EP2830620A1 - 4-aminopyridin als therapeutisches mittel für spinale muskelatrophie - Google Patents

4-aminopyridin als therapeutisches mittel für spinale muskelatrophie

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Publication number
EP2830620A1
EP2830620A1 EP13769101.0A EP13769101A EP2830620A1 EP 2830620 A1 EP2830620 A1 EP 2830620A1 EP 13769101 A EP13769101 A EP 13769101A EP 2830620 A1 EP2830620 A1 EP 2830620A1
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EP
European Patent Office
Prior art keywords
smn
neurons
motor
mutants
sma
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EP13769101.0A
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English (en)
French (fr)
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EP2830620A4 (de
Inventor
Brian Mccabe
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of EP2830620A1 publication Critical patent/EP2830620A1/de
Publication of EP2830620A4 publication Critical patent/EP2830620A4/de
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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • SMA Spinal muscular atrophy
  • SMA is an autosomal recessive disease characterized by degeneration of motor neurons in the anterior horn of the spinal cord, leading to muscular paralysis and atrophy. SMA is traditionally categorized into three types, according to the age and severity: Infantile SMA-
  • Type 1 or Werdnig-Hoffmann disease (generally 0-6 months) is the most severe form, and manifests in the first year of life resulting in an inability to ever maintain an independent sitting position.
  • Intermediate SMA-Type 2 (generally 7-18 months) describes those children who are never able to stand and walk, but who are able to maintain a sitting position at least some time in their life. The onset of weakness is usually recognized sometime between 6 and 18 months.
  • Juvenile SMA-Type 3 or Kugelberg-Welander disease (generally >18 months describes those who are able to walk at some time.
  • Adult SMA-Type 4 is associated with weakness that usually begins in late adolescence in tongue, hands, or feet then progresses to other areas of the body. The course of disease is much slower and has little or no impact on life expectancy. Additionally, for prenatal onset of very severe symptoms of SMA and early neonatal death results, SMA is categorized as type 0 (Eur J Paediatr Neurol 1999; 3:49-51; Lancet 1995; 346: 1162;
  • SMA occurs in approximately 1 in 6000-10000 live births and has a carrier frequency of 1 in 50. It is the second most common autosomal recessive inherited disorder in humans and the most common genetic cause of infant mortality (Semin Neurol 1998; 18: 19-26).
  • Linkage mapping identified the Survival of Motor Neuron (SMN) gene as the genetic locus of SMA (Lefebvre et al., Cell 80, 1-5).
  • SMA Motor Neuron
  • SMNl and SMN2 two nearly identical SMN genes exist on chromosome 5ql3. Deletions or mutations within SMNl but not the SMN2 gene cause all forms of proximal SMA (Lefebvre et al., Cell 80, 1-5).
  • SMNl encodes a ubiquitously expressed 38 kDa SMN protein that is necessary for snRNP assembly, an essential process for cell survival (Wan, L., et al. 2005. Mol. Cell. Biol. 25:5543-5551).
  • SMNl and SMN2 differ by a critical C to T substitution at position 6 of exon 7 (C6U in transcript of SMN2) (Lorson, C. L., et al. 1999. Proc. Natl. Acad. Sci. USA 96:6307-6311;
  • SMN is a multifunctional protein that has been implicated in a variety of cellular processes linked to RNA metabolism (Pellizzoni, 2007).
  • Certain embodiments of the invention are directed to methods comprising, identifying a subject who has spinal muscular atrophy, and administering to the subject a therapeutically effective amount of a K+ channel antagonist, including a broad-based K+ channel antagonist, and antagonists selected from the group comprising 4-aminopyridine, 4- (dimethylamino)pyridine, 4-(methylamino)pyridine, and 4-(aminomethyl)pyridine.
  • K+ channel antagonists for us in the present methods include dofetilide, sotalol, ibutilide),
  • the therapeutically effective amount is an amount ranging from about 0.5 mg to 100 mg per administration and the antagonist is administered from one to three times per day.
  • Other embodiments are directed to pharmaceutical formulations, comprising 4-AP and one or more of the enumerated therapeutic agents, preferably formulated for delivery across the blood brain barrier or for administration directly into the epidural venous plexus, brain, spinal column or cerebrospinal fluid.
  • the Present methods can be used to treat any form of SMA: types 1, 2 or 3.
  • FIG. 1 smn mutants have reduced muscle size, decreased locomotion, defective motor rhythm and aberrant neuromuscular junction (NMJ) neurotransmitter release.
  • A-B Sample images of muscles from segment A3 of control (A) and SMNX7 mutant (B) third instar larvae labeled with TRITC phallodin show a reduction muscle surface area (C) that is fully rescued by ubiquitous expression of UAS-flag-SMN driven by Da-Gal4 (genotype: Da- Gal4/UAS::flagSMN; S MNX7/S MNX7 ) .
  • D-F 10 sample superimposed 60 second larval locomotion path traces from control (D) and SMNX7 mutants (E).
  • smn mutant larvae have reduced velocity compared to controls that corrected by ubiquitous expression of transgenic SMN (F).
  • G-I Recordings from muscle 6 in segment Al of semi-intact larval preparations where the brain, ventral nerve cord and motor neurons are intact. Control larva produce a regular motor rhythm with periodic bursting activity corresponding to peristaltic muscle contractions (G). In contrast, smn mutant larvae have an irregular motor pattern with short and uncoordinated bursts (H) as shown by an increase in the average inter-spike interval (I) that is rescued by ubiquitous expression of SMN.
  • J-L Representative traces recorded from muscle 6 of segment A3 in control (J) and SMNX7 mutant (K) larva.
  • eEPSP evoked Excitatory Postsynaptic Potential
  • A-D. Sample images of muscles from segment A3 of (A) control, (B) SMNX7 mutant, (C) SMNX7 mutants with transgenic SMN expression*n only in muscles (G14- Gal4/UAS::flagSMN; SMNX7/ SMNX7) or (D) neurons (nsyb-Gal4/UAS::flagSMN;
  • SMNX7/SMNX7) Restoration of SMN expression in muscles has no effect on muscle size however restoration in neurons fully rescues muscle surface area.
  • FIG. 3. SMN expression is required in cholinergic neurons and not motor neurons.
  • A-D Representative traces of control (A), SMNX7 mutant (B), transgenic SMN expressed in the motor neurons of smn mutants (OK371-Gal4/UAS::SMN; S MNX7/S MNX7 ) (C), transgenic SMN expressed in the cholinergic neurons of smn mutants (Cha-Gal4/UAS::SMN;
  • SMNX7/SMNX7) (D). Expression of transgenic SMN in motor neurons does not restore normal neurotransmitter release in smn mutants however expression of SMN on cholinergic neurons restores normal eEPSP amplitude.
  • E-F Quantification of muscle surface area (E), locomotion (F), motor rhythm (G) and NMJ eEPSP amplitude (H) normalized to controls. Expression of transgenic SMN in the motor neurons of smn mutants with OK371-Gal4 or OK6-Gal4 or in GABAergic neurons with GAD1-Gal4 does not rescue any phenotype.
  • FIG. 4. SMN is required in both proprioceptive and central cholinergic neurons.
  • A. Expression of pattern cholinergic neuron Gal4 lines (dotted). Cha-Gal4 is expressed in both central and sensory cholinergic neurons. Clh201-Gal4 in only expressed in md and es sensory neurons. 1003.3-Gal4, ppk-Gal4 and ppk-Gal4 are expressed in subsets of md, es or ch sensory neurons. Diagonal hatchlines indicate the ability to rescue of smn mutant phenotypes. B,C.
  • Expression of transgenic SMN in both central and sensory cholinergic neurons in smn mutants with Cha-Gal4 fully rescues all phenotypes.
  • FIG. 5 Restoration of SMN after embryogenesis rescues smn mutants.
  • A Schematic of transgenic SMN induction in the nervous system. RU486 is required for the activation of transgene induction by geneswitch Gal4. Elav::geneswitch/UAS::flagSMN; SMNX7/ SMNX7 larva were transferred to either vehicle media or RU486 containing media immediately after hatching, 48 hours after hatching or 96 hours after hatching.
  • B Representative traces recorded from smn mutants that were cultured on either vehicle media or RU486 media 0, 48 or 96 hours after hatching. Induction of SMN at every each time -point fully restored normal eEPSP amplitude.
  • C-F Schematic of transgenic SMN induction in the nervous system. RU486 is required for the activation of transgene induction by geneswitch Gal4. Elav::geneswitch/UAS::flagSMN; SMNX7/
  • C muscle surface area
  • D locomotion
  • E motor rhythm
  • F NMJ eEPSP amplitude
  • Muscle size, locomotion and motor rhythm is fully rescued if transgenic SMN is induced immediately after hatching, but if SMN induction is delayed rescue is incomplete. In contrast, induction of SMN for only 48 hours is sufficient to completely restore normal neurotransmitter release the NMJ.
  • FIG. 6 Inhibiting cholinergic neuron activity mimics smn mutant phenotypes.
  • FIG. 7 Genetic or pharmacological inhibition of K+ channels ameliorates smn mutant phenotypes.
  • A-C Locomotion path traces from (A) control, (B) smn mutants and (C) smn mutants expressing a UAS dominant negative Shaker K+ channel (UAS-SDN) in cholinergic neurons with Cha-Gal4. Expressing SDN increases rescues the locomotion of smn mutants.
  • D-G Quantification of muscle surface area (D), locomotion (E), motor rhythm (F) and NMJ eEPSP amplitude (G) normalized to controls.
  • FIG. 8 shows that SMNX7 mutants have ⁇ 6 SMN protein levels compared to controls.
  • FIG. 9 A NMJ mEPSP amplitude of smnX7 mutants is similar to wildtype (WT) controls.
  • B NMJ mEPSP frequency is increased in smnX7 mutants compared to controls
  • C NMJ quantal content is increased in smnX7 mutants compared to controls.
  • D smnX7 heterozygous mutants have similar NMJ eEPSP amplitude to controls.
  • Transallelic combinations of smnX7 with smn73Ao or smnE33 have increased NMJ eEPSP amplitude similar to smnX7 homozygous mutants.
  • E E.
  • the present invention is based on the discovery that SMN must be restored in both proprioceptive neurons and cholinergic interneurons in order to rescue smn mutant phenotypes. It was further discovered that increasing the excitability of central cholinergic neurons in an animal model of SMA increased motor network activity and altered smn mutant phenotypes.
  • certain embodiments of the invention are directed to methods of treatment of SMA by administering therapeutically effective amounts of one or more potassium channel
  • 4-aminopyridine hereinafter as 4-AP
  • 4-AP 4-aminopyridine
  • 4-(dimethylamino)pyridine 4- (methylamino)pyridine
  • 4-(aminomethyl)pyridine herein collectively "the therapeutic agents.”
  • Other embodiments are directed to new pharmaceutical formulations comprising two or more potassium channel antagonists.
  • SMA is the most common inherited cause of infant mortality (Pearn, 1978), and it is both recessive and monogenic. SMA is characterized by motor neuron functional alterations and degeneration.
  • SMN is ubiquitously expressed and highly conserved across evolution with orthologs found in mouse, zebrafish, fruit flies, nematodes and yeast (Schmid and DiDonato, 2007). In genetic models, complete removal of all SMN protein results in loss of cell viability. In contrast, the reduced level of SMN found in SMA patients does not appear to significantly perturb the majority of organ systems (Crawford and Pardo, 1996). However, SMA patients develop motor problems and muscle weakness, with the proximal limb and trunk muscles stereotypically the most severely affected, progressing eventually to respiratory insufficiency and death (Swoboda et al., 2005). Postmortem studies show SMA patients have pathologically abnormal motor neurons and evidence of motor neuron loss (Simic, 2008), however it is currently unclear if this is the primary origin of motor system dysfunction or a terminal consequence.
  • NMJ neuromuscular junctions
  • the studies herein use the Dwsophila SMN Mutant Model to study the neurocircuitry and physiology of the central sensory neurons, peripheral sensory neurons, and motor neurons in the pathway associated with SMA.
  • Dwsophila smn mutants that have reduced muscle size and defective locomotion, motor rhythm and motor neuron neurotransmission; were exploited to determine the essential cellular site and requirement for SMN in the motor system.
  • motor neurons are exclusively glutamatergic while both peripheral sensory neurons and the majority of excitatory interneurons are cholinergic Baines, 2006; Salvaterra and Kitamoto, 2001).
  • Robust phenotypic rescue of Dwsophila smn mutants was produced by genetic inhibition of Voltage Gated Potassium Channels (Kv channels) which increases the amplitude and duration of synaptic neurotransmitter release.
  • Kv channels Voltage Gated Potassium Channels
  • Proprioceptive neurons provide essential inputs to motor circuits (Hughes and Thomas, 2007) and cholinergic interneurons are critical for Dwsophila CNS function (Kitamoto et al., 2000), including synaptic output onto motor neurons (Baines et al., 2001).
  • Restoration of SMN after the completion of nervous system development is sufficient to rescue SMN-dependent phenotypes, arguing that is not the connectivity but rather the function of motor circuits that is disrupted by the depletion of SMN.
  • Two lines of evidence further support this. Firstly, inhibiting the activity of cholinergic neurons can mimic a number of smn mutant phenotypes including non-autonomous effects on motor neurons.
  • Example 1 validate the Drosophila model and show also that Drosophila smn mutants have increased NMJ evoked neurotransmitter release that is accompanied by defects of muscle growth, locomotion and motor rhythm. (.FIGs 1 and 2).
  • Example 2 shows that in contrast to muscle restoration of SMN, pan- neuronal restoration of SMN fully rescued the muscle surface area of smn mutants to control levels (FIG. 2 B,D,E) and also completely restored their locomotor velocity, rhythmic motor output and NMJ eEPSP amplitudes (FIG. FIG. 2F-H). These results showed that the defects of muscle growth in smn mutant larvae are due to a non-autonomous requirement for normal SMN levels in the nervous system rather than in muscle fibers themselves.
  • Example 3 The results in Example 3 show that SMN is required in cholinergic neurons and not in glutaminergic motor neurons, and that SMN is required in both proprioceptive and central cholinergic neurons.
  • Expression of transgenic SMN levels in central cholinergic neurons completely rescued the muscle growth, locomotion and rhythmic activity defects of smn mutants (FIG. 3E-G).
  • expression of SMN in cholinergic neurons also fully rescued eEPSP amplitudes at the NMJ terminals of smn mutants to control levels (FIG. 3D, H).
  • FIG. 3D, H control levels
  • expression of SMN only in cholinergic neurons is sufficient to fully rescue smn mutant phenotypes and can nonautonomously rescue the SMN-dependent defects of both motor neurons and muscles.
  • neurotransmitter release properties of motor neurons consistent with cholinergic sensory neurons in the motor circuit having reduced function in smn mutants.
  • Example 4 Building upon the hypothesis that motor circuits have functional deficits in smn mutants, experiments were designed to test whether increasing the excitability of central cholinergic neurons in these animals could increase motor network activity and alter smn mutant phenotypes. The results of various experiments showed that pharmacological inhibition of K+ channels (using the FDA-approved broad-spectrum K+ channel antagonist 4-AP) positively benefitted smn mutant phenotypes, a result that is consistent with the defective excitability of motor circuits by their intemeuron or sensory neuron inputs being a critical consequence of SMN depletion.
  • K+ channels using the FDA-approved broad-spectrum K+ channel antagonist 4-AP
  • results presented here establish that restoration of SMN in at least two groups of motor circuit neurons (bd and type I md sensory neurons) results in a full rescue of larval phenotypes.
  • the bd and type I md sensory neurons are essential components of a proprioceptive sensory feedback circuit necessary for coordinated contractile locomotion of Drosophila larvae (Hughes and Thomas, 2007).
  • Both the bd and type I md subsets of sensory neurons express the mechanosensitive NompC mechanosensitive NompC TRP channel that is essential for proprioception (Cheng et al., 2010).
  • cholinergic neurons have a particular and conserved sensitivity to the reduced levels of SMN.
  • Treatment with 4-AP has been linked to improvement in function in patients with spinal cord injury, myasthenia gravis and Lambert-Eaton syndrome (Hayes, 2007) and can improve muscle twitch tension in a canine hereditary motor neuron disease (Pinter et al., 1997).
  • a sustained release preparation of 4-AP was recently approved by the FDA for human clinical use in multiple sclerosis (Chwieduk and Keating, 2010). However, until the present discovery, it was not known that SMA involved central sensory neurons at any level.
  • SMA can be treated by administering therapeutically effective amounts of 4-AP (or biologically active derivatives or variants thereof) formulated to cross the blood brain barrier.
  • Other potassium channel antagonists can be used in the present invention, including 4-(dimethylamino)pyridine, 4-(methylamino)pyridine and 4- (aminomethyl)pyridine, to treat SMA, either alone or in combination with one another.
  • the therapeutic agents can be administered on the same day or on different days, as discussed below in “pharmaceutical formulations.”
  • K+ channel antagonists for use in the present invention include Dofetilide, Sotalol, Ibutilide (which is approved by the Food and Drug Administration for acute conversion of atrial fibrillation to sinus rhythm), Azimilide, Bretylium. Clo ilium. E-4Q3L Nit ' ekalant. Tedisamil. and Sematilide.
  • Certain other embodiments are directed to formulations of more than one therapeutic agent, including formulations that optimize the ability of the drugs to cross the BBB.
  • 4-Aminopyridine is also known as INN fampridine and dalfampridine (Acorda).
  • 4-AP is an organic compound with the chemical formula C 5 H 4 N-NH 2 .
  • the molecule is one of the three isomeric amines of pyridine.
  • 4-AP is a relatively selective blocker of members of Kvl (Shaker. KCNA) family of voltage-activated K+ channels. At concentration of 1 mM it selectively and reversibly inhibits Shaker channels without significant effect on other sodium, calcium, and potassium conductances.
  • Fampridine (4-AP) has also been used clinically in patients with spinal cord injury, myasthenia gravis and Lambert-Eaton syndrome (Hayes, 2007), and can improve muscle twitch tension in a canine hereditary motor neuron disease (Pinter et al., 1997). It is a broad based potassium channel antagonist that prolongs action potentials and consequently increases neurotransmitter release from neurons. The drug has also been shown to reverse tetrodotoxin toxicity in animal experiments. MS patients treated with 4-AP exhibited a response rate of 29.5% to 80%. A long-term study (32 months) indicated that 80-90% of patients who initially responded to 4-AP exhibited long-term benefits.
  • Treatment with 4-AP has been linked to improvement in function in patients with spinal cord injury, myasthenia gravis and Lambert- Eaton syndrome (Hayes, 2007) and can improve muscle twitch tension in a canine hereditary motor neuron disease (Pinter et al., 1997).
  • Therapeutically effective amounts will vary depending on various factors including: 1. whether one or more than one agents is administered; 2. the efficacy of the agent, alone or combined with other agents; 3. the type of formulation, such as a sustained-release formulation that may have higher amounts since the drug is released slowly; 4. the age of the patient, severity of the disease; 5. the frequency of administration; and 6. the individual subject's tolerance of and response to the agent.
  • multiple therapeutically effective doses of one or more the therapeutic agents are administered in a single day, or over the course of weeks or months, as needed to ameliorate one or more symptoms of the disease.
  • the therapeutic agents can be administered individually (i.e. treatment with only one specific agent), or more than one agent can be administered either separately or in combination.
  • the agents can be administered once or more than once per day.
  • the therapeutically effective amounts of the different agents may vary depending on the specific agent and whether the agent is administered alone or together with another agent. The therapeutically effective amount will also vary based on the particular formulation.
  • compositions for use in the present methods include therapeutically effective amounts of one or more of the therapeutic agents, i.e., an amount sufficient to prevent or treat the diseases described herein in a subject, formulated for local or systemic
  • the subject is preferably a human but can be non-human as well.
  • a suitable subject can be an individual who is suspected of having, has been diagnosed as having, or is at risk of developing one of the described diseases.
  • Active agents for therapeutic administration are preferably low in toxicity and cross the blood brain barrier. The progress of this therapy is easily monitored by conventional techniques and assays that may be used to adjust dosage to achieve a desired therapeutic effect.
  • a composition of the therapeutic agents can also include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antiviral agents, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds can also be incorporated into the compositions.
  • Other topical formulations are described in Sheele et al., 7,151,091.
  • Therapeutic compositions may contain, for example, such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain l%-95% of active ingredient, preferably 2 -70 active ingredient.
  • the therapeutic agents can also be mixed with diluents or excipients which are compatible and physiologically tolerable as selected in accordance with the route of
  • compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
  • the therapeutic compositions of the present invention are prepared either as liquid solutions or suspensions, or in solid forms, preferably for oral administration.
  • the formulations may include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • Solutions, suspensions, or sustained release formulations typically contain 1 %-95% of active ingredient, preferably 2%-70%.
  • the formulations may also contain more than one therapeutic agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the therapeutic agents, which matrices are in the form of shaped articles, e.g., films, or microcapsule.
  • sustained release matrices include, but are not limited to, polyesters, hydro gels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT (injectable micro spheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
  • the therapeutic agents of the present invention may be formulated for administration by any suitable means as long as they cross the blood brain barrier (BBB).
  • BBB blood brain barrier
  • o Cerebral vasodilatation stimulation of the sphenopalatine ganglion, nitric oxide inhalation
  • An alternative for delivering drugs to the brain is direct administration to the brain or spinal cord, thus bypassing the BBB. This can be accomplished for example by injection into epidural venous plexus or direct introduction of drugs into the brain, spinal column or the cerebrospinal fluid (CSF). Drug pumps could facilitation continual administration of the therapeutic agents in the methods of the present invention.
  • CSF cerebrospinal fluid
  • Liposomes can be useful in delivery of small molecules to the brain.
  • Particularly useful liposomes can be generated by, for example, the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
  • phosphatidylethanolamine PEG-PE
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Polypeptides of the present invention can be conjugated to the liposomes as described in, for example, Werle et al., Int. J. Pharm. 370(1-2): 26-32 (2009).
  • the pharmaceutical compositions are preferably administered orally or parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. Stadler, et al., U.S. Pat. No. 5,286,634.
  • the appropriate dosage will depend on the severity of the disease, whether the drug is administered for protective or therapeutic purposes, previous therapy, the patient's clinical history and response to the drugs and the discretion of the attending physician.
  • the resulting pharmaceutical preparations may be sterilized by conventional, well known sterilization techniques.
  • the aqueous solutions can then be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the lipidic suspension may include lipid-protective agents which protect lipids against free- radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as a- tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • compositions of this invention may be in a variety of forms, which may be selected according to the preferred modes of administration. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • compositions of this invention may, for example, be placed into sterile, isotonic formulations with or without cofactors which stimulate uptake or stability.
  • the formulation is preferably liquid, or may be lyophilized powder.
  • the compositions of the invention may be diluted with a formulation buffer comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20.
  • This solution can be lyophilized, stored under refrigeration and reconstituted prior to administration with sterile Water-For-Injection (USP).
  • Suitable Solvates include Hydrates.
  • Suitable salts include those formed with both organic and inorganic acids or bases.
  • Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexylamine and N- methyl-D-glucamine.
  • Formulations of use in the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Therapeutic agents of the present invention may be administered simultaneously meaning the administration of medicaments such that the individual medicaments are present within a subject at the same time.
  • simultaneous administration may include the administration of the medicaments (via the same or an alternative route) at different times.
  • the terms "individual,” “subject,” and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • a “subject” as used herein generally refers to any living multicellular organism.
  • Subjects include, but are not limited to animals ⁇ e.g., cows, pigs, horses, sheep, dogs and cats) and plants, including hominoids ⁇ e.g., humans, chimpanzees, and monkeys).
  • the term includes transgenic and cloned species.
  • the term "patient” refers to both human and veterinary subjects.
  • administering shall mean delivering in a manner which is affected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • Administering can be performed, for example, orally, or intravenously, via implant,
  • transmucosally transdermally, intradermally, intramuscularly, subcutaneously, or
  • the phrase "therapeutically effective amount” means an amount sufficient to produce a therapeutic result.
  • the therapeutic result is an objective or subjective improvement of a disease or condition, achieved by inducing or enhancing a physiological process, blocking or inhibiting a physiological process, or in general terms performing a biological function that helps in or contributes to the elimination or abatement of the disease or condition. For example, eliminating or reducing or mitigating the severity of a disease or set of one or more symptoms.
  • the full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
  • Treating means taking steps to obtain beneficial or desired results, including clinical results, such as mitigating, alleviating or ameliorating one or more symptoms of a disease; diminishing the extent of disease; delaying or slowing disease progression; ameliorating and palliating or stabilizing a metric (statistic) of disease.
  • Treatment refers to the steps taken.
  • Mitigating means reducing or ameliorating a disease or symptom of a disease.
  • mitigation can be achieved by administering a therapeutic agent before the phenotypic expression of the disease (i.e. prior to the appearance of symptoms of the disease)
  • Mitigation includes making the effects of disease less severe by avoiding, containing, reducing or removing it or a symptom of it.
  • Mitigating an enumerated disease as described herein comes within the definition of "treating” an enumerated disease before symptoms occur. Amounts of therapeutic agents that mitigate a disease are herein referred to as "therapeutically effective amounts.”
  • smnKl (Chang et al., 2008) is small deletion that removes the entire smn coding region from 93 bp upstream of the transcription start site up to the last 44 bp of the 3' UTR without disrupting other loci.
  • the number of homozygous smnKl mutants that survive to the third instar is few consistent with previous reports (Chang et al., 2008), however if stocks were cultured at low density, an increase the numbers that survived to this stage could reliably be identified.
  • smn73Ao is a strong loss-of-function point mutant allele that produces an unstable
  • SMN73Ao allele which reported a decrease in evoked neurotransmitter release at the NMJ of these animals (Chan et al., 2003). This finding was replicated in one SMN73Ao stock (B#4802) , however, this defect could not be rescued by transgenic SMN (data not shown).
  • a different SMN73Ao stock (Gift from Greg Matera, UNC), confirmed to have low SMN, and which had gone through multiple backcrosses to a wild-type background had an similar increase in eEPSP amplitude to smnKl and other smn mutant alleles both in homozygous and in transheterozygous combinations (Supplementary Figure 2D).
  • UAS-PLTXII Membrane tethered PLTXII assembled from N to C terminus as a secretory signal sequence, the mature cleaved PLTX-II peptide sequence, a hydrophilic linker sequence with an embedded c-Myc epitope tag, and a GPI targeting sequence (B.C, Michael Nitabach and BDM unpublished) Gal4 lines: nsyb-Ga ⁇ 4 (Bushey et al., 2009), OK6-Gal4, G14-Gal4 (Aberle et al., 2002), OK371- Gal4 (Mahr and Aberle, 2006), Actin-Gal4 (Ito et al, 1997), Cha-Gal4 (
  • Spontaneous motor rhythm was recorded as previously described (Fox et al., 2006). Briefly, recordings were made from muscle 6 of abdominal segment Al in standard saline (Jan and Jan, 1976) where CNS and motor neurons were intact. To measure the average inter event interval, the peak detection feature of MiniAnalysis (Synaptosoft, Inc.) was used detect all spontaneous eEPSPs events that occurred over a 3 minute period. Locomotion: Assays were essentially performed as previously described (Suster and Bate, 2002). Briefly, single larva were placed on 1% agarose plates on a lightbox at 25°C and 70% humidity.
  • Drosophila stocks smnXl (Chang et al., 2008), smn73Ao (Chan et al., 2003), smnE33 (Rajendra et al., 2007).
  • Muscle measurement Muscle area measurements were carried out at muscle 6 of segment A3 of phallodin stained muscle fillet preparations (Brent et al., 2009).
  • Locomotion Larval locomotion assays were essentially performed as previously described (Suster and Bate, 2002) (see supplemental information).
  • Motor rhythm Spontaneous motor rhythm was recorded as previously described (Fox et al., 2006). To measure the average inter-spike interval, the peak detection feature of
  • NMJ electrophysiology Intracellular recordings from muscle 6, segment A3 were performed as previously described (Imlach and McCabe, 2009).
  • Drosophila smn mutant larvae were smaller than control animals. In order to examine if this was associated with a reduction in muscle size, smn mutant and control larval muscles were labeled with Phalloidin. The results showed that smn mutants had a 46% (P ⁇ 0.001) reduction in muscle surface area compared to controls (FIG. 1A-C, see also Table S I). This defect was fully rescued by ubiquitous expression of transgenic SMN. Smn mutant larvae were sluggish and moved less frequently than controls. To quantify this defect, video capture and tracing software was used to measure the locomotion of smn mutant and control larvae.
  • results showed that smn mutants showed a 63% (P ⁇ 0.001) decrease in locomotion velocity compared to control animals which was restored to control levels by ubiquitous expression of transgenic SMN (FIG. 1D-F).
  • Drosophila with low levels of SMN have muscle and locomotion defects.
  • Locomotion of Drosophila larvae has been linked to the rhythmic activity of segmental central pattern-generating networks (CPGs) in the ventral nerve cord (VNC) (Fox et al., 2006) which receive inputs from both the brain hemispheres (Cattaert and Birman, 2001) and proprioceptive sensory neurons (Cheng et al., 2010; Hughes and Thomas, 2007; Song et al., 2007) and output activity to motor neurons.
  • CPGs segmental central pattern-generating networks
  • VNC ventral nerve cord
  • Example 2 Restoration of SMN in the nervous system rescues smn mutant phenotypes.
  • SMN restoration was tested only in the nervous system of smn mutants using the neuron- specific nsyb-Gal4 driver.
  • pan-neuronal restoration of SMN fully rescued the muscle surface area of smn mutants to control levels (FIG. 2 B,D,E) and also completely restored their locomotor velocity, rhythmic motor output and NMJ eEPSP amplitudes (FIG. 2F-H).
  • Example 3 SMN is required in cholinergic neurons and not in motor neurons.
  • the Drosophila VNC like the human spinal cord, is populated by neurons with diverse neurotransmitter expression. All Drosophila motor neurons in addition to a subset of central interneurons are glutamatergic (Daniels et al., 2008). Because there are presynaptic defects in neurotransmitter release at the NMJ in smn mutants, the ability of transgenic SMN expression in motor neurons to rescue smn mutants was tested. OK371-Gal4 was used as an enhancer trap inserted in the vesicular glutamate transporter promoter to express transgenic SMN only in the glutamatergic neurons of smn mutants.
  • Inhibitory inputs are important regulators of motor circuit function (Featherstone et al., 2000) so the glutamic acid decarboxylase 1 promoter Gal4 was used to restore of SMN in gabaergic neurons, however no significant rescue of any smn mutant phenotype was seen (FIG. 3EH).
  • the majority of excitatory neurons in the Drosophila nervous system are cholinergic (Salvaterra and Kitamoto, 2001) and motor neurons receive synaptic input from cholinergic neurons (Baines, 2006). Therefore transgenic SMN was restored in smn mutants using choline acetyltransferase (Cha) promoter-driven Gal4.
  • SMN is required in both proprioceptive and central cholinergic neurons
  • Drosophila larval sensory neurons in addition to the majority of excitatory central neurons are cholinergic (Salvaterra and Kitamoto, 2001). To dissect the requirement for normal SMN levels between these two populations, the ability of transgenic SMN expression in sensory neurons alone to rescue smn mutant phenotypes was determined. Drosophila sensory neurons are categorized into three major types - multiple dendrite neurons (md) of which there are 5 subclasses (bd, I, II, III and IV), external sense organ neurons (es) and chordotonal neurons (ch). A panel of sensory neuron Gal4 drivers (FIG. 4A) was used to restore SMN only in major types of sensory neurons.
  • md multiple dendrite neurons
  • es external sense organ neurons
  • ch chordotonal neurons
  • SMN expression in the nervous system of smn mutants was delayed until progressively later larval stage.
  • transgenic SMN was induced in smn mutants at 48 or 96 hours after embryo hatching, intermediate phenotypes were found where muscle volume, motor rhythm defects and locomotion where only partially restored compared to controls (FIG. 5C-D).
  • NMJ eEPSP amplitudes were completely restored in smn mutants to control levels by only 48 hours of SMN expression (FIG. 5B,F).
  • Inhibiting cholinergic neuron activity mimics aspects of SMN depletion.
  • PLTXII which inhibits synaptic N-type voltage gated calcium channels
  • B.C. Michael Nitabach and B.D.M, unpublished.
  • Kir2.1 reduced eEPSP amplitudes by 32% (P ⁇ 0.001) while expression of PLTXII reduced eEPSP amplitudes by 96% (P ⁇ 0.001) indicating the both transgenes were capable of partially inhibiting neurotransmission.
  • Kir2.1 in choliner.gic neurons produced a 50% increase (P ⁇ 0.001) in NMJ eEPSP amplitudes while expression of PLTX induced a 45% increase (P ⁇ 0.001) (FIG. 6F). Therefore, inhibition of cholinergic neuron activity replicated a number of the features of smn mutants including non-cell autonomous effects on the neurotransmitter release properties of motor neurons, consistent with cholinergic neurons in the motor circuit having reduced function in smn mutants.
  • Example 4 Increasing neuronal excitability rescues smn mutant phenotypes.
  • 4-aminopyridine (4-AP), an FDA approved small molecule inhibitor of voltage activated vertebrate (Hayes, 2007) and Dwsophila K+ channels (Wicher et al., 2001) was tested. 4-AP was added to larval media and titrated the compound to identify the maximum dosage at which the drug could be tolerated without lethality in wild-type larvae (2 mM). The effects of exposure of 4-AP throughout the larval period was examined fin both control and smn mutants.
  • Postsynaptic PKA controls quantal size and reveals a retrograde signal that regulates presynaptic transmitter release in Drosophila. Neuron 20, 305-315.
  • Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse. Neuron 27, 71-84.
  • Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect.
  • the Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124, 761-771.
  • SMNDelta7 the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum Mol Genet 14, 845-857.
  • Amyloid-beta-induced neuronal dysfunction in Alzheimer's disease from synapses toward neural networks. Nat Neurosci 13, 812-818.
  • SSN motor neuron
  • Non-synaptic ion channels in insects basic properties of currents and their modulation in neurons and skeletal muscles. Prog Neurobiol 64, 431-525.

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