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Title:  Therapy for injured muscles
United States Patent: 
7,108,857
Issued: 
September 19, 2006

Inventors: 
Brooks; Gregory F. (Irvine, CA), Aoki; Kei Roger (Coto de Caza, CA)
Assignee: 
Allergan, Inc. (Irvine, CA)
Appl. No.: 
10/981,212
Filed: 
November 3, 2004


 

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Abstract

Methods for treating an injured muscle by local administration of a neurotoxin, such as a botulinum toxin, to promote healing and/or to reduce the pain associated with an injured muscle.

SUMMARY OF THE INVENTION

In accordance with the present invention, an effective method for treating an injured muscle includes the step of in vivo, local administration of a therapeutically effective amount of a neurotoxin into or to the vicinity of the injured muscle. The neurotoxin functions to provide a temporary chemodenervation of the injured muscle and to reduce the muscle's contractions. An objective of the present invention is therapy is to facility healing and a speedy return to function of an injured muscle. The injured muscle may be, for example, a strained muscle. In one embodiment, the neurotoxin is administered intramuscularly or subcutaneously. In another embodiment, the step of administering a neurotoxin is preceded by and/or followed by physical therapy and/or surgery.

Further in accordance with the invention, the step of administering the neurotoxin is immediately after the muscle is injured, or is as soon thereafter as is practical. In one embodiment, the neurotoxin is effective to immobilize or to substantially immobilize the injured muscle during at least phase 1 and/or phase 2 of the repair process of the injured muscle.

In accordance with the invention, the neurotoxin can include a targeting component, a therapeutic component and a translocation component. The targeting component can bind to a presynaptic motor neuron. In one embodiment, the targeting component can comprise a carboxyl end fragment of a heavy chain of a butyricum toxin, a tetani toxin, or of a botulinum toxin type A, B, C.sub.1, D, E, F, G or a variant thereof. The therapeutic component can interfere with or modulate the release of a neurotransmitter from a neuron or its processes. In one embodiment, the therapeutic component comprises a light chain of a butyricum toxin, a tetani toxin, or of a botulinum toxin type A, B, C.sub.1, D, E, F, G or a variant thereof. The translocation component can facilitate the transfer of at least a part of the neurotoxin, for example the therapeutic component, into the cytoplasm of the target cell. In one embodiment, the translocation component can comprise an amino end fragment of a heavy chain of a butyricum toxin, a tetani toxin, or of a botulinum toxin type A, B, C.sub.1, D, E, F, G or variants thereof.

Still further in accordance with the invention, the neurotoxin is a botulinum toxin type A, B, E and/or F. In a preferred embodiment, the neurotoxin used to treat an injured muscle is botulinum toxin type A. In fact, the use of botulinum toxin type A is preferred because of its commercial availability, known clinical uses, and successful application to treat muscle injury according to the present invention, as disclosed herein. Use of from about 0.1 U/kg to about 30 U/kg of a botulinum toxin type A and from about 1 U/kg to about 150 U/kg of a botulinum toxin type B is within the scope of a method practiced according to the present disclosed invention. With regard to the other botulinum toxin serotypes (including toxin types E and F) the U/kg dosage to be used is within the range of about 0.1 U/kg to about 150 U/kg, as set forth herein.

Still further in accordance with the invention, the neurotoxin can be recombinantly produced.

A detailed embodiment of the present invention is a method for treating (as by promoting the healing of) an injured muscle by in vivo, local administration of a therapeutically effective amount of a botulinum toxin to an injured muscle, thereby treating the injured muscle. The botulinum toxin can be botulinum toxin type A. Significantly, the present invention also encompasses a method for treating pain associated with an injured muscle by in vivo, local administration of a therapeutically effective amount of a botulinum toxin to an injured muscle, thereby reducing the pain associated with an injured muscle.

Each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present invention provided that the features included in such a combination are not mutually inconsistent.

DESCRIPTION OF THE INVENTION

In a broad embodiment, an effective method for treating an injured muscle according to the present invention can include the step of locally administering a therapeutically effective amount of a neurotoxin into an injured muscle. Preferably, the injured muscle is a strained muscle.

A strain injury of the skeletal muscle may be classified as a shearing injury. In shearing injury, not only the myofibers but also the mysial sheaths are torn. Almost immediately after the injury of the muscle, a repair process of muscle begins. The repair process of the shearing injury may be divided into three phases.

Phase 1 is the destruction phase, which is characterized by hematoma formation, myofiber necrosis, and inflammatory cell reaction. The site of rupture of an otherwise healthy muscle often occurs close to its distal myotendinous junction (MTJ) after a strain. The ruptured myofibers contract and a gap is formed between the stumps. Because skeletal muscle is richly vascularized, hemorrhage from the torn vessels is inescapable and the gap becomes filled with a hematoma, later replaced by scar tissue. In shearing injuries the mechanical force tears the entire myofiber, damaging the myofiber plasma membrane and leaving sarcoplasm open at the ends of the stumps. Because myofibers are very long, string-like cells, the necrosis initiated at this site extends all along the whole length of the ruptured myofiber. The blood vessels are also torn in shearing injuries; thus, blood-borne inflammatory cells gain immediate access to the injury site to induce an inflammation. Phase 1 persists for about 2 to 3 days following the injury.

Phase 2 is the repair phase, which consists of phagocycosis of the necrotized tissue, regeneration of the myofibers, production of connective tissue scar, and capillary ingrowth. The key step in the regeneration of injured muscle tissue is the vascularization of the injured area. The restoration of vascular supply is a necessary for the regeneration of an injured muscle. The new capillaries sprout from surviving trunks of blood vessels and pierce coward the center of injured area. These new capillaries help provide adequate oxygen supply to the regenerating area.

Phase 3 is the remodeling phase, which consists of maturation of the regenerated myofibers, contraction and reorganization of the scar tissue, and restoration of the functional capacity of the repaired muscle. Phase 2 (repair) and 3 (remodeling) often occur simultaneously and persists for about 2 days to about six weeks following phase 1.

In one embodiment of the present invention, the neurotoxin is locally administered, preferably intramuscularly, to immobilize the injured muscle to facilitate healing. Local administration of a neurotoxin according to the present disclosed invention can also reduce the pain experienced due to a muscle injury. Preferably, the administration of the neurotoxin is immediately at the time of injury or closely thereafter. In one preferred embodiment, the neurotoxin is effective to immobilize the injured muscle during the destruction phase (phase 1) to prevent re-rupturing of the muscle.

Without wishing to limit the invention to any particular theory of mechanism of operation; it is believed that mobilization during the repair and/or remodeling phases is beneficial in that such mobilization induces more rapid and intensive capillary ingrowth to the injured area, as well as better muscle fiber regeneration and orientation. Therefore, in one embodiment, the immobilizing effect of the neurotoxin is absent during the repair phase (phase 2) and/or remodeling phase (phase 3). In a more preferred embodiment, the neurotoxin is administered and is effective to immobilize the injured muscle during phase 1, but not during phases 2 and 3 of the repair process. For example, if the neurotoxin is injected, preferably intramuscularly, immediately to the muscle following an injury, it is preferable that the neurotoxin immobilizes the injured muscle for about 3 days after the time of administration. Alternatively, the neurotoxin can have its immobilization effect only up to the point where the patient experiences little or no pain in the use of the injured muscle in basic movements. When this critical point is reached, the patient should be encouraged to start active, progressive mobilization.

In another embodiment of the present invention, the neurotoxin is effective to immobilize the injured muscle for all of the phase 1 3 periods and for a subsequent muscle injury recovery period thereafter.

Neurotoxins, such as certain of the botulinum toxins, which can require from less than about one day to about seven days to exhibit significant clinical muscle paralysis effect and/or and where the muscle paralysis effect is sustained post injection for a period of several months, are within the scope of the present invention, as such neurotoxins can be used to treat relatively serious or long lasting muscle injuries or where a long period of muscle immobilization is indicated for proper healing.

In a broad embodiment, the neurotoxin is a neuromuscular blocking agent. Table 1 shows a non-limiting list of neuromuscular blocking agents and their potential site of actions. In an embodiment, neuromuscular blocking agents having the ability to immobilize muscles, preferably injured muscles, for at least about 5 days, and preferably for at least about 3 days are administered to treat injured muscles. In a preferred embodiment of the present invention, the neurotoxin is a botulinum toxin because of the known uses and clinical safety of a botulinum toxin, such as botulinum toxin type E to treat muscle disorders, such as muscle spasms. In a particularly preferred embodiment of the present invention, especially for severe, or third degree muscle injuries, the locally administered botulinum toxin is a botulinum toxin type E. botulinum toxin type A can also be used in both these embodiments.

TABLE-US-00001 TABLE 1 Compound Site of Action Relative to NMJ Pharmacological Class Acetylcholine Synaptic ACh Esterase Inducers Esterase Inducers Aconitine Presynaptic Sodium Channel Activator Adenoregulin (from the frog Presynaptic Adenosine Receptor Regulator Phyllomedeusa bicolor) Adenosine Agonist Pre & Post Synaptic Adenosine Adenosine Antagonist Pre & Post Synaptic Adenosine Adenosine Pre & Post Synaptic Adenosine Regulating Agent Adrenergics Presynaptic Alpha Adrenergic Anatoxin-A Postsynaptic AChR Agonist Antiepileptics CNS Antiepileptics Antisense Pre & Post Synaptic Antisense technology for specific proteins or messages important in neurotransmitter release, receptor production. Anxiolytics CNS Anxiolytics Antiepileptic Atacurium Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant Atracurium besylate (Tracurium) Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant Baclofen (Lioresal .RTM., Geigy; Intrathecal, Presynaptic GABA analog Medtronic Neurological; generic, Athena, Biocraft, Warner Chilcott) Bacterial, Plant and Fungal Products Batrachotoxin Presynaptic Sodium Channel Activator Benzylpiperidines Synaptic Cleft ACh Esterase Inhibitors (nontraditional) Botanical Pre and Post varies Neurotoxins Synaptic as well as Synaptic Cleft Bungarotoxin-.beta. (.beta.-BuTX) Presynaptic PLA2 and voltage sensitive potassium channel blocker. Snake toxin from Bungarus multicinctus. Bupivacain Pre and Post Synaptic Local Anesthetic Myotoxin Captopril (Capoten .RTM., Presynaptic Antihypertensive ACE Inhibitor zinc Squibb; Capzide .RTM., Squibb) endopeptidase inhibitor Choline+ acetyl Pre Synaptic CAT Inhibitors transferase inhibitors Cholinesterase Inhibitors Synaptic Cleft ACh Esterase Inhibitors Ciguatoxins Presynaptic Sodium Channel Conotoxin MI (alpha Conotoxin) Postsynaptic AChR Antagonist Conotoxin-.mu. (mu-CT) GIIIA Na+ channel blocker Conotoxin-.OMEGA. (omega-CT) GVIA Ca2+ channel blocker in neutrons only Curare Postsynaptic AChR Antagonist Nondepolarizing Dantrolene Sodium (Dantrium, P & G) Postsynaptic Skeletal Muscle Relaxant Dauricine Post Synaptic AChR antagonist Decamethonium Bromide Presynaptic Ganglionic blocker Dendrotoxin Pre and Post Synaptic Potassium Channel blocker Diaminopyridine (3-DAP) Presynaptic Botulinum toxin intoxication Reversal Diazepam CNS Anxiolytic Doxacurium chloride (Nuromax Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant .RTM., Burroughs Wellcome) Doxorubicin (Adriamyocin, Adria; Postsynaptic Myotoxin Chemo Myectomy Rubex, Immunex; Cetus Onoclogy) Epibatidine Dihydrochloride Postsynaptic AChR Agonist Felbamate (Felbatol, Carter- Presynaptic CNS Antiepileptic Wallace lic to Schering-Plough) Foroxymithine Presynaptic Angiotensin I Converting Enzyme inhibitor Gabapentin (Neurontin, Parke-Davis) Presynaptic CNS Antiepileptic GABA Analog Gallamine Postsynaptic AChR Antagonist Grayantoxin Presynaptic Sodium Channel Activator Hexahydroazepinyl Presynaptic ACh Releaser Acetamides and other chemical classes Huperzine A Synaptic Cleft ACh Esterase Inhibitor Insect Venoms Ion Channel Blockers Pre and Post Synaptic Channel Blockers Ion Channel Stimulants Pre and Post Synaptic Channel Stimulants Latrotoxin-.alpha. Presynaptic Calcium Ionophore black widow spider venom component Lidocaine, procaine, mepivacain, etc. Presynaptic Local Anesthetics Linopirdine (DuP 996, Dupont Merck) Presynaptic ACh Release Enhancer Lophotoxin and analogs Postsynaptic AChR Antagonist Irreversible Marine Natural Products Methocarbamol (Robaxin, Robins Co.) CNS Depression, muscle relaxation. Methyllycaconitine Mivacurium chloride (Mivacro .RTM., Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant BW-BW1090U, Burroughs Wellcome) Modified Clostridial Toxins Pre Synaptic ACh Release Inhibitor receptor, agrin, neurotransmitters, Monoclonal antibodies against plasma membrane components, inactivating enzymes, etc. NMJ components Muscarinic Agonist and Antagonists Pre and Post Muscarinic CNS Agonist Antagonist Synaptic, Neosaxitoxin Neosurugatoxin Presynaptic Sodium Channel Blocker Autonomic Ganglionic AChR Blocker. (no effect @ NMJ) Neuromuscular Blocking Agents Postsynaptic AChR Antagonists AChR Depolarizing Neurotoxins from reptile, Pre and Post Synaptic as varies insects, and other sources well as Synaptic Cleft Pancuronium Bromide (Organon) Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant Pancuronium-3-OH Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant metabolites (Organon) Papverine HCl (30 mg/ml) Smooth Muscle Relaxants Physostigmine and Analogs Synaptic Cleft ACh Esterase inhibitor Pipercuronium Postsynaptic AChR Antagonist (Arduan, Organon) Nondepolarizing muscle relaxant Presynaptic Nerve Terminal Recpetors Pre Synaptic any extra or intraneuronal recpetors on nerve terminal Short Neurotoxin alpha Postsynaptic AChR Antagonist .beta.-Bungarotoxin (.beta.-BuTX) Presynaptic Snake toxin from Bungarus multicinctus. Succinylcholine chloride Postsynaptic AChR Receptor Agonist Depolarizing skeletal (Anectine, Burroughs Wellcome) muscle relaxant Tetanus Toxin Presynaptic EAA release inhibitor Tetanus Toxin Transporter Presynaptic Tetrahydroamino-acridine (THA) Synaptic Cleft ACh Esterase Inhibitor Tetrodoxtoxin Pre and Post Synaptic Sodium Channel Blocker Tiagabine (Novo Nordisk) CNS Antiepileptic GABA uptake inhibitor Transglutaminase inhibitors or Pre and Post Synaptic Enzyme induction prevention Valium diazepam CNS Anxiolytic Vecuronium (Norcuron, Organon) Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant Vecuronium-3-OH metabolites Postsynaptic AChR Antagonist Nondepolarizing muscle relaxant (Organon) Veratridine Presynaptic Sodium Channel Activator Vigabatrin (Sabril, Marion Presynaptic CNS Antiepileptic GABA metabolism inhibitor (irreversible) Merrell Dow) Vesamicol and other drugs Presynaptic ACh Vesicle transport inhibitor with the same mechanism. Zinc Endopeptidase and other proteases Pre Synaptic Enyzmes. reduce neurotransmitter release delivered by Botulinum toxin or tetanus toxin transporter

In a broad embodiment, the neurotoxin can comprise a targeting component, a therapeutic component and a translocation component. The targeting component can bind to a presynaptic motor neuron. In one embodiment, the targeting component can comprise a carboxyl end fragment of a heavy chain of a butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C1, D, E, F, G or a variant thereof. In a preferred embodiment, the targeting component can include a carboxyl end fragment of a botulinum toxin type A.

The therapeutic component can substantially interfere with or modulate the release of neurotransmitters from a cell or its processes. In one embodiment, the therapeutic component comprises a light chain of a butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C.sub.1, D, E, F, G or a variant thereof. In a preferred embodiment, the therapeutic component may include a light chain of a botulinum toxin type which has a short biological persistence, for example less than about 5 days, preferably less than about 3 days. Preferably, such light chain can be a light chain of a botulinum toxin type E or F. Alternately, the light chain can be a light chain of a botulinum toxin type A.

The translocation component can facilitate the transfer of at least a part of the neurotoxin, for example the therapeutic component into the cytoplasm of the target cell. In one embodiment, the translocation component comprises an amino end fragment of a heavy chain of a butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C.sub.1, D, E, F, G or variants thereof. In a preferred embodiment, the translocation component comprises an amino end fragment of a heavy chain of a botulinum toxin type A.

In one embodiment, the targeting component comprises a carboxyl end fragment of a heavy chain of a botulinum toxin type E or F, the therapeutic component comprises a light chain of a botulinum toxin type E or F and the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type E or F. In a preferred embodiment, the neurotoxin comprises a botulinum toxin type E. In another preferred embodiment, the neurotoxin comprises a botulinum toxin type F. In yet another embodiment, the neurotoxin comprises a mixture of botulinum toxin type E and F.

In one embodiment, the targeting component comprises a carboxyl end fragment of a heavy chain of a botulinum toxin type A, the therapeutic component comprises a light chain of a botulinum toxin type A and the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type A. In a preferred embodiment, the neurotoxin of the present invention comprises a botulinum toxin type A. A suitable botulinum toxin type A to use herein is BOTOX.RTM. (Allergan, Inc., Irvine, Calif.)

Although the neurotoxins of the present invention treats injured muscles by immobilizing them, in one embodiment, the neurotoxin may also be administered to injured muscles to reduce pain and/or spasm. In another embodiment, the neurotoxin is able to immobilize the injured muscle and to reduce pain associated with that injured muscle. In a preferred embodiment, a neurotoxin, for example a botulinum toxin type E, pr most preferably type A, is administered to a strained muscle to immobilize the muscle and/or to reduce pain associated with that muscle.

Of course, an ordinarily skilled medical provider can determine the appropriate dose and frequency of administration(s) to achieve an optimum clinical result. That is, one of ordinary skill in medicine would be able to administer the appropriate amount of the neuromuscular blocking agent at the appropriate time(s) to effectively immobilize the injured muscle(s). The dose of the neurotoxin to be administered depends upon a variety of factors, including the size of the muscle, the severity of the muscle injury. In a preferred embodiment, the dose of the neurotoxin administered immobilizes the injured muscle(s) for no longer than the duration of phase 1 of the repair process. In the various methods of the present invention, from about 0.1 U/kg to about 15 U/kg, of botulinum toxin type A can be administered to the injured muscle. Preferably, about 1 U/kg to about 20 U/kg of botulinum toxin type A may be administered to the injured muscle. Use of from about 0.1 U/kg to about 30 U/kg of a botulinum toxin type A and from about 1 U/kg to about 150 U/kg of a botulinum toxin type B is within the scope of a method practiced according to the present disclosed invention. With regard to the other botulinum toxin serotypes (including toxin types E and F) the U/kg dosage to be used is within the range of about 0.1 U/kg to about 150 U/kg, as set forth herein.

Although intramuscular injection is the preferred route of administration, other routes of local administration are available, such as subcutaneous administration.

In another broad embodiment, the method of treating injured muscle according to this invention further includes other steps described below. These other steps may be taken prior to, in conjunction with or following the step of administering a neurotoxin, preferably to the injured muscle. For example, the present recommended treatment for strained muscle includes resting, icing, compression and elevating. These four steps (or procedures) have the same objective. They minimize bleeding from ruptured blood vessels to rupture site. This will prevent the formation of a large hematoma, which has a direct impact on the size of scar tissue at the end of the regeneration. A small hematoma and the limitation of interstitial edema accumulation on the rupture site also shorten the ischemic period in the granulation tissue, which in turn accelerates regeneration.

Other additional steps may be employed in the treatment of injured muscles. In one embodiment, the additional step include an administration of nonsteroidal anti-inflammatory drugs (NSAIDs), therapeutic ultrasound, hyperbaric oxygen, and in severe injuries, surgery may also be employed. NSAIDs should be a part of early treatment and should he started immediately after the injury. Short-term use of NSAIDs in the early phase of healing decreases the inflammatory cell reaction, and has no adverse effects on tensile or contractile properties of injured muscle.

In another embodiment, the additional step includes the use of therapeutic ultrasound. Therapeutic ultrasound is widely recommended and used in the treatment of muscle strains. It is thought that therapeutic ultrasound promotes the proliferation phase of myoregeneration.

In another embodiment, the additional step includes the use of hyperbaric oxygen. It is known that hyperbaric oxygen therapy in rabbits during the early phase of the repair substantially improves the final outcome. It is believed that such hyperbaric oxygen therapy in other mammals, for example human beings, may be helpful, such as by speeding up muscle regeneration.

In another embodiment, the additional step includes surgical intervention. Surgical treatment of muscle injuries should be reserved for the most serious injuries, because in most cases conservative treatment results in a good outcome. Surgical treatment is indicated only in cases of (1) large intramuscular hematomas, (2) third-degree strains or tears of muscles with few or no agonise muscles, and (3) second-degree strains, if more than half of the muscle belly is torn.

In another broad aspect of this invention, recombinant techniques are used to produce at least one of the components of the neurotoxins. The technique includes steps of obtaining genetic materials from either DNA cloned from natural sources, or synthetic oligonucleotide sequences, which have codes for one of the components, for example the therapeutic, translocation and/or targeting component(s). The genetic constructs are incorporated into host cells for amplification by first fusing the genetic constructs with a cloning vectors, such as phages or plasmids. Then the cloning vectors are inserted into hosts, preferably E. coli's. Following the expressions of the recombinant genes in host cells, the resultant proteins can be isolated using conventional techniques. The protein expressed may comprise all three components of the neurotoxin. For example, the protein expressed may include a light chain of botulinum toxin type E (the therapeutic component), a heavy chain, preferably the H.sub.N, of a botulinum toxin type B (the translocation component), and an H.sub.C of botulinum toxin type A, which selectively binds to the motor neurons. In one embodiment, the protein expressed may include less than all three components of the neurotoxin. In such case, the components may be chemically joined using techniques known in the art.

There can be many advantages to producing these neurotoxins recombinantly. For example, production of neurotoxin from anaerobic Clostridium cultures is a cumbersome and time-consuming process including a multi-step purification protocol involving several protein precipitation steps and either prolonged and repeated crystallization of the toxin or several stages of column chromatography. Significantly, the high toxicity of the product dictates that the procedure must be performed under strict containment (BL-3). During the fermentation process, the folded single-chain neurotoxins are activated by endogenous Clostridial proteases through a process termed nicking. This involves the removal of approximately 10 amino acid residues from the single-chain to create the dichain form in which the two chains remain covalently linked through the intrachain disulfide bond.

The nicked neurotoxin is much more active than the unnicked form. The amount and precise location of nicking varies with the serotypes of the bacteria producing the toxin. The differences in single-chain neurotoxin activation and, hence, the yield of nicked toxin, are due to variations in the type and amounts of proteolytic activity produced by a given strain. For example, greater than 99% of Clostridial botulinum type A single-chain neurotoxin is activated by the Hall A Clostridial botulinum strain, whereas type B and E strains produce toxins with lower amounts of activation (0 to 75% depending upon the fermentation time). Thus, the high toxicity of the mature neurotoxin plays a major part in the commercial manufacture of neurotoxins as therapeutic neurotoxins.

The degree of activation of engineered Clostridial toxins is, therefore, an important consideration for manufacture of these materials. It would be a major advantage if neurotoxins such as botulinum toxin and tetanus toxin could be expressed, recombinantly, in high yield in rapidly-growing bacteria (such as heterologous E. coli cells) as relatively non-toxic single-chains (or single chains having reduced toxic activity) which are safe, easy to isolate and simple to convert to the fully-active form.

With safety being a prime concern, previous work has concentrated on the expression in E. coli and purification of individual H and L chains of tetanus and botulinum toxins; these isolated chains are, by themselves, non-toxic; see Li et al., Biochemistry 33:7014 7020 (1994); Zhou et al., Biochemistry 34:15175 15181 (1995), hereby incorporated by reference herein. Following the separate production of these peptide chains and under strictly controlled conditions the H and L subunits can be combined by oxidative disulphide linkage to form the neuroparalytic di-chains.
 


Claim 1 of 10 Claims

1. A method for treating an injured muscle, the method comprising the step of local administration of a therapeutically effective amount of a botulinum toxin to an injured muscle, thereby facilitating the healing of the injured muscle, wherein the injured muscle is selected from the group consisting of a biceps, knee and shin muscle.

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