AU2003200441B2 - Compositions and methods for modulating neural sprouting - Google Patents

Description

Our Ref:7776281 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Address for Service: Invention Title: The following statement is a performing it known to me:- Allergan Sales, Inc.

2525 Dupont Drive Irvine California 92612 United States of America DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Compositions and methods for modulating neural sprouting full description of this invention, including the best method of IP Australia Documents were received on: 1 1 FEB 2003 M atcn No: COMPOSITIONS AND METHODS FOR MODULATING NEURAL SPROUTING This application is a divisional of parent application No. 37484/99.

Field of the Invention The present invention is directed towards methods and compositions for inhibiting neural sprouting in neurons that have been subjected to botulinum toxin- Also disclosed are methods and compositions for extending the period of time during which treatment of nerve cells with botulinum toxin is effective to prevent innervation of a cell or tissue, such as muscle cells or tissue. Such methods and compositions are effective in the treatment of spasms or muscular tetanus. Disclosed as well are meth6ds and compositions for stimulating neural outgrowth.

Background of the invention Neurotoxins, such as those obtained from Clostridium botulinum and Clostridium tetanus, are highly potent and specific poisons of neural cells. These Gram positive bacteria secrete two related but distinct toxins, each comprising two disulfide-linked amino acid chains: a light chain of about 50 KDa and a heavy chain of about 100 KDa, which are wholly responsible for the symptoms of these diseases.

The tetanus and botulinum toxins are among the most lethal substances known to man, having a lethal dose in humans of between 0.1 ng and 1 ng per kilogram of body weight. Tonello et al., Adv. Exp. Med. Biol. 389:251-260 (1996). Both toxins function by inhibiting neurotransmitter release in affected neurons. The tetanus neurotoxin (TeNT) acts mainly in the central nervous system, while botulinum neurotoxin (BoNT) acts at the neuromuscular junction by inhibiting acetylcholine release from the axon of the affected neuron into the synapse, resulting in a localized flaccid paralysis. The effect of intoxication on the affected neuron is long-lasting and has been thought to be irreversible.

The tetanus neurotoxin (TeNT) is known to exist in one immunologically distinct type; the botulinum neurotoxins (BoNT) are known to occur in seven different immunogenic types, termed BoNT/A through BoNT/G. While all of these types are produced by isolates of C. botulinumn, two other species, C. baratii and C. butyricum also produce toxins similar to /F and respectively. See Coffield et al., The Site and Mechanism of Action of Botulinum Neurotoxin in Therapy with Botulinum Toxin 3-13 (Jankovic J.

Hallett M. eds. 1994), the disclosure of which is incorporated herein by reference.

Regardless of type, the molecular mechanism of intoxication appears to be similar. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain and a cell surface receptor; the receptor is thought to be different for each type of botulinum toxin and for TeNT. The carboxy terminus of the heavy chain appears to be important for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This last step is thought to be mediated by the amino terminus of the heavy chain, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower.

Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin then translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy and light chain. The entire toxic activity of botulinum and tetanus toxins is contained in the light chain of the holotoxin; the light chain is a zinc endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmittercontaining vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. TxNT, BoNT/B BoNT/D, BoNT/F, and BoNT/G cause degradation of synaptobrevin 2 (also called vesicleassociated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.

BoNT/A and /E selectively cleave the plasma membraneassociated protein SNAP-25; this protein is bound to and present on the cytosolic surface of the plasma membrane.

BoNT/C cleaves syntaxin, an integral protein having most of its mass exposed to the cytosol. Syntaxin interacts with the calcium channels at presynaptic terminal active zones.

See Tonello et al., Tetanus and Botulism Neurotoxins in Intracellular Protein Catabolism 251-260 (Suzuki K Bond J. eds. 1996), the disclosure of which is incorporated by reference as part of this specification. BoNT/C also cleaves SNAP-25 at a peptide bond next to that cleaved by BoNT/A.

Both TeNT and BoNT are taken up at the neuromuscular junction. BoNT remains within peripheral neurons, and blocks release of the neurotransmitter acetylcholine from these cells. Through its receptor, TeNT enters vesicles that move in a retrograde manner along the axon to the soma, and is discharged into the intersynaptic space between motor neurons and the inhibitory neurons of the spinal cord. At this point, TeNT binds receptors of the inhibitory neurons, is again internalized, and the light chain enters the cytosol to block the release of the inhibitory neurotransmitters 4-aminobutyric acid (GABA) and glycine from these cells. Id.

Because of its specifically localized effects, dilute preparations of BoNT have been used since 1981 as therapeutic agents in the treatment of patients having various spastic conditions, including strabismus (misalignment of the eye), bephlarospasm (involuntary eyelid closure) and hemifacial spasm. See Borrodic et al., Pharmacology and Histology Botulinum Toxin in Therapy with Botulinum Toxin 3-13 (Jankovic J. Hallett M. eds. 1994), incorporated by reference herein. Of the seven toxin types, BoNT/A is the most potent of the BoNTs, and the most well characterized. Intramuscular injection of spastic tissue with dilute preparations of BoNT/A has been also used effectively to treat spasticity due to brain injury, spinal cord injury, stroke, multiple sclerosis and cerebral palsy.

The extent of paralysis depends on both the dose and dose volume delivered to the target site. Typically, the neurotoxin is administered in a preparation that also contains several non-toxic proteins as well, including hemagglutins and associated glycoproteins that assist in maximizing its stability and presentation to the target motor neuron.

Typically there is a 24 to 72 hour delay between the administration of the toxin and the onset of the clinical effect. Exposure to the toxin causes denervation atrophy.

See Dutton Acute and Chronic Effects of Botulinum Toxin in the Management of Blepharospasm, in Therapy with Botulinum Toxin at 199, incorporated by reference herein.

Although the therapeutic application of BoNT has been extraordinarily effective, what side effects have been observed are mainly immediate effects associated with the treatment event itself. Peripheral effects causing weakness in adjacent muscles may sometimes occur; these effects usually do not persist beyond 1 to two weeks. The specific manifestations of these effects upon adjacent muscle groups depend upon the particular indication being treated. For example, patients treated for blepharospasm sometimes experience ptosis, and swallowing problems may occur after injection of neck muscles for torticollis.

Other possible consequences of treatment include the potential for overdosage through miscalculation or differences in activity between different preparations of the toxin, generalized fatigue, and the potential for allergic reactions.

A feature of treatment with BoNT/A, and other clostridial neurotoxin types, is that the paralytic action is temporary with symptoms reappearing in patients within a few months after toxin injection. This characteristic has been thought to be associated with the observed sprouting of nascent, synaptically active processes at the neuromuscular junction (NMJ). The production of such sprouts following BoNT/A therapeutic treatment has appeared to contribute to the reinervation of the treated tissue and therefore the need for repeated serial injections of the toxin.

The duration of paralytic action caused by clostridial neurotoxin and the extent of sprouting, appears to depend on the neurotoxin subtype studied and therefore appears to be related to the specific SNARE target cleaved by each neurotoxin. Thus, BoNT/A and BoNT/C,, which cleave the t- SNARE protein SNAP-25 within one amino acid of each other, cause a long lasting paralysis with a long average sprout length observed. BoNT/F, which cleaves the v-SNARE protein VAMP, causes a paralysis of shorter duration and sprouts of shorter average length. BoNT/E, which cleaves SNAP-25 at a different position than that of BoNT/A and BoNT/C, (thereby liberating a SNAP fragment of different size from the plasma membrane), has a short duration period of about 5 days, and virtually no neural sprouting is observed. Without wishing to be bound by theory, these observations suggest that neural sprouting and duration of paralysis are normally related events, and that the cleavage products of BoNT proteolytic digestion the liberated fragment or the membrane-bound fragment) can directly or indirectly regulate, or be coregulated with, neural sprouting.

It would therefore be advantageous to design a method whereby the sprouting phenomenon may be uncoupled from duration of therapeutic effect, and thus delayed, blocked or attenuated so as to prolong the effects of injection of tissue with toxin. Although the experiments described herein utilize a BoNT/A preparation, it will be recognized by those of skill in the art that the methods shown herein will be suitable for employment using other clostridial toxins, such as BoNT/B through and TeNT, in which a sprouting pathway can be observed.

Additionally, it would be advantageous to provide herein compositions effective for the inhibition or prevention of the sprouting phenomenon. Such compositions and methods would lessen the need for patients to undergo repeated neurotoxin treatment.

Summary of the Invention The present invention concerns methods for increasing the period of time between therapeutic treatments of neural tissue with a clostridial neurotoxin; thus the method provides a method of increasing the effectiveness of such treatments. A direct advantage of such methods is an increased therapeutic "life" and a concomitant lessening in the required frequency of treatment of the patient with neurotoxin. Reducing frequency of treatment would provide less opportunity for a patient to experience the side effects described above that are observed following treatment, but which tend to subside long before the effectiveness of the toxin in the target area has subsided.

Additionally, reduced frequency of treatment provides less opportunity for miscalculation of dosage amount and other treatment-specific risks.

Accordingly, an aspect of the invention concerns a composition comprising a first agent comprising a clostridial neurotoxin for use as a therapeutic agent and a second agent able to extend the duration of therapeutic benefit of said first agent, wherein the second agent is effective to attenuate the production of nerve terminal sprouts following treatment of a neuromuscular junction with the clostridial neurotoxin.

In a particular embodiment of this aspect of the invention, the first and second agent may comprise a single entity which is provided the patient in a single treatment session. For example, the entity may comprise a single io molecule, or a disulfide-linked multichain polypeptide.

Additionally or alternatively, the entity may comprise one or more adsorbed or linked heterogroup, such as a small organic molecule or a nucleic acid linked thereto. The entity preferably comprises both the receptor binding and translocation activities of a clostridial heavy chain and an active portion of a clostridial toxin light chain. The light chain may also comprise an auxiliary enzymatic activity, such as a ribonuclease, which specifically cleaves a nucleic acid encoding an intraneuronal factor which is responsible for the expression, activation and/or secretion of neurotrophic factors or cell adhesion molecules. In a preferred embodiment, such an auxiliary activity is provided by a ribozyme. By ribozyme is meant a nucleic acid or nucleic acid analog having a sequence-specific nuclease activity; the construction and use of ribozymes are well known in the art; see Cech, Science 236:1532- 1539(1987); Cech, T. Curr. Opin. Struct. Biol. 2:605-609 (1992); and Usman et al., Nucleic Acids Mol. Biol. 10:243- 264 (1996), the disclosures of which are hereby incorporated by reference herein. By nucleic acid analog is meant a polymeric molecule able to form a sequence-specific hybrid with a target single-stranded nucleic acid; such analogs may contain modified nucleotides (or ribonucleotides) such as methyl nucleotides, phosphorothioate modified nucleotides, methylphosphonate nucleotides, or nucleotide bases separated by a peptide-like bond.

Alternatively, a nucleic acid or nucleic acid analog 0o comprised in the single entity referred to above may be an antisense agent able to selectively bind to a nucleic acid encoding an intraneuronal factor which is responsible for the expression, activation and/or secretion of neurotrophic factors or cell adhesion molecules. This antisense agent may further provide a double-stranded substrate for the action of an intracellular RNAse H activity. Details concerning certain embodiments of these aspects of the invention are contained in Dolly et al., International Publication No. W095/32738, entitled Modification of Clostridial Toxins for Use as Transport Proteins and Uherek et al., J. Biol. Chem. 273:8835-8841 (1998). These two references are incorporated by reference as part of the present application.

A nucleic acid moiety linked polypeptide portion of the entity may encode a protein or polypeptide having the ability to be expressed within a neuron and to directly or indirectly regulate the expression, activation and/or secretion of neurotrophic factors or cell adhesion molecules.

In preferred aspects of the invention, the second agent is selected from the group consisting of agents able to compete with, down-regulate, or neutralize the effects of: IGFI, IGF II, a neurotrophic factor, leukemia inhibitory factor, a nerve cell adhesion molecule and neural agrin. In a more preferred aspect of the invention, the neurotrophic factor is selected from the group consisting of: ciliary neurotrophic factor, NT-3, NT-4, and brain-derived neurotrophic factor and/or the nerve cell adhesion molecule is selected from the group consisting of tenascin-C, ninjurin, neural cell adhesion molecule.

Applicants have surprisingly discovered that recovery of neural function following poisoning of nerve terminals with clostridial neurotoxin involves two distinct and apparently coordinated events. First, the poisoned endplate becomes synaptically inactive. Shortly thereafter the endplate elaborates thin nascent axon neural processes.

These processes or "sprouts" are synaptically competent after about 14 days following treatment with clostridial neurotoxin. The sprouts continue growing, reaching a maximal length and level of complexity after about 42 days following treatment with neurotoxin. During this time, the endplate remains synaptically inactive.

Secondly, after about 42 days, the sprouts begin to regress, shortening in length and decreasing in complexity.

At the same time, the original endplate begins to become synaptically active, undergoing synaptic vesicle turnover.

The increase in such turnover reaches that of the unpoisoned endplate after about 91 days post-treatment, at approximately the same time that the sprouting phenomenon has completely regressed, and no sprouts can be observed.

These findings are reported in DePaiva et al., Proc. Nat'l.

Acad. Sci. 96:3200-3205 (March 1999), which is hereby incorporated by reference herein.

These observations are diametrically opposed to the prevailing wisdom in the art, in which it has largely been assumed that the original endplate is permanently inactivated upon treatment with clostridial toxin, and that all return of synaptic activity is due to extension of sprouts to compensate for the lack of a neurologically functional endplate. However, as indicated, the old paradigm has been shown by the present Applicants to be in error, in that the poisoned endplate regains neurological function over time, while the axon sprouts regress so that after a given time period the nerve terminal appears essentially as it did prior to treatment. Therefore, Applicants have discovered that, far from being a permanent feature, the axonal sprouts assume a temporary role in the rehabilitation of the poisoned endplate.

Although not wishing to be limited by theory, Applicants believe that these results indicate that the two events outlined above are temporally coordinated, in that a blockage of the neural sprouting phenomenon would delay or block the recovery of the functional endplate. Such temporal coordination could be due to the secretion of one or more factor by the damaged neural endplate (or the inactive muscle fiber) that has neurotrophic effects resulting in the formation of neural sprouts; these sprouts may then elaborate a factor (either the same or different from the first factor) which promotes continued sprouting.

This factor may be produced during neurite sprouting in amounts sufficient for the reinervation of the neurotoxindamaged endplate even after treatment with clostridial toxin. Thus, treatment of cells with an agent able to block neural sprouting would also delay or otherwise attenuate the ability of the treated endplate to experience return of neurological function, and in fact may well block such return altogether.

As indicated, the signaling event indicating the initiation of the neural sprouting phenomenon appears to be mediated by a cytokine or other intercellular messenger. One such agent, agrin, appear to be an important player, if not the key molecule, in the formation of the neuromuscular junction in development, and in neuromuscular regeneration.

See Ruegg M.A. and Bixby J. Trends in Neurol. Sci.

21:22-27 (1998), the disclosure of which is incorporated herein by reference. Agrin appears to be present in a number of isoforms, which result from alternative mRNA splicing.

Soluble agrin isolated from synaptic basal lamina extracts (to which it binds following secretion) is able to induce the aggregation of acetylcholine receptors in the postsynaptic portions of muscle cells. Agrin present in motor neuron terminals (n-agrin) contains an insert, relative to other agrin species, in a region termed the B/z region; this insert is important in conferring the ability on n-agrin to aggregate acetylcholine receptors in postsynaptic tissue. Neural agrin is released by the motornerve terminal and is believed by Applicants to induce postsynaptic specialization and up-regulation of other factors, such as muscle-diffusable factors, involved in the neural sprouting response.

It is anticipated that methods which interfere with the sprouting phenomenon (such as by the preventing the action of agrin) would delay the return of innervation to cells which are controlled by neurons which have been therapeutically poisoned by BoNT or TeNT) or otherwise damaged. This is because, as indicated above, the rate of return of neural function appears to be partly dependent upon the presence of synaptically active sprouts.

Additionally, agents to be used in the inhibition of sprouting may very well also delay or prevent the recovery of neural activity of the endplate following poisoning.

Thus utilization of such methods would therefore be expected to extend the effective period of treatment of tissue with a clostridial toxin, by delaying the regeneration of active neuromuscular synapses, both through inhibition of sprouting and of recovery of the poisoned endplate.

According to a further aspect of the invention as claimed there is provided a method for extending the therapeutic effect of a pharmaceutically acceptable therapeutic dose of a clostridial toxin, comprising: contacting affected tissue with a pharmaceutically acceptable composition comprising an inhibitor of neural sprouting.

Detailed Description of the Invention This invention is drawn to methods and compositions for increasing the therapeutic effectiveness of treatment of tissue with clostridial neurotoxin. This increase in effectiveness is made possible by the surprising discovery that regeneration of neural tissue damaged by treatment with clostridial neurotoxin is a complex occurrence in which two coordinated events take place.

In the first of these events, the poisoned neuromuscular endplate becomes synaptically inactive, demonstrating no exocytosis of synaptic vesicles and thus no transport of intracellular acetylcholine. In four days after treatment, the endplate begins to form neural sprouts that are shown to release and regenerate synaptic vesicles.

These sprouts grow in length and complexity until approximately 42 days following treatment with the neurotoxin; at this point the neural sprouts begin to regress and shorten. At ninety-one days following treatment, the neural sprouts can no longer be seen.

In the second event, simultaneously with the beginning of regression of the neural sprouts, the synaptically inactive endplate begins to regain the ability to release acetylcholine and begin to recycle synaptic vesicles. This ability, which begins at relatively low levels, increases over the time period indicated above. At approximately 91 days following treatment with clostridial neurotoxin the endplate is histologically and synaptically indistinguishable from the condition of the endplate before treatment with clostridial neurotoxin.

These findings indicate that one may therapeutically intervene at one of the major steps of the sprouting phenomenon to prevent or attenuate the neural sprouting as a method of extending the effective period during which tissue treated with the toxin remains paralyzed. In an initial step, the muscle cells surrounding the neural endplate respond, either sensing the inactive muscle or in response to a signal from the poisoned nerve terminal, by producing muscle-derived diffusable factors. The expression of a number of muscle-derived signaling factors appears to be upregulated by muscle inactivation; such factors include insulin-like growth factors (IGF-1 and IGF-2). A factor such as neural agrin is believed to be the initial signal directing the muscle cell to produce the IGF molecules.

Reports have demonstrated that IGF I and IGF II effect neurite outgrowth in cultured BoNT/A treated dorsal root ganglia, and also are able to stimulate the initial sprouting response in paralyzed mouse gluteus muscle. See Caroni, P. and Schneider, C. J. Neurosci. 14:3378-3388 (1994) and Caroni, et al. J. Cell Biol. 125:893-902 (1994) Thus, blocking the effects of such muscle derived diffusable factors that positively affect neurite outgrowth and sprouting may attenuate not only clostridial neurotoxininduced sprouting, but may also delay the eventual recovery of neurotransmission at the poisoned nerve terminals. Such blocking may occur through the use of antibodies specific for the muscle derived diffusable factor in question, or that are common to such muscle derived diffusable factors.

Alternatively, there are naturally occurring binding proteins, such as the IGF binding proteins IGF-BP 4 and IGF- BP 5, which can bind to, and therefore block, the neurotrophic effect of such diffusable factors.

IGF-BP 4 has an amino acid sequence (from the amino terminus) of: MLPLCLVAALLLAAGPGPSLGDEAI

HCPPCSEEKLRCRPPVGCEELVREPGCGCCATCA

LGbGMPCGVYTPRCGSGLRCYPPRGEKPLHTLHGQGVCMEAEIEAIQESLOPSDKDE GDHPNNSFS PCSAIIDRRCLQKIIFAKI

RDRSTSGGKMKVNGAPREDARPVPQGSCQSELHR

ALERIAA.SQSRTUEDLYI I PI PNCDRNGNFHPKQCHPALDGQRGKCVRKTGVKLPGG

LEPKGELDCHQLADSFRE

(SEQ ID NO: 1) This is encoded within the nucleotide base sequence (from to of: GTGCCCTCCG

C

GCCCCGCGCCC

CGTGGCCGCC

AAGCCATCCA

CCCCCCGTGG

CGCCACTTGC

GTTGCGGCTC

CTGCACACAC

CGAGGCCATC

ACCCCAACAA

CAGAAGCACT

GAAGGTCAAT

CCTGCCAGAG

AGCCGCACCC

CAACGGCAAC

GTGGCAAGTG

GGCCTGGAGC

TCGAGAGTGA

:CGCTCGCCC GCGCGCCCGC GCTCCCCGCC

TGCGCCCAGC

~GCGCCCCAG

:TGCTGCTGG

2TGCCCGCCC 3CTGCGAGGA

JCCCTGGGCT

.,GGCCTGCGC

rGATGCACGG

CAGGAAAGCC

CAGCTTCAGC

TCGCCAAAAT

GGGGCGCCCC

CGAGCTGCAC

ACGAGGACCT

TTCCAC CC CA

CTGGTGTGTG

CAAAGGGGGA

GGCCTGCCAC

TCCTCGGGCG

CCGCCGGGCC

TGCTCCGAGG

GCTGGTGCGA

TGGGGATGCC

TGCTACCCGC

GCAAGGCGTG

TGCAGCCCTC

CCCTGTAGCG

TCGAGACCGG

GGGAGGATGC

CGGGCGCTGG

CTACATCATC

,AGCAGTGTCA

;GACCGGAAG1

LGCTGGACTGC

;CAGGCCAGGC

CGGGCCGAGC

AGAACCTGGC

GAGCCGGGCT

CTGCGGGGTG

CCCGAGGGGT

TGCATGGAGC

TGACAAGGAC

CCCATGACCG

AGCACCAGTG

CCGGCCTGTG

AGCGGCTGGC

*CCCATCCCCA

LCCCAGCTCTG

LCGGGGGTGAA

CACCAGCTGG

;ACTCAGCGTC

-AGAGTGGAG'I

CTGGGCGACG

GCGCTGCCGC

GCGGCTGTTG

TACACCCCCC

GGAGAAGCCC

TGGCGGAGAT

GAGGGTGACC

CAGGTGCCTG

GGGGCAAGAT

CCCCAGGGCT

CGCTTCACAG

ACTGCGACCG

GATGGGCAGC

GCTTCCGGGG

CTGACAGCTT

ICCCTGCTACT

ICTGAGTCTGA

CCTGTGCTCT GGAGGCTGCA

GAGCTGACCC

GTCCTGTCTC

TGCCTGCGGC

CGTGTGCGTG

TGCGTGCGTG

TGGGGTAAGC CAGAGCCT1GG AGGACTGAGA

CTGGCACTTA

CAGATCGATC

CTGGATTCAC

ACCTAAAAAC

ATTTACTGAC

CCTTGGGAGG

TTTTATTCTG

TCCTATAAGG

AGAGTTCAAG

AGTCAGAGGA

GAAGAGACAT

TAGCCAGAGG

GTGGGAGCCT

GGTCACGAGG

TCCAGACCCA

TTCTCTTCTT

CCCCAGGTCC

CTTGGTTGGC

TGGCAGCTGA

AAAGGAAGAC

TTGAAGCACA

GAGCAGTCAG

GGTGGGAAGA

ATGGAGAAGA

CCCACGTGCT

CCTACCCCAT

TTGTGGTCAC

TTCCAGTGGC

TCGCTCCCTG

CCCTACACCT

CCCTCCCCAC

TTGACTGGAT

GGAAGGAGAC

CCAGAAGTTT

TGTGTGTGTT

GGTGTTCTCT

GCCCAACAGG

TCACTCACTC

CATGTACTAC

ACTTCCTCTG

CGTGTGGGAG

GTACCTTGAC

AAGGAAGCGT

CTCCCAAAGC

TTCCTTTAGG

GAGCCCTGCT

GAGGGCTAGG

AAGAATGCAA

AGGGGATGAG

AGCCATGAAG

TAGCTCTGCC

IACCTCCCTAC

TTAGGAACCT

CCCTCAAATG CGCGTGTGCA TGTGAGCATG

GGTGTGCCCT

TTGGTGTTAC

ACAGCCCAAG

TCTGAGCCCT

GGTGTGTTTC

ATTCCTTCAC

TCATCCAGCC

GTGCCAGCTC

TAGTTTTCAG

ATTTTGGCAT GTGGAGACAC TAGAAAAATC

TCATTCCCAG

CATCGTCCTT CCTCTCAAGC GGGGTAGCAG

ATGGAGTAAT

TCAGACTTGC

CAGGCTCCCT

TCTGGTTGTT

GCACCATCTG

GTGGGAGAGC

GAAGGGGGTC

GAGGTGGGGT

ACATTTCTCT

GAGTGGACTG

AATGTGCCTA

GGGCTTCCTG

GGTCCTGTTC

TCACCGGGAT

GAACCTATCC

TCCCTCTCCA

TATCTCCTTC

TCCCCTGGGC

ATCTTCTGGC

ACCAGTTGGC CATGATGTCT

TTTCT

(SEQ ID NO: 3) IGFBP 5 has an amino acid sequence (from the amino terminus) of: MVLLTAVLIJLLAAYAGPAQSLGS

FVHCEPCDEKAILSMCPPSPLGCELVKEPGCGCCMTCA

LAEGQSCGVYTERCAQGLRCLPRQDEEKPLHALLHGRGVCLNERSYhEQVKI

ERDSREHE

EPTEAEYPIRKTIEK

AKDRKTSFGANAPISA

PEMRQESEQGPCRRHMEASLQELKASPRPAWLPNCDRKGFYKOCKPSRGRRGI

CWCVDKYGMKLPGMEYVDGDFQCHTFDSSV

(SEQ ID No. 2) and is encoded within a nucleotide base sequence (from 5' to of: GGGGAAAAGA GCTAGGAAAG AGCTGCAAAG CAGTGTGGGC

TTTTTCCCTT

TTTTTGCTCCT TT'TCATTAC CCCTCCTCCG TTTCACCCT

TCTCCGGACT

TCGCGTAGAA CCTGCGAATT TCGAAGAGGA GGTGGCAAAG

TGGGAGAAAA

GAGGTGTTAG

CTTGATTTCA

ACCTGTTCTG

GGTGTATTTT

CTCCCACCCC

GTTGCTTGGG

CCCGCTCAGG

GCGCTGCCCT

GGGACCTGCT

AAGTGTCTGC

AAACAAAAAC

CACTCTCGCT

GATGGTGTTG

CGGCCCAGAG

GCCCTCTCCA

GCCGGGCTGC

GCGGCGTCTP.

CAGGACGAGC

CCTCAACGA-P

GTGAGCACG1

CCCAAGATCI

AGCAGTGAAC

GGGGAGCCGJ

AGACAGGAG

GCAGGAGCT(

CCAATTGT&

CGTGGCCGC

GCTGCCAGG'

ACAGCAGCA

CTCCCACCC

GCCACTCAT

GGAAAAAAA

GGTTTGGGGT

ACATTTTCTC

CGGCGCCGCG

AGATTTTAAG

CAACGCCATC

GGTGAACAAT

CTTGAGGGTT

GCTTCCCCCA

CGCAGGCACC

GGGAGGGCAC

AAAAAAATCT

CTCCTGCCCC

CTCACCGCGG

CCTGGGCTCC

TGTGCCCCCC

GGCTGCTGCA

CACCGAGCGC

AGAAGCCGC'I

AAGAGCTACC

GGAGCCCACC

7TCCGGCCCA?

AAGGACCGCI

k. GAACACTGC( r CTGAGCAGG :AAAGCCAGC4 P, CCGCAAAGG, Pi AGCGTGGCA' C ATGGAGTAC A CGTTGAGTG C CAGCCCCGA T TCATCTCAT A AAAAAAAAA TTTTTTGTTT T CCACCCTCTC

G

CACCGCTGGC

P

CAAAAATTTT TCCACTGCAT

C

TTTGTGGCTT

I

TCTCCGGCCT

ACCTGTTGCA

CCAGCCCTCC

CTGCTCTACC

CCGGGGGCCC

ACCCCGAGCT

TCCTCCTGCT

TTCGTGC.ACTI

CAGCCCCCTG

TGACCTGCGC

TGCGCCCAGG

GCACGCCCTG

GCOAGCAAGT

ACCTCTGAGA

k ACACACCCGC

SGAAAGAAGCT

2CACCCCCGGA 3CCCCTGCCGC

ZCACGCATGGT

k TTCTACAAGA r CTGCTGGTGC G TTGACGGGGA A TGCGTCCCCC C TCCAGCCAGC T TAAGGGAAAA A AA 'TGTTTTTGT T ~GCTGCAGCC

A

LGCTGAGGGT

T

LAAGATAAAT

C

:CGATCTCAT

T

?TTTTCCCCTA

:CGCrrCACTG

C

\GGCTTTAAT

TI

kCCTCTCTCT

P

FGCCAGAAAT I1 rCTTGGCCCC I3 !AAGGGGGCG

I

SCTGGCCGCC

SCGAGCCCTG

GGCTGCGAGC

CCTGGCCGAG

GGCTGCGC!TG

CTGCACGGCC

CAAGATCGAG

TGGCCGAGGA

ATCTCCGAGC

GACCCAGTCC

TCATCTCTGC

AGACACATGG

GCCCCGTGCT

GAAAGCAGTG

GTGGACAAGT

CTTTCAGTGC

CCCAACCTTT

GCCTCCCTCC

ATATATATOT

TTTTAATTT

ACGCCTCTT

AGAAAGCGG

CATTTTTCT

'ATTTCGGTG

TAATTCTGA

:GTGCACCTG

'CTTGCAACT

£ATTTTTGC

~TTAAAACAA

?TTATCCCTG

~CTAAGAGAA

PATGCGGGGC

:GACGAGAAA

rGGTCAAGGA 3GGCAGTCGT

CCTCCCCCGG

SCGGGGTTTG

AGAGACTCCC

GACCTACTCC

TGAAGGCTGA

AAGTTTGTCG

ACCTGAGATG

AGGCTTCCCT

GTGTACCTGC

CAAACCTTCC

ACGGGATGAA

CACACCTTCG

CCCTCACCCC

ACCCCAGGAC

ATCTATTTGA

(SEQ ID NO: 4) These binding proteins can be made synthetically or cloned and produced for therapeutic purposes, while a cell line producing a desired monoclonal antibody can be maintained for relatively large-scale antibody production.

Cloning and general antibody methodologies are commonplace in the art; such methodologies are disclosed within Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor Laboratory Press 1989), the disclosure of which is hereby incorporated by reference as part of this disclosure.

Rather than using competitive methods, another aspect of the invention involves the use of a cholinergic special transporter to insert a gene which produces inactive receptors for one or more factor involved in promotion of neural sprouting. Such receptors would maintain high specific binding constants to their ligands, but the biological activity of the receptors would be abrogated; such receptors could be easily be generated and screened through the introduction of mutations in the nucleotide sequence encoding the protein, and assaying the mutants for binding strength and biological activity. Alternatively, the neurotrophic activities of factors produced by the neural endplate or nascent sprouts may be inhibited through intracellular targeting and delivery of a competitive inhibitor, ribozyme, transcriptional suppresser or other agent specifically able to block or attenuate such activities, as described above. Such intracellular targeting is disclosed in references such as, Dolly et al., International Patent Publication No. W095/32738, previously incorporated by reference herein.

The second point at which intervention in the sprouting phenomenon may be made is during the stage of axonal outgrowth and arbor development. At this stage such outgrowth has already been initiated, but auxiliary factors appear to be necessary in order to maintain axonal growth. A number of factors are known to affect the rates of outgrowth, and may also effect the survivability of many types of neurons. Such factors include ciliary neurotrophic factor (CNTF); neurotrophins, including NT-3, NT-4, and brain-derived neurotrophic factor (BDNF); and leukemia inhibitory factor (LIF). Not only are these factors important in establishing an initial rate of axonal outgrowth, but they appear to either directly or indirectly stimulate their own production therefore blocking the initial outgrowth of sprouts may be essential in preventing further propagation through the continued expression of such factors.

The same methods as disclosed above can be used to prevent one or more of the agents listed above to manifest its activity. As would be expected by those of skill in the art in light of the present disclosure, such agents, destroy or bind to, and therefore attenuate the activity of these factors or their block their ability to bind to their receptors, or inactive forms of their receptors, would be useful when used in conjunction with a clostridial neurotoxin to prevent or reduce the rate of sprouting of neurites from the poisoned or damaged endplate.

The third stage at which the sprouting phenomenon may be attenuated or inhibited concerns the binding of axons to the extracellular matrix. Axons are guided to cellular processes containing the appropriate neurotransmitter receptors by binding to components of the extracellular matrix. Such binding involves a variety of cell-borne or matrix associated adhesion molecules. Tenascin-C is an extracellular matrix component derived from Schwann cells that appears to bind neural processes. Ninjurin is a cell surface adhesion molecule that is up-regulated following peripheral nerve injury and thought to be involved in exonal guidance. See Araki, et al., J. Biol. Chem. 272:21373- 21380 (1997), incorporated by reference herein. Similarly, neural-cell adhesion molecule (N-CAM), is an adhesion molecule which is thought to be involved in binding of neural sprouts to the extracellular matrix. The same techniques indicated above may be employed to prevent the expression or inhibit the activity of these molecules when used as part of a clostridial neurotoxin therapy.

Thus, in one aspect the present invention is drawn to a method for extending the effective period during which tissue treated with clostridial toxin is paralyzed, comprising: Contacting said tissue with a composition comprising an agent able to prevent the neuroregenitive activity of a polypeptide selected from the group consisting of IGF-1, IGF-2, cilary neurotrophic factor, NT-3, NT-4, brain-derived neurotrophic factor, leukemia inhibitory factor, tenascin-C, ninjurin, neural cell adhesion molecule, and neural agrin.

In one preferred embodiment, the agent comprises a polypeptide able to bind to IGF-1 and/or IGF-2 in a manner that prevents an IGF molecule from binding to or activating a cell surface receptor involved in the initiation of neural sprouting. In a most preferred embodiment the polypeptide comprises at least a portion of a amino acid sequence selected from the group consisting of: IGFBP-4 (SEQ ID NO: 1) or IGFBP5 (SEQ ID NO: Preferably said portion comprises at least 10 contiguous amino acids of said sequence; more preferably said portion comprises at least contiguous nucleotides of said.sequence. Most preferably, the portion comprises an amino acid sequence selected from the group consisting of the entire amino acid sequence of IGFBP-4 or Treatment of cells with such a composition may be accomplished either before or simultaneously with treatment with clostridial toxin. Preferably, the clostridial toxin is a botulinum toxin. Even more preferably, the botulinum toxin comprises BoNT/A. In other embodiments, the clostridial toxin is TeNT. Agent which are able to bind to any of these factors in a manner that inhibits their neurotrophic activity, or which bind to the receptors for such factors, would, in light of the present application, be expected to function as agents for extending the effective period between treatments of tissue with a neurotoxin.

Another embodiment comprises a cholinergic specific transporter joined to a gene encoding a gene which produces an inactive receptor for one or more factor involved in promotion of neural sprouting when delivered to a neural cell in vivo. Preferably the receptors maintain high specific binding constants to said factor(s) and the biological activity of the receptor is reduced or absent.

In a particularly preferred embodiment the transporter comprises some or all of a clostridial neurotoxin heavy chain, although other transporters such as the diphtheria toxin transporter may be effective in this regard as well.

In another embodiment the invention comprises a cholonergic specific transporter that is covalently or noncovalently joined to a nucleic acid which comprises a ribozyme or antisense nucleic acid able to specifically destroy the nucleic acids encoding neurotrophic agents or their receptors. Said joining may be made through methods including, but not limited to, covalent bonding or electrostatic forces.

In another embodiment, the present invention is drawn to the methods for stimulating the outgrowth of neural sprouts from damaged neural tissue. Such methods could be effective ways of increasing the rate at which reinnervation occurs after a neural injury. These methods comprise: Contacting said tissue with a composition comprising a polypeptide which comprises a neurotrophically active domain derived from an agent selected from a group consisting of IGF-1, IGF-2, cilary neurotrophic factor, NT-3, NT-4, brain derived neurotrophic factor, leukemia inhibitory factor, tenascin-C, ninjurin, neural cell adhesion molecule, and neural agrin. Such damage may be a result of neurotoxin poisoning or due to a traumatic event, including but not limited to nerve or spinal cord crush injuries, traumatic brain injuries, glaucoma-induced damage to the retina and/or optic nerve, or surgical trauma or injury.

The following examples illustrate various embodiments of the present invention, and are not intended to limit the scope of the invention, which is solely defined by the claims which conclude this specification.

Example 1 Blepharospasm is a medical condition characterized by uncontrolled eyelid movement. In its early stages, the condition is characterized by excessive blinking or fluttering of the eyelids. The condition is generally a progressive one, in which excessive blinking is replaced in the later stages with spasms of eye closure that interfere with visual function. The spasms become more frequent and severe, and involve the preseptal, pretarsal, and orbicularis oculi muscle. The condition often results in functional blindness relatively quickly (in a matter of two to three years) after the symptoms are first encountered.

A patient suffering from moderate idiopathic blepharospasm is treated with injections of BoNT/A toxin preparation containing non-toxic proteins and hemagglutins in sterile saline. Alternatively, the same toxin preparation without hemagglutins may be used. The injections are generally in the volume of 100 gIl; and each injection contains 1.25 to 2.5 units of the toxin preparation. The injections are made into the pretarsal orbicularis oculi of the upper lid laterally and medially and in the lower lid laterally and medially. Additionally, unit injections (100 pl each) are made lateral to the lateral canthus and into the brow medially. Total amount of BoNT/A toxin injected is roughly 6.25 to 12.5 units per eye. The Bo/A toxin is provided in a sterile, preservativefree saline, and the same solution is used to dilute the BoNT/A toxin if the master preparation of it is too concentrated.

Following injection the treated muscles are sufficiently paralyzed due to this treatment to alleviate the major symptoms of blepharospasm. Some mild concomitant weakness in the surrounding muscle tissue is observed; these side effects are mild and tolerated well by the patient.

The effect of this treatment lasts approximately 8 weeks, and must be repeated at the end of this time to maintain the beneficial effects.

Example 2 A patient with blepharospasm is pre-treated with BoNT/A toxin as indicated in Example 1 with the following difference. Prior to injection with BoNT/A toxin preparation, the patient is given a ten-fold excess of IGFBP-4, having an amino acid sequence of SEQ ID NO: 1. The binding protein preparation is dissolved in sterile, preservative-free saline. Each injection is in the same area as the toxin injections that follow the pre-treatment; the volume of each injection is 100 pl. The BoNT/A toxin preparation is injected ten minutes after the injection of the IGFBT 4 injection.

The patient's therapeutic response to the BoNT/A toxin is similar to that seen in Example 1. The duration of the benefit provided by the BoNT/A toxin treatment is extended to 12 weeks or more, during which time no further injection need be made.

Example 3 A patient with blepharospasm is pre-treated with BoNT/A toxin as indicated in Example 1 with the following difference. The BoNT/A toxin has been modified to have joined thereto a nucleic acid comprising a ribozyme specifically targeted to enzymatically destroy neural agrin mRNA. No supplemental injections are made.

The patient's therapeutic response to the BoNT/A toxin is similar to that seen in Example 1. The duration of the benefit provided by the BoNT/A toxin treatment is extended to 12 weeks or more, during which time no further injection need be made.

Example 4 A patient with blepharospasm is pre-treated with BoNT/A toxin as indicated in Example 1 with the following difference. The BoNT/A toxin has been modified to have joined thereto a nucleic acid encoding an inactive neurotrophin receptor which retains the ability to bind its target neurotrophin. No supplemental injections are made.

The patient's therapeutic response to the BoNT/A toxin is similar to that seen in Example 1. The duration of the benefit provided by the BoNT/A toxin treatment is extended beyond that seen with BoNT/A alone, during which time no further injection need be made.

It will be understood that, while reference is made to BoNT/A in the Examples above, any other of the species of botulinum toxins BoNT/B through G) could be substituted therefor, with appropriate adjustments possibly necessary due to differences in specific activity of the toxin. Additionally, the light chain segment could be derived from any clostridial neurotoxin (or other neurotoxin) with the heavy chain retaining the motor neuron receptor binding and exo-vescular transport activities retained from the BoNT heavy chain.

These Examples are not intended to exhaust the embodiments of the present invention, and the invention is not to be seen as limited thereby. Further embodiments will be disclosed within the claims that conclude this specification.

Throughout this ,specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

The reference to any prior art in this specification is not, and should not be ,taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

SEQUENCE LISTING <110> Dolly, Oliver J.

Aoki, Kei Roger De Paiva, Anton <120> Compositions and Methods for Modulating Neural Sprouting <130> 17259 <150> 60/083,472 <151> 1998-04-29 <160> 4 <170> FastSEQ for Windows Version <210> 1 <211> 258 <212> PRT <213> Homo sapiens <400> 1 Met Leu 1 Gly Pro Glu Lys Arg Glu Met Pro Tyr Pro Gin Gly Leu Gin Ser Pro 130 Lys Ile 145 Ala Pro Glu Leu His Glu Asn Phe 210 Lys Cys 225 Leu Glu Pro Leu Ser Leu Leu Ala Pro Gly Cys Gly Pro Arg Val Cys 100 Pro Ser -115 Cys Ser Arg Asp Arg Glu His Arg 180 Asp Leu 195 His Pro Trp Cys Pro Lys Cys 5 Gly Arg Cys val Gly Met Asp Ala Arg Asp 165 Ala Tyr Lys Val Gly 245 Leu Asp Cys Gly Tyr 70 Val Glu Lys His Ser 150 Ala Leu Ile Gin Asp 230 Glu Val Ala Ala Leu Leu Leu Ala Ala Gly Pro 10 Glu Ala Arg Pro 40 Cys Cys 55 Thr Pro Glu Lys Leu Ala Asp Glu 120 Asp Arg 135 Thr Ser Arg Pro Glu Arg Ile Pro 200 Cys His 215 Arg Lys Leu Asp Ile His Cys 25 Pro Val Gly Ala Thr Cys Arg Cys Gly 75 Pro Leu His 90 Glu Ile Glu 105 Gly Asp His Arg Cys Leu Gly Gly Lys 155 Val Pro Gin 170 Leu Ala Ala 185 Ile Pro Asn Pro Ala Leu Thr Gly Val 235 Cys His Gin 250 Pro Cys Ala Ser Thr Ala Pro Gin 140 Met Gly Ser Cys Asp 220 Lys Pro Glu Leu Gly Leu Ile Asn 125 Lys Lys Ser Gin Asp 205 Gly Leu Cys Glu Gly Leu Met Gin 110 Asn His Val Cys Ser 190 Arg Gin Pro Ser Leu Leu Arg His Glu Ser Phe Asn Gin 175 Arg Asn Arg Gly Glu Val Gly Cys Gly Ser Phe Ala Gly 160 Ser Thr Gly Gly Gly 240 Leu Ala Asp Ser .Phe 255 Arg Glu <210> <211> <212> <213> 2 272

PRT

Homo Sapiens <400> 2 Met 1 Pro Lys Lys Gin Leu Arg Glu Glu Ser 145 Thr Ile Arg Met Tyr 225 Cys Val Val Ala Ala Glu Ser Pro Gly Arg Glu 130 Glu Gin lle Arg Val 210 Lys Trp Asp Leu Gin Leu Pro Cys Arg Val Asp 115 Thr Leu Ser Ser His 195 Pro Arg Cys Gly Leu Ser Ser Gly Gly Gin Cys 100 Ser Tyr Lys Lys Ala 180 Met Arg Lys Val Asp 260 Thr 5 Leu Met Cys Val Asp Leu Arg Ser Ala Phe 165 Pro Glu Ala Gin Asp 245 Phe Ala Gly Cys Gly Tyr 70 Glu Asn Glu Pro Glu 150 Val Glu Ala Val Cys 230 Lys Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly Ser Pro Cys 55 Thr Glu Glu His Lys 135 Ala Gly Met Ser Tyr 215 Lys Tyr Phe Pro 40 Cys Glu Lys Lys Glu 120 Ile Val Gly Arg Leu 200 Leu Pro Gly Val 25 Ser Met Arg Pro Ser 105 Glu Phe Lys Ala Gin 185 Gin Pro Ser Met 10 His Pro Thr Cys Leu 90 Tyr Pro Arg Lys Glu 170 Glu Glu Asn Arg Lys 250 Cys Leu Cys Ala 75 His Arg Thr Pro Asp 155 Asn Ser Leu Cys Gly 235 Leu Glu Gly Ala Gln Ala Glu Thr Lys 140 Arg Thr Glu Lys Asp 220 Arg Pro Pro ys Leu Gly Leu Gin Ser 125 His Arg Ala Gin Ala 205 Arg Lys Gly Cys Glu Ala Leu Leu Val 110 Glu Thr Lys His Gly 190 Ser Lys Arg Met Asp Leu Glu Arg His Lys Met Arg Lys Pro 175 Pro Pro Gly Gly Glu 255 Glu Val Gly Cys Gly Ile Ala Ile Leu 160 Arg Cys Arg Phe Ile 240 Tyr Gin Cys His Thr Phe Asp Ser Ser Asn Val Glu 265 270 <210> 3 <211> 1955 <212> DNA <213> Homo Sapiens <400> 3 gtgccctccg ccgctcgccc gcgcgcccgc gctccccgcc tgcgcccagc gccccgcc cgcgccccag tcctcgggcg gtcatgctgc ccctctgcct cgtggccgcc ctgctgcttgg 120 ccgccgggcc cgggccgagc ctgggcgacg aagccatcca ctgcccgccc tgctccgagg 180 agaagctggc gcgctgccgc ecccccgtgg gctgcgagga gctggtgcga gagccgggct 240 gcggetgt tg 300 gt tgcggct c 360 tgatgcacgg 420 tgcagooctc 480 cccatgaccg 540 ggggcaagat 600 ectgeeagag 660 a cgaggacc t 720 ageagtgtca 780 cgggggtgaa 840 ctgacagctt 900 cctgtgctct 960 tgeetgcggc 1020 tgtgtgtgtt 1080 ttggtgttac 1140 ggtgtgttte 1200 aeetaaaaac 1260 ttttattetg 1320 cctgtgggag 1380 catcgtcctt 1440 atggagtaat 1500 ttotettctt 1560 tggeagctga 1620 gagggctagg 1680 gagtggactg 1740 ggt eotgt to 1800 egeeacttgc gggcctgcgc gcaaggcgtg tgaeaaggac caggtgcetg gaaggteaat cgagctgcae ctacatcatc eccagctctg gcttccgggg tcgagagtga ggaggctgea ccagaagttt tgtgageatg acagcccaag eagategatc atttactgac aetteetctg tagaaaaate c t ctc aagc ggtcacgagg ccecaggtcc gagcectgct gaggtggggt aatgtgccta cctaceccat gccc tgggct tyctacccgc tgcatggage gagggtga cc cagaagcact ggggcgccce cgggegetgg eccatcccca gatgggcagc ggcctggage ggcctgccag gag ctgaccc ccctcaaatg ggtgtgccct aggactgaga ctgga ttoao catgtactac attttggcat tea tt cccag tagccagagg tccagaecca tt cctttagg gtgggagagc aeatttctct atggagaaga ttgtggtcac tggggatgcc cccgaggggt tygeggagat accccaacaa tcgceaaaat gggaggatge agcggctggc actgcgaccg gtggeaagtg caaaggggga caggecaggy agagtggagt cgcgtgtgea tggggtaagc ctggcactta toacteacto gtgooagctc gtggagacae agtcagagga gtgggagcct ctcccaaagc tctggttgt t gaagggggtc gagcagtcag cc cacg tget agceatgaag c tgcggggtg ggagaagccc cgaggecate cag ott cagc t egagac egg ceggectgtg cgcttcacag eaa egg caa c Ctggtgtgtg gctggactgc actcagcgtc ctgagtetga cgtgtgcgtg cagagcctgg gcccaagagg attoetteac tagtt ttcag tcctataagg gaagagaeat aaggaagcgt tcagaettge gcaccatctg aaaggaagae ggtgggaaga aggggatgag tcaccgggat tacacecce ctgcacaeac eaggaaagcc cectgtagcg agoaccagtg ccccagggct agcegeaccc ttccacecca gaooggaaga cacoagotgg coctgetaet gtcctgtctc tgegtgcgtg ggtgttet ct tetgagccct tcatccagcc ccttgggagg agagtteaag gtaccttgac ggggtagcag caggctcet.

cttggttggc t tgaag ca ca aagaatgcaa gggcttcctg gaacctatcc ttcoagtggc 1860 tcgctccctg tagctctgcc tccctctcca tatctccttc cetacacet ccctccccac acctccctac tcccctgggc atcttctggc ttgactggat ggaaggagac 1920 ttagyaacct accagttggc catgatgtct tttct 1955 c210> 4 c211> 1722 c212> DNA c213> Homo sapiens c400> 4 ggggaaaaga ttttcattac 12 0 tcgaagagga 180 ttgtttttgt 240 aacgcctctt 300 ggtgtatttt 360 caa cgc cat c 420 tttgtggctt 480 ccgctcactg 540 tcttgcaact 600 aagtgtctgg 660 aaaaaaatct 720 accccgaggt 780 gctggccgcc 840 cgacgagaaa 900 gccgggctgc 960 caccgagcgc 1020 gcacgccctg 1080 caagatcgag 1140 gacctactcc 1200 agcagtgaag 1260 gaa ca ctgc c 1320 cccctgccgc 1360 gctaggaaag ccctcctccg ggtggcaaag tttttaattt acctgttctg agattttaag tccactgcat tttttcccct cgtgcacctg gggacctgct gggaggycac ccgggggccc aaagggggcg tatgcggggc gcctttcca ggc tgctgca tgcgcccagg ctgcacggcc agagactccc cccaagatct aaggaccgca cacccccgga agacacatgg agctgcaaag ttttcaccct tgggagaaa cttgatttca cggcgccgcg caaaaatttt ccgatctcat ataattctga gcgctgccct cgcaggcacc ctgctctacc tcttggcccc ac taagagaa cggcccagag tgtgcccCCC tgacctgcgc ggctgcgc tg gcggggtttg gtgagcacga tc cggcc caa gaaagaagct tcatctctgc *aggcttccct cagtgtgggc tctc cggact gaggtgttag acattttctc ca cC5 atgo aaagataaat tatttcggtg cccgctcagg gcttccccca ccagccctcc tgccagaaat tttatccctg gatggtgttg cctgggct cc c ag ccc cc tg cctggccgag CCtCCCCC99 cctcaacgaa ggag cccac c acacacccgc gacccagtcc acctgagatg gcaggagctc tttttccctt tcgcgtagaa ggtttggggt ccaccctctc agctgagggt ccatttttct gt tgct tggg cttgagggtt acctg'ttgca acctctctct tttaaaacaa cactctcgct ctcaccgcgg ttcgtgcact ggctgcgagc gggcagt cgt caggacgagg aagagctacc acct ctgaga atctccgagc aagtttgtcg agacaggagt aaagccagcc tttttgctcc cctgcgaatt ttttttgttt ggctgcagcc tagaaagcgg ctcccacccc ggtgaacaat tctccggcct aggctttaat acatttttgc aaacaaaaac ctcctgccc tcctcctgct gcgagccctg tggtcaagga gcggcgtcta agaagccgc t gcgagcaagt tggccgagga tgaaggctga ggggagccga ctgagcaggg cacgcatggt gccccgtgct gtgtacctgc ccaattgtga ccgcaaagga ttctacaaga gaaagcagtg 1440 caaaccttcc cgtggccgca agcgtggcat ctgctggtgc gtggacaagt acgggatgaa 1500 gctgccaggc atggagtacg ttgacgggga ctttcagtgc cacaccttcg acagcagcaa 1560 cgttgagtga tgcgtccccc cccaaccttt ccctcacccc ctcccacccc cagccccgac 1620 tccagccagc gcctccctcc acoccaggac gccactcatt tcatctcatt taagggaaaa 1680 atatatatct atctatttga ggaaaaaaaa aaaaaaaaaa aa 1722

Claims (1)

1. 26-06-'06 12:51 FROM-DCC SYDNEY +61292621080 T-351 P008014 F-347 P:WVPDOCSiWSWtr \17776281( &c-2&0O6li cN SThe claims defining the invention are as follows: -n 1) A method for extending a therapeutic effect of a VO c-i clostridial toxin treatment in a human, said method comprising the step of administering to said human pharmaceutically acceptable composition comprising: j a) a first agent comprising a clostridial toxin; and o b) a second agent comprising an inhibitor of neuronal C sprouting. C 2) A method for extending a therapeutic effect of a clostridial toxin treatment in a human, said method comprising the steps of: a) administering to said human pharmaceutically acceptable composition comprising a clostridial toxin; and b) subsequently administering to said human pharmaceutically acceptable composition comprising an inhibitor of neuronal sprouting. 3) A method for extending a therapeutic effect of a clostridial toxin treatment in a human, said method comprising the steps of: a) administering to said human pharmaceutically acceptable composition comprising an inhibitor of neuronal sprouting; and b) subsequently administering to said human pharmaceutically acceptable composition comprising a clostridial toxin. 4) The method of any one of claims 1, 2, or 3, wherein said clostridial toxin comprises BoNT. COMS ID No: SBMI-03975435 Received by IP Australia: Time 12:55 Date 2006-06-26 .26-06-'06 12:51 FROM-DCC SYDNEY +61292621080 T-351 P009/014 F-347 0 0 cN The method of any one of claims 1, 2, or 3, wherein said clostridial toxin comprises BoNT/A. \O ci 6) The method of any one of claims 1, 2, or 3, wherein P-H said inhibitor is an inhibitor of a polypeptide having Sneurotrophic activity, said -inhibitor selected from the o group consisting of: a) an antibody able to selectively bind said polypeptide having neurotrophic activity, C b) a competitive inhibitor of said polypeptide having neurotrophic activity, c) a compound able to selectively prevent the expression of a gene encoding said polypeptide having neurotrophic activity, d) a binding protein, other than an antibody, able to selectively bind said polypeptide having neurotrophic activity, e) a ribozyme able to cleave a nucleic acid molecule encoding an interneuronal factor responsible for the expression, activation and/or secretion of said polypeptide having neurotrophic activity, and f) a nucleic acid encoding an inactive growth factor receptor able to bind said polypeptide having neurotrophic activity. 7) The method of claim 6 wherein said inhibitor is an antibody able to selectively bind said polypeptide having neurotrophic activity. 8) The method of claim 6 wherein said inhibitor is a competitive inhibitor of said polypeptide having neurotrophic activity. -31- COMS ID No: SBMI-03975435 Received by IP Australia: Time 12:55 Date 2006-06-26 26-06-'06 12:52 FROM-DCC SYDNEY +61292621080 T-351 P010/014 F-347 P \WPDCS S5pts 2\77627I).d.s2&6&'6O Va 9) The method of claim 6 wherein said inhibitor is a (N compound able to prevent the expression of a gene encoding said polypeptide having neurotrophic activity. The method of claim 6 wherein said inhibitor is a o binding protein, other than an antibody, able to selectively bind said polypeptide having neurotrophic activity. (c 11) The method of claim 10 wherein said polypeptide having neurotrophic activity is selected from the group consisting of IGF I and IGF II, and said binding protein is selected from the group consisting of IGF-BP4 and 12) The method of any one of claims 1, 2, or 3, wherein said inhibitor is an inhibitor of a muscle-derived signaling factor. 13) The method of any one of claim 12, wherein said muscle- derived signaling factor is selected from the group consisting of an IGF I, an IGF II and a neural agrin. 14) The method of any one of claims 1, 2, or 3, wherein said inhibitor is an inhibitor of an auxiliary factor necessary for axonal growth maintenance. The method of any one of claim 14, wherein said auxiliary factor is a ciliary neurotrophic factor. 16) The method of any one of claim 14, wherein said auxiliary factor is a neurotrophic factor. -32- COMS ID No: SBMI-03975435 Received by IP Australia: Time 12:55 Date 2006-06-26 26-06-'06 12:52 FROM-DCC SYDNEY +61292621080 T-351 F011014 F-347 PwMDocs\HijW Spt* \m6776Lt().dc.2OOa(6 cN S17) The method -of any one of claim 16, wherein said neurotrophic factor is selected from the group consisting of VO C-q a NT-3, a NT-4, a leukemia inhibitory factor and a brain- derived neurotrophic factor, S18) The method of any one of claims 1, 2, or 3, wherein o said inhibitor is an inhibitor of a nerve cell adhesion molecule. Ci 19) The method of any one of claim 18, wherein said cell adhesion molecule is selected from the group consisting of a tenascin-C, a ninjurin and a neural cell adhesion molecule. The method of claim 1, wherein said first agent and said second agent comprise a single entity. DATED this Twenty sixth day of June, 2006 Allergan, Inc. By its Patent Attorneys DAVIES COLLISON CAVE -33- COMS ID No: SBMI-03975435 Received by IP Australia: Time 12:55 Date 2006-06-26 SEQUENCE LISTING <110> Dolly, Oliver J. Aoki, Kei Roger De Paiva, Anton <120> Compositions and Methods for Modulating Neural Sprouting <130> 17259 <150> 60/083,472 <151> 1998-04-29 <160> 4 <170> FastSEQ for Windows Version <210> 1 <211> 258 <212> PRT <213> Homo sapiens <400> 1 Met Leu 1 Gly Pro Glu Lys Arg Glu Met Pro Tyr Pro Gin Gly Leu Gin Ser Pro 130 Lys Ile 145 Ala Pro Glu Leu His Glu Asn Phe 210 Lys Cys 225 Leu Glu Pro Leu Ser Leu Leu Ala Pro Gly Cys Gly Pro Arg Val Cys 100 Pro Ser -115 Cys Ser Arg Asp Arg Glu His Arg 180 Asp Leu 195 His Pro Trp Cys Pro Lys Cys 5 Gly Arg Cys val Gly Met Asp Ala Arg Asp 165 Ala Tyr Lys Val Gly 245 Leu Asp Cys Gly Tyr 70 Val Glu Lys His Ser 150 Ala Leu Ile Gin Asp 230 Glu Val Ala Ala Leu Leu Leu Ala Ala Gly Pro 10 Glu Ala Arg Pro 40 Cys Cys 55 Thr Pro Glu Lys Leu Ala Asp Glu 120 Asp Arg 135 Thr Ser Arg Pro Glu Arg Ile Pro 200 Cys His 215 Arg Lys Leu Asp Ile His Cys 25 Pro Val Gly Ala Thr Cys Arg Cys Gly 75 Pro Leu His 90 Glu Ile Glu 105 Gly Asp His Arg Cys Leu Gly Gly Lys 155 Val Pro Gin 170 Leu Ala Ala 185 Ile Pro Asn Pro Ala Leu Thr Gly Val 235 Cys His Gin 250 Pro Cys Ala Ser Thr Ala Pro Gin 140 Met Gly Ser Cys Asp 220 Lys Pro Glu Leu Gly Leu Ile Asn 125 Lys Lys Ser Gin Asp 205 Gly Leu Cys Glu Gly Leu Met Gin 110 Asn His Val Cys Ser 190 Arg Gin Pro Ser Leu Leu Arg His Glu Ser Phe Asn Gin 175 Arg Asn Arg Gly Glu Val Gly Cys Gly Ser Phe Ala Gly 160 Ser Thr Gly Gly Gly 240 Leu Ala Asp Ser .Phe 255 Arg Glu <210> <211> <212> <213> 2 272 PRT Homo Sapiens <400> 2 Met 1 Pro Lys Lys Gin Leu Arg Glu Glu Ser 145 Thr Ile Arg Met Tyr 225 Cys Val Val Ala Ala Glu Ser Pro Gly Arg Glu 130 Glu Gin lle Arg Val 210 Lys Trp Asp Leu Gin Leu Pro Cys Arg Val Asp 115 Thr Leu Ser Ser His 195 Pro Arg Cys Gly Leu Ser Ser Gly Gly Gin Cys 100 Ser Tyr Lys Lys Ala 180 Met Arg Lys Val Asp 260 Thr 5 Leu Met Cys Val Asp Leu Arg Ser Ala Phe 165 Pro Glu Ala Gin Asp 245 Phe Ala Gly Cys Gly Tyr 70 Glu Asn Glu Pro Glu 150 Val Glu Ala Val Cys 230 Lys Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly Ser Pro Cys 55 Thr Glu Glu His Lys 135 Ala Gly Met Ser Tyr 215 Lys Tyr Phe Pro 40 Cys Glu Lys Lys Glu 120 Ile Val Gly Arg Leu 200 Leu Pro Gly Val 25 Ser Met Arg Pro Ser 105 Glu Phe Lys Ala Gin 185 Gin Pro Ser Met 10 His Pro Thr Cys Leu 90 Tyr Pro Arg Lys Glu 170 Glu Glu Asn Arg Lys 250 Cys Leu Cys Ala 75 His Arg Thr Pro Asp 155 Asn Ser Leu Cys Gly 235 Leu Glu Gly Ala Gln Ala Glu Thr Lys 140 Arg Thr Glu Lys Asp 220 Arg Pro Pro ys Leu Gly Leu Gin Ser 125 His Arg Ala Gin Ala 205 Arg Lys Gly Cys Glu Ala Leu Leu Val 110 Glu Thr Lys His Gly 190 Ser Lys Arg Met Asp Leu Glu Arg His Lys Met Arg Lys Pro 175 Pro Pro Gly Gly Glu 255 Glu Val Gly Cys Gly Ile Ala Ile Leu 160 Arg Cys Arg Phe Ile 240 Tyr Gin Cys His Thr Phe Asp Ser Ser Asn Val Glu 265 270 <210> 3 <211> 1955 <212> DNA <213> Homo Sapiens <400> 3 gtgccctccg ccgctcgccc gcgcgcccgc gctccccgcc tgcgcccagc gccccgcc cgcgccccag tcctcgggcg gtcatgctgc ccctctgcct cgtggccgcc ctgctgcttgg 120 ccgccgggcc cgggccgagc ctgggcgacg aagccatcca ctgcccgccc tgctccgagg 180 agaagctggc gcgctgccgc ecccccgtgg gctgcgagga gctggtgcga gagccgggct 240 gcggetgt tg 300 gt tgcggct c 360 tgatgcacgg 420 tgcagooctc 480 cccatgaccg 540 ggggcaagat 600 ectgeeagag 660 a cgaggacc t 720 ageagtgtca 780 cgggggtgaa 840 ctgacagctt 900 cctgtgctct 960 tgeetgcggc 1020 tgtgtgtgtt 1080 ttggtgttac 1140 ggtgtgttte 1200 aeetaaaaac 1260 ttttattetg 1320 cctgtgggag 1380 catcgtcctt 1440 atggagtaat 1500 ttotettctt 1560 tggeagctga 1620 gagggctagg 1680 gagtggactg 1740 ggt eotgt to 1800 egeeacttgc gggcctgcgc gcaaggcgtg tgaeaaggac caggtgcetg gaaggteaat cgagctgcae ctacatcatc eccagctctg gcttccgggg tcgagagtga ggaggctgea ccagaagttt tgtgageatg acagcccaag eagategatc atttactgac aetteetctg tagaaaaate c t ctc aagc ggtcacgagg ccecaggtcc gagcectgct gaggtggggt aatgtgccta cctaceccat gccc tgggct tyctacccgc tgcatggage gagggtga cc cagaagcact ggggcgccce cgggegetgg eccatcccca gatgggcagc ggcctggage ggcctgccag gag ctgaccc ccctcaaatg ggtgtgccct aggactgaga ctgga ttoao catgtactac attttggcat tea tt cccag tagccagagg tccagaecca tt cctttagg gtgggagagc aeatttctct atggagaaga ttgtggtcac tggggatgcc cccgaggggt tygeggagat accccaacaa tcgceaaaat gggaggatge agcggctggc actgcgaccg gtggeaagtg caaaggggga caggecaggy agagtggagt cgcgtgtgea tggggtaagc ctggcactta toacteacto gtgooagctc gtggagacae agtcagagga gtgggagcct ctcccaaagc tctggttgt t gaagggggtc gagcagtcag cc cacg tget agceatgaag c tgcggggtg ggagaagccc cgaggecate cag ott cagc t egagac egg ceggectgtg cgcttcacag eaa egg caa c Ctggtgtgtg gctggactgc actcagcgtc ctgagtetga cgtgtgcgtg cagagcctgg gcccaagagg attoetteac tagtt ttcag tcctataagg gaagagaeat aaggaagcgt tcagaettge gcaccatctg aaaggaagae ggtgggaaga aggggatgag tcaccgggat tacacecce ctgcacaeac eaggaaagcc cectgtagcg agoaccagtg ccccagggct agcegeaccc ttccacecca gaooggaaga cacoagotgg coctgetaet gtcctgtctc tgegtgcgtg ggtgttet ct tetgagccct tcatccagcc ccttgggagg agagtteaag gtaccttgac ggggtagcag caggctcet. cttggttggc t tgaag ca ca aagaatgcaa gggcttcctg gaacctatcc ttcoagtggc 1860 tcgctccctg tagctctgcc tccctctcca tatctccttc cetacacet ccctccccac acctccctac tcccctgggc atcttctggc ttgactggat ggaaggagac 1920 ttagyaacct accagttggc catgatgtct tttct 1955 c210> 4 c211> 1722 c212> DNA c213> Homo sapiens c400> 4 ggggaaaaga ttttcattac 12 0 tcgaagagga 180 ttgtttttgt 240 aacgcctctt 300 ggtgtatttt 360 caa cgc cat c 420 tttgtggctt 480 ccgctcactg 540 tcttgcaact 600 aagtgtctgg 660 aaaaaaatct 720 accccgaggt 780 gctggccgcc 840 cgacgagaaa 900 gccgggctgc 960 caccgagcgc 1020 gcacgccctg 1080 caagatcgag 1140 gacctactcc 1200 agcagtgaag 1260 gaa ca ctgc c 1320 cccctgccgc 1360 gctaggaaag ccctcctccg ggtggcaaag tttttaattt acctgttctg agattttaag tccactgcat tttttcccct cgtgcacctg gggacctgct gggaggycac ccgggggccc aaagggggcg tatgcggggc gcctttcca ggc tgctgca tgcgcccagg ctgcacggcc agagactccc cccaagatct aaggaccgca cacccccgga agacacatgg agctgcaaag ttttcaccct tgggagaaa cttgatttca cggcgccgcg caaaaatttt ccgatctcat ataattctga gcgctgccct cgcaggcacc ctgctctacc tcttggcccc ac taagagaa cggcccagag tgtgcccCCC tgacctgcgc ggctgcgc tg gcggggtttg gtgagcacga tc cggcc caa gaaagaagct tcatctctgc *aggcttccct cagtgtgggc tctc cggact gaggtgttag acattttctc ca cC5 atgo aaagataaat tatttcggtg cccgctcagg gcttccccca ccagccctcc tgccagaaat tttatccctg gatggtgttg cctgggct cc c ag ccc cc tg cctggccgag CCtCCCCC99 cctcaacgaa ggag cccac c acacacccgc gacccagtcc acctgagatg gcaggagctc tttttccctt tcgcgtagaa ggtttggggt ccaccctctc agctgagggt ccatttttct gt tgct tggg cttgagggtt acctg'ttgca acctctctct tttaaaacaa cactctcgct ctcaccgcgg ttcgtgcact ggctgcgagc gggcagt cgt caggacgagg aagagctacc acct ctgaga atctccgagc aagtttgtcg agacaggagt aaagccagcc tttttgctcc cctgcgaatt ttttttgttt ggctgcagcc tagaaagcgg ctcccacccc ggtgaacaat tctccggcct aggctttaat acatttttgc aaacaaaaac ctcctgccc tcctcctgct gcgagccctg tggtcaagga gcggcgtcta agaagccgc t gcgagcaagt tggccgagga tgaaggctga ggggagccga ctgagcaggg cacgcatggt gccccgtgct gtgtacctgc ccaattgtga ccgcaaagga ttctacaaga gaaagcagtg 1440 caaaccttcc cgtggccgca agcgtggcat ctgctggtgc gtggacaagt acgggatgaa 1500 gctgccaggc atggagtacg ttgacgggga ctttcagtgc cacaccttcg acagcagcaa 1560 cgttgagtga tgcgtccccc cccaaccttt ccctcacccc ctcccacccc cagccccgac 1620 tccagccagc gcctccctcc acoccaggac gccactcatt tcatctcatt taagggaaaa 1680 atatatatct atctatttga ggaaaaaaaa aaaaaaaaaa aa 1722