Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
  • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/08—Clostridium, e.g. Clostridium tetani
• • 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
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration

Definitions

• the present invention relates to methods for treating muscle injuries.
• the present invention relates to a method for treating an injured muscle by administration of a neurotoxin to the injured muscle.
• Injuries to muscles include acute injuries to skeletal muscles such as contusions (bruises), lacerations, ischemia, strains, and complete ruptures. These injuries may cause tremendous pain and can incapacitate the affected person, preventing them from being able to go to work or even to participate in normal daily activities.
• strain also known as stretch-induced injuries
• strains can account for up to 30% of all injuries treated by occupational or sports medicine professionals. Garrett et al. Am J Sports Med, 24(6):S2-S8, 1996.
• a muscle strain injury is characterized by a disruption of a muscle- tendon unit. The disruption of the muscle-tendon unit may occur anywhere on the muscle. This type of injury most commonly occurs near the myotendinous junction (MTJ) of the superficial muscles working across two joints, such as the rectus femoris, semitendinousus and gastroenemius muscles.
• MMTJ myotendinous junction
• Muscle strain may result from an eccentric exercise, or uncommon use of the muscle.
• eccentric contractions employ fewer active motor units to generate higher forces.
• the overextended muscle units experience excessive tension during lengthening.
• the excessive tension may cause microscopic damages to the contractile element of the muscle, centering on what appears to be random disruptions of the Z-lines.
• the affected person may experience a delayed onset muscle soreness, characterized by pain, weakness and a limited range of motion. The pain is most intense for about 1 to 2 days after the muscle injury and the weakness and limited range of motion can last for a week or more. If a minor strain of the skeletal muscles is treated inappropriately, a more serious injury can occur.
• muscle strains There are three classifications of muscle strains, based on the severity of the injury and the nature of the hematoma: (1) mild, (first degree) strain; a tear of a few muscle fibers; minor swelling and discomfort with no or only minimal loss of strength and restriction of movement; (2) moderate, (second degree) strain; a greater damage of muscle fibers with a clear loss of strength, and; (3) severe (third degree) strain; a tear extending across the whole muscle belly, resulting in a total loss of muscle function.
• mild, (first degree) strain a tear of a few muscle fibers; minor swelling and discomfort with no or only minimal loss of strength and restriction of movement
• moderate, (second degree) strain a greater damage of muscle fibers with a clear loss of strength
• severe (third degree) strain a tear extending across the whole muscle belly, resulting in a total loss of muscle function.
• Intramuscular and intermuscular hematomas Two different types of hematomas occur in the injured muscle: intramuscular and intermuscular hematomas.
• the first type intramuscular hematomas, is limited in size by the intact muscle fascia. There, the extravasation of blood increases the intramuscular pressure, compressing and limiting the size of the hematoma. Such type of hematoma causes pain and loss of function of the muscle.
• the second type, intermuscular hematomas develops when the muscle fascia is ruptured and extravasated blood spreads into the intermuscular spaces without significantly increasing the pressure within the muscle. This type of hematoma may not cause significant pain if the pressure within the muscle does not increase.
• a known method for immobilization of an injured/strained muscle requires use of a physical restraint or cast.
• a cervical collar can be used to immobilize an injured cervical flexor or extensor.
• the use of a restraint is often cumbersome and uncomfortable.
• it is not practical or possible to use a physical restraint for injuries of certain muscle groups, it is not practical or possible to use a physical restraint. For example, it is very difficult to immobilize a strained upper trapezius or gluteus maximus muscle with a restraint.
• Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism.
• the spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism.
• the effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores.
• the botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
• Botulinum toxin type A (“BoNT/A”) is the most lethal natural biological neurotoxin known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) serotype A is a LD 50 in mice. One unit (U) of botulinum toxin is defined as the LD 50 upon intraperitoneal injection into female Swiss Webster mice weighing 18-20 grams each. Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C ⁇ , D, E, F and G each of which is distinguished by neutralization with serotype- specific antibodies.
• botulinum toxin serotypes vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that BoNt A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin serotype B (BoNT/B). Additionally, BoNt/B has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD 50 for BoNt A. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
• Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles.
• BoNt/A has been approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm.
• Non-serotype A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to BoNt/A.
• Clinical effects of intramuscular of a botulinum toxin, such as BoNt/A can be noted in a matter of hours.
• botulinum toxins can, upon intramuscular injection, produce significant muscle paralysis within one day of the injection, as measured, for example, by the mouse Digit Abduction Score (DAS).
• DAS Digit Abduction Score
• the typical duration of symptomatic relief from a single intramuscular injection of BoNt/A averages about three months.
• Botulinum toxins including botulinum toxin type A, with reduced periods of in vivo biological activity are set forth in co- pending U.S. patent application serial number 09/620840, which application is incorporated herein by reference in its entirety.
• botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites.
• botulinum serotypes A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein.
• BoNT/B, D, F and G act on vesicle- associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site.
• VAMP vesicle- associated protein
• botulinum toxin serotype Ci (B0NT/C 1 ) has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.
• the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages.
• the toxin binds with high affinity to the presynaptic membrane of the target neuron through a specific interaction between the H chain and a cell surface receptor; the receptor is thought to be different for each serotype of botulinum toxin and for tetanus toxin.
• the carboxyl end segment of the H chain, H c appears to be important for targeting of the toxin to the cell surface.
• 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 end segment of the H chain, HN, 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 cleavage of the critical intracellular exocytosis proteins by the L chain.
• the entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane.
• VAMP vesicle-associated membrane protein
• Each toxin specifically cleaves a different bond.
• the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins.
• the BoNt/A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms.
• BoNT/ B and Ci are apparently produced as only a 500 kD complex.
• BoNT/D is produced as both 300 kD and 500 kD complexes.
• BoNT/E and F are produced as only approximately 300 kD complexes.
• the complexes i.e. molecular weight greater than about 150 kD
• These two non-toxin proteins may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested.
• botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
• botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
• BoNt/A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C-i, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture.
• Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form.
• the proteolytic strains that produce, for example, the BoNt/B serotype only cleave a portion of the toxin produced.
• the exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the BoNt/B toxin is likely to be inactive, possibly accounting for the known significantly lower potency of BoNt/B as compared to BoNt/A.
• BoNt/B has, upon intramuscular injection, a shorter duration of activity and is also less potent than BoNt/A at the same dose level.
• BoNt/A has been used in clinical settings as follows:
• BOTOX® 1 per intramuscular injection (multiple muscles) to treat cervical dystonia
• BOTOX® 5-10 units of BOTOX® per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);
• flexor carpi radialis 15 U to 60 U
• biceps brachii 50 U to 200 U.
• Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® by intramuscular injection at each treatment session.
• BoNt/A botulinum toxin serotypes
• BoNT/A preparations BOTOX ® and Dysport ®
• BoNT/B and F both obtained from Wako Chemicals, Japan
• DAS mouse digit abduction scoring assay
• the therapeutic index was calculated as LD 50 /ED50 Separate groups of mice received hind limb injections of BOTOX ® (5.0 to 10.0 units/kg) or BoNt B (50.0 to 400.0 units/kg), and were tested for muscle weakness and increased water consumption, the later being a putative model for dry mouth. Antigenic potential was assessed by monthly intramuscular injections in rabbits (2.0 or 8.7 Units/kg for BoNt/B or 3.0 Units/kg for BOTOX ® ). Peak muscle weakness and duration were dose related for all serotypes. DAS ED 50 values (units/kg) were as follows: BOTOX ® : 6.7, Dysport ® : 24.7, BoNt/B: 11.8 to 244.0, BoNT/F: 4.3.
• BOTOX ® had a longer duration of action than BoNt/B or BoNt/F.
• Therapeutic index values were as follows: BOTOX ® : 10.5, Dysport ® : 6.3, BoNt/B: 4.8. Water consumption was greater in mice injected with BoNt/B than with BOTOX ® , although BoNt/B was less effective at weakening muscles. After four months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of 4 (where treated with 6.5 ng/kg) rabbits developed antibodies against BoNt B. In a separate study, 0 of 9 BOTOX ® treated rabbits demonstrated antibodies against BoNt/A.
• DAS results indicate relative peak potencies of BoNt/A being equal to BoNt/F, and BoNt/F being greater than BoNt/B. With regard to duration of effect, BoNt/A was greater than BoNt/B, and BoNt/B duration of effect was greater than BoNt/F.
• BoNt/A was greater than BoNt/B
• BoNt/B duration of effect was greater than BoNt/F.
• the two commercial preparations of BoNt/A (BOTOX ® and Dysport ® ) are different.
• the increased water consumption behavior observed following hind limb injection of BoNt/B indicates that clinically significant amounts of this serotype entered the murine systemic circulation.
• the results also indicate that in order to achieve efficacy comparable to BoNt/A, it is necessary to increase doses of the other serotypes examined. Increased dosage can comprise safety.
• serotype B was more antigenic than was BOTOX ® , possibly because of the higher protein load injected to achieve
• the tetanus neurotoxin acts mainly in the central nervous system, while botulinum neurotoxin acts at the neuromuscular junction; both act by inhibiting acetylcholine release from the axon of the affected neuron into the synapse, resulting in paralysis.
• the effect of intoxication on the affected neuron is long-lasting and until recently has been thought to be irreversible.
• the tetanus neurotoxin is known to exist in one immunologically distinct serotype. Acetylcholine
• the neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postgangiionic neurons of the parasympathetic nervous system, and by some of the postgangiionic neurons of the sympathetic nervous system.
• acetylcholine has an excitatory effect.
• acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of the heart by the vagal nerve.
• the efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system.
• the preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord.
• the preganglionic sympathetic nerve fibers extending from the cell body, synapse with postgangiionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postgangiionic neurons. Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors.
• the muscarinic receptors are found in all effector cells stimulated by the postgangiionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postgangiionic cholinergic neurons of the sympathetic nervous system.
• the nicotinic receptors are found in the synapses between the preganglionic and postgangiionic neurons of both the sympathetic and parasympathetic.
• the nicotinic receptors are also present in many membranes of skeletal muscle fibers at the neuromuscular junction. Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane.
• non-neuronal secretory cells such as, adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and insulin, respectively, from large dense-core vesicles.
• the PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development.
• Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell.
• Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
• a neuromuscular junction is formed in skeletal muscle by the proximity of axons to muscle cells.
• a signal transmitted through the nervous system results in an action potential at the terminal axon, with activation of ion channels and resulting release of the neurotransmitter acetylcholine from intraneuronal synaptic vesicles, for example at the motor endplate of the neuromuscular junction.
• the acetylcholine crosses the extracellular space to bind with acetylcholine receptor proteins on the surface of the muscle end plate. Once sufficient binding has occurred, an action potential of the muscle cell causes specific membrane ion channel changes, resulting in muscle cell contraction.
• the acetylcholine is then released from the muscle cells and metabolized by cholinesterases in the extracellular space. The metabolites are recycled back into the terminal axon for reprocessing into further acetylcholine.
• 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.
• the neurotoxin is administered intramuscularly or subcutaneously.
• the step of administering a neurotoxin is preceded by and/or followed by physical therapy and/or surgery.
• the step of administering the neurotoxin is immediately after the muscle is injured, or is as soon thereafter as is practical.
• 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.
• the neurotoxin can include a targeting component, a therapeutic component and a translocation component.
• the targeting component can bind to a presynaptic motor neuron.
• 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, Ci, 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.
• the therapeutic component comprises a light chain of a butyricum toxin, a tetani toxin, or of a botulinum toxin type A, B, C-i, 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.
• 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, Ci, D, E, F, G or variants thereof.
• the neurotoxin is a botulinum toxin type A, B, E and/or F.
• the neurotoxin used to treat an injured muscle is botulinum toxin type A.
• 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.
• 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.
• 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.
• 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.
• Heavy chain means the heavy chain of a clostridial neurotoxin. It preferably has a molecular weight of about 100 kDa and may be referred to herein as H chain or as H.
• HN means a fragment (preferably having a molecular weight of about 50 kDa) derived from the H chain of a Clostridial neurotoxin which is approximately equivalent to the amino terminal segment of the H chain, or the portion corresponding to that fragment in the intact in the H chain. It is believed to contain the portion of the natural or wild type clostridial neurotoxin involved in the translocation of the L chain across an intracellular endosomal membrane.
• He means a fragment (about 50 kDa) derived from the H chain of a clostridial neurotoxin which is approximately equivalent to the carboxyl terminal segment of the H chain, or the portion corresponding to that fragment in the intact H chain. It is believed to be immunogenic and to contain the portion of the natural or wild type Clostridial neurotoxin involved in high affinity, presynaptic binding to motor neurons.
• “Injured muscle” includes a strained, torn or pulled muscle, as well as a muscle with a contusion (bruise), laceration, ischemia or rupture.
• “Light chain” means the light chain of a clostridial neurotoxin. It preferably has a molecular weight of about 50 kDa, and can be referred to as L chain, L or as the proteolytic domain (amino acid sequence) of a clostridial neurotoxin. The light chain is believed to be effective as an inhibitor of neurotransmitter release when it is released into a cytoplasm of a target cell.
• Local administration means direct administration of a pharmaceutical at or to the vicinity of a site on or within an animal body, at which site a biological effect of the pharmaceutical is desired. Local administration excludes systemic routes of administration, such as intravenous or oral administration.
• Neuron means a chemical entity that is capable of interfering with or modulating at least one function of a neuron.
• the "neurotoxin” can be naturally occurring or man-made.
• the “neurotoxin” can be a small molecule, a large molecule, a polypeptide, a conjugated- polypeptide or mixtures thereof.
• Variant means a chemical entity which is slightly different from a parent chemical entity but which still has a biological effect.
• the biological effect of the variant may be substantially the same or better than that of the parent.
• a variant light chain of a botulinum toxin having at least one amino acid replaced, modified, deleted or added may have the same or better ability to prevent the release of neurotransmitter vesicles.
• the biological effect of a variant may be decreased.
• a variant light chain of a botulinum toxin type A having a leucine-based motif removed may have a shorter biological persistence than that of the parent (or native) botulinum toxin type A light chain.
• 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.
• the injured muscle is a strained muscle.
• a strain injury of the skeletal muscle may be classified as a shearing injury.
• 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.
• MTJ myotendinous junction
• 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.
• 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.
• 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.
• the administration of the neurotoxin is immediately at the time of injury or closely thereafter.
• the neurotoxin is effective to immobilize the injured muscle during the destruction phase (phase 1) to prevent re-rupturing of the muscle.
• the immobilizing effect of the neurotoxin is absent during the repair phase (phase 2) and/or remodeling phase (phase 3).
• 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.
• 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.
• 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.
• 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.
• the neurotoxin is a neuromuscular blocking agent.
• Table 1 shows a non-limiting list of neuromuscular blocking agents and their potential site of actions.
• 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.
• 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.
• the locally administered botulinum toxin is a botulinum toxin type E.
• Botulinum toxin type A can also be used in both these embodiments.
• Captopril Presynaptic Antihypertensive (Capoten .RTM., ACE Inhibitor Squibb; Capzide .RTM. zinc endopeptidase Squibb) inhibitor
• Gabapentin Presynaptic Antiepileptic (Neurontin, Parke- CNS GABA Analog Davis) 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 Pre and Post Channel Blockers Blockers Synaptic Ion Channel Pre and Post Channel Stimulants Stimulants Synapti Latrotoxin- Presynaptic Calcium Ionophore black widow spider venom component
• AChR Antagonists Blocking Agents AChR Depolarizing Neurotoxins from Pre and Post varies reptile, insects, Synaptic as and other sources well as Synaptic Cleft
• Presynaptic Nerve Pre Synaptic any extra or intraneuronal Terminal Recpetors recpetors on nerve terminal Short Neurotoxin Postsynaptic AChR Antagonist alpha ⁇ -Bungarotoxin Presynaptic Snake toxin from Bungarus ( ⁇ -BuTX) multicinctus .
• Nondepolarizing muscle Organon relaxant Vecuronium-3-OH Postsynaptic AChR Antagonist metabolites Nondepolarizing muscle
• Zinc Endopeptidase Pre Synaptic Enyzmes . and other proteases reduce neurotransmitter delivered by release Botulinum toxin or tetanus toxin transporter
• the neurotoxin can comprise a targeting component, a therapeutic component and a translocation component.
• the targeting component can bind to a presynaptic motor neuron.
• 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.
• 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.
• the therapeutic component comprises a light chain of a butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C-i, D, E, F, G or a variant thereof.
• 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.
• such light chain can be a light chain of a botulinum toxin type E or F.
• 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.
• 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-i, D, E, F, G or variants thereof.
• the translocation component comprises an amino end fragment of a heavy chain of a botulinum toxin type A.
• 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
• the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type E or F.
• the neurotoxin comprises a botulinum toxin type E.
• the neurotoxin comprises a botulinum toxin type F.
• the neurotoxin comprises a mixture of botulinum toxin type E and F.
• 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
• the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type A.
• the neurotoxin of the present invention comprises a botulinum toxin type A.
• a suitable botulinum toxin type A to use herein is BOTOX ® (Allergan, Inc., Irvine, California)
• 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.
• the neurotoxin is able to immobilize the injured muscle and to reduce pain associated with that injured muscle.
• 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.
• 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.
• a botulinum toxin type A 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.
• 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.
• 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.
• 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.
• NSAIDs nonsteroidal anti-inflammatory drugs
• therapeutic ultrasound a part of early treatment and should he started immediately after the injury.
• hyperbaric oxygen a gas stream containing a spasmodic agents
• 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.
• 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.
• 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.
• 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.
• 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.
• the protein expressed may include a light chain of botulinum toxin type E (the therapeutic component), a heavy chain, preferably the HN, of a botulinum toxin type B (the translocation component), and an H c of botulinum toxin type A, which selectively binds to the motor neurons.
• 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.
• 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.
• the high toxicity of the product dictates that the procedure must be performed under strict containment (BL-3).
• BL-3 strict containment
• 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.
• Clostridial toxins 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.
• Example 1 Treatment of a Ruptured Biceps Tendon Ruptures of the biceps brachii commonly occur at the proximal end and involve the long head of the biceps. The muscle may rupture at the distal insertion onto the radius, but is rare.
• a 45 year old man presents with a bulge in the lower arm after lifting heavy boxes. He reports a history of sudden sharp pain in the upper arm, often accompanied by an audible snap. The man is diagnosed as having a ruptured biceps tendon and is at the beginning of phase 1 of the repair process. The rupture may be classified as a mild second degree strain.
• the patient is treated by a bolus injection of between about 0.1 U/kg to about 25 U/kg of a neurotoxin intramuscularly to the biceps.
• the neurotoxin is botulinum toxin type E and/or F, more preferably type A.
• the particular dose and frequency of administrations depend upon a variety of factors, and are to be determined by the treating physician.
• the patient is further instructed to rest and apply ice and compression to the biceps.
• Within about three days after the administration of the neurotoxin the patient is able to bend his arm. Also, after about three days, the patient experiences a reduction in inflammation, which is a sign that the patient is entering into phase 2 and 3 of the repair process. The patient also experiences a significant pain reduction.
• Local administration of from about 10 units to about 200 units of botulinum toxin type A can also be used for long term (2-4 months) muscle immobilization and pain reduction.
• Rupture of the extensor mechanism of the knee occurs in one of two ways: in younger patients as a result of a sudden or violent force (such as jumping, heavy lifting); and in older patients as a result of relatively trivial force.
• a sudden or violent force such as jumping, heavy lifting
• older patients as a result of relatively trivial force.
• This condition affects older patients who have typically been somewhat sedentary and have suddenly increased their activity level, or patient who have had some preexisting or co-existing condition such as diabetes mellitus, rheumatoid arthritis, and other systemic inflammatory disorders, or prior knee surgery.
• a 22 year old female soccer player presents with an inability to extend her knee.
• the patient also is also unable to do straight leg raise, but is able to walk if she keeps a hand on her thigh and maintain her knee in extension.
• a plain radiograph shows that the patella is in a lower than usual location. The patient is diagnosed with a severe rupture of the quadriceps.
• the patient After determining the injury is severe (third degree), the patient agrees to undergo reparative surgery. Post-operationally, the patient is treated by a bolus injection of between about 0.1 U/kg to about 25 U/kg of a neurotoxin (such as about 10 units to about 400 units of botulinum toxin type A) intramuscularly to the quadriceps.
• a neurotoxin such as about 10 units to about 400 units of botulinum toxin type A
• the neurotoxin is botulinum toxin type A.
• the particular dose and frequency of administrations depend upon a variety of factors, and are to be determined by the treating physician.
• the patient is further instructed to rest and apply ice and compression to the quadriceps. Within about 15 days after the administration of the neurotoxin, gradual movement and activity of the injured muscle is possible.
• botulinum toxin therapy would facilitate her early return to this activity.
• Local administration of from about 10 units to about 200 units of botulinum toxin type A can be used for long term (2-4 months) muscle immobilization.
• Runners commonly experience shin splits in the lower limb which causes pain and restricts this activity.
• the lower leg pain resulting from shin splits is caused by very small tears in the leg muscles at their point of attachment to the shin.
• strained muscle such as a shin splint
• five steps are recommended: (1) Protect the injured muscle from further injury by using splints, pads and/or crutches; (2) Restrict activity, usually for 48 to 72 hours to allow the healing process to begin.
• Suitable botulinum toxins, including botulinum toxin type A, with reduced periods of in vivo biological activity suitable for use herein are set forth in co-pending U.S.
• Chromosomal DNA from C. botulinum serves as a template in the amplification reaction.
• the PCR amplification is performed in a 100 ⁇ l volume containing 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCI 2 , 0.2 mM of each deoxynucleotide triphosphate (dNTP), 50 pmol of each primer, 200 ng of genomic DNA and 2.5 units of Taq-polymerase (Promega).
• the reaction mixture is subjected to 35 cycles of denaturation (1 minute at 94° C), annealing (2 minutes at 37°C) and polymerization (2 minutes at 72°C). Finally, the reaction is extended for an additional 5 minutes at 72°C.
• the PCR amplification product is digested with Stu I and EcoR I, purified by agarose gel electrophoresis, and ligated into Sma I and EcoR I digested pBluescript II SK* to yield the plasmid, pSAL.
• Bacterial transformants harboring this plasmid are isolated by standard procedures.
• the identity of the cloned L chain polynucleotide is confirmed by double stranded plasmid sequencing using SEQUENASE (United States Biochemicals) according to the manufacturer's instructions. Synthetic oligonucleotide sequencing primers are prepared as necessary to achieve overlapping sequencing runs.
• the cloned sequence is found to be identical to the sequence disclosed by Binz, et al., in J. Biol. Chem.
• a small-scale SDS-PAGE analysis confirmed the presence of a 90 kDa protein band in samples derived from IPTG-induced bacteria.
• This M r is consistent with the predicted size of a fusion protein having MBP ( ⁇ 40 kDa) and BoNT/A-L chain ( ⁇ 50 kDa) components.
• the IPTG-induced clones contained substantially larger amounts of the fusion protein.
• the MBP-L chain fusion proteins encoded by the pCAL and pCAL- TyrU7 expression plasmids are purified from bacteria by amylose affinity chromatography. Recombinant wild-type or mutant L chains are then separated from the sugar binding domains of the fusion proteins by site- specific cleavage with Factor X 2 . This cleavage procedure yielded free MBP, free L chains and a small amount of uncleaved fusion protein. While the resulting L chains present in such mixtures have been shown to possess the desired activities, we have also employed an additional purification step. Accordingly, the mixture of cleavage products is applied to a second amylose affinity column that bound both the MBP and uncleaved fusion protein. Free L chains are not retained on the affinity column, and are isolated for use in experiments described below.
• This Example describes a method to produce and purify wild-type recombinant BoNT/A light chains from bacterial clones.
• Pellets from 1 liter cultures of bacteria expressing the wild-type BoNT/A-L chain proteins are resuspended in column buffer [10 mM Tris-HCI (pH 8.0), 200 mM NaCI, 1 mM EGTA and I mM DTT] containing 1 mM phenyl-methanesulfonyl fluoride (PMSF) and 10 mM benzamidine, and lysed by sonication.
• the lysates are cleared by centrifugation at 15,000 x g for 15 minutes at 4°C.
• Supematants are applied to an amylose affinity column [2x10 cm, 30 ml resin] (New England BioLabs; Hitchin, UK). Unbound proteins are washed from the resin with column buffer until the eluate is free of protein as judged by a stable absorbance reading at 280 nm. The bound MBP-L chain fusion protein is subsequently eluted with column buffer containing 10 mM maltose. Fractions containing the fusion protein are pooled and dialyzed against 20 mM Tris-HCI (pH 8.0) supplemented with 150 mM NaCI, 2 mM, CaCI 2 and 1 mM DTT for 72 hours at 4°C.
• Tris-HCI pH 8.0
• Fusion proteins are cleaved with Factor X 2 (Promega; Southampton, UK) at an enzyme:substrate ratio of 1 :100 while dialyzing against a buffer of 20 mM Tris-HCI (pH 8.0) supplemented with 150 mM NaCI, 2 mM, CaCI 2 and 1 mM DTT. Dialysis is carried out for 24 hours at 4°C. The mixture of MBP and either wild-type or mutant L chain that resulted from the cleavage step is loaded onto a 10 ml amylose column equilibrated with column buffer. Aliquots of the flow through fractions are prepared for SDS-PAGE analysis to identify samples containing the L chains.
• a sensitive antibody-based assay is developed to compare the enzymatic activities of recombinant L chain products and their native counterparts.
• the assay employed an antibody having specificity for the intact C-terminal region of SNAP-25 that corresponded to the BoNT/A cleavage site.
• Western Blotting of the reaction products of BoNT/A cleavage of SNAP-25 indicated an inability of the antibody to bind SNAP- 25 sub-fragments.
• the antibody reneurotoxin employed in the following Example detected only intact SNAP-25.
• the loss of antibody binding served as an indicator of SNAP-25 proteolysis mediated by added BoNT/A light chain or recombinant derivatives thereof.
• This Example describes a method to demonstrate that both native and recombinant BoNT/A-L chains can proteolyze a SNAP-25 substrate.
• a quantitative assay is employed to compare the abilities of the wild-type and their recombinant analogs to cleave a SNAP-25 substrate.
• the substrate utilized for this assay is obtained by preparing a glutathione-S- transferase (GST)-SNAP-25 fusion protein, containing a cleavage site for thrombin, expressed using the pGEX-2T vector and purified by affinity chromatography on glutathione agarose.
• GST glutathione-S- transferase
• the SNAP-25 is then cleaved from the fusion protein using thrombin in 50 mM Tris-HCI (pH 7.5) containing 150 mM NaCI and 2.5 mM CaCI 2 (Smith et al., Gene 67:31 (1988)) at an enzyme:substrate ratio of 1:100. Uncleaved fusion protein and the cleaved glutathione- binding domain bound to the gel. The recombinant SNAP-25 protein is eluted with the latter buffer and dialyzed against 100 mM HEPES (pH 7.5) for 24 hours at 4°C. The total protein concentration is determined by routine methods.
• CANQRATKMLGSG SEQ ID#3
• This peptide corresponded to residues 195 to 206 of the synaptic plasma membrane protein and an N-terminal cysteine residue not found in native SNAP-25.
• the synthetic peptide is conjugated to bovine serum albumin (BSA) (Sigma; Poole, UK) using maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as a cross- linking neurotoxin (Sigma; Poole, UK) to improve antigenicity (Liu et al., Biochemistry 18:690 (1979)1.
• Affinity purification of the anti-peptide antibodies is carried out using a column having the antigenic peptide conjugated via its N-terminal cysteine residue to an aminoalkyl agarose resin (Bio-Rad; Hemel Hempstead, UK), activated with iodoacetic acid using the cross-linker ethyl 3-(3-dimethytpropyl) carbodiimide.
• the peptide-specific antibodies are eluted using a solution of 100 mM glycine (pH 2.5) and 200 mM NaCI, and collected in tubes containing 0.2 ml of 1 M Tris-HCI (pH 8.0) neutralizing buffer. All recombinant preparations containing wild-type L chain are dialyzed overnight at 4°C into 100 mM HEPES (pH 7.5) containing 0.02% Lubrol and 10 ⁇ M zinc acetate before assessing their enzymatic activities. BoNT/A, previously reduced with 20 mM DTT for 30 minutes at 37°C, as well as these dialyzed samples, are then diluted to different concentrations in the latter HEPES buffer supplemented with 1 mM DTT.
• Reaction mixtures include 5 ⁇ l recombinant SNAP-25 substrate (8.5 ⁇ M final concentration) and either 20 ⁇ l reduced BoNT/A or recombinant wild-type L chain. All samples are incubated at 37°C for 1 hour before quenching the reactions with 25 ⁇ l aqueous 2% trifluoroacetic acid (TFA) and 5 mM EDTA (Foran et al., Biochemistry 33:15365(1994)). Aliquots of each sample are prepared for SDS-PAGE and Western blotting with the polyclonal SNAP-25 antibody by adding SDS-PAGE sample buffer and boiling. Anti-SNAP-25 antibody reactivity is monitored using an ECL detection system and quantified by densitometric scanning.
• TFA trifluoroacetic acid
• Purified H Chain Native H and L chains are dissociated from BoNT/A (List Biologicals Inc.; Campbell, USA) with 2 M urea, reduced with 100 mM DTT and then purified according to established chromatographic procedures (Kozaki et al., Japan J. Med. Sci. Biol. 34:61 (1981); Maisey et al., Eur. J. Biochem. 177:683 (1988)). Purified H chain is combined with an equimolar amount of either native L chain or recombinant wild-type L chain.
• Reconstitution is carried out by dialyzing the samples against a buffer consisting of 25 mM Tris (pH 8.0), 50 ⁇ M zinc acetate and 150 mM NaCI over 4 days at 4°C. Following dialysis, the association of the recombinant L chain and native H chain to form disulfide-linked 150 kDa dichains is monitored by SDS-PAGE and quantified by densitometric scanning. The proportion of dichain molecules formed with the recombinant L chains is lower than that obtained when native L chain is employed. Indeed, only about 30% of the recombinant wild-type or mutant L chain is reconstituted while >90% of the native L chain reassociated with the H chain. In spite of this lower efficiency of reconstitution, sufficient material incorporating the recombinant L chains is easily produced for use in subsequent functional studies.

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