WO2008157753A1

WO2008157753A1 - Methods of treatment for spinal muscular atrophy

Info

Publication number: WO2008157753A1
Application number: PCT/US2008/067652
Authority: WO
Inventors: Meg Winberg
Original assignee: Sma Foundation
Priority date: 2007-06-21
Filing date: 2008-06-20
Publication date: 2008-12-24

Abstract

The present invention discloses a method of treating spinal muscular atrophy by administering an epidermal growth factor receptor antagonist to a subject. The invention also discloses a second method of treating spinal muscular atrophy by administering a motor neuron neurotrophic factor to a subject. The invention also includes a method for screening compounds for efficacy in treating spinal muscular atrophy using a double transgenic mouse model of the disease.

Description

Methods of Treatment for Spinal Muscular Atrophy

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. Provisional Application No. 60/945,492, filed on June 21, 2007 and entitled "Methods of Treatment for Spinal Muscular Atrophy", which is herein incorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive disorder which is the leading hereditary cause of infant death in humans. The disease is characterized by a progressive muscle weakness from proximal to distal with lower limbs more greatly affected than upper limbs. Three types of SMA have been described based on disease severity and age of onset. Type I affects approximately fifty percent of SMA patients with symptoms presenting within the first six months after birth. Death typically occurs within the first two years due to respiratory failure. Type II SMA has an onset between six months and eighteen months of age, and length of survival is dependent on the severity of respiratory impairment. Type III SMA patients, who have symptom onset between eighteen months and early childhood, usually do not have a decrease in life expectancy, although most are wheel chair bound at some point in their disease progression.

[0003] SMA is characterized by loss of alpha-motor neurons in the anterior horn of the spinal cord, which is correlated with muscle paralysis and atrophy (Crawford and Pardo (1996) Neurobiol. Dis. 3: 97-110). Motor neuron degeneration is thought to be due to low levels of the survival motor neuron protein (Coovert et al. (1997) Hum. MoI. Genet. 6: 1205-1214; Lefebvre et al., (1997) Nat. Genet. 16: 265-269). Homozygous mutations of the telomeric copy of the survival motor neuron (SMNl) gene located on chromosome 5q cause SMA (Lefebvre et al. (1995) Cell 80: 155-165). The majority of the population have a centromeric copy of the survival motor neuron (SMN2) gene, which can partially compensate for the loss of the SMNl gene product in SMA patients.

[0004] The essential difference between the SMNl and SMN2 genes is a C to T transition in exon 7 of the SMN2 gene, which causes this exon to be frequently omitted during transcription (Lorson et al. (1999) Proc. Natl. Acad. Sci. USA 96: 6307-6311; Monani et al. (1999) Hum. MoI.

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66408 v2/DC Genet. 8: 1177- 1183). Although transcription from SMNl produces full-length transcripts, the majority of transcripts produced from SMN2 lack exon 7 and result in the expression of a truncated protein product. The truncated protein does not oligomerize as well as full-length protein and is quickly degraded (Lorson et al. (1998) Nat. Genet. 19: 63-66; Le et al. (2000) Neurogenetics 3: 7-16). However, the ability of the SMN2 gene to generate low levels of full- length transcript and in turn full-length protein may explain why SMN2 copy number modulates the disease phenotype (Parsons et al. (1998) Am. J. Hum. Genet. 63: 1712-1723). [0005] Currently, there are no effective therapeutics for SMA disease. In addition, the development of appropriate animal models to test potential treatments is difficult since the presence of the SMN2 gene is unique to humans. Most other organisms only have one copy of the SMN gene, disruption of which results in an embryonic lethal phenotype. Therefore, designing an animal model that resembles the SMA disease condition in humans and obtaining a viable organism in which to test candidate drug compounds has been challenging.

SUMMARY OF THE INVENTION

[0006] The present inventor has developed a method for screening compounds for efficacy in treating SMA using a double transgenic mouse model of the disease, which has an extended lifespan compared to some other mouse models. The novel method entails treating transgenic mice with potential therapeutic compounds and evaluating their performance on several physiological measures to include various motor tasks. By using this drug screening method, the inventor has identified two classes of compounds as well as specific examples that improve function in the double transgenic mouse model of SMA. These classes of compounds may be useful in treating SMA in humans.

[0007] The present invention provides for a method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of an epidermal growth factor receptor antagonist, wherein at least one symptom of spinal muscular atrophy is alleviated following administration. In one embodiment, said epidermal growth factor receptor antagonist is erlotinib.

[0008] The present invention also provides for a method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of a motor neuron neurotrophic factor, wherein at least one symptom of spinal muscular atrophy is

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66408 v2/DC alleviated following administration. In one embodiment, said motor neuron neurotrophic factor is troglitazone.

[0009] In some embodiments, the methods may further comprise administering a pharmaceutically effective amount of a SMN modulating compound. In one embodiment, the SMN modulating compound increases the expression of SMN protein. SMN modulating compounds include valproic acid, phenylbutyrate, sodium butyrate, hydroxyurea, trapoxin, and trichostatin A.

[0010] The present invention also encompasses a method of screening compounds for efficacy in treating spinal muscular atrophy comprising administering at least one dose of a compound to a mouse of the double transgenic mouse model of spinal muscular atrophy (SMNA7 ; SMN2 ; Smn ^") and measuring at least one physiological parameter in said treated mouse. In one embodiment, said mouse is a neonate. In another embodiment, several doses of said compound are administered. In another embodiment, said physiological parameters include body weight, survival, and motor performance. In some embodiments, the method further comprises selecting a compound that produces an increase in body weight, survival, or motor performance, wherein said selected compound is identified as a compound efficacious for treating spinal muscular atrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows the performance of wild-type (S2 +/+_D+/+_S1+/+), heterozygote (S2 +/+_D+/+_S1+/-), and knockout (S2 +/+_D+/+_S 1 -/-) mice of the double transgenic mouse model of SMA (SMNA7; SMN2; Smn ^~'^~) on the geotaxis motor test. The time to complete the test in seconds is plotted against age at test (postnatal day 4, 6, 8, 10, and 12). The knockout mice take approximately twenty seconds longer than either wild-type or heterozygote mice to complete the task at all ages tested.

[0012] Figures 2 A to 2D show the results of all four parameters measured on the tube test. The test is performed in two consecutive trials on each day tested. Each trial is plotted separately. Figure 2A. Time spent hanging in seconds is plotted against age at test (postnatal day 2, 4, 6, 8, 10, and 12) for wild-type (WT; SMNA7; SMN2; Smn ^+/+), heterozygote (HET; SMNA7; SMN2; Smn ^+/~), and knockout (KO; SMNA7; SMN2; Smn ^~!~) mice of the double transgenic mouse model of SMA. Figure 2B. Number of pulls is plotted against age at test (postnatal day 2, 4, 6, 8, 10,

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66408 v2/DC and 12). A pull is recorded when the animal attempts to pull up and escape the tube. Figure 2C. Hind limb strength score (HLSS) is plotted against age at test (postnatal day 2, 4, 6, 8, 10, and 12). The score is assessed on a scale of 0 (indicating paralysis) to 4 (normal tone). Figure 2D. Tube test score (TTS) is plotted against age at test (postnatal day 2, 4, 6, 8, 10, and 12). TTS is calculated based on the values of the other three parameters according to the following equation: TTS = [(time spent hanging) + (#pulls x 10)] x (HLSS score + 1) / 4. The knockout mice show impaired performance on all four parameters compared to wild-type and heterozygote animals. Heterozygote animals do not differ significantly from wild-type animals. [0013] Figure 3 depicts the Kaplan Meier survival curve of SMA knockout mice (male and female combined) either untreated or treated with 25 or 50 mg/kg of erlotinib, or vehicle from postnatal day 0 to 21. Mice dosed with 50 mg/kg of erlotinib were less likely to die before P12 (P = 0.0753, a trend) in comparison to vehicle treated controls. The survival curve of the 50 mg/kg group was significantly different from the 25 mg/kg dose.

[0014] Figures 4A to 4D show the results of the tube test for SMA knockout mice (KO, SMNΔ7; SMN2; Smn ^~'^~) receiving either 25 or 50 mg/kg erlotinib or vehicle once a day. The tube test was performed at postnatal day 6, 8, 10 and 12. Figure 4A: Time spent hanging at the edge of the tube. KO mice treated with erlotinib at either 25 or 50 mg/kg showed a significant improvement in the hanging time at P8 during the first and second trial (P < 0.05). Figure 4B: Number of pulls to escape the tube. Figure 4C: Hind-limb strength score (HLSS). 0 indicates paralysis and 4 indicates normal tone. Figure 4D: Tube test score. The tube test score is equal to [(time hanging) + (number of pulls x 10)] x (Hind limb strength score+l)/4. [0015] Figures 5A and 5B. Figure 5A: Kaplan Meier survival curve of SMA knockout mice (male and female combined) treated twice a day with either 100 or 150 mg/kg Troglitazone or vehicle from postnatal day 0 to 21. The untreated group was not run in parallel with the current study so it is shown only for reference. Figure 5B: Bar graph showing the mean survival values for SMA knockout mice treated with either Troglitazone (100 or 150 mg/kg bid) or vehicle split by gender. ANOVA indicated a significant main effect for treatment (P = 0.0466) and no significant treatment x gender interaction (P = 0.7708) indicating that both male and females benefited from Troglitazone treatment.

66408 v2/DC DETAILED DESCRIPTION

[0016] The invention provides a method for screening compounds for efficacy in treating SMA. The screening method entails administering at least one dose of a compound to a double transgenic mouse model of SMA and measuring several physiological parameters to assess changes in phenotype. The invention also includes two classes of compounds, epidermal growth factor receptor antagonists and motor neuron neurotrophic factors, that were identified using this screening method to improve symptoms of SMA.

Classes of compounds useful for treating SMA

[0017] Epidermal growth factor receptor (EGFR) antagonists, such as erlotinib (marketed as Tarceva®), have been shown to block the inhibition of neurite outgrowth induced by myelin proteins and chondroitin sulfate proteoglycans (Koprivica et al. (2005) Science 310: 106-110). Therefore, inhibition of the EGFR may facilitate the neuronal axon regeneration process. [0018] Erlotinib inhibits the autophosphorylation of the EGFR and prevents downstream signal transduction. The EGFR is a receptor tyrosine kinase that is activated upon binding of epidermal growth factor. Activation of the receptor via autophosphorylation leads to the activation of a downstream signaling cascade that regulates cell growth, survival, proliferation, and differentiation. For a complete description of the EGFR signaling cascade and its function see recent reviews (Bublil and Yarden (2007) Curr. Opin. Cell Biol. 19: 124-134; Oda et al. (2005) Molecular Systems Biology 1:2005.0010). Mutations of the EGFR can cause constitutive phosphorylation of the receptor and persistent signal transduction that can lead to aberrant DNA synthesis and cell proliferation, which has been observed in some forms of cancer. For this reason, erlotinib is effective in treating some forms of cancer and has been approved by the Food and Drug Administration for this use.

[0019] Thus, the present invention provides a method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of an epidermal growth factor receptor antagonist, wherein at least one symptom of spinal muscular atrophy is alleviated following administration. EGFR antagonists suitable for use in the methods of the invention include, but are not limited to, erlotinib (Tarceva), gefitinib (Iressa), EKB-569, HKI-272, CI-1033, ZD 6474, 4-(3-Chloroanilino)-6, 7-dimethoxyquinazoline (AG1478), 4-[(3- Bromophenyl)amino]-6-acrylamidoquinazoline (PD 168393), 5-Amino-[(N-2,5-

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66408 v2/DC dihydroxybenzyl)-N'-2-hydroxybenzyl]salicylic Acid (Lavendustin A), Cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide, N8-(3- Chloro-4-fluorophenyl)-N2-(l-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, 2HCl (BIBXl 382BS), 4-(4-Benzyloxyanilino)-6,7-dimethoxyquinazoline, N-(4-((3-Chloro-4- fluorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)2-butynamide, and 4-[(3-Bromophenyl)amino]- 6,7-dimethoxyquinazoline (AG1517). In a preferred embodiment, the epidermal growth factor receptor antagonist is erlotinib (Tarceva®).

[0020] The EGFR can also be phosphorylated by "trans-activation" mechanisms independent of ligand binding. Various signaling molecules, including calcium, protein kinase C (PKC), cytoplasmic tyrosine kianses (Src and Pyk2), and G-protein coupled receptors, have been reported to participate in the trans-activation of the EGFR (Zwick et al. (1999) Trends Pharmacol. Sci. 20: 408-412; Andreev et al. (2001) J. Biol. Chem. 276: 20130-20135; Rosen and Greenberg (1996) Proc. Natl. Acad. Sci. USA 93: 1113-1118). The present invention encompasses compounds that would inhibit the trans-activation of the EGFR as well as those that directly inhibit the kinase activity of the EGFR itself. Furthermore, inhibitors of downstream signaling molecules, such as mitogen-activated protein (MAP) kinase, phosphatidyl inositol 3 (PI3) kinase, and phospholipase C-γ (PLC-γ), would also be within the scope of the invention since they would prevent cellular events triggered downstream of the EGFR. [0021] Another class of compounds that would be useful for treating SMA is motor neuron neurotrophic factors. The present invention encompasses a method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of a motor neuron neurotrophic factor, wherein at least one symptom of spinal muscular atrophy is alleviated following administration. A motor neuron neurotrophic factor is any agent that promotes the survival, axonal regeneration, or growth of motor neurons. Troglitazone, a member of the thiazolidinedione class of compounds, has been reported to promote motor neuron survival in rats (Nishijima et al. (2001) J. Neurochem. 76: 383-390). Pioglitazone and rosiglitazone, two other members of the thiazolidinedione class, have been shown to reduce motor neuron loss after spinal cord injury in rats (Park et al. (2007) J. Pharmacol. Exp. Ther. 320: 1002-1012).

[0022] The thiazolidinediones, whose members include troglitazone, rosiglitazone, and pioglitazone, are agonists of the peroxisome proliferator-activated receptor gamma (PP ARγ).

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66408 v2/DC When activated, PP ARγ translocates to the nucleus and functions as a transcription factor to induce expression of several genes, which leads to decrease in insulin resistance among other effects. In addition, the thiazolidinediones have been shown to have an anti-inflammatory effect (Park et al. (2007) J. Pharmacol. Exp. Ther. 320: 1002-1012; Aljada et al. (2001) J. Clin. Endocrinol. Metab. 86: 3250-3256).

[0023] Other classes of motor neuron neurotrophic factors that may be used in the methods of the present invention include the neurotrophin family of growth factors, insulin-like growth factor-I (IGF-I), glial cell line-derived neurotrophic factor (GDNF), and fibroblast growth factors (FGF). The neurotrophin family of growth factors includes nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4). In a preferred embodiment, the motor neuron neurotrophic factor is troglitazone. [0024] In some embodiments, the methods may further comprise administering a pharmaceutically effective amount of a second therapeutic compound with the EGFR antagonist or the motor neuron neurotrophic factor. A second therapeutic compound may be a compound typically prescribed to treat SMA symptoms or a compound shown to improve motor function in SMA patients. A second therapeutic compound may be a neuroprotective compound, a myotherapeutic compound or a SMN modulating compound. A neuroprotective compound prevents the degeneration and promotes the survival of neurons. Examples of suitable neuroprotective compounds that may be combined with the EGFR antagonist or the motor neuron neurotrophic agent include, but are not limited to, riluzole, gabapentin, L-carnitine, acetyl-L-carnitine and cardiotrophin- 1. A myotherapeutic compound prevents muscle atrophy or promotes muscle growth. Examples of suitable myotherapeutic compounds that may be combined with the EGFR antagonist or the motor neuron neurotrophic agent include, but are not limited to, albuterol, terbutaline, losartan/Cozaar, insulin-like growth factor-I (IGF-I), follistatin or other agents that inhibit the myostatin pathway, and glucocorticoid corticosteroids such as dexamethasone or prednisolone.

[0025] A SMN modulating compound is an agent that increases levels of full-length SMN protein by increasing expression of the SMN2 gene, promoting exon 7 inclusion in SMN2 transcripts, and/or stabilizing SMN protein. Histone deacetylase inhibitors or HDAC inhibitors have been shown to increase expression of SMN protein in cells derived from Type 1 SMA patients. Exemplary HDAC inhibitors include valproic acid, phenylbutyrate, sodium butyrate,

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66408 v2/DC suberoyl anilide hydroxamic acid, trapoxin, and trichostatin A. Hydroxyurea has also been shown to increase SMN protein levels in SMA patient-derived cells. Compounds that act to stabilize SMN protein include, but are not limited to, indoprofen, aminoglycosides, and proteasome inhibitors. Nucleic acids encoding the SMNl gene and vectors comprising such nucleic acids are also contemplated as SMN modulating compounds.

[0026] In one embodiment, the methods of treating spinal muscular atrophy in a subject in need thereof further comprise administering a pharmaceutically effective amount of a SMN modulating compound with an EGFR antagonist or a motor neuron neurotrophic factor. In another embodiment, the SMN modulating compound increases expression of SMN protein. In another embodiment, the SMN modulation compound is valproic acid, phenylbutyrate, sodium butyrate, suberoyl anilide hydroxamic acid, hydroxyurea, trapoxin, or trichostatin A.

Formulation and methods of administration

[0027] Methods of administration of a pharmaceutically effective amount of a compound of the invention include, but are not limited to, parenteral administration (e.g. intravenous, intramuscular, subcutaneous, intradermal, and intrathecal), oral, intranasal, rectal, inhalational, topical, or epidural. As used herein, the term "pharmaceutically effective amount" means an amount that improves one or more symptoms of SMA. Symptoms of SMA include but are not limited to muscle weakness, muscle atrophy, motor neuron loss, decreased life expectancy, poor muscle tone, decreased or absent deep tendon reflexes, twitching of leg, arm or tongue muscles, abnormal gait, or difficulty breathing. In some embodiments of the invention, at least one symptom of SMA is alleviated following administration of an epidermal growth factor receptor antagonist, a motor neuron neurotrophic factor, or combinations of such compounds with SMN modulating agents as described herein.

[0028] Formulation of a compound for treatment purposes would comprise combining pharmaceutically effective amounts of the compound of the invention with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the

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66408 v2/DC material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Therapeutic proteins of the invention may be produced as fusion proteins to modulate or extend the half-life of the protein. Such fusion proteins may include human serum albumin, transferrin, other serum proteins, etc. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present compounds. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated.

[0029] The compound can be administered using oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. J. Rhodes. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. [0030] Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used. Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

[0031] In general, the composition of the treatment is formulated to be compatible with the route of administration. A solution for parenteral, intradermal, or subcutaneous administration can include: a sterile diluent such as water, saline, glycerin, fixed oils, polyethylene glycols, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent; a

66408 v2/DC buffering agent such as acetate or phosphate. The solution can be stored in ampoules, disposable syringes, or plastic or glass vials.

[0032] In some embodiments, the compounds of the invention (e.g. epidermal growth factor receptor antagonists and motor neuron neurotrophic factors) may be administered with a second SMA therapeutic compound, such as a SMA modulating compound, as described above. Combination treatments may be formulated together into a single formulation or prepared as separate formulations. If the two or more compounds are prepared as separate formulations, the formulations may be mixed prior to administration to a subject.

[0033] Dosages for administration to human subjects of the epidermal growth factor receptor antagonist or motor neuron neurotrophic factor can be estimated from effective doses in transgenic mouse models of SMA, such as the double transgenic model described in Example 1. Appropriate dosages will depend upon the compound to be administered, the route of administration, severity of SMA disease, other therapeutic compounds that are co-administered, and characteristics particular to each patient, such as body weight, age, health, and tolerance of the treatment regimen. A physician is able to optimize the dosages based on changes in symptoms of SMA and any other related changes in health during the course of treatment. Estimated dosages of erlotinib for human administration based on results with the mouse model described herein may be from about 25 mg per day to about 500 mg per day, preferably from about 100 mg per day to about 400 mg per day, and more preferably from about 150 mg per day to about 250 mg per day. Estimated dosages of troglitazone for human administration may be from about 200 mg per day to about 1000 mg per day, preferably from about 300 mg per day to about 800 mg per day, and more preferably from about 400 mg per day to about 600 mg per day.

Screening of potential therapeutic compounds

[0034] The present invention also contemplates a method of screening compounds for efficacy in treating spinal muscular atrophy utilizing a double transgenic mouse model of spinal muscular atrophy. As described herein, this double transgenic mouse model carries the human SMN2 gene and the human SMN2 gene lacking exon 7 in a mouse Smn knockout background. These mice were found to have an extended life span compared to mice carrying only the human SMN2 gene and more closely mimicked the interaction of SMN splice variants (e.g. full length SMN and SMNΔexon7) found in SMA patients. In one embodiment of the invention, the method

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66408 v2/DC comprises administering at least one dose of a compound to a mouse of the double transgenic mouse model of spinal muscular atrophy (SMNA7 ^+/+; SMN2^+/+; Smri^) and measuring at least one physiological parameter in said treated mouse.

[0035] As a first step to testing a compound for efficacy in treating SMA, the maximal tolerable dose may be estimated. As used herein, the term "maximal tolerable dose" is defined as a dose which produces observable but mild to moderate behavioral and non-behavioral side effects such as change in body weight, without seizures or other major physiological changes. In one embodiment, wild-type litter mates (SMNA7; SMN2; Smn ^{+ +}) of the double transgenic mice described in Example 1 are given at least two doses of the compound of interest by oral gavage or subcutaneous injection at least once daily for eight consecutive days. Visual inspection of the mice for changes in the animals' behavior (e.g. respiration, motor activity, grooming), appearance, or survival is performed daily to determine toxicity. The doses to be used for subsequent testing in the double transgenic mouse model described in Example 1 may be estimated from the maximal tolerable dose.

[0036] To test the efficacy of a compound for ameliorating the symptoms associated with SMA, at least one dose may be administered by either oral administration, oral gavage, or subcutaneous injection to a mouse of the double transgenic mouse model (SMNA7; SMN2; Smn ^~'^~) described in Example 1. In one embodiment the mouse is a neonate. A "neonate" is a mouse between the ages of birth (postnatal day 0) and about postnatal day 12. Dosing may begin at postnatal day 3 and continue until the knockout animals are dead. In another embodiment, several doses of the compound may be administered to obtain a dose response curve for the compound. [0037] Several physiological parameters may be used to evaluate the effect of the compound on the transgenic animal's phenotype. These tests include skin color, body temperature, milk content, muscle tone, respiratory rate, presence of gasping, body weight gain, survival, and motor tasks (e.g. geotaxis and tube test as described in Example 2). Measurements of body weight and survival may be recorded daily during the course of the study. Motor function may be assessed as described in Example 2 on postnatal day 6 (P6), P8, PlO, P12, and P14. Transgenic animals treated with the compound may be compared to transgenic animals treated with vehicle alone. Statistical differences in the measurements described above may be used to indicate a significant effect of the compound tested.

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66408 v2/DC [0038] In one embodiment, the physiological parameters that are measured include body weight, survival, and motor performance. In some embodiments, the method further comprises selecting those compounds that produce an increase in body weight, survival, or motor performance, wherein said selected compound is identified as a compound efficacious for treating spinal muscular atrophy.

[0039] This invention is further illustrated by the following additional examples that should not be construed as limiting.

EXAMPLES Example 1

Generation of the double transgenic mouse model of SMA (SMNA7;SMN2; Smn -/-) [0040] A SMN construct lacking exon 7 was produced by performing RT-PCR from total RNA isolated from a type I SMA patient fibroblast cell line (number 3813), using the primers 5'- CAGGATCCCTATGGCGATGAGCAGC-3' and 5'-CGGAATTCAGT ACAATGAAC A- GCCATGTCCA-3 ' . The amplified 1.37 kb fragments were digested with Bam HI and Eco RI from sites engineered into the primers, and subcloned into pcDNA3. The clones produced from the preceding procedure were amplified with primers directed to exons 4 and 8 as previously described (Parsons et al. (1998) Am. J. Hum. Genet. 63: 1712-1723; Andreassi et al. (2001) Hum. MoI. Genet. 10: 2841-2849) to identify clones lacking exon 7. In addition, the SMNΔ7 clones were sequenced to verify that the clones did not contain additional mutations. [0041] A Kpn 1-Sac II SMN promoter fragment (-3.4 kb, Monani et al. (1999) Biochim. Biophys. Acta. 1445: 330-336) was cloned into the pEGFP (BD Biosciences Clontech, San Diego, CA) vector and subsequently excised using Kpn I and Bam HI. This excised fragment was directionally ligated to the SMNΔ7 cDNA in the pcDNA3 and the CMV promoter removed as described previously (Monani et al. (2003) J. Cell Biol. 160: 41-52). The resulting 5.4 kb promoter- SMNΔ7 construct was excised with Kpn I and Dra III and microinjected into fertilized mouse oocytes from the FVB/N strain. Copy number of the SMNΔ7 transgene was determined from the three founder lines (4299, 4352, and 4353) using quantitative Southern Blot and realtime PCR as previously described (Schaeffeler et al. (2003) Hum. Mutat. 22:476-485) except using two copies of the mouse β-globin gene as the control gene. Line 4299 was estimated to

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66408 v2/DC have six copies of SMNΔ7, line 4352 had 17 copies of SMNΔ7, and line 4353 had two copies of SMNΔ7. Mice from line 4299 were subsequently crossed with mice containing the human SMN2 gene and a mouse Smn knockout allele (Schrank et al. (1997) Proc. Natl. Acad. Sci. USA 94: 9920-9925) to obtain double transgenic mice SMNM; SMN2; Smn ^+/~ on a FVB/N background. These mice were then interbred to obtain Smn ^{' "} mice carrying the SMN2 and SMNA7 genes (SMNM; SMN2; Smn ^''^'). The triple mutant was backcrossed to a FVB/N background for at least six generations.

[0042] The triple mutants (SMNΔ7 SMA) had a maximum survival of 17 days with a mean survival time of 13 days. The SMNΔ7 SMA mice had reduced body weights compared to their normal littermates. By postnatal day 5, the SMNΔ7 SMA mice had difficulty righting themselves when placed on their backs, and muscle weakness became more progressive over the following week. By postnatal day 10, the SMNΔ7 SMA mice displayed an abnormal gait, shakiness in the hind limbs, and a tendency to fall over. Loss of spinal motor neurons in the lumbar region was apparent at postnatal day 9 in the SMNΔ7 SMA mice (Le et al. (2005) Hum. MoI. Genet. 14: 845-857).

Example 2

Development of motor tasks for evaluating performance of SMA double transgenic mice

[0043] Geotaxis. The geotaxis test evaluates the animal's ability to orient itself when placed on an inclined platform. Performance in this task measures motor coordination and vestibular system function. The mouse was placed face down (towards the bottom) on a forty-five degree inclined platform. The animal was given sixty seconds to orient itself towards the top of the platform. The time the animal took to perform the re-orientation was recorded. In addition, one of the following scores was given to each animal: Y = animal is able to reposition itself, N = animal did not reposition itself, and F = animal failed to physically perform the task. Measurements taken at postnatal day 4 (P4), P8, PlO and P12 revealed that the SMNΔ7 SMA knockout mice (SMNΔ7; SMN2; Smn ^~'^~) required an average of twenty seconds more to perform the task at each of the time points than either their heterozygous (SMNΔ7; SMN2; Smn ^+/~) or wild-type (SMNA7; SMN2; Smn ^+/+) litter mates (see Figure 1).

[0044] Tube test. The tube test was designed to test the hind limb strength and motor tone of neonates (postnatal day 0 to postnatal day 12). Each animal performed two consecutive trials of

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66408 v2/DC the test. In each trial, the animal was placed head first into a conical 50 mL plastic tube so that it was hanging into the tube by its hind limbs. The following three parameters were measured: time spent hanging, number of pulls, and hind limb strength score(HLSS, Figure 2). A pull was recorded when the animal attempted to pull itself up to escape the tube. The hind limb strength score was a value from 0 (indicating paralysis) to 4 (normal tone) that was given to each animal based on the separation of the hind limbs of each animal hanging in the tube. At the conclusion of the test, an overall tube test score (TTS, see Figure 2D) was calculated according to the following equation:

TTS = [(time hanging) + (# of pulls x 10)] x (HLSS + l)/4

On all three parameters, the performance of the SMNΔ7 SMA knockout mice was significantly different from their heterozygous and wild-type littermates. The knockout mice spent significantly less time hanging in the tube than wild-types or heterozygotes. The performance of the knockout mice continued to deteriorate from P2 to P12 (see Figure 2A). Similar results were observed with the measure of hind limb strength. The HLSS score of the SMNΔ7 SMA knockout mice was considerably lower than heterozygote or wild-type mice. Like the time spent hanging measure, HLSS also decreased as the knockout animals aged (see Figure 2C). The SMNΔ7 SMA knockout mice showed less than two pulls from the tube at all ages tested (P2, P4, P6, P8, PlO, and P 12), while heterozygotes and wild-types frequently displayed greater than two pulls at all ages tested with a maximum at approximately 10 pulls (age PlO, see Figure 2B). These results suggest that the tube test is sensitive enough to detect motor impairments in neonates and thus would be useful in evaluating the effectiveness of treatments in improving motor symptoms associated with SMA. Detailed description of the experimental procedure is also provided by El-Khodor et al, 2008, Experimental Neurology.

Example 3

Validation of the method for screening compounds for treatment of SMA

[0045] Valproic acid, a histone deacetylase inhibitor, and hyrdroxyurea, an inhibitor of DNA replication, have been shown to be effective for improving symptoms associated with SMA (US Pat. No. 6,376,508, US Pat. No. 6,573, 300, respectively). To test the validity of the screening method, these two compounds were assessed to determine whether improvement in the physiological parameters of the SMNΔ7 SMA knockout mice (SMNA7; SMN2; Smn ^~'^~) could be

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66408 v2/DC observed. Either valproic acid (50 mg/kg), hyrdoxyurea (5 mg/kg) or vehicle alone (water) was administered twice a day by oral gavage to SMNΔ7 SMA knockout mice (SMNA 7; SMN2; Smn ^~'^~) starting at postnatal day 3 (P3) until the animals died. Survival and body weight were monitored daily during the course of the study. Motor performance assessment using the geotaxis and tube tests was performed on P6, P8, PlO, P12, and P14.

[0046] SMNΔ7 SMA knockout mice treated with hydroxyurea had a statistically significant higher body weight at P7, P8, P9, PlO, and PI l compared to vehicle-treated control. Survival curves in hydroxyurea-treated knockout mice were shifted to the right, indicating a trend to extended lifespan in these animals. Hydroxyurea treatment was also able to improve some measures of motor performance. At P6, SMNΔ7 SMA knockout mice treated with hydroxyurea completed the geotaxis test faster and had an increased number of pulls in the tube test than vehicle-treated animals.

[0047] Valproic acid produced some improvement in motor function in the double transgenic mouse model of SMA. Mice treated with 50mg/kg valproic acid performed and completed the geotaxis test faster than the vehicle group. In the tube test, the valproic acid-treated mice showed a significant increase in the number of pulls and tube test score (TTS) at P6 and P8. In addition, the survival curve in knockout mouse treated with valproic acid was shifted to the right demonstrating a trend toward longer survival.

[0048] The results with hydroxyurea and valproic acid demonstrate that improvement of physiological parameters measured in SMNΔ7 SMA knockout mice treated with the compounds in the screening method of the invention can reliably predict compounds that are beneficial to SMA patients.

Example 4

Use of erlotinib in the double transgenic mouse model of SMA

[0049] The effect of the epidermal growth factor receptor (EGFR) antagonist, erlotinib (also known as Tarceva), was tested for effectiveness in the double transgenic mouse model of SMA. Erlotinib (either 25 mg/kg or 50 mg/kg) or vehicle alone (0.2% carboxymethylcellulose) was administered once a day by oral gavage to SMNΔ7 SMA knockout mice (SMNA 7; SMN2; Smn ^~'^~ ) starting at postnatal day 3 (P3) until the animals died. Survival and body weight were

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66408 v2/DC monitored daily during the course of the study. Motor performance assessment using the geotaxis and tube tests was performed on P6, P8, PlO, P12, and P14.

[0050] Knockout animals treated with 50 mg/kg erlotinib showed a significant delay in death onset (first death at P 12) compared to knockout animals treated with the lower erlotinib dose (first death at P8) or vehicle alone (first death at P5, see Figure 3). Knockout mice treated with either dose of erlotinib (25 mg/kg or 50 mg/kg) showed a significant increase in hang time in the tube test at P 8 compared to animals treated with vehicle alone (Figure 4A), indicating an improvement in motor function at an early time point.

Example 5

Treatment of SMA double transgenic mice with troglitazone

[0051] Troglitazone, a compound that has been reported to have neurotrophic effects on rat motor neurons (Nishijima et al. (2001) J. Neurochem. 76: 383-390), was administered to SMNΔ7 SMA knockout mice (SMNΔ7; SMN2; Smn ^~'^~) to determine whether the compound could ameliorate survival and/or motor function in this double transgenic model of SMA. Troglitazone was dissolved in 10% DMSO and given to knockout animals by oral gavage at either 100 mg/kg or 150 mg/kg twice a day starting at P3 and continuing until the knockout animals died. Control animals received vehicle (10% DMSO in water) alone twice a day. Body weight and survival were recorded daily during the course of the study, while motor function was assessed at P6, P8, PlO, P12 and P14 using the geotaxis and tube tests.

[0052] Troglitazone at both doses showed a beneficial effect on mean survival for both female and male knockout mice (Figure 5A). Male knockout mice treated with either 100 mg/kg (16.00 ± 0.50 days) or 150 mg/kg (16.25 ± 0.68 days) twice a day showed a significant improvement in mean survival time compared to male knockout animals treated with vehicle alone (14.78 ± 0.66 days, see Figure 5B). Female knockout mice treated with 100 mg/kg twice a day had a mean survival time of 15.43 ± 0.84 days, and female knockout mice treated with 150 mg/kg twice a day had a mean survival time of 14.86 ± 1.01 days compared to vehicle -treated controls (13.10 ± 0.91 days, see Figure 5B).

Example 6

Troglitazone and erlotinib do not affect SMN expression

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66408 v2/DC [0053] To test whether troglitazone and erlotinib affected the expression of SMN protein, motor neuron and fibroblast cultures derived from wild-type and SMN deficient cells were exposed to a range of dosages of each of the two compounds.

[0054] Motor neuron cultures were prepared from both SMN-deficient mouse embryonic stem cells (A2 cells) and wild-type embryonic stem cells (HB9 cells). The SMN-deficient mouse embryonic stem cells were isolated from mice containing the human SMN2 gene in a mouse Smn knockout background (Schrank et al. (1997) Proc. Natl. Acad. Sci. USA 94: 9920-9925). Confluent plates of embryonic stem cells were dissociated, and plated in 15 cm dishes to induce embryoid body formation. On Day 2 and Day 3, cultured embryoid bodies were treated with retinoic acid (RA) and sonic hedge hog (Shh) to induce motor neuron formation. On Day 6, embryoid bodies were dissociated and transferred to 96-well plates. Motor neuron cultures were fed every three days by removing half of the media and replenishing it with Ix media supplemented with 2x the concentration of each growth factor (BDNF, GDNF, and CNTF). On Day 7, each sample of troglitazone or erlotinib was diluted in media to one of seven concentrations ranging from 0 μM to 15 μM and added to a well containing motor neurons. Motor neuron cultures were incubated with compound for either 48 or 72 hours. Following compound incubation, cell cultures were fixed with cold methanol and incubated overnight with mouse anti-SMN (1:200 dilution; Pharmingen BD) and anti-GFP antibodies conjugated to Alexa-647 (1 :1000 dilution; InVitrogen). GFP fluorescence was used to identify motor neurons as the GFP gene was under the control of the HB9 promoter. The following day, anti-mouse antibody conjugated to Alexa-Fluor 488 (1 :2000) was added to visualize SMN protein. Positive controls which corresponded to motor neuron cultures exposed to compounds known to upregulate SMN expression were also evaluated to ensure the integrity of the cultures. Samples were evaluated on an Evotec Opera automated confocal microscope. Dose response curves for each of the two compounds for both wild- type (HB9) and SMN-deficient (A2) cells were constructed by quantifying the fluorescence corresponding to SMN protein in motor neurons using image analysis software. Troglitazone and erlotinib had no effect on nuclear or cytoplasmic levels of SMN protein in either SMN-deficient or wild-type motor neurons. [0055] The effects of both of these compounds on SMN protein expression levels were also assessed in fibroblast cultures. Fibroblasts derived from a human SMA patient or their unaffected parent were plated in 384-well plates (500 cells/well), or in 96-well plates. On day 3 each

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66408 v2/DC sample of troglitazone or erlotinib was diluted in media to one of twelve concentrations ranging from 0 μM to 10 μM and added to a well containing fibroblasts. In one experiment, concentrations of up to 50 μM troglitazone were tested. Fibroblast cultures were incubated with compound for 18, 24, 48, or 72 hours. Following compound incubation cell cultures were fixed with 4% PFA or cold methanol and acetone, and incubated with mouse anti-SMN diluted 1 :8000 (Pharmingen BD) followed by incubation with anti-mouse antibody conjugated to Alexa-Fluor 488 and Hoechst dye (both diluted 1 :2000). Samples were evaluated on an Evotec Opera automated confocal microscope. Dose response curves for each of the two compounds for parent fibroblasts and SMA patient fibroblasts were constructed. Similar to the results obtained in the motor neuron cultures, troglitazone and erlotinib did not significantly increase nuclear or cytoplasmic SMN levels in the parental or patient fibroblast cell lines. These results suggest that the mechanism by which troglitazone and erlotinib improve motor function and survival in the mouse model of SMA is distinct from upregulation of SMN protein.

[0056] All publications, patent applications, and references cited herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0057] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

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Claims

Claims:

1. A method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of an epidermal growth factor receptor antagonist, wherein at least one symptom of spinal muscular atrophy is alleviated following administration.

2. The method of claim 1, wherein said epidermal growth factor receptor antagonist is erlotinib.

3. The method of claim 1, wherein said at least one symptom is selected from the group consisting of muscle weakness, muscle atrophy, motor neuron loss, decreased life expectancy, poor muscle tone, decreased deep tendon reflexes, abnormal gait, and difficulty breathing.

4. The method of claim 1, further comprising administering a pharmaceutically effective amount of a SMN modulating compound.

5. The method of claim 4, wherein said SMN modulating compound increases expression of SMN protein.

6. The method of claim 5, wherein said SMN modulating compound is selected from the group consisting of valproic acid, phenylbutyrate, sodium butyrate, hydroxyurea, trapoxin, and trichostatin A.

7. A method of treating spinal muscular atrophy in a subject in need thereof comprising administering a pharmaceutically effective amount of a motor neuron neurotrophic factor, wherein at least one symptom of spinal muscular atrophy is alleviated following administration.

8. The method of claim 7, wherein said motor neuron neurotrophic factor is troglitazone.

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9. The method of claim 7, wherein said at least one symptom is selected from the group consisting of muscle weakness, muscle atrophy, motor neuron loss, decreased life expectancy, poor muscle tone, decreased deep tendon reflexes, abnormal gait, and difficulty breathing.

10. The method of claim 7, further comprising administering a pharmaceutically effective amount of a SMN modulating compound.

11. The method of claim 10, wherein said SMN modulating compound increases expression of SMN protein.

12. The method of claim 11, wherein said SMN modulating compound is selected from the group consisting of valproic acid, phenylbutyrate, sodium butyrate, hydroxyurea, trapoxin, and trichostatin A.

13. A method of screening compounds for efficacy in treating spinal muscular atrophy comprising: i) administering at least one dose of a compound to a mouse of the double transgenic mouse model of spinal muscular atrophy (SMNA7 ^{+ +}; SMN2^{+ +}; Smn^") and ii) measuring at least one physiological parameter in said treated mouse.

14. The method of claim 13, wherein said mouse is a neonate.

15. The method of claim 13, wherein several doses of said compound are administered.

16. The method of claim 13, wherein said physiological parameters include body weight, survival, and motor performance.

17. The method of claim 16, further comprising selecting a compound that produces an increase in body weight, survival, or motor performance, wherein said selected compound is identified as a compound efficacious for treating spinal muscular atrophy.

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