcpS protein on the array (in the presence or absence of unlabeled annotated small molecules).
  • FIG. 4 B is a bar graph depicting the quantitation of the array images depicted in FIG. 4 A.
  • the quantified competition binding data was normalized against signals arising from I-streptavidin binding to the biotinylated control protein. Standard deviations for the replicate assays are indicated.
  • FIG. 5A is a pair of photographs of autoradiograms depicting the effect of C5- substituted quinazolines on DcpS decapping activity in vitro.
  • Radiolabeled m7Gp*pppG was incubated with purified human DcpS (5 nM) in the presence of increasing concentrations of C5- quinazolines (Cpd. 1 or Cpd. 4; left photograph) or a positive control for DcpS inhibition (Cap structure) or negative vehicle (DMSO) control (right photograph).
  • “*" in the name of a radiolabeled compound denotes that the phosphate group ("p") preceding the "*” is detectably labeled (e.g., radioisotope labeled).
  • FIG. 5B is a line graph depicting the results of the experiment described in FIG. 5 A (numerical data for the relative decapping rate vs. concentration of compounds (nM)).
  • the Y- axis represents the % of DcpS decapping activity and the X-axis depicts the concentration of the compound used in the experiment (nM).
  • FIG. 5C is a line graph depicting the results of an experiment to determine the effect of Cpds. 1, 4, 5, 6, 7, and 8 on DcpS decapping activity (in vitro).
  • the Y-axis represents the % of DcpS decapping activity and the X-axis depicts the concentration of the compound used in the experiment (nM).
  • the structures of Cpds. 1, 4, 5, 6, 7, and 8 are depicted in Table 1.
  • FIG. 5D is a line graph depicting the linear correlation between SMN2 promoter activity and DcpS inhibition (e.g., as determined in FIGs. 1C and 5C).
  • the Y-axis represents the DcpS Assay IC 50 values (nM) and the X-axis represents the SMN2 promoter assay EC 50 values (nM).
  • FIG. 6A is a picture of a representative immunoblot showing SMN and ⁇ -actin protein expression in spinal cord extracts from SMN ⁇ 7 SMA mice treated with either Cpd. 1 or vehicle for 5 days.
  • FIG. 6B is a bar graph showing changes in SMN protein levels relative to ⁇ -actin protein in Cpd. 1- or vehicle-treated spinal cord extracts.
  • FIG. 7A is a Kaplan-Meier plot showing survival of SMN ⁇ 7 SMA mice receiving either vehicle or Cpd. 1 (3 mg/kg/d) beginning at either PND04 or PND09.
  • FIG. 7B is a Kaplan-Meier plot showing onset of body mass loss for SMN ⁇ 7 SMA mice receiving either vehicle or Cpd. 1 (3 mg/kg/d) beginning at either PND04 or PND09.
  • FIG. 7C is body mass curves for SMN ⁇ 7 SMA mice receiving either vehicle or cpd. 1 (3 mg/kg/d) beginning at PND04.
  • FIGS. 8A - 8E are bar graphs showing that oral administration of Cpd. 1 improved the motor phenotype of SMN ⁇ 7 SMA mice and reduced motor neuron loss in the lumbar spinal cord.
  • FIG. 9A is a line graph showing Cpd. 1 levels in prenatal mouse brains whose dams were dosed with Cpd. 1 (0-60 mg/kg/d) beginning at EDl 1.5.
  • FIG. 9B is a bar graph showing ⁇ -galactosidase activity — a marker for mSmn promoter activity — in the brains of mice whose dams received Cpd. 1 (0-60 mg/kg/d) beginning at EDl 1.5.
  • FIG. 1OA is a Kaplan-Meier plot showing survival of SMN ⁇ 7 SMA mice receiving either vehicle or Cpd. 1 (3 mg/kg/d) beginning EDl 1.5 and continuing after birth or ending at birth.
  • FIG. 1OB is a Kaplan- Meier plot showing onset of body mass loss for SMN ⁇ 7 SMA mice receiving either vehicle or Cpd. 1 beginning EDl 1.5 and continuing after birth or ending at birth.
  • FIG. 1OC is body mass curves for SMN ⁇ 7 SMA mice receiving either vehicle or Cpd. 1 (3 mg/kg/d) beginning at EDl 1.5.
  • FIG. 11 is a bar graph showing therapeutic window of opportunity for protective effects of SMN2 induction by Cpd.l.
  • the disclosure relates to methods and compositions (e.g., compounds and pharmaceutical compositions thereof) useful for increasing expression of SMN in a cell (e.g., in vitro or in vivo).
  • a deficiency in SMN can result in the development of an SMA condition in a subject
  • the methods and compositions described herein can also be used to treat, or prevent, an SMA condition in a subject. Exemplary methods and compositions are set forth below.
  • SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • SMA condition an SMA condition in a subject
  • R 2 and R 3 are each independently H, or lower alkyl
  • X and Y are each independently CH or N, so that the ring that contains X and Y is cyclohexane, piperidine, or piperazine;
  • R 14 and R 15 are each independently H, or lower alkyl; and R 1 is H, alkyl, alkoxy, carbocycle, substituted carbocycle, heteroaryl or substituted heteroaryl, heterocyclyl or substituted heterocyclyl, fluoroalkyl, and alkoxyalkyl.
  • Substituted carbocycles such as monsubstituted or 2,6-dihalo substituted phenyls, are preferred for Ri.
  • R 1 is not 3,4-dichlorophenyl.
  • R 1 is H or lower alkyl; in certain embodiments, R x -R 3 and R -R are each independently H or lower alkyl.
  • a compound useful for increasing expression of SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • a compound useful for increasing expression of SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • an SMA condition in a subject can have the formula (II),
  • R and R are each independently H, or lower alkyl
  • R 14 and R 15 are each independently H, or lower alkyl
  • R 1 is selected from the group consisting of: H; alkyl; alkoxy; carbocycle; heterocyclyl; substituted heterocyclyl; substituted carbocycle; fluoroalkyl and alkoxyalkyl.
  • a compound useful for increasing expression of SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • a compound useful for increasing expression of SMN can have the formula (III),
  • R and R are each independently H, or lower alkyl; and R is selected from the group consisting of H, alkyl, alkoxy, carbocycle, substituted carbocycle, heteroaryl or substituted heteroaryl, heterocyclyl, and substituted heterocyclyl, fluoroalkyl, and alkoxyalkyl.
  • R is not 3,4-dichlorophenyl.
  • a compound useful for increasing expression of SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • SMN e.g., SMN protein or an mRNA encoding an SMN protein
  • Exemplary compounds can have the following formulae:
  • R is selected from the group consisting of:
  • Alkyl includes linear, branched, or cyclic saturated hydrocarbon structures and combinations thereof (e.g., t-butyl-cyclohexyl).
  • Lower alkyl refers to alkyl groups having from 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl.
  • Preferred alkyl groups are those of having 20 carbon atoms or fewer.
  • Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups having single rings, as well as polycyclic hydrocarbons having two or more rings that share an edge or bridgeheads.
  • a polycyclic ring system is one in which two or more rings have two or more carbons in common.
  • Preferred cycloalkyl groups have single rings from 3 to 8 carbon atoms, and two rings with from 7 to 10 carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and the like.
  • polycyclic hydrocarbons include ring systems such as norbornyl, adamantyl, and decalin.
  • alkenyl or “alkenylene” includes linear, branched, or cyclic unsaturated hydrocarbon structures having the specified number of carbon atoms and one or more carbon- carbon double bonds at any point.
  • alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, and the like.
  • Cycloalkenyl is a subset of alkenyl and includes non-aromatic cyclic hydrocarbon groups having one or more double bonds in single rings, as well as polycyclic hydrocarbons having one or more double bonds and two or more rings that share an edge or bridgeheads.
  • Preferred cycloalkenyl groups have single rings from 3 to 8 carbon atoms, and two rings with from 7 to 10 carbon atoms. Examples of cycloalkenyls include cyclohexenyl, nobornenyl, and the like.
  • Alkynyl or “alkynylene” includes linear, branched, or cyclic unsaturated hydrocarbon structures having one or more carbon-carbon triple bonds at any point. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
  • Alkoxy or alkoxyl refers to an alkyl group attached to a parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to alkoxy groups containing one to six carbons.
  • Oxaalkyl refers to alkyl groups in which one or more carbon atoms together with its bonded hydrogens has been replaced by oxygen. Examples include methoxypropoxy, 3,6,9- trioxadecyl and the like.
  • Acyl refers to hydrocarbon structures, attached to a parent structure through a carbonyl functionality. One or more carbons in the acyl group may be independently replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers to acyl groups containing one to six carbons.
  • Aryl means a 5- or 6-membered aromatic ring, a bicyclic 9- or 10-membered aromatic ring system, or a tricyclic 12-, 13- or 14-membered aromatic ring system.
  • the aromatic 6- to 14- membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene.
  • Heteroaryl means a 5- or 6-membered heteroaromatic ring containing 0-3 heteroatoms independently selected from O, N, or S; a bicyclic 9- or 10-membered heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S; or a tricyclic 12-, 13- or 14-membered heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S.
  • the 5- to 10-membered aromatic heterocyclic rings include, e.g., pyrrole, imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, and pyrazole.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • Heterocycle means heteroaryl group, or a cycloalkyl group in which from one to three carbons is independently replaced by a heteroatom selected from the group consisting of N, O and S.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • heterocycles that fall within the scope of the definitions include (without limitation) pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like.
  • heterocyclyl residues additionally include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxo-pyrrolidinyl, 2-oxoazepinyl, azepinyl, 4- piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazol
  • Carbocycle is the complement of heterocycle.
  • Carbocycle as used herein means a cycloalkyl or aryl residue in which all of the ring atoms are carbon. It includes polycyclic rings. Examples include cyclohexane, benzene, cyclopentadiene, naphthalene, phenanthrene, fluorene, norbornane, bicycloheptadiene, indane, and bicyclooctane.
  • Substituted alkyl, substituted aryl, substituted cycloalkyl, substituted heterocyclyl, etc. refer respectively to alkyl, aryl, cycloalkyl, or heterocyclyl, etc.
  • each group is independently replaced with an atom or group selected from: halogen, haloalkyl, hydroxy, lower alkoxy, carboxylic acid, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), cyano, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, halobenzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, benzoyl, halobenzoyl, or loweralkylhydroxy.
  • up to three hydrogen atoms are replaced by substituents.
  • hydrocarbon includes alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, cycloalkynyl, aryl and combinations thereof. Examples include phenethyl, cyclohexylmethyl, camphoryl and naphthylethyl.
  • halogen means fluorine, chlorine, bromine or iodine.
  • substitution includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, automerization, dissociation, etc.
  • solvate refers to a compound of Formulae (I), (II), or (III) in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate.
  • solvates are formed by dissolving the compound in an appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • Co-crystals are combinations of two or more distinct molecules arranged to create a unique crystal form whose physical properties are different from those of its pure constituents.
  • Pharmaceutical co-crystals have recently become of considerable interest for improving the solubility, formulation and bioavailability of such drugs as itraconazole (see, e.g., Remenar et al. (2003) J. Am. Chem. Soc, 125:8456-8457) and fluoxetine.
  • Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19th Ed. (1995) volume 1, page 176-177, which is incorporated herein by reference in its entirety.
  • the most commonly employed inclusion complexes are those with cyclodextrins, and all cyclodextrin complexes, natural and synthetic, are specifically encompassed within the claims.
  • salts refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, benzenesulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like.
  • suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.
  • a DcpS inhibitor can be any compound capable of inhibiting the activity or the expression of a DcpS protein.
  • a DcpS inhibitor can be a compound that binds to an active site of a DcpS protein and inhibit the activity of the DcpS protein.
  • a DcpS inhibitor can be a compound that: (i) destabilizes a DcpS protein; (ii) destabilizes an mRNA encoding a DcpS protein; (iii) inhibits the transcription of an mRNA encoding a DcpS protein; or (iv) inhibits the translation of an mRNA encoding a DcpS protein.
  • the DcpS protein, or mRNA encoding the DcpS protein can be, e.g., a mammalian (e.g., a rodent, a non-human primate, or a human) form of the DcpS protein or mRNA.
  • a mammalian e.g., a rodent, a non-human primate, or a human
  • An exemplary amino acid sequence for a human form of a DcpS protein is as follows:
  • a subject can be, e.g., an animal such as an insect, a fish, a bird, a reptile, or a mammal (e.g., a rodent (e.g., a mouse, rat, hamster, or rabbit), cat, dog, goat, cow, goat, whale, non-human primate (e.g., a chimpanzee or macaque) or a human).
  • a rodent e.g., a mouse, rat, hamster, or rabbit
  • cat dog, goat, cow, goat, whale, non-human primate (e.g., a chimpanzee or macaque) or a human).
  • compositions provided herein contain therapeutically effective amounts of one or more of the compounds described herein that are useful in increasing the expression of SMN in a cell and/or in the prevention, treatment, or amelioration of one or more of the symptoms of Spinal Muscular Atrophy (SMA) condition.
  • SMA conditions include, without limitation, e.g., infantile SMA (Werdnig-Hoffmann disease); severe infantile SMA; intermediate SMA; juvenile SMA (Kugelberg-Welander disease); and adult SMA.
  • compositions suitable for administration of the compounds described above include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • the compositions contain one or more compounds described above.
  • the compounds are, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition, (1985), 126).
  • compositions effective concentrations of one or more compounds or pharmaceutically acceptable derivatives thereof is (are) mixed with a suitable pharmaceutical carrier.
  • the compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms of an SMA condition.
  • the compositions are formulated for single dosage administration.
  • the weight fraction of a compound is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.
  • the active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems such as those described herein and then extrapolated therefrom for dosages for humans.
  • the concentration of active compound in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the amount that is delivered is sufficient to ameliorate one or more of the symptoms of any SMA condition that is known in the art or described herein.
  • the active ingredient can be administered once, or can be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data (for example, test data collected from the murine models described in the accompanying Examples). It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated.
  • Derivatives of the compounds such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
  • the resulting mixture may be a solution, suspension, emulsion, or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple- dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art.
  • the contemplated compositions may contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, in another embodiment 75-85%.
  • compositions for oral administration are provided.
  • Oral pharmaceutical dosage forms are either solid, gel or liquid.
  • the solid dosage forms are tablets, capsules, granules, and bulk powders.
  • Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film- coated.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.
  • the formulations are solid dosage forms, in one embodiment, capsules or tablets.
  • the tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating.
  • binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.
  • Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.
  • Glidants include, but are not limited to, colloidal silicon dioxide.
  • Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.
  • Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
  • Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • Emetic- coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • the compound, or pharmaceutically acceptable derivative thereof could be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • the dosage unit form when it is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics.
  • the active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.
  • tablets and capsules formulations can be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in- water or water-in-oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.
  • Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
  • Solvents include glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol.
  • non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
  • emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
  • Organic acids include citric and tartaric acid.
  • Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.
  • Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
  • the solution or suspension in for example propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule.
  • a gelatin capsule Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the solution e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603.
  • such formulations include, but are not limited to, those containing a compound provided herein, a dialkylated mono- or poly- alkylene glycol, including, but not limited to, 1 ,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
  • BHT buty
  • compositions include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal.
  • Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol.
  • Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal. Injectables, solutions, and emulsions
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene- vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., poly
  • Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • aqueous vehicles include sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection.
  • Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p- hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. [0102] The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
  • the unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration.
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.
  • Injectables are designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).
  • the compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.
  • lyophilized powders which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.
  • the sterile, lyophilized powder is prepared by dissolving a compound provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents.
  • the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those skilled in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial will contain a single dosage or multiple dosages of the compound.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 0 C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the lyophilized powder is added to sterile water or other suitable carrier.
  • the precise amount depends upon the selected compound. Such amount can be empirically determined.
  • Topical mixtures are prepared as described for the local and systemic administration.
  • the resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
  • the compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma).
  • These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.
  • the compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • solutions particularly those intended for ophthalmic use, may be formulated as 0.01% - 10% isotonic solutions, pH about 5-7, with appropriate salts.
  • compositions for other routes of administration are provided.
  • Transdermal patches including iontophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983,
  • rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients.
  • Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used.
  • spermaceti and wax agents to raise the melting point of suppositories include spermaceti and wax.
  • Rectal suppositories may be prepared either by the compressed method or by molding.
  • the weight of a rectal suppository in one embodiment, is about 2 to 3 g.
  • Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
  • Targeted Formulations are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
  • the compounds provided herein, or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a particular tissue (e.g., a motor neuron), receptor, or other area of the body of the subject to be treated.
  • a particular tissue e.g., a motor neuron
  • receptor e.g., a motor neuron
  • All such targeting methods are contemplated herein for use in the instant compositions.
  • For non-limiting examples of targeting methods see, e.g., U.S. Patent Nos.
  • liposomal suspensions including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.
  • tissue-targeted liposomes such as tumor-targeted liposomes
  • liposome formulations may be prepared according to methods known to those skilled in the art.
  • liposome formulations may be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLVs) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask.
  • MLVs multilamellar vesicles
  • a solution of a compound provided herein in phosphate buffered saline lacking divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed.
  • PBS phosphate buffered saline lacking divalent cations
  • Methods for increasing expression of SMN in a cell can include the step of contacting a cell with (or introducing into a cell) a DcpS inhibitor (such as a compound described herein) in an amount effective to increase the expression of SMN in the cell.
  • a DcpS inhibitor such as a compound described herein
  • Such contacting can occur in vitro (e.g., in cell culture) or in vivo.
  • Suitable in vivo methods for contacting a cell with a DcpS inhibitor include, e.g., any of the methods of treatment described below under "Methods for Treating an SMA Condition.”
  • Suitable in vitro methods for contacting a cell with a DcpS inhibitor include, e.g., plating a cell (or a population of cells) on solid support matrix (e.g., a plastic tissue culture plate, or a multiwell (96 or 386-well) tissue culture plate) and culturing the cell at a physiological temperature (e.g., 37°C) in an aqueous buffered medium (e.g., a tissue culture medium such as Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) medium) containing the DcpS inhibitor.
  • a tissue culture medium such as Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) medium
  • the cell can be transfected with a nucleic acid encoding the protein, siRNA, or antisense RNA and the cell is then cultured under conditions that permit the expression of the protein, siRNA, or antisense RNA.
  • the transfection step can be accomplished by any standard means used for such nucleic acid delivery including, e.g., calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. (See, e.g., Sambrook et al. (2001) Molecular Cloning, a Lab Manual, 3rd Edition). Alternatively, liposomes or polymeric microparticles can be used. It is understood that the delivery method will depend, at least in part, on the type of cell being transfected.
  • the cell can be any cell that expresses, or is capable of expressing, SMN protein or an mRNA encoding an SMN protein.
  • the cell can endogenously express (or be capable of endogenously expressing) SMN, or the cell can exogenously express (or be capable of exogenously expressing) SMN.
  • the cell can contain an endogenous nucleic acid (or gene) that encodes an SMN protein (that is, the cell can naturally express SMN) or the cell can contain a trans gene (e.g., an expression vector) encoding a recombinant SMN.
  • the cell can be a prokaryotic or eukaryotic cell.
  • the cell can be a bacterial cell, a fungal cell (e.g., a yeast cell), a plant cell, or an animal cell (e.g., a cell from a fish, a reptile, or a mammal (e.g., a mouse, rat, guinea pig, dog, cat, pig, horse, goat, cow, a non- human primate (e.g., a chimpanzee or a macaque) or a human)).
  • a mammal e.g., a mouse, rat, guinea pig, dog, cat, pig, horse, goat, cow, a non- human primate (e.g., a chimpanzee or a macaque) or a human
  • the cell can be of any of a variety of histological types including, without limitation, an epithelial cell, a lymphoid cell, a macrophage, a monocyte, a dendritic cell, a motor neuron, a keratinocyte, or a muscle cell.
  • the cell can also be obtained from any of a diverse group of tissues such as, but not limited to, lung, breast, colon, pancreas, kidney, stomach, liver, bone, blood, brain, skin, thyroid, ovary, testes, cervix, vagina, or bladder.
  • an increased expression of SMN is an increase in the amount or stability of SMN protein or an increase in the amount or stability of an mRNA encoding an SMN protein. It is understood that an increase in the amount or stability of an mRNA encoding a SMN protein can result in an increase in the amount of SMN protein.
  • a DcpS inhibitor can increase the expression of SMN protein by increasing the stability of a capped mRNA encoding the SMN protein.
  • the methods can include the steps of detecting the presence or amount of SMN protein or mRNA in the cell and/or determining whether an increase in SMN expression has occurred in the cell.
  • Numerous methods can be used for detecting SMN expression in a cell. Such methods depend on, e.g., whether mRNA or protein expression is being detected.
  • one suitable method for detecting SMN protein expression in a cell is a solid-phase immunoassay (e.g., a "sandwich" type immunoassay), wherein a first anti-SMN antibody is adhered to a solid-phase matrix (e.g., sepharose, agarose, magnetic beads, or a multi- well assay plate).
  • a SMN protein-containing sample e.g., a cell lysate containing or suspected of containing SMN protein
  • a SMN protein-containing sample is then added to the antibody-coupled matrix and incubated for a time sufficient to allow binding of the SMN protein (if present) to the immobilized anti-SMN antibody. Unbound proteins, if any, are removed in subsequent wash steps.
  • SMN proteins, if bound by the immobilized anti-SMN antibody can then be detected using a second anti-SMN antibody.
  • the second antibody can optionally have a different epitope specificity than the first antibody.
  • the second anti-SMN antibody can be directly coupled to a detection moiety.
  • Detection moieties include, for example, fluorescent labels (e.g., cy5, cy3, green fluorescent protein, or fluorescein). Detection moieties can also be radioisotope labels, such as 35 S, 32 P, 33 P, 3 H, or 125 I. Detection labels can also be enzymes, e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), luciferase, or chloramphenicol acetyl transferase (CAT). Alternatively, it is often useful that the detection moiety be coupled to a secondary antibody that specifically recognizes the first, detection antibody (for example, to amplify the assay signal strength).
  • fluorescent labels e.g., cy5, cy3, green fluorescent protein, or fluorescein
  • Detection moieties can also be radioisotope labels, such as 35 S, 32 P, 33 P, 3 H, or 125 I. Detection labels can also
  • the detection antibody or secondary antibody can be conjugated to a first member of a binding pair (e.g., biotin or streptavidin) and the detection moiety can be linked to a second member of a binding pair (e.g., streptavidin or biotin).
  • a binding pair e.g., biotin or streptavidin
  • SMN protein can be detected by western blotting using anti-SMN antibodies described herein. Western blotting methods are described in, for example, Sambrook et al. (supra).
  • a sample containing SMN protein can be suspended in a denaturing buffer (e.g., Laemmli's buffer) containing both detergent (e.g., sodium dodecyl sulfate) and a reducing agent (e.g., DTT or beta-mercaptoethanol).
  • a denaturing buffer e.g., Laemmli's buffer
  • detergent e.g., sodium dodecyl sulfate
  • a reducing agent e.g., DTT or beta-mercaptoethanol
  • PAGE SDS-polyacrylamide gel electrophoresis
  • PAGE resolved proteins, separated by size can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to western blot techniques using antibodies specific to SMN.
  • the level of SMN in a sample can be determined by comparison to a control or reference sample containing a known amount of SMN.
  • the western technique sometimes called a dot-blot
  • Methods for detecting the expression of an mRNA encoding SMN protein include, e.g., northern blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Such methods are well known in the art and described in detail in Sambrook et al. (supra).
  • Methods of assessing the level of a SMN protein or SMN mRNA in a sample can be quantitative, semi-quantitative, or qualitative.
  • the level of SMN in a sample can be determined as a discrete value.
  • the level of SMN can be measured as a numerical value by correlating the detection signal derived from the quantitative assay to the detection signal of a known concentration of SMN protein or the signal presence of SMN protein in a reference sample provided from a second cell.
  • the level of SMN protein can be assessed using any of a variety of semi-quantitative/qualitative systems.
  • the level of expression of SMN protein useful in the immunoassay in a sample can be expressed as, for example, (a) one or more of “very high,” “high,” “average,” “low,” and /or “very low” or (b) one or more of "++++,” “+++,” “++,” “+,” “+/-,” and/or “-.”
  • the level of SMN protein in a cell can be expressed relative to the SMN protein levels in a reference sample (e.g., from a reference cell).
  • the DcpS inhibitor can be a compound selected from a variety of chemical classes, so long as the compound possesses the appropriate activity.
  • Compounds can be biomolecules including, but not limited to, peptides, polypeptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs (e.g., non-naturally-occurring amino acids), saccharides, fatty acids, steroids, purines, pyrimidines, derivatives or structural analogues thereof, polynucleotides, and polynucleotide analogs (e.g., non-naturally-occurring polynucleotides).
  • Compounds can be both small or large molecule compounds.
  • small molecule compounds are relatively small organic molecules having a molecular weight in the range of about 50 to 2,500 Daltons.
  • These compounds can comprise functional groups necessary for structural interaction with proteins (e.g., hydrogen bonding), and can include at least an amine, carbonyl, hydroxyl, or carboxyl group, and preferably at least two of the functional chemical groups.
  • These compounds can often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures (e.g., purine core) substituted with one or more of the above functional groups.
  • Exemplary DcpS inhibitors include compounds containing, or having, the structure of any one of Formulas (I), (II), or (III). Exemplary DcpS inhibitors also include compounds that contain, or have, the structure of one of any of the compounds depicted in Table 1. Compounds can also be any of those described under the section herein titled “Exemplary Compounds and Pharmaceutical Compositions Thereof.”
  • nucleic acid aptamers are relatively short nucleic acid (DNA, RNA or a combination of both) sequences that bind with high avidity to a variety of proteins and inhibit the binding to such proteins of ligands, receptors, and other molecules.
  • Aptamers are generally about 25 - 40 nucleotides in length and have molecular weights in the range of about 6 - 18 kDa.
  • Aptamers with high specificity and affinity for targets can be obtained by an in vitro evolutionary process termed SELEX (systemic evolution of ligands by exponential enrichment) (see, for example, Zhang et al., Arch. Immunol. Ther.
  • nucleic acid aptamers see Zhang, et al. (2004) and Brody et al. (Reviews in Molecular Biotechnology, (2000) 74:5-13, the disclosure of which is incorporated herein by reference in its entirety).
  • Molecules that are targeted to an mRNA encoding the DcpS protein are useful for the methods described herein, e.g., enhancing the expression of a DcpS protein, e.g., for treating an SMA condition.
  • nucleic acids include siRNAs.
  • Other such molecules that function using the mechanisms associated with RNAi can also be used, including chemically modified siRNAs and vector driven expression of hairpin RNA that are then cleaved to siRNA.
  • nucleic acid molecules or constructs that are useful as described herein include dsRNA (e.g., siRNA) molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is complementary to the first strand.
  • dsRNA e.g., siRNA
  • 16-30 e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand
  • one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical,
  • the dsRNA molecules can be chemically synthesized, can transcribed be in vitro from a DNA template, or can be transcribed in vivo from, e.g., shRNA.
  • the dsRNA molecules can be designed using methods known in the art, e.g., Dharmacon.com (see, siDESIGN CENTER) or "The siRNA User Guide,” available on the Internet at mpibpc.gwdg.de/ e- n/100/105/sirna.html.
  • Negative control siRNAs generally have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome.
  • Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. Controls can also be designed by introducing an appropriate number of base mismatches into the selected siRNA sequence.
  • a pool of siRNA' s is used to modulate the expression of a target gene.
  • the pool is composed of at least 2 (e.g., 3, 4, 5, 8, or 10) different sequences targeted to the target gene.
  • Antisense nucleic acids are also useful for inhibiting the expression of a DcpS protein by decreasing the stability of an mRNA encoding the DcpS protein.
  • Such antisense nucleic acid molecules i.e., nucleic acid molecules whose nucleotide sequence is complementary to all or part of an mRNA encoding a DcpS protein.
  • An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding an inhibitor protein.
  • the non-coding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences that flank the coding region and are not translated into amino acids.
  • a "gene walk" comprising a series of oligonucleotides of 15-30 nucleotides spanning the length of a nucleic acid (e.g., a target nucleic acid) can be prepared, followed by testing for inhibition of expression of the gene.
  • gaps of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides or more in length.
  • An antisense nucleic acid described herein can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • An antisense nucleic acid molecule can be an alpha-anomeric nucleic acid molecule.
  • An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids Res., 15:6625-6641, (1987)).
  • the antisense nucleic acid molecule can also comprise a 2 -o-methylribonucleotide (Inoue et al., Nucleic Acids Res., 15:6131-6148, 1987) or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett., 215:327-330, 1987).
  • Large molecule compounds can include large proteins (e.g., a dominant negative form of a DcpS protein) or macromolecular complexes comprising two or more proteins.
  • Large molecule compounds, particularly those that are composed of more than one polypeptide, can be cov alently joined or non-covalently joined, e.g., by hydrogen bonding, Van der Waals forces, or hydrophobic interactions.
  • DcpS inhibitors can be identified from a number of potential sources, including: chemical libraries, natural product libraries, and combinatorial libraries comprised of systematically randomized peptides, oligonucleotides, or organic molecules.
  • Chemical libraries can consist of random chemical structures, some of which are analogs of known compounds or analogs or compounds that have been identified as "hits” or “leads” in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry.
  • Natural product libraries are collections of compounds from microorganisms, animals, plants, or marine organisms, which can be obtained by, e.g.: (1) fermentation and extraction of broths from soil, plant, or marine microorganisms, or (2) extraction of plants or marine organisms.
  • Natural product libraries include polypeptides, non- ribosomal peptides, and variants (non-naturally occurring) thereof.
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-pep tide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
  • Suitable methods for determining whether a compound is a DcpS inhibitor include in vitro DcpS decapping assays described in the accompanying Examples. A variety of compounds such as Cpd. 1 (herein) can be used as positive controls in such assays.
  • a condition e.g., an SMA condition
  • methods for treating a condition can include the step of administering to a subject a compound that inhibits DcpS (e.g., any of the compounds or compositions described herein).
  • a compound that inhibits DcpS e.g., any of the compounds or compositions described herein.
  • cells contacted with DcpS inhibitors exhibited an increased expression of SMN mRNA and SMN protein.
  • the methods described herein can be used for treating any condition (e.g., a genetic disorder) that results from, or whose symptoms or progression are exacerbated by, an insufficient expression of a gene product, the steady-state mRNA level of the gene product being regulated by DcpS.
  • the gene product can be, e.g., the SMN protein.
  • the condition can be one that is characterized (and/or whose symptoms or progression is exacerbated) by the absence, or low levels, of expression of SMN protein in a cell (or in many cells in a multicellular organism) such as an SMA condition (e.g., Proximal SMA; see further herein), Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease, see, e.g., Biochem. Biophys. Res. Commun., (2007) Dec. 28; 364(4): 850-5.; Neurology 2006 Oct 10;67(7):l 147-50, Neurology 2005 Sep 27;65(6):820-5), or other motor neuron diseases.
  • effective amounts of any of the compounds or compositions described herein having the appropriate activity are administered. Such amounts are sufficient to achieve a therapeutically effective concentration of the compound or active component of the composition in vivo.
  • Proximal SMA condition is a term applied to a number of different disorders, all having mutations in the SMNl gene (resulting in a reduction in SMN protein expression) and the manifestation of muscle weakness and atrophy due to loss of the motor neurons of the spinal cord and brainstem.
  • Proximal SMA conditions can include, without limitation, any of the SMA conditions known in the art such as, but not limited to infantile SMA (Werdnig-Hoffmann disease); severe infantile SMA; intermediate SMA; juvenile SMA (Kugelberg-Welander disease); and adult SMA. In some embodiments, the Proximal SMA condition is not adult SMA.
  • the compound can be administered to a pregnant mother carrying a child having, suspected of having, or at risk of developing, a condition that is characterized by the absence, or low levels, of expression of SMN in a cell (or in many cells in a multicellular organism) such an any of the SMA conditions described herein.
  • a subject "suspected of having an SMA condition” is one who exhibits one or more symptoms of an SMA condition.
  • Symptoms of an SMA condition can include one or more of muscle weakness; poor muscle tone; weak cry; limpness or a tendency to flop; difficulty sucking or swallowing; accumulation of secretions in the lungs or throat; tongue fasciculation; legs that tend to be weaker than the arms; hypotonia; areflexia; multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells; feeding difficulties; increased susceptibility to respiratory tract infections; or failure to reach developmental milestones, such as lifting the head or sitting up.
  • Symptoms of an SMA condition can appear at any stage of development; however, In general, the earlier the symptoms appear, the shorter the life span for the subject. The onset is often sudden and dramatic.
  • a subject "at risk of developing" an SMA condition is one with a genetic predisposition for developing an SMA condition.
  • mutations in the SMN gene e.g., a complete loss of function mutation or deletion in both copies of the SMN gene.
  • a subject having a mutation in the SMN gene can be at risk of developing an SMA condition.
  • the methods can include the step of determining whether a subject has an SMA condition.
  • Methods for determining whether a subject has an SMA condition include any of a number of qualitative and quantitative tests.
  • a medical practitioner e.g., a doctor or nurse
  • a medical practitioner can include genetic tests to determine whether one or both copies of the SMN gene in a subject are present or mutated.
  • tests such as an electromyography (EMG) test or muscle biopsy can be performed as a diagnostic test.
  • EMG electromyography
  • a compound useful for treating, preventing, or ameliorating one or more symptoms of an SMA condition can be administered to a subject, e.g., a human subject, by a variety of methods.
  • the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection.
  • IV intravenous injection or infusion
  • SC subcutaneous injection
  • IP intraperitoneally
  • intramuscular injection e.g., intrathecal, intracerebroventricular (ICV), intracerebral, or intracranial.
  • the compound can be administered as a fixed dose, or in a mg/kg dose.
  • administration can be oral (e.g., administered by inhalation), transdermal (topical), transmucosal, or rectal. (See, e.g., the section herein entitled "Pharmaceutical Compositions").
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the dose can also be chosen to reduce or avoid production of antibodies against the compound.
  • the route and/or mode of administration of the agent can also be tailored for the individual case.
  • Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or, when administered in combination with another agent, a combinatorial therapeutic effect.
  • the dosage regimen can, for example, be capable of treating, preventing, or ameliorating one or more symptoms of an SMA condition.
  • the dose of a compound can be, optionally, formulated separately or together with an appropriate dose of a second therapeutic agent can be used to provide a subject with the agent.
  • Suitable dosages and/or dose ranges for the compound include an amount sufficient to treat, prevent, or ameliorate one or more symptoms of an SMA condition.
  • Such dosages can include, e.g., about 0.001 ⁇ g/kg to 10,000 ⁇ g/kg body weight of the subject, per dose.
  • the dosage can be about 1 ⁇ g/kg to 100 ⁇ g/kg body weight of the subject, per dose.
  • the dosage can be about 1 ⁇ g/kg to 30 ⁇ g/kg body weight of the subject, per dose, e.g., from 3 ⁇ g/kg to 10 ⁇ g/kg body weight of the subject, per dose.
  • a dose of a compound required to treat, prevent, or ameliorate one or more symptoms of an SMA condition can depend on a variety of factors including, for example, the age, sex, and weight of a subject to be treated. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the subject's SMA condition. For example, a patient with advanced form of an SMA condition can require a administration of a different dosage of a compound than a patient with a milder form of an SMA condition.
  • Other factors can include, e.g., other disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will depend upon the judgment of the treating medical practitioner. The amount of any active ingredients will also depend upon the particular described compound and the presence or absence and the nature of the additional therapeutic agents in a composition comprising the compound.
  • the dose of a compound that inhibits DcpS can be determined by the efficacy of the particular compound employed and the condition of the subject, as well as the gender or body weight of the subject to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound in a particular subject.
  • the physician can evaluate factors such as the circulating plasma levels of the compound, compound toxicities, and/or the progression of the disease, etc.
  • Toxicity and therapeutic efficacy of such compounds can be determined by known pharmaceutical procedures in cell cultures or experimental animal models (e.g., animal models of an SMA condition; see, e.g., Monani et al. (2000) Human Molecular Genetics 9:2451-2457 or the working Examples). These procedures can be used, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 . Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies generally within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography. Methods for determining an IC 50 for a DcpS inhibitor in cell culture are detailed in the accompanying Examples.
  • Dosage unit form or "fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect (e.g., treating, preventing, or ameliorating one or more symptoms of an SMA condition) in association with the required pharmaceutical carrier and optionally in association with the other agent. Suitable administration frequencies are described elsewhere herein.
  • the efficacy of the treatment in ameliorating one or more symptoms of an SMA condition can be assessed by comparing the number and/or severity of one or more symptoms presented by a patient before and after treatment.
  • treatment efficacy can be assessed as a delay in presentation of, or a failure to present, one or more symptoms of an SMA condition.
  • the efficacy of a treatment (e.g., a compound or composition described herein) over time can be determined by assessing, e.g., the number or severity of one or more symptoms at multiple time points following treatment.
  • a subject e.g., a patient
  • the effect of treatment on ameliorating one or more symptoms of an SMA condition can be assessed at various time points after the final treatment. For example, following the last administration of a dose of one or more compounds, the number or severity of a patient's symptoms can be assessed at 1 month (e.g., at 2 months, at 6 months, at one year, at two years, at 5 years or more) subsequent to the final treatment.
  • the efficacy of a treatment with one or more compounds (or compositions) described herein on one or more symptoms of an SMA condition can be assessed as a monotherapy or as part of a multi-therapeutic regimen.
  • the compound(s) can be administered in conjunction with other clinically relevant treatments for an SMA condition including, but not limited to, physical or respiratory therapy, a decongestant, butyrates, valproic acid, hydroxyurea (HU), histone deacetylase (HDAC) inhibitors, methylase inhibitors, and/or Rilutek® (riluzole).
  • HDAC inhibitors include, but are not limited to, valproic acid, hydroxybutyrate, phenylbutyrate, phenylbutyrate derivatives, butyrate prodrugs, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), AR-42 (OSU-HD AC42; Arno Therapeutics, Fairfield, NJ), and those described in, e.g., Herman et al., Nat Chem Biol, (2006 Oct.); 2(10):551-8), the disclosure of which is incorporated herein by reference.
  • An exemplary methylase inhibitor is 5- azacytidine.
  • Additional clinically relevant agents for an SMA condition include, without limitation: polyphenol botanical compounds (see, e.g., Hum Genet. (2008) 122(6):635-43); PPl inhibitors (Hum MoI Genet. (2008) 17(l):52-70); tetracycline derivatives (e.g., alcarubicin; see Hum MoI Genet. (2001) 10(24):2841); aminoglycosides (see, e.g., Hum Genet. (2006) 120(4):589-601); 5-(N-ethyl-N-isopropyl)-amiloride (see, e.g., Ann Neurol.
  • polyphenol botanical compounds see, e.g., Hum Genet. (2008) 122(6):635-43
  • PPl inhibitors Hum MoI Genet. (2008) 17(l):52-70
  • tetracycline derivatives e.g., alcarubicin; see Hum MoI Genet. (2001) 10(24):2841
  • a "therapeutically effective amount" of a DcpS inhibitor is an amount of the DcpS inhibitor that is capable of producing a medically desirable result (e.g., amelioration of one or more symptoms of an SMA condition and/or increasing the expression of SMN in a cell) in a treated subject.
  • a therapeutically effective amount of a DcpS inhibitor i.e., an effective dosage
  • a compound or pharmaceutical composition thereof described herein can be administered to a subject as a combination therapy with another treatment (another active ingredients), e.g., a treatment for an SMA condition.
  • the combination therapy can include administering to the subject (e.g., a human patient) one or more additional agents that provide a therapeutic benefit to the subject who has, or is at risk of developing, (or suspected of having) an SMA condition.
  • the compound or pharmaceutical composition and the one or more additional agents are administered at the same time.
  • the compound can be administered first in time and the one or more additional agents administered second in time.
  • the one or more additional agents can be administered first in time and the compound administered second in time.
  • the compound can replace or augment a previously or currently administered therapy.
  • administration of the one or more additional agents can cease or diminish, e.g., be administered at lower levels.
  • Administration of the previous therapy can also be maintained.
  • a previous therapy can be maintained until the level of the compound (e.g., the dosage or schedule) reaches a level sufficient to provide a therapeutic effect.
  • the two therapies can be administered in combination.
  • a previous therapy is particularly toxic (e.g., a treatment for an SMA condition that carries significant side-effects) or poorly tolerated by a subject (e.g., a patient)
  • administration of the compound can be used to offset and/or lessen the amount of the previous therapy to a level sufficient to give the same or improved therapeutic benefit, but without the toxicity.
  • the first therapy is halted.
  • the subject can be monitored for a first pre-selected result, e.g., an improvement in one or more symptoms of an SMA condition such as any of those described herein (e.g., see above).
  • a first pre-selected result e.g., an improvement in one or more symptoms of an SMA condition such as any of those described herein (e.g., see above).
  • treatment with the compound is decreased or halted.
  • the subject can then be monitored for a second pre-selected result after treatment with the compound is halted, e.g., a worsening of a symptom of an SMA condition.
  • administration of the compound to the subject can be reinstated or increased, or administration of the first therapy is reinstated, or the subject is administered both a compound and first therapy, or an increased amount of the compound and the first therapeutic regimen.
  • assessing the effect of a therapy on subject having an SMA condition can be done by assessing, e.g., whether an increase in the expression of SMN in one or more affected cells in a subject having (or suspected of having) an SMA condition occurred.
  • the DcpS inhibitor can also be administered with a treatment for one or more symptoms of an SMA condition.
  • the DcpS inhibitor can be co-administered (e.g., at the same time or by any combination regimen described above) with, e.g., a pain medication, an antibiotic, or a decongestant.
  • the methods can be used to prevent an SMA condition, or one or more symptoms of an SMA condition, from occurring in a subject.
  • a subject When the terms “prevent,” “preventing,” “prevention,” or “prophylaxis” are used herein in connection with a given treatment for a given SMA condition, they mean that the treated subject either does not develop a clinically observable level of an SMA condition at all (e.g., the subject does not exhibit one or more symptoms of the an SMA condition), or the condition develops more slowly and/or to a lesser degree (e.g., as a result of increased expression of SMN) in the subject than it would have absent the treatment.
  • a treatment will be said to have "prevented” an SMA condition if it is given prior to the onset of any symptoms of the SMA condition in a subject at risk of developing an SMA condition and results in the subject's experiencing fewer and/or milder symptoms of the SMA condition than otherwise expected.
  • a treatment can "prevent” an SMA condition when the subject displays only mild overt symptoms of an SMA condition. "Prevention” does not imply that there must have been no symptoms of an SMA condition nor that those symptoms are entirely absent in a treated subject.
  • articles of manufacture that include: a container; and a composition contained within the container, wherein the composition comprises an active ingredient for increasing the expression of SMN in a cell, wherein the active ingredient comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or 10 or more) of any of the compounds described herein, and wherein the container has a label indicating that the composition is for use in increasing the expression of SMN in a cell and/or treating (or preventing) an SMA condition in a subject (e.g., a human).
  • the label can further indicate that the composition is to be administered to a subject having, suspected of having, or at risk of developing, an SMA condition such as any of those described herein.
  • the label can further indicate that the composition is to be given to a pregnant woman carrying a child having, suspected of having, or at risk of developing, an SMA condition.
  • the composition of the article of manufacture can be dried or lyophilized and can include, e.g., one or more solutions (and/or instructions) for solubilizing a dried or lyophilized composition.
  • the articles of manufacture can also include instructions for administering the composition to a subject (e.g., any of the methods described above under "Methods for Treating an SMA Condition").
  • kits Such articles of manufacture or the various components thereof can also be included in kits.
  • the samples were eluted using 20-70% eluent B over 50min, (eluent A 0.1% TFA in water, eluent B 1% TFA in ACN).
  • Four 0.85 mL column fractions were collected and to the fractions was added 0.2 mL water.
  • the samples were then re-injected into the HPLC. Two fractions (having a total activity of about ⁇ 6 mCi) were collected.
  • a sample of the product co- eluted with authentic cold standard, Cpd. 3.
  • a solution made with 400 mg sucrose, 7.6 mL water and 0.4 mL ethanol was used as a medium for freeze drying the isolated compounds.
  • a quality control check was performed by analyzing one of the samples on a Jupiter 7.5 cm C4 column, and indicated that the purified compounds had ⁇ 0.1% free iodine and > 98.1% radiochemical purity (specific radioactivity 2000 Ci/mmol). Five 230 ⁇ Ci samples were shipped on cold pack for biological evaluations. Radioiodination was performed at GE Healthcare, Bio-Science Division, Woburn, MA.
  • Cpd. 1 and TSA were formulated in DMSO and added to a culture of NSC-34 cells (which cells resulted from a fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma) at a final concentration of 500 nM/50 nM and 100 nM, respectively.
  • the final concentration of DMSO was 0.5%.
  • the cells were incubated for 18 hours and then harvested for RNA isolation using RNeasy Mini Kit (Qiagen) according to manufactures protocol. RNA quality and quantity was measured on an Agilent 2100 Bioanalyzer (Agilent Technologies).
  • cDNA synthesis was performed using High Capacity cDNA Archive kit (Applied Biosystems), according to manufactures protocol. Gene expression analysis was performed using TaqMan Assays-on- Demand Gene Expression Products (Applied Biosystems) according to manufactures protocol. Each assay was run in triplicate and the data was normalized against an internal housekeeping gene, B-actin. The relative difference in expression was calculated using the equation 2e, ( ⁇ Ac ⁇ ⁇ Statistical comparisons between groups in the gene expression studies, was done by using Students t-test, an associated probability of ⁇ 5% was considered significant.
  • NSC-34 cells are a hybrid cell line resulting from the fusion of mouse neuroblastoma N18TG2 cells with motor neuron-enriched embryonic day 12-14 cells harvested from mouse spinal cord (Cashman et al. (1992) Dev. Dyn. 194:209-21), the cells contain the mouse SMN gene in its normal chromosomal context. Therefore, an effort was made to ascertain if the C5-quinazolines would act on the NSC-34 cells to increase endogenous mouse SMN mRNA. To that end, real-time PCR studies were carried out to measure the mRNA levels in cells treated with Cpd. 1 (see FIG.
  • SMN2 Diminution of DcpS levels led to an increase in SMN2 that was not further accentuated by Cpd. 1.
  • SMN2 mRNA levels were determined with qPCR as described previously (Britcha et al. (2008) Hum. Genet. 123:141-53).
  • SMN2 levels in the DcpS knockdown cell increased approximately 3 fold and the addition of the Cpd. 1 did not further increase the levels strongly (FIG. ID), indicating that Cpd. 1 functions through DcpS.
  • C5-quinazolines were also tested for their ability to enhance SMN protein expression in human fibroblasts obtained from a type I SMA patient (hereinafter referred to as "3813" cells). 3813 cells were treated with Cpd. 1 or Cpd. 12, each at 10, 100, or 1000 nM for 5 days.
  • Cpd. 12 refers to 5-[l-(3-chlorobenzyl)piperidin-4-ylmethoxy]quinazoline-2,4-diamine
  • Cpd 1. refers to 5-[l-(2-fluorobenzyl)piperidin-4-ylmethoxy]quinazoline-2,4-diamine.
  • the structure of Cpd. 12 is as follows:
  • Cpd. 12 A set of cells was also treated with DMSO, the vehicle, as a control. Following treatment with the compounds, cell lysates were prepared from the cells. Sixty ⁇ g of each cell lysate was subjected to SDS-PAGE and SMN protein was detected by western blotting using mouse anti-SMN monoclonal antibody (MANSMA2) (FIG. 2). As a protein loading control, ⁇ - tubulin was also detected using a mouse anti- ⁇ -tubulin antibody. An increase in the amount of SMN protein in 3813 cells was observed in cells treated with each of Cpds. 1 and 12, but not with DMSO. These results demonstrated that the C5-quinazolines are able to enhance SMN protein expression in human fibroblasts.
  • MANSMA2 mouse anti-SMN monoclonal antibody
  • Protein microarrays were blocked for 1 hour in Tris buffer (50 mM Tris-HCl pH 7.5, 5 mM MgSO 4 , 0.1% v/v Tween20) with low-speed orbital shaking.
  • Tris buffer 50 mM Tris-HCl pH 7.5, 5 mM MgSO 4 , 0.1% v/v Tween20
  • arrays were washed three times with the aforementioned Tris buffer using 28 ml per wash. After washing, arrays were placed into a slide holder and spun for 2 minutes at 2000 rpm in a plate centrifuge. Dried arrays were exposed to a phosphorimager screen, images were acquired using a PerkinElmer Phosphorimager, and data were extracted with microarray data acquisition software (GenePix Pro, Molecular Devices).
  • Protein microarrays were assayed as described above with probing solutions comprised of 100 nM I-Cpd. 3 mixed with 10 nM I-streptavidin for positional mapping, in the presence or absence of 10 ⁇ M unlabeled competitor compound.
  • Unlabeled competitor molecules included Cpd. 3, Cpd. 1, staurosporine, and tertbutylquinone. All assays were carried out in duplicate.
  • Cpd. 3 a tin derivative of Cpd. 3 was first prepared (Cpd. 2), which supported radioiodination to the corresponding I labeled analog using the iododestannylation reaction (FIG. 3). This reaction with 100 mole percent incorporation of iodine under carrier-free conditions provided a theoretical specific activity of 2200 Ci/mmol. Additional quinazolines with potent SMN2 promoter activity to use as unlabeled competitors of I-Cpd. 3 in binding experiments were selected and tested as follows.
  • High density human protein arrays were used as the test bed for probing with I- Cpd. 3 tracer compound. Recombinant human proteins were expressed as fusion proteins with N-terminal glutathione-S -transferase (GST) purified from Baculovirus-infected insect cells. Represented on the protein microarrays were soluble proteins of potential therapeutic interest including kinases, phosphatases, nuclear receptors, and enzymes of intermediary metabolism. Proteins were purified under non-denaturing conditions and printed as adjacent duplicate spots on chemically modified glass slides. Additionally, the arrays contain control elements suitable for positional mapping of the data acquisition grid as well as positive and negative assay controls.
  • GST N-terminal glutathione-S -transferase
  • the Human ProtoArray" Protein Microarray v3.0 mg containing more than 5,000 human proteins was probed with 100 nM of the I-Cpd. 3 tracer in the presence of I- streptavidin to facilitate accurate positional mapping. High resolution images were generated through phosphoimaging. Pixel intensity data was extracted from these images and the resultant
  • RNA corresponding to the pcDNA3 (Invitrogen) polylinker (pcP) was transcribed in vitro by SP6 polymerase according to the manufacturer's directions (Promega) from a PCR- generated template using T7 and SP6 promoter primers. Cap labeling of the RNA was carried out with the vaccinia virus capping enzyme in the presence of [ ⁇ - P] GTP and S-adenosyl- methionine (SAM) as previously described (Wang et al. (1999) MoI.
  • SAM S-adenosyl- methionine
  • Decapping assays were carried out with 5 ng Flag- tagged recombinant DcpS protein and 20 nM unlabeled cap structure (purchased from New England Biolab) spiked with radioactive cap structure in decapping buffer (10 mM Tris-HCl pH 7.5, 100 mM KOAc, 2 mM Mg(OAc) 2 , 2 mM DTT) for 30 seconds at room temperature. Decapping reactions were terminated with the addition of 1.7 N formic acid.
  • mice For survival and phenotype analyses, SMN ⁇ 7 SMA mice (SMN2 +/+ ; Smn ⁇ 7 +/+ ; mSmn -/- ) were used (Butchbach et al. (2007) Neurobiol. Dis. 27:207-219). These mice were generated from males and females of the genotype SMN2 +/+ ; Smn ⁇ 7 ; mSmn (line 4299; FVB.Cg-Tg(SMN2*delta7)4299Ahmb Tg(SMN2)89Ahmb Smnl tmlMsd ). These mice originated from our colony but can be obtained from Jackson (#005025).
  • Neonatal offspring were genotyped using a PCR-based assay on genomic DNA from tail biopsies as described previously (Butchbach et al. (2007) Neurobiol Dis 27:207-219, incorporated herein by reference). Only pups of the genotypes SMN2 +/+ ;Smn ⁇ 7 +/+ ;mSmn +/ ⁇ (carrier) and SMN2 +/+ ;Smn ⁇ 7 +/+ ;mSmn ⁇ / ⁇ (SMA) were used in these experiments.
  • Carrier and SMA littermate mice were treated with Cpd. 1 (3 mg/kg/d) or the appropriate vehicle via oral administration beginning at PND04 — with birth being defined as PNDOl — or PND09 as described previously (Butchbach et al. (2007) J. Neurosci. Methods 161:282-290). Mice were monitored daily for changes in body mass and for death. SMN ⁇ 7 carrier mice received differing doses of Cpd. 1 (0.1-30 mg/kg/d) or the appropriate vehicle beginning at PND04. Cpd. 1 or vehicle were delivered via oral administration as described previously (Butchbach et al. (2007) J. Neurosci. Methods 161:282-290).
  • Treatment lasted for 5 days for the maximum tolerable dose (MTD) analysis at which time the pups were euthanized 1 hour after final dosing. Serum samples were taken and the brains were dissected and rapidly frozen in liquid nitrogen. Tail snips were also taken for genotyping.
  • MTD maximum tolerable dose
  • Serum and forebrain samples were obtained from neonatal mice treated with differing doses and processed as described elsewhere (Thurmond et al. (2008) J Med Chem 51:449-469, incorporated herein by reference). Drug levels were measured using LC/MS/MS. Protein concentrations were measured for each sample after LC-MS-MS analysis; serum and forebrain drug levels were expressed as ng/mg protein.
  • ⁇ -Galactosidase activity was measured using the Galacto-Light chemiluminescent reporter gene assay (Applied Biosystems, Bedford, MA) according to manufacturer's directions except that the lysis buffer contained 0.1% Triton X-100. Luminescence was monitored using a Luminoskan Ascent luminometer.
  • mice The maximum tolerable dose (MTD) for Cpd. 1 was first determined in SMN ⁇ 7 carrier (SMN2 + + ;SMN ⁇ 7 + + ;mSmn + ⁇ ) mice. These mice received different oral doses of Cpd. 1 (0.1-30 mg/kg/d) or the appropriate vehicle (UdH 2 O) — for 5 days beginning at postnatal day (PND) 04. No adverse effects were observed for Cpd. 1 at any of the doses tested.
  • Cpd. 1 was able to cross the blood-brain barrier. Brain Cpd. 1 levels increased with dose in a linear manner at lower doses while serum Cpd. 1 levels were low and fairly constant.
  • mice with Cpd. 1 increased mSmn promoter activity in forebrain and spinal cord extracts in a dose-dependent manner to a maximum at 3 mg/kg/d; mSmn promoter activity then decreases with increasing dose of Cpd. 1. Based on these observations and the MTD analysis, the optimal dose for Cpd. 1 is 3 mg/kg/d.
  • Tissue protein extract 100 ⁇ g was mixed with 0.16 volume of 6xloading dye (10.28% SDS, 36% glycerol and 0.012% bromophenol blue in 350 mM TrisHCl, pH 6.8) containing freshly added 100 mM DTT and resolved through a 12% polyacrylamide gel containing 0.1% SDS. Samples were then transferred onto a PVDF membrane via electroblotting. The resultant blots were incubated in lxblocking buffer (5% nonfat milk, 1% BSA in PBST (0.2% Tween-20 in PBS)) overnight at 4 0 C and then with a primary antibody diluted in 0.2xblocking buffer for 1 hour at room temperature.
  • 6xloading dye 10.28% SDS, 36% glycerol and 0.012% bromophenol blue in 350 mM TrisHCl, pH 6.8
  • Samples were then transferred onto a PVDF membrane via electroblotting.
  • the resultant blots were incubated in lxblocking buffer (5%
  • SMN protein For detection of SMN protein, a mixture containing equivalent amount of the following mAbs was used: 8F7 (MANSMA2), 1F1(MANSMA21), 5E3 (MANSMA13) and 7Q12 (MANSMA19) (e.g., Young et al. (2000), Exp. Cell Res., 256:365-374, incorporated herein by reference). This SMN cocktail was used at a titer of 1:500.
  • the blots were incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:1000) diluted in 0.2xblocking buffer for 1 hour at room temperature, washed extensive as above and the bound antibodies were detected by chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Biosciences). To confirm equal loading of protein in each lane, the blots were stripped and reprobed using a mouse anti- ⁇ -actin mAb (1:20 000; clone AC-15, Sigma- Aldrich).
  • Cpd. 1 The effect of Cpd. 1 treatment on SMN protein levels was examined in the spinal cord of SMN ⁇ 7 SMA mice.
  • oral administration of Cpd. 1 resulted in a -70% increase in SMN protein levels in the spinal cord (FIG. 6B).
  • Cpd. 1-treated SMN ⁇ 7 SMA mice had spinal cord SMN proteins that were approximately 25% of the levels observed in SMN ⁇ 7 carrier mice. Therefore, Cpd. 1 can increase SMN protein levels in the CNS of SMA mice in vivo but not to those levels observed in carrier mice.
  • each pup was placed in the center of a gridded (with 28 2.5-cm 2 grids) arena and the number of grids crossed in 1 min was counted as well as the latency for walking a distance greater than its body length (vectorial movement latency).
  • each pup was placed in the center of a gridded arena and the number of times the pup turned 90 0 C (pivots) during a 1-min time frame was counted.
  • Spontaneous locomotor activity and pivoting were monitored on PND07, PNDl 1 and PND 14. To minimize the stress on the pup, spontaneous locomotor activity and pivoting tests was conducted simultaneously.
  • SMN ⁇ 7 SMA mice received oral Cpd. 1 (3 mg/kg/d) beginning at PND04 and continued to receive drug for the duration of their lives.
  • SMN ⁇ 7 SMA mice develop a progressive impairment of neonatal motor behaviors including loss of surface righting and reduced spontaneous locomotion (Butchbach et al. (2007) Neurobiol Dis 27:207-219). Since Cpd. 1 improved survival of SMN ⁇ 7 SMA mice when administered prior to motor neuron loss, the effect of this quinazoline on the amelioration of the SMA motor phenotype was examined in these mice. Gross observation of Cpd. 1 -treated SMN ⁇ 7 SMA mice showed an improvement in motor activity when compared to vehicle-treated SMN ⁇ 7 SMA mice. Surface righting reflex responses are impaired in SMN ⁇ 7 SMA mice; treatment with Cpd. 1 partially reduced the righting reflex latency (FIG.
  • SMN ⁇ 7 SMA mice have a progressive loss of motor neurons in the lumbar spinal cord starting at -PND09 (e.g., Le et al. (2005) Hum MoI Genet 14:845-857). The effect of Cpd. 1 administration on the number of motor neurons in the lumbar spinal cord was examined. Dosing of SMN ⁇ 7 SMA mice began at PND04 and continued daily until PND 11. At PND 11 , vehicle-treated SMN ⁇ 7 SMA mice had -41% fewer motor neurons in the lumbar spinal cord than carrier mice (FIG. 8E); Cpd. 1 -treated SMN ⁇ 7 SMA mice, however, had motor neuron counts similar to those observed in carrier mice. Treatment of SMN ⁇ 7 SMA mice with Cpd. 1 starting at PND04 delayed the loss of motor neurons at PNDl 1.
  • carrier dams received either Cpd. 1 (0.3-60 mg/kg/d for MTD and 3 mg/kg/d for survival analysis) or vehicle via oral administration (e.g., Butchbach et al. (2007) J. Neurosci. Methods 161:285-290) beginning at embryonic day 11.5 (ED11.5).
  • Treatment lasted for 5 days for the MTD analysis at which time the dams were euthanized 1 hour after final dosing and the brains of the feti were dissected and rapidly frozen in liquid nitrogen. Tail snips from the feti were also taken for genotyping. Additionally, the forebrains of the dams were dissected and rapidly frozen in liquid nitrogen.
  • dosing continued through birth and the resultant pups were treated with or vehicle via oral administration beginning at PND02 until death; dosing of the dam ceased after PNDOl.
  • Cpd. 1 As Cpd. 1 was able to penetrate the blood-brain barrier, the effect of Cpd. 1 on survival of SMN ⁇ 7 SMA mice when the drug was administered during prenatal development was determined.
  • SMN ⁇ 7 SMA mice received either Cpd. 1 (3 mg/kg/d) or vehicle beginning at EDl 1. Treatment continued through birth and was orally administered once the pups were born.

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Abstract

L'invention porte sur des procédés et des compositions (par exemple, des composés et des compositions pharmaceutiques de ceux-ci) utiles pour augmenter l'expression de SMN dans une cellule (par exemple, in vitro ou in vivo). Étant donné qu'une déficience en SMN peut conduire au développement d'une condition de SMA chez un sujet, les procédés et les compositions décrits ici peuvent également être utilisés, par exemple, pour traiter ou prévenir un état de SMA chez un sujet.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011130515A1 (fr) * 2010-04-14 2011-10-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Arylthiazolyl pipéridines et composés associés comme modulateurs de la production de protéine de neurone moteur de survie (smn)
WO2016040748A1 (fr) * 2014-09-12 2016-03-17 Ionis Pharmaceuticals, Inc. Compositions et procédés de détection d'une protéine smn chez un patient et traitement d'un patient
US9717750B2 (en) 2009-06-17 2017-08-01 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US9926559B2 (en) 2013-01-09 2018-03-27 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US10357543B2 (en) 2015-11-16 2019-07-23 Ohio State Innovation Foundation Methods and compositions for treating disorders and diseases using Survival Motor Neuron (SMN) protein
US11198867B2 (en) 2016-06-16 2021-12-14 Ionis Pharmaceuticals, Inc. Combinations for the modulation of SMN expression
US11299737B1 (en) 2020-02-28 2022-04-12 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating SMN2
US11535848B2 (en) 2014-04-17 2022-12-27 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050288314A1 (en) * 2004-06-08 2005-12-29 Decode Chemistry, Inc. 2,4-diaminoquinazolines for spinal muscular atrophy
US20090042900A1 (en) * 2006-08-01 2009-02-12 Decode Chemistry, Inc. 2,4-diaminoquinazolines for spinal muscular atrophy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050288314A1 (en) * 2004-06-08 2005-12-29 Decode Chemistry, Inc. 2,4-diaminoquinazolines for spinal muscular atrophy
US20090042900A1 (en) * 2006-08-01 2009-02-12 Decode Chemistry, Inc. 2,4-diaminoquinazolines for spinal muscular atrophy

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAVIES ET AL.: 'Spinal muscular atrophies-distinctions and therapeutic progress.' DRUGS OF THE FUTURE vol. 31, no. 4, 2006, pages 365 - 371 *
LIU ET AL.: 'The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases.' EMBO J. vol. 21, no. 17, 2002, pages 4699 - 4708 *
THURMOND ET AL.: 'Synthesis and biological evaluation of novel 2,4-diaminoquinazoline derivatives as SMN2 promoter activators for the potential treatment of spinal muscular atrophy.' J MED CHEM. vol. 51, no. 3, 14 February 2008, pages 449 - 469 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9717750B2 (en) 2009-06-17 2017-08-01 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
WO2011130515A1 (fr) * 2010-04-14 2011-10-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Arylthiazolyl pipéridines et composés associés comme modulateurs de la production de protéine de neurone moteur de survie (smn)
US9926559B2 (en) 2013-01-09 2018-03-27 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US11535848B2 (en) 2014-04-17 2022-12-27 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
WO2016040748A1 (fr) * 2014-09-12 2016-03-17 Ionis Pharmaceuticals, Inc. Compositions et procédés de détection d'une protéine smn chez un patient et traitement d'un patient
US10436802B2 (en) 2014-09-12 2019-10-08 Biogen Ma Inc. Methods for treating spinal muscular atrophy
US12013403B2 (en) 2014-09-12 2024-06-18 Biogen Ma Inc. Compositions and methods for detection of SMN protein in a subject and treatment of a subject
US10357543B2 (en) 2015-11-16 2019-07-23 Ohio State Innovation Foundation Methods and compositions for treating disorders and diseases using Survival Motor Neuron (SMN) protein
US11198867B2 (en) 2016-06-16 2021-12-14 Ionis Pharmaceuticals, Inc. Combinations for the modulation of SMN expression
US11299737B1 (en) 2020-02-28 2022-04-12 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating SMN2

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